relatório actividade

Transcrição

RELATÓRIO
DE
ACTIVIDADE
2013
Relatório de Actividade
Introdução
Produção Científica
Research Groups
Cancer Biology
Cancer Drug Resistance
Cancer Genetics
Differentiation and Cancer
Expression Regulation in Cancer
Genetic Diversity
Glycobiology in Cancer
Population Genetics
Proteolysis in Diseases
Post-Graduation Unit
Outreach Activities
Science Diffusion
Public Awareness of Cancer
IPATIMUP Diagnostics
Innovation & Translation
IPATIMUP Innovation
IPATIMUP Translation
Internal Services
Animal Model Service
Cell Lines Bank
In vivo CAM Assays
Proteomics Service
Sequencing Service
Core Services
Informatics
Programs Office
Risk Management System
Secretary General
Technical Body
ANNEXES
Recent PhDs
Research Projects
Scientific Papers
Members of IPATIMUP at Editorial Boards
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Introdução
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Introdução
O Ipatimup tem como objectivos fundamentais da sua actividade a investigação nas áreas da Oncologia (cancro e lesões
precancerosas da tireóide, estômago, colon e mama, entre outros) e nas da Genética Populacional e Forense.
O Ipatimup tem 10 grupos de investigação, que contabilizam aproximadamente 200 pessoas entre investigadores,
docentes e bolseiros.
No ano de 2013, o IPATIMUP atingiu o número de 163 artigos publicados em revistas internacionais indexadas. Dos 163
artigos, foram publicados 27 em revistas com Factor de Impacto (FI) superior a 6; 74 em revistas com FI entre 3 a 6; 31
em revistas com FI entre 1 e 3.
Em 2013, doutoraram-se 13 elementos do Ipatimup, 11 pela Universidade do Porto através de quatro das suas Faculdade e dois pela Universidade de Leeds. Apesar deste indicador ter diminuído (em 2012 tivemos 20 doutoramentos)
mantém-se muito elevado, se tivermos em conta o número de investigadores séniores que trabalham no Ipatimup.
Os primeiros resultados da constituição da Unidade Ipatimup Translação e da Unidade Ipatimup Inovação são visíveis em
2013. O Ipatimup demonstrou ser capaz de completar o ciclo de inovação e de produzir diversos “outputs” económicos
a partir das suas actividades de investigação.
A Unidade de Inovação requereu seis registos de patentes, dois dos quais na fase internacional e assinou dois acordos
de licenciamento, um a uma empresa dinamarquesa e o outro a uma empresa espanhola. Quanto ao acesso a capital de
risco e para projectos de base tecnológica em fases de “proof of concept, seed e/ou early stage” a Unidade obteve quatro
financiamentos: dois projectos QREN (Vales Inovação e Empreendedorismo), um projecto exploratório da Fundação
para a Ciência e a Tecnologia (FCT) em colaboração com o INEB, e obteve o prémio prova de conceito no concurso
de inovação Arrisca C. Em 2013, foram criadas duas novas empresas “spin-off”, que actuam na área de prestação de
serviços de apoio a actividades de investigação.
A Unidade de Translação contratualizou três projectos de investigação, dois com a indústria farmacêutica e um terceiro
com uma empresa dedicada à área da genómica e em conjunto com a organização chinesa BGI (Beijing Genomics Institute). Estes projectos vieram contribuir para o objectivo da diversificação das fontes de financiamento do Instituto,
procurando colmatar algumas dificuldades decorrentes da redução de financiamento da FCT. Esta Unidade assegurou
ainda em 2013 a organização do Porto Cancer Meeting, sobre o tema Translational Research in Cancer, onde participaram cientistas, clínicos e profissionais da indústria dedicados ao desenvolvimento e aplicação de novos medicamentos
para doentes com cancro.
O Ipatimup reforçou as suas competências na área da Sequenciação de Nova Geração e registou, em 2013, um aumento
significativo de pedidos de análise provenientes de vários hospitais e outras instituições nacionais e estrangeiras. Este
aumento é em parte devido às vantagens na utilização das novas tecnologias no que se refere à complexidade das
doenças de base genética que podem ser estudadas e à possibilidade de desenvolvimentos científicos tendo em vista
a medicina personalizada.
Em 2013 foi extinto um grupo de investigação, Proteolysis in Diseases, e terminaram os contratos de trabalho com os
10 investigadores doutorados celebrados no âmbito do Contrato-Programa com a Fundação para a Ciência e Tecnologia
para Contratação de Doutorados para o SCTN – Ciência 2007. Sete destes investigadores continuam actualmente no
Ipatimup com diversos tipos de contrato de trabalho ou contratos de bolsa.
Em 2013 foi criado o grupo de investigação, Genetic Dynamics of Cancer Cells (Leader: José Carlos Machado), que visa
contribuir para o esclarecimento do modo como as mutações genéticas ocorrem nas células tumorais e como são transmitidas as subpopulações de “células-filhas” e como essa dinamica influencia, e é influenciada, por eventos clínicos
relacionados com a terapeutica e/ou a progressão da doença.
Iniciaram-se em 2013, 23 projectos de investigação dos quais 12 financiados pela FCT, um financiado pela Agência de
Inovação, três resultantes de contratos de investigação contratada com a indústria, uma Marie Curie Training Network,
dois projectos contratados com outras entidades internacionais e quatro financiados pelo ON.2. Refira-se a importância destes últimos para a manutenção dos níveis de actividade de investigação no IPATIMUP, num ano de descida do financiamento estratégico proveniente da Fundação para a Ciência e a Tecnologia em 25% e do término dos contratos de
Investigador celebrados no âmbito do Programa Ciência 2007. Os três projectos que compõem o Programa Integrado
de IC&DT - do Eixo Prioritário I - Competitividade, Inovação e Conhecimento do ON.2 - Programa Operacional Regional
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do Norte, com o título “Translational research on cancerous and pre-cancerous lesions” permitiram a contratação de
investigadores e a atribuição de bolsas, o que contribuiu decisivamente para a sustentação do ambiente científico do
Ipatimup.
O Ipatimup celebrou quatro consórcios com entidades nacionais para criação de redes de partilha de infra-estruturas
científicas, a integrar no Roteiro Nacional de Infra-estruturas de Investigação de Interesse Estratégico, no âmbito do
concurso público lançado pela Fundação para a Ciência e a Tecnologia. O Ipatimup integrou candidaturas nas áreas
da genómica, microscopia avançada, proteómica e biobancos, tendo três delas obtido parecer favorável para uma
segunda fase do concurso, onde se decidirá a atribuição do financiamento.
A instituição de acolhimento Ipatimup “ganhou”, em 2013, cinco posições no âmbito do Concurso Investigador FCT,
cujos contratos se iniciaram já em 2014.
O Ipatimup participou em oito programas doutorais da Universidade do Porto, destacando-se, desde 1996, o GABBA Graduate Program on Basic and Applied Biology Areas – em conjunto com as Faculdades de Medicina e de Ciências da
Universidade do Porto, o ICBAS e o IBMC. Participou na primeira edição, em 2013/2014, do BiotechHealth - Programa
Doutoral em Biotecnologia Molecular e Celular Aplicada às Ciências da Saúde – em conjunto com o ICBAS e a Faculdade
de Farmácia da Universidade do Porto, o INEB, o IBMC, o REQUIMTE e o Centro Hospitalar do Porto.
O Ipatimup continuou a acolher e a apoiar a Associação Portuguesa de Investigação em Cancro- ASPIC, enquanto rede
cientifica nacional ligada à instituição europeia com maior visibilidade na área do cancro, a EACR - European Association
for Cancer Research. A primeira Conferência da ASPIC realizou-se no Porto em Novembro de 2013, sob o tema “Public
Benefit of Cancer Research”.
O Ipatimup continuou a manter uma estreita colaboração com o Health Cluster Portugal (HCP) - Pólo de Competitividade em Saúde, quer isoladamente, quer em articulação com o IPO-Porto (Consórcio IPATIMUP–IPO) e o Centro Hospitalar de S. João (Protocolo de colaboração).
O Ipatimup promoveu, em Março de 2013, a site visit anual regular do seu External Advisory Board.
Finalmente, intensificaram-se em 2013 os trabalhos de preparação da construção e da implementação do I3S - Instituto
de Investigação e Inovação em Saúde da U.Porto. Progrediu a obra de construção do edifício I3S e foi assinado o Contrato de Consórcio entre a UP e IBMC, INEB e Ipatimup, tendo por objectivo a finalização da construção do edifício-sede
do I3S, o equipamento do edifício e o modelo de articulação das competências existentes na U.Porto no domínio das
Ciências da Saúde. O Consórcio começou a preparar o futuro sistema para a cooperação interinstitucional na promoção
da formação pós-graduada, da cultura científica, do desenvolvimento tecnológico e da inovação no universo da biomedicina, fomentando a qualificação dos recursos humanos e a internacionalização.
A FCT lançou um aviso para avaliação das unidades de investigação científica que beneficiam do estatuto de Laboratório
Associado, com a opção de, seguindo critérios de aproveitamento racional de recursos e infraestruturas, as unidades
poderem optar por manter a composição e organização existentes ou reorganizarem-se noutra configuração. O
Ipatimup, o IBMC e o INEB optaram por apresentar uma candidatura conjunta, que integrou ainda a Universidade do
Porto, sobretudo através da respectiva Faculdade de Medicina. Desta candidatura conjunta resultou o registo de uma
nova Unidade de I&D, o Instituto de Investigação e Inovação em Saúde, tendo como instituição responsável pela Gestão
a Universidade do Porto.
Este concurso propiciou a aceleração dos trabalhos de preparação para implementação do I3S, quer fisicamente, quer
em termos de organização científica, administrativa e logística. Foram criados grupos de trabalho interinstitucionais
para áreas específicas com o objectivo de proporem os futuros modelos de organização e governação ao Conselho de
Gestão e Orientação do I3S.
Da preparação desta candidatura resultou a elaboração de um Programa Estratégico para o período 2015-2020,
agrupado em três linhas:
1)
2)
3)
Programa Integrado no domínio do Cancro
Programa Integrado no domínio da Resposta e Interacção do Hospedeiro com Microorganismos
Programa Integrado no domínio da Neurobiologia e Doenças Neurológicas.
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Reescrevemos aqui o Abstract apresentado na candidatura à FCT de forma a proporcionar uma visão de conjunto.
“O Instituto de Investigação e Inovação em Saúde (I3S) visa dar resposta a problemas de saúde importantes,
nomeadamente o cancro, as doenças neurodegenerativas e infecciosas e a regeneração de tecidos, reunindo, para tal,
investigadores com reconhecimento internacional em investigação básica, de translação e clínica”.*
Os grandes desafios que se colocam nestas áreas, e que serão abordados por equipas multidisciplinares agrupadas
em três grandes Programas Integrados, são: (i) o controlo do crescimento anormal dos tecidos no cancro; (ii) a
modulação da resposta do hospedeiro no contexto de infecção, inflamação e reparação/regeneração de tecidos; (iii) a
promoção do crescimento e da regeneração nervosa após lesão ou neurodegeneração.
Para atingir estes objectivos, o I3S reúne três instituições (Instituto de Biologia Molecular e Celular – IBMC; Instituto de
Engenharia Biomédica – INEB; e Instituto de Patologia e Imunologia Molecular da Universidade do Porto – Ipatimup), e
outros grupos de investigação da Universidade do Porto.
O Programa Integrado em Cancro envolverá grupos de investigação que trabalham em divisão celular, ciclo celular,
oncobiologia, patologia, biologia molecular e celular, genética do cancro e genética populacional e metabolismo,
em articulação estreita com grupos de investigação em bioengenharia. Este Programa tem como objectivo
identificar fatores de risco que levam à transformação de células e tecidos, bem como os mecanismos celulares e
moleculares envolvidos no desenvolvimento e progressão do cancro. O Programa desenvolverá, ainda, sistemas
baseados em nanopartículas funcionalizadas com sondas moleculares, capazes de visar especificamente células
tumorais invasivas, com o objectivo de promover um diagnóstico precoce*. O Programa Integrado em Resposta e
Interação do Hospedeiro abrange grupos de investigação que trabalham em infecção, imunidade, genética e medicina
regenerativa. Este Programa tem como objetivo decifrar e modular a interação que ocorre entre o sistema imune do
hospedeiro e entidades externas, nomeadamente agentes patogénicos e materiais implantados. Serão desenvolvidos
sistemas inovadores de prevenção, diagnóstico e terapêuticos, baseados em libertação controlada de fármacos, para
combater infeções, bem como terapias regenerativas baseadas em células e biomateriais. O Programa Integrado
em Neurobiologia e Doenças Neurológicas envolverá grupos de investigação que desenvolvem a sua atividade em
neurociências, genética, estrutura de proteínas e medicina regenerativa. O Programa focar-se-á na compreensão da
biologia dos neurónios e células da glia, nas causas e nos mecanismos da disfunção e degeneração neuronal, explorando
ainda o uso de biomateriais na regeneração nervosa e no desenvolvimento de estratégias terapêuticas para aliviar ou
retardar o início de doenças neurológicas.
O I3S continuará também a implementar uma política de valorização da investigação e dos serviços para a
comunidade, envolvendo o público em geral e organizações de pacientes, além de desempenhar um papel central
na promoção da cultura científica, dentro de um contexto que envolve escolas e sociedade.”*
*Apenas estão sublinhadas as secções que se prendem com o cancro e as doenças pré-cancerosas, embora os investigadores dos grupos na área da genética populacional estejam também
integrados nos outros dois Programas.
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No ano de 2013, o IPATIMUP atingiu o número de 163 artigos publicados em revistas internacionais indexadas. Dos 163 artigos, foram
publicados 27 em revistas com Factor de Impacto (FI) superior a 6; 74 em revistas com FI entre 3 a 6; 31 em revistas com FI entre 1 e 3.
O gráfico seguinte ilustra a evolução do número de artigos publicados e de citações desde 2003, de acordo com o Factor de Impacto
das respectivas revistas:
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Research Groups
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Cancer Biology
Objectives
The general subject of study of the group is the identification of molecular mechanisms of human cancer with potential applications
in the diagnosis, prognosis and therapy, using as biologic models, thyroid and other neuroendocrine tumors. Besides the component
of translational research, the group has basic research interests such as oncogenic signaling, survival mechanisms and mechanisms/
molecules involved in mobility and invasion. Within this frame, a particular attention is paid to: a)Signalling induced by genetic
alterations in tyrosine kinase receptors and signal transducing molecules involved in the MAPK and the PI3K/mTOR pathway; b)
survival mechanisms of cancer cells, including telomerase reactivation and apoptosis dysregulation; c) molecular mechanisms of
metabolic alterations secondary to mitochondrial DNA mutations/deletions or to mutations in nuclear genes encoding metabolic
enzymes .
In addition the group has a fundamental, scientific interest in some aspects of cell cycle, tumour-microenvironment interactions and
motility/invasiveness processes in cancer.
Main research topics:
1. Oncobiology and genetics of familial and sporadic thyroid tumours
2. Clinico-pathologic and molecular characterization of thyroid cancers (PTC and Hurthel cell tumours) and of some neuroendocrine
tumours (GEP-NET, paraganglioma, melanomas)
3. Role of mitochondrial alterations in the etiopathogenesis of sporadic, familial and irradiation-induced thyroid tumours
4. Role of mitochondrial alterations in the cancer-associated metabolic switch
Main Achievements
Clinico-pathological studies on thyroid tumors
Thyroglobulin (Tg) levels measured at the time of remnant ablation after thyroid hormone withdrawal (THW) were shown to have
prognostic value in predicting disease-free status. We performed a prospective observational study in order to determine whether
stimulated Tg levels, measured at the time of remnant ablation performed under recombinant human TSH (rhTSH) stimulation, has
value in predicting absence of detectable disease 1 year after radioiodine therapy Our results show that when rhTSH was used,
stimulated Tg at ablation had independent predictive value for disease-free status 1 year later. A low stimulated Tg at rhTSH-aided
ablation may be considered a favorable prognosis factor (Melo M et al, J Clin Endocrinol Metab. 2013).
Clinico-pathological studies on rare variants of thyroid tumors
The rare reports of primary, nonneuroendocrine small cell carcinomas of the thyroid have not provided enough evidence to
support the recognition of these tumors as an entity or to understand their etiopathogenesis. We report two cases of a primary,
nonneuroendocrine small cell carcinoma of the thyroid displaying diffuse expression of cytokeratins, CD99, and p63, in the absence of
vimentin expression, in a 24-year-old male who is alive without any signs of disease 13 years after total thyroidectomy and radioactive
iodine. The tumor disclosed the EWSR1-FLI1 rearrangement, and we propose to designate it as a carcinoma of the thyroid with Ewing
family tumor elements. (Eloy C, Int J Surg Pathol. 2013).
The small cell group of thyroid tumors that includes lymphoma, poorly differentiated carcinoma, medullary carcinoma, secondary
neoplasms, as well as tumors with uncertain histogenesis, remains as a valid diagnostic cul-de-sac due to its heterogeneous
constitution. The existence of small cell thyroid tumors with EWSR1-FLI1 rearrangement together with neuroendocrine and/or
carcinomatous differentiation raises not only differential diagnostic problems but also a very interesting therapeutic dilemma (Eloy
C, Int J Surg Pathol. 2013).
Cribriform-morular variant of papillary thyroid carcinoma (CMVPTC) usually occurs in the setting of familial adenomatous polyposis
(FAP) although it can rarely arise sporadically. Poorly differentiated thyroid carcinoma (PDTC) is a follicular cell-derived neoplasm with
more aggressive behavior than well-differentiated carcinomas such as CMVPTC. We reported for the first time a case of a sporadic
CMVPTC with transformation into PDTC. The tumor was composed of CMVPTC and PDTC components that shared the same somatic
APC gene mutation (p.Cys520Tyr_fsX534). Although the majority of CMVPTCs carry an indolent clinical outcome, the coexistence of
poorly differentiated areas may justify the aggressiveness of the CMVPTC reported here (Nakazawa T et al, I
Dissecting molecular pathways in BRAF activated tumours
Somatic mutations in GNAQ gene were described as being the main oncogenic activation in uveal melanomas, whereas mutations
in BRAF gene have been described as a key genetic alteration that contributes to skin melanoma development. We have previously
reported differential activation of the MAPK and AKT/mTOR signalling pathways in uveal and skin melanomas harbouring, respectively,
GNAQ and BRAF mutations. We compare the functional effect of GNAQ and BRAF mutations in mTOR and MAPK pathway activation,
cell proliferation and apoptosis and treated melanoma cell lines displaying different BRAF and GNAQ mutational status with the mTOR
inhibitor RAD001 and with the MEK1/2 inhibitor U0126 to evaluated the effects in the growth of the cell lines and in mTOR and MAPK
pathway effectors expression. Our results indicate that GNAQ and BRAF activation drive distinct intracellular signalling pathways that
may be useful for therapeutic decisions in human melanomas. (Populo H et al, PeerJ. 2013)
Dissecting the genetics of human cancers
Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood.
We reported the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%),
bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter
mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in
glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid
cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. In summary, our data
identify TERT mutations as common events in human cancers and support the assumption that TERT promoter mutations may be one
of the mechanisms that underlies telomerase reactivation in several types of human tumours (Vinagre J et al, Nat Commun. 2013)
Irradiation and cancer
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An elevated prevalence of BCC has been associated with radiation, namely after the Tinea capitis epilation treatment. We evaluate
BCC histological subtypes in individuals subjected to X-ray epilation for Tinea capitis treatment and compare to non-irradiated
patients. Moreover we also evaluate mitochondrial D-Loop instability in both groups of BCCs in order to compare the frequency of
D-Loop mutations in post-irradiation BCC versus sporadic BCC.The infiltrative subtype of BCC, considered to be more aggressive, was
significantly more frequent in irradiated patients. BCC D-Loop D310 mutation rate was significantly higher in irradiated BCCs than in
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the non-irradiated ones. Moreover, it was associated with a higher irradiation dose. Our results suggest that radiation-induced BCCs
may be considered to be more aggressive tumors. Further studies are needed to clarify the role of mtDNA D-Loop mutations in tumors
from irradiated patients (Boaventura P et al, J Dermatol Sci.2013) .
Exposure to ionizing radiation at young age is the strongest risk factor for the occurrence of papillary thyroid carcinoma (PTC). RET/PTC
rearrangements are the most frequent genetic alterations associated with radiation-induced PTC, whereas BRAF and RAS mutations
and PAX8-PPARG rearrangement have been associated with sporadic PTC. We search for such genetic alterations in PTCs of patients
subjected in childhood to scalp irradiation. The prevalence of BRAF(V600E) mutation in our series is the highest reported in series of
PTCs arising in radiation-exposed individuals. The prevalence of RET/PTC1 rearrangement fits with the values recently described in a
similar setting (Boaventura P et al, Eur J Endocrinol. 2013) .
Mitochondrial alterations and cancer - Role of mtDNA mutations in tumourigenesis and in oncocytic
transformation
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, disclosing
somatic KIT, PDGFRA and BRAF mutations. Loss of function of the mitochondrial proteins of the succinate dehydrogenase (SDH)
complex is an alternative molecular mechanism in GISTs, namely in carriers of germline mutations of the SDH complex that develop
Carney-Stratakis dyad characterized by multifocal GISTs and multicentric paragangliomas (PGLs). We studied a series of 25 apparently
sporadic primary wild-type (WT) KIT/PDGFRA/BRAF GISTs occurring in patients without personal or familial history of PGLs, reevaluated clinicopathological features and analyzed molecular alterations and immunohistochemistry expression of SDH complex.
SDHB expression was absent in 20% and SDHB germline mutations were detected in 12% of WT GISTs. Germline SDHB mutations were
significantly associated to younger age at diagnosis. Our results confirm the occurrence of germline SDH genes mutations in isolated,
apparently sporadic WT GISTs. WT KIT/PDGFRA/BRAF GISTs without SDHB or SDHA/SDHB expression may correspond to CarneyStratakis dyad or Carney triad (Celestino R et al, Eur J Hum Genet. 2013)
Internationalization/Networking
National Collaborations
Ana Teixeira - Animal Cell Technology Laboratory, Institute of Experimental and Technological Biology, Oeiras, Portugal
Arnaldo Videira - ICBAS, Porto, Portugal.
Carlos Palmeira & Ana Bela Rolo- Centre for Neuroscience and Cell Biology (CNC), Coimbra, Portugal.
Cristina Rego - Faculty of Medicine and Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
Isabel Bento - Instituto de Tecnologia Química e Biológica (ITQB), Oeiras, Portugal.
Isabel Torres – Department of Endocrinology, Portuguese Institute of Oncology, Porto, Portugal
Maria Oliveira - Biomaterials Division, NEWTherapies Group, Institute of Biomedical Engineering, Porto
Micaela Fernandes - Instituto Tecnológico e Nuclear/Instituto Superior Técnico, Lisboa.
Nuno Carinhas - Institute of Experimental and Technologic Biology (IBET), Oeiras.
Raquel Soares - Biochemistry Department, Faculty of Medicine of the University of Porto
Rui Vaz –Department of Neurosurgery, Hospital S. João, Porto
International Collaborations
Lothe RA, Skotheim RI- Department of Cancer Prevention of the Institute for Cancer Research of the Norwegian Radium Hospital of
Oslo University Hospital.
Keshav K. Singh - Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, USA
John Blenis – Department of Cell Biology, Harvard Medical School, Boston, USA
Keshav Singh - Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, USA.
Dhan Kalvakolanu - Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore.
Daniele de Sanctis - European Synchrotron Radiation Facility (ESRF), Grenoble, France.
Cameselle-Teijeiro JM and Clara Alvarez - University of Santiago de Compostela, Santiago de Compostela.
Etel Gimba - Universidade Federal Fluminense and Instituto Nacional do Câncer, Rio de Janeiro - Brazil
Robert Hofstra from the University Medical Center Groningen, Holland.
Luca Scorrano - Department of Cell Physiology and Metabolism of the University of Genev
Future Research
Tert promoter mutations in human cancer: cellular and clinical implications.
In the field of molecular characterization human tumours, we have recently identified, for the first time, TERT promoter mutations in
several human cancers including thyroid, melanoma and gliomas. These mutations, that create new binding sites for ELK transcription
factors, seem to cooperate with BRAF in the transformation of thyroid cells and melanocytes. We will focus our efforts in the
understanding of the effect of these mutations in terms of cancer cell biology, as well as in the diagnostic and prognostic value of
these alterations. This work will use several tumour models (thyroid, melanoma, glioma and bladder)
Non-canonical BRAF signalling in thyroid cancer: Cellular and animal models
Our studies in non-canonical BRAF signalling, namely in the AKT/mTOR pathway, will be pursued by exploring the relationship between
BRAF/mTOR activation in thyroid tumours and the expression of genes involved in iodine metabolism (NIS). We have also recently
started a project concerning the role of BRAF mutations in tumor microenvironment modification, namely the changes in matrix
proteins such as osteopontin. We have been developing an animal model of BRAF induced thyroid cancer - BRAF-transgenic zebrafis
- in collaboration with IGC.
LRP1B in cancer: a preditive therapeutic marker?
Following the discovery of LRP1B as a putative tumour suppressor gene in thyroid cancer, we intend to pursue this work by developing
new diagnostic and therapeutics tools. Our findings open the possibility of using LRP1B activity as a tool for exploring a novel
therapeutic approach that is not centered on the cancer cell itself, but rather in its environment. The future challenge, is therefore to
use the newly identified LRP1B to design strategies for therapeutic intervention. We are developing and testing specific monoclonal
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antibodies for detection of LRP1B. These antibodies have potential for clinical use since can be predictive for lipossomal therapeutic
strategies response.
Irradiation and cancer
We are still following-up a cohort of individuals that suffered epilation by scalp X-ray irradiation for Tinea capitis treatment. We will
continue to collect clinical data, concerning the incidence of neoplasias in these individuals as well as other (cardiovascular) health
disturbances. Additional studies will be conducted in order toidentify and characterize immunological and genetic risk factors for
radiation-associated diseases
Cancer metabolism and epigenetic changes
We would like to prove that OXPHOS/Krebs cycle dysfunction modifies the epigenetic landscape of the nuclear genome, such as DNA
methylation and protein acetylation. Since the enzymes that carry out these epigenetic modifications use, as substrates, several
metabolites whose levels are dynamically regulated by alterations in cellular metabolism, it is tempting to predict that mitochondrial
dysfunction is able to alter the epigenetic landscape of the cell. We already have preliminary results which show that OXPHOS/Krebs
cycle dysfunction change the levels of DNA methylation. We are also analyzing the metabolic alterations associated with the induction
of EMT, namely in a model of ERK2 and c-met induction.
Mitochondrial dysfunction in cancer and Warburg effect
Concerning the role of mitochondria and metabolism in cancer and using the cybrid cell lines with OXPHOS deficiency, we want to:
verify if the mutation/downregulation of Krebs cycle and OXPHOS proteins underlie the Warburg effect in tumor cells; verify if the
Warburg effect confers selective advantage to tumor cells; thoroughly characterize the metabolic pathways that are on the basis of
the Warburg effect and may serve as new targets for cancer therapy. We intend to do an in depth metabolic characterization, in vitro
and in human thyroid tumors, and to correlate these data with the presence/absence of activated oncogenes
Participation in PhD Programs
Valdemar Máximo, Jorge Lima and Manuel Sobrinho-Simões.Metabolism and Cancer integrated in the Oncobiology module of the
Doctoral Programme in Molecular Medicine and Oncology (IPATIMUP, February 2013)
Paula Soares, Jorge Lima, Valdemar Máximo and Manuel Sobrinho-Simões.Cell Growth (proliferation, apoptosis and metabolism)
integrated in the Oncobiology module of the 13th Edition of the GABBA Doctoral Programme (IPATIMUP, April 2013)
Paula Soares, Valdemar Máximo, Jorge Lima and Manuel Sobrinho-Simões.Oncobiology II – PhD Programs in Medicine and Molecular
Oncology and in Biomedicine, FMUP.
Paula Soares, Valdemar Máximo, Jorge Lima and Manuel Sobrinho-Simões. Oncobiology - PhD Program in Pathology and Molecular
Genetics, ICBAS.
Paula Soares, Valdemar Máximo, João Vinagre.Cell cycle and Apoptosis II - PhD Program in Pathology and Molecular Genetics, ICBAS.
Valdemar MáximoCoordinator, in partnership with Professor Fátima Carneiro, of the Oncobiology II Course units of the PhD
Programmes in Medicine and Molecular Oncology and Biomedicine - FMUP.
Manuel Sobrinho-Simões Coordinator of the PhD Programme in Medicine and Molecular Oncology - FMUP.
Participation in Master Programs
Valdemar Máximo. Coordinator, in partnership with Professor Fátima Carneiro, of the Oncobiology I and Oncobiology II Course units
of the PhD Programmes in Medicine and Molecular Oncology and Biomedicine - FMUP.
Paula Soares, Valdemar Máximo, João Vinagre.Cell Cell cycle and Apoptosis I - Master Program in Medicine and Molecular Oncology
and in Biomedicine, FMUP.
Paula Soares, Valdemar Máximo, Jorge Lima and Manuel Sobrinho-Simões . Master Program in Medicine and Molecular Oncology,
FMUP
Invited Talks
Helena Pópulo. Relevância da via de sinalização do mTOR em melanoma, com enfase nas implicações em prognóstico e terapêutica .
1º Simpósio Nacional de Melanoma. Lisbon, Portugal. 29/06/2013.
Ricardo Celestino. Wild-type gastrointestinal stromal tumours (GIST): molecular alterations and expression of succinate dehydrogenase
complex. IV Simpósio Técnico de Anatomia Patológica. Porto. 27/09/2013.
Prazeres H, Viegas M, Martins T. Status mutacional no cancro do pulmão, quando?. 1as Jornadas Cancro do Pulmão do IPO de Coimbra.
Hotel Tivoli de Coimbra. 01/12/2013.
Jorge Lima. Paragangliomas: da genética à clínica. II Reunião Nacional de Tumores Endócrinos. Porto. 23/02/2013.
Valdemar Máximo. Mitochondria in cancer cells. 25th European Congress of Pathology - ECP . Lisbon. 31/08/2013.
Valdemar Máximo. Alterações mitocondriais em cancro. Reuniões científicas do Centro de Investigação do IPOP. Porto, Portugal.
15/02/2013.
Miguel Melo. Carcinoma da tiróide- patologia molecular para a clínica. III Curso – Patologia Molecular em Medicina, Faculdade de
Medicina da Universidade de Coimbra. Coimbra, Portugal. 01/06/2013.
Miguel Melo. Marcadores moleculares no carcinoma da tiróide – que interesse?. Jornadas Internacionais de Cirurgia dos HUC/CHUC.
Coimbra, Portugal. 19/04/2013.
Miguel Melo. Decisão clínica com base na citologia e testes moleculares. 4º Curso De Cirurgia Endócrina Do H. S. João. Porto, Portugal.
18/10/2013.
Sobrinho-Simões M. Actualização sobre Patologia Molecular em Citologia e Histologia da Tireoide. 3º Congresso ALGEDM. Porto,
Portugal. 12/01/2013.
Sobrinho-Simões M. Health Sciences . XXI Century Challenges and Opportunities . Macau. 18/01/2013.
Sobrinho-Simões M. Practical problems in the diagnosis of thyroid tumours. FORPATH asbl Workshop. Brussels. 02/02/2013.
Sobrinho-Simões M. Relevância Clinica da Expressão Molecular do Cancro da Tireoide. 12ª Reunião Internacional de Cirurgia. Lisboa.
19/02/2013.
Sobrinho-Simões M. Investigar em tempos de crise. XX Congresso de Pneumologia do Norte. Porto. 08/03/2013.
14
2013
Sobrinho-Simões M. Tríptico: luz, corpo e MORTE. O que é a vida antes da morte? “Is there life before death?”. Serralves. Porto.
14/03/2013.
Sobrinho-Simões M. Cancer genes: Beyond the classic dual division. GABBA. Porto. 01/04/2013.
Sobrinho-Simões. Interacção genético-ambiental na saúde e na doença. Conferência Prof. Alberto Aguiar. Porto. 18/04/2013.
Sobrinho-Simões M. SAVED by the BELL. SEAP Congress . Cadis. 22/05/2013.
Sobrinho-Simões M. Thyroid tumours with a follicular pattern of growth. Long Course, SEAP Congress. Cadis. 23/05/2013.
Sobrinho-Simões M. • Thyroid tumours with a follicular pattern of growth • Poorly differentiated and undifferentiated (anaplastic)
carcinoma of the thyroid • Papillary carcinoma and its variants - Thyroid Pathology for the practicing pathologist . Paris. 06/06/2013.
Sobrinho-Simões M. Diagnostico Histopatológico Molecular em Oncologia. San Sebastian. Espanha. 03/07/2013.
Sobrinho-Simões M. Welcome Address and Chairman of the Organizing Committee . 25º Congresso Europeu de Patologia. Lisboa.
31/08/2013.
Sobrinho-Simões M. A scientific and scenic tour of Sicily • Oncocytic lesions of thyroid, kidney, and salivary glands • The importance
of understanding • Papillary thyroid microcarcinoma (PTmC): How to diagnose it and how to manage this epidemics • Rare flowers in
thyroid gland: The case of small cell tumours - International Pathology Meeting in Sicily. Sicilia. 06/10/2013.
Sobrinho-Simões M. Quem somos, de onde vimos e para onde vamos. Arouca. Portugal. 19/10/2013.
Sobrinho-Simões M. Inovação e Avanços em Oncologia: Exploração das potencialidades da interacção genetico-ambiental. Faculdade
de Medicina, Universidade do Algarve. Algarve. 08/11/2013.
Sobrinho-Simões M. O que somos? Uma viagem dos genes ao ambiente. Fundação Inês de Castro, Quinta das Lágrimas, Coimbra.
Coimbra. 07/11/2013.
Sobrinho-Simões M. O superorganismo Homem . 25 anos do Hospital da Prelada SCM. Porto. 22/11/2013.
Sobrinho-Simões M. Um em cada dois portugueses nascidos nas próximas décadas terá cancro…. GESS. Porto. 22/11/2013.
Sobrinho-Simões M. Ciência em tempo de crise. Encontro “Ciência, Economia e Crise” . Porto. 22/11/2013.
Sobrinho-Simões M. Das IDLE ao VOMIT - Os excessos a que a cancerofobia tem levado. Ordem dos Farmacêuticos. Porto. 28/11/2013.
Sobrinho-Simões M. A SAÚDE NO SEC. XXI . Desafios e oportunidades. Lisboa. 29/11/2013.
Sobrinho-Simões M. Medicina no séc. XXI: Desafios, Oportunidades e Riscos. Conferência Cunha Vaz. Vilamoura. 06/12/2013.
Oral Presentations
Catarina Eloy, Alexandra Betts & Manuel Sobrinho-Simões. Angiomatoid papillary thyroid carcinoma. 25th European Congress of
Pathology. Lisbon. 09/2013.
Catarina Eloy & Manuel Sobrinho Simões. Parathyroid hyperplasia and neoplasia - Update in differential diagnosis of parathyroid
pathology: “new and old markers”. 25th European Congress of Pathology. Lisbon. 09/2013.
Catarina Salgado, Hugo Prazeres, Paula Soares. Exome-Sequencing in familial thyroid carcinoma: genetic and functional
characterization. XXXVIII Portuguese Genetics Conference. Porto. 2013.
Pereira. S.S., Morais,T., Costa, M., Monteiro, M.P., Pignatelli, D. The use of a morphometric computerized analysis tool in the differential
diagnosis of adrenocortical tumors. European Joint Congress of Clinical Anatomy. Lisbon. 2013.
Dias A, Batista R, Lima J, Soares P, Máximo V. Insights into the regulation of GRIM-19 expression in renal cell carcinoma. ECCO17ESMO38 - ESTRO32 European Cancer Congress. Amsterdam. 2013.
Mendes A, Alvelos I., Carvalho D., Capela J., Soares P.. Sporadic Primary Hyperparathyroidism: Molecular Alterations in a Series of
Parathyroid Sporadic Lesions. XIV Congresso Português de Endocrinologia. Porto. 2013.
M. Melo, G. Costa, C. Ribeiro, F. Carrilho, M.J. Martins, A. Gaspar da Rocha, P. Soares, M. Carvalheiro. Valor preditivo da tiroglobulina
no momento da terapêutica ablativa com 131i utilizando tsh humana recombinante. XIV Congresso Português de Endocrinologia.
Porto. 01/2013.
João Vinagre. TERT promoter mutations in human cancers. I Mini Simpósio de Patologia Endócrina Celular e Molecular. Rio de Janeiro,
Brasil. 21/10/2013.
Prizes
Miguel Melo, Gracinda Costa, Cristina Ribeiro, Francisco Carrilho, Maria João Martins, Adriana Gaspar da Rocha, Manuel SobrinhoSimões, Manuela Carvalheiro, Paula Soares.Valor preditivo da tiroglobulina no momento da terapêutica ablativa com 131I utilizando
TSH humana recombinante.
1st Prize in Clinical Investigation (ex-Aequo) da Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo , XIV Congresso
Português de Endocrinologia, 2013
Pereira. S.S., Morais,T., Costa, M., Monteiro, M.P., Pignatelli, D.Immunohistochemistry markers in the differential diagnosis of
adrenocortical tumors
Prémio Prof. Manuel Pinheiro Hargreaves. Melhor Póster na área da endocrinologia, Diabetes e Metabolismo no XIX Curso Pós
Graduado de Endocrinologia, Diabetes e Metabolismo, Março de 2013
15
2013
Cancer Drug Resistance
Objectives
The Cancer Drug Resistance group is mainly focused on translating basic science findings related to drug resistance into validation
of potentially new molecular targets for cancer therapy, such as anti-apoptotic molecules and some microRNAs, using several in
vitro models for different cancer types. Also, in an attempt to counteract cancer drug resistance, we are involved in testing newly
synthesized compounds (“small molecules”) in particular sets of cancer cell lines.
Main Achievements
Understanding the role of microRNAs in Drug Resistance/Drug response
This work was aimed at investigating the role of selected microRNAs (miRs) in drug resistance/drug response, in acute myeloid
leukaemia, in collaboration with Hospital São João. The project was financed by Fundação Calouste Gulbenkian (co-PIs: Profs. M.H.
Vasconcelos and J.E. Guimarães). We found out that targeting miR-21 with antimiRs induces autophagy and chemosensitivity of
leukemia cells (Seca et al., Curr Drug Targets 2013). In addition, we have studied the role of miR-128-1 in the response of acute myeloid
leukemia cells to chemotherapeutic drugs (submitted for publication) and this work was presented at the 55th American Society
of Hematology Annual Meeting, New Orleans, December 7-10 of 2013, USA. Also, a PhD thesis was completed under the theme
“Increasing sensitivity to drugs in leukemias by modulation of microRNA expression” (Hugo Seca, 2013).
Moreover, the subject of “miRNAs in cancer drug resistance and drug sensitivity” was reviewed in a book chapter to be published by
Springer (Seca H. et al., 2014).
Role of P-glycoprotein (P-gp) in drug resistance and identification of anti-Pgp small molecules
Protocols for extracellular vesicles (EVs) isolation have been established and compared (submitted for publication). Studies on the
significance of P-gp found in EVs isolated from drug resistant cells have been initiated. In particular, we have initiated studies on how
P-gp may be responsible for the intercellular transfer of drug resistance from resistant (P-gp overexpressing) cells to drug sensitive
cells. Furthermore, the topic of interactions between P-gp and microRNAs has been reviewed (Lopes-Rodrigues V. et al., Int J Cancer,
in press).
Finally, we have continued the study of molecules, from the CEQUIMED-UP library, having dual activity: anti-Pgp and antitumor
activity (Sousa E. et al., Med Chem Res 2013).
Molecular targets involved in drug resistance in Cancer Stem Cells
The presence of targets related to drug resistance in cancer stem cells was investigated within an FCT funded project (PTDC/EBBBIO/099672/2008), in which the PI was Gabriela Almeida. This work was performed in collaboration with CEQUIMED-UP and with
Dr. L.F. Santos-Silva at IPATIMUP. Enrichment of cancer stem like-cells by incubation with chemotherapeutic agents was performed
and the cell populations selected with this strategy showed increased drug resistance, increased expression of the stemness marker
ABCG2, as well as increased expression of Notch1, Nanog, and Oct4 stemness genes and XIAP, Bcl2 and P-glycoprotein chemoresistance
proteins. The subject of “therapy-induced enrichment of putative lung cancer stem-like cells” was reviewed (Freitas D.P. et al., Int J
Cancer 2014).
Two members of this research group (G.M. Almeida and M.H. Vasconcelos) were in the organizing Committee of the 1st Working
Group Meeting of the COST Action CM1106 on “Chemical approaches to targeting drug resistance in cancer stem cells”. This meeting
was organized at IPATIMUP, February 21-22 of 2013, Portugal.
Development of small molecules with antitumor potential
In an attempt to counteract cancer drug resistance, the group was involved in testing newly synthesized compounds in particular sets
of cancer cell lines.
This work involved: A) screening of compounds with potential antitumor activity; and B) investigation of the cellular mechanism of
action of the best compounds.
Most of this work was carried out as collaboration with CEQUIMED-UP, funded by FCT (FFUP as the Proponent Institution, PI Prof. M.
Pinto, PTDC/SAU-FCF/100930/2008), or as collaboration with University of Minho (Prof. M.J. Queiroz) and Instituto Politécnico de
Bragança (Prof. Isabel Ferreira). We have identified some compounds capable of decreasing human tumor cell growth (Azevedo C. et
al., Bioorgan Med Chem 2013; Azevedo C. et al., Eur J Med Chem 2013). Other compounds were further found to interfere with cell
cycle and/or apoptosis (Pereira C. et. al., Eur J Pharmaceut Sci 2014; Queiroz M.J. et al., Eur J Med Chem 2013; Lima R.T. et al., Anticancer Agents Med Chem 2013). Additional work showed that one compound interferes with autophagy (unpublished data); this work
was presented at the 11th International Congress on Targeted Anticancer Therapies - TAT 2013, 4-6 March, Paris, France. In addition,
we have identified small molecules that were found to be inhibitors of EBV (Lima R.T. et al., Chemical Biology & Drug Design 2013). A
review on “EBV-associated cancers: strategies for targeting the virus” was written for a book chapter published by Formatex Research
Center (Lima R.T. et al., 2013).
Finally, we have been searching for small molecule modulators of p53 family proteins (project having ICETA-Porto/UP as the Proponent
Institution, PI Prof. Lucília Saraiva, PTDC/SAU-FAR/110848/2009). In this framework, the group has tested nanoparticles to improve
the effect of a new inhibitor of p53-MDM2 interaction (Paiva A. et al., Int J Pharmaceut 2013).
Searching for natural products with antitumor potential
Several studies have been carried out, in collaboration with CEQUIMED-UP or with Instituto Politécnico de Bragança, to identify
natural products with antitumor potential. Part of this work was funded by FCT (Instituto Politécnico de Bragança as Proponent
Institution, PI Prof I.C.F.R. Ferreira, PTDC/AGR-ALI/098402/2008). Several publications resulted from this work (dos Santos T. et al., J.
Funct Foods 2014; dos Santos T. et al., Food Res Int 2013; Cazal C.M. et al., Anti-cancer Agents Med Chem 2013).
European COST Action BM1202 “European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD)” - M.H.
Vasconcelos was a substitute member of the Management Committee.
European COST Action CM1106 “Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells” - .M. Almeida was a member
of the Management Committee. In addition, G.M. Almeida and M.H. Vasconcelos were members of the Organizing Committee of the
1st Working Group Meeting of COST Action CM1106 on “Chemical approaches to targeting drug resistance in cancer stem cells”, 21-22
February 2013, Porto, Portugal.
16
2013
Department of Food, Environmental and Nutritional Sciences, University of Milan, Italy (Prof. Sabrina Dallavalle) - Collaboration
through COST Action CM1106.
University of Leicester & Leicester University Hospitals, UK (Dr. Dean Fennell) - Collaboration through an FCT funded project, Reference
PTDC/EBB-BIO/099672/2008 (Dr. Dean Fennel is consultant of the project)
University of Nebraska Medical Center (Prof. Arnold Angelo Rizzino) - Collaboration through an FCT funded project, Reference PTDC/
EBB-BIO/099672/2008 (Prof. Rizzino is consultant of the project).
Department of Cancer Studies and Molecular Medicine Biocentre, University of Leicester (Prof. Margaret Manson) - Collaboration
through an FCT funded project, Reference PTDC/AGR-ALI/098402/2008 (Prof. Manson is consultant of the project).
Dublin City University and National Institute for Cellular Biotechnology, Ireland (Dr. Robert O’Connor) - Dr. O’ Connor is collaborator
of a PhD student’s project (V. Rodrigues).
CEQUIMED-UP (Prof. Madalena Pinto, Prof. Emília Sousa, Prof. Honorina Cidade, Prof. Carlos Afonso, Prof. Maria São José Nascimento,
Prof. Maurício Barbosa, Prof. Maribel Teixeira) - Collaboration through a PhD student project (V. Rodrigues), a post-doc project (R.T.
Lima) and projects financed by FCT (PTDC/SAU-FCF/100930/2008 and PTDC/EBB-BIO/099672/2008).
Hospital São João (Prof. José Eduardo Guimarães e Prof. Manuel A. Sobrinho Simões) - Collaboration through a PhD student project (H.
Seca), a visiting researcher (R. Bergantim) and a project financed by Fundação Calouste Gulbenkian.
Instituto Politécnico de Bragança (Prof. Isabel Ferreira and Prof. Anabela Martins) - Collaboration through a project financed by FCT
(PTDC/AGR-ALI/098402/2008).
Instituto Superior de Ciências da Saúde - Norte (Prof. Hassan Bousbaa and Prof. Madalena Pedro) - Collaboration through a project
financed by FCT (PTDC/SAU-FCF/100930/2008).
Universidade Fernando Pessoa (Prof. Fátima Cerqueira) - Collaboration through a project financed by FCT (PTDC/SAU-FCF/100930/2008).
Universidade do Minho, Braga (Prof. Maria João Queiroz) - Collaboration through research papers.
Universidade da Madeira, Departamento de Química e Centro de Química da Madeira (Prof. Miguel Xavier Fernandes) - Collaboration
through one project financed by FCT (PTDC/SAU-FAR/110848/2009).
REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences of FFUP (Prof. Lucília Saraiva) - Collaboration through one
project financed by FCT (PTDC/SAU-FAR/110848/2009).
Future Research
Study the role of microRNAs shed by extracellular vesicles in cancer drug resistance
We aim to confirm if some of the microRNAs that are responsible for cancer drug resistance are transferred via extracellular vesicles
from drug resistant to drug sensitive tumor cells.
Understanding the molecular mechanisms and identifying novel inhibitors of P-gp mediated cancer drug
resistance
We aim to better understand the molecular mechanisms of P-gp mediated cancer drug resistance, particularly: i) the role of P-gp in
cellular resistance to apoptosis and ii) P-gp involvement in the intercellular transfer of resistant phenotype mediated by extracellular
vesicles. In addition, we aim to identify inhibitors of these processes (PhD project of V. Rodrigues).
Identification and validation of small molecule p53 modulators
We aim to continue our collaboration with Prof. Lucília Saraiva in order to identify small molecules which are modulators of p53 family
proteins (project approved having ICETA-Porto/UP as the Proponent Institution, PI Prof. Lucília Saraiva, PTDC/SAU-FAR/110848/2009).
Investigation of the mechanism of action of a leader small molecule which induces programmed cell death
We aim to further investigate the mechanism of action of one leader molecule from the CEQUIMED-UP library, which has potent
human tumor cell growth inhibitory potential (post-doctoral project of R.T. Lima). This work is as collaboration between CEQUIMEDUP and IPATIMUP.
Identification of natural products with antitumor potential
We aim to continue identifying natural products with antitumor potential.
M. Helena VasconcelosPrograma de doutoramento em Ciências da Saúde da Faculdade de Medicina da Universidade de Coimbra
(participação no Módulo de Patologia Molecular).
M. Helena VasconcelosPrograma Doutoral em Segurança e Saúde Ocupacionais, Faculdade de Engenharia da Universidade do Porto
(participação na Unidade Curricular “Seminários Multidisciplinares”).
M. Helena VasconcelosPrograma Doutoral em Medicina e Oncologia Molecular, Faculdade de Medicina da Universidade do Porto
(participação na Unidade Curricular “Técnicas de Biologia Molecular II”).
M. Helena VasconcelosPrograma Doutoral GABBA, “Programa Graduado em Áreas da Biologia Básica e Aplicada” da Universidade do
Porto (participação no módulo de Oncobiologia).
R.T. LimaMestrado em Medicina Legal, Instituto de Ciências Biomédicas Abel Salazar.
M. Helena VasconcelosMestrado em Genética Molecular, Departamento de Biologia, Universidade do Minho (participação no curso
de pós-graduação “Mammalian and yeast as complementary cell models in programmed cell death”).
M. Helena VasconcelosMestrado em Medicina e Oncologia Molecular, Faculdade de Medicina da Universidade do Porto (participação
na Unidade Curricular “Técnicas de Biologia Molecular I”).
Invited Talks
M. Helena Vasconcelos. Aplicações práticas da cultura de células na investigação em oncobiologia. Cell Culture Course. Bragança,
Portugal. 12/04/2013.
M. Helena Vasconcelos. Resistência à terapêutica antineoplásica. VII Jornadas de Ciências. Porto, Portugal. 20/04/2013.
17
2013
R.T. Lima. História de uma pequena molécula: Autofagia na terapia do cancro. Conferências em Oncobiologia 2013 - Faculdade de
Farmácia da Universidade do Porto. Porto, Portugal. 28/05/2013.
Oral Presentations
M. Helena Vasconcelos. A new small molecule with antitumour and chemosensitizing potential as P-gp inhibitor. 1st Working
Group Meeting of COST Action CM1106 “Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells”. Porto, Portugal.
22/02/2013.
Prizes
Hugo Seca, Raquel T. Lima, Gabriela M. Almeida, Manuel Sobrinho-Simões, Rui Bergantim, José E. Guimarães, M. Helena
Vasconcelos“miR-128 induces DNA damage and sensitizes AML cells to etoposide and doxorubicin” - 2º Prémio - Poster trabalho
experimental – Reunião Anual da Sociedade Portuguesa de Hematologia 2013
18
2013
Cancer Genetics
Objectives
Epithelial homeostasis mostly depends on cellular junctions, which control highly integrated networks and constitute key protein
receptors in outside-in cellular signaling, with significant consequences on cell behavior.
The long-term goal of the Group is to uncover how epithelial cell-cell and cell-matrix junctions, as well as the surrounding
microenvironment, can influence cancer progression.
More specifically, and based on three common epithelial-derived cancers (gastric, breast and colorectal), the Group aims at establishing
the contribution of adhesion molecules (E- and P-cadherins), infections (Helicobacter pylori and the microbiota), and non-neoplastic
components of the tumor tissue (fibroblast-like cells, the cancer cell secreted peptides and the elements of the extracellular matrix),
to altered epithelial homeostasis and to cancer development.
The accomplishment of these research goals will contribute to the development of new tools for cancer screening, prevention and
patient surveillance, as well as therapeutic strategies based on the modulation of cancer cell interactions.
Main Achievements
E-cadherin-integrin-ECM crosstalk in gastric cancer
Cell-cell and cell-extracellular matrix adhesions are crucial physical interactions for tissue homeostasis and architecture. Cadherins
are central molecules in cell-cell junctions, whereas integrins play a major role in the interaction between cells and the extracellular
matrix (ECM). In cancer, E-cadherin loss and integrin dysfunction are frequent events and have been implicated in the initiation,
progression and metastasis of solid tumours. Aiming at disclosing the molecular mechanisms underlying the regulation of E-cadherinintegrin-ECM crosstalk in gastric cancer, and using an in vitro cell model, we showed that the expression of E-cadherin leads to
decreased expression of Integrin b4 and Integrin b1. We verified that Laminin g2, which is known to interact with both b1 and b4
integrins, is also downregulated in the presence of E-cadherin. Furthermore, we demonstrated that modulation of Laminin g2 by
siRNA enhances E-cadherin expression through a mechanism involving its trafficking machinery. Arf6, a key protein involved in
E-cadherin endocytosis and recycling is decreased. Simultaneously, the inhibition of Laminin g2 stimulates the expression of PIPKIg,
which is described to bind to E-cadherin promoting its transport to the plasma membrane. We present evidence of a direct influence
of E-cadherin in the expression of integrins and ECM components and the reverse, the effect of an ECM component in E-cadherin
expression and in its intracellular trafficking.
Ectopic laminin expression at the Ecad+/Ecad- interface promotes invasion and survival
We used two independent models (in vivo and in vitro) to understand what triggered laminin 332 overexpression in the context
of silencing of E-cadherin and to unveil the functional significance of its deregulation. In the Drosophila tissue context, cells with
reduced E-cadherin start dying and expressing MMP1 to degrade the basement membrane whereas, at the invasive front, cells sense
sharp differences in E-cadherin levels and ectopically express Laminin A (the fly orthologue of LAMA3). In the fly, this phenomenon
seems to be linked to integrin signaling and non-autonomous JNK activation. This was further validated with human cell lines. We
demonstrated that AGS parental cells (negative for E-cadherin) overexpressed Laminin g2 (332), MMP2, integrins (b1 and b4) when
compared to the same line stably transfected with E-cadherin. We concluded that, at the interface, the cells expressing Laminin and
integrin are those that lost E-cadherin. Laminin g2 overexpression leads to an increase in the invasive capacity and an increase in cell
survival, in accordance with pSrc and pAKT upregulation and caspase 3 dowregulation. In summary, Laminin expression at the Ecad+/
Ecad- interface is key for Ecad- cells to invade and survive.
The gastric molecular program that allows cell survival upon loss of E-cadherin-mediated adhesion
Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome caused by germline mutations in the E-cadherin
encoding gene, CDH1. What is striking is that CDH1 germline mutations confer more than 80% lifetime risk to specifically develop
gastric cancer, despite that E-cadherin is expressed in all epithelial tissues. We hypothesized that CDH1 biallelic inactivation is only
tolerated in gastric epithelium due to a favorable gastric-specific expression program that allows cell-autonomous survival.
We have characterized the survival response of gastric, breast, and colon epithelium upon in E-cadherin downregulation by siRNA. The
results obtained corroborate our hypothesis that stomach epithelium is more resistant to loss of adhesion acquiring cell-autonomous
survival capacity. Moreover, basal cell death levels upon apoptosis induction with staurosporine and taxol indeed suggest there must
be a favorable gastric-specific expression program that allows and creates the tissue specific conditions to overcome apoptosis.
Accordingly, we evaluated the expression profiles and functional significance in resistance to apoptosis induction of a series of
candidate stomach-specific genes previously selected using bioinformatics tools. Our data supports a role for S100P and CTSE in a
gastric-specific molecular program that may ultimately encompass oncogenic potential. Furthermore, through a proteomic profiler
approach, we observed a striking upregulation of anti-apoptotic proteins upon CDH1 downregulation in gastric epithelium. We
have subsequently performed functional analysis of candidate molecules and found that PON2 and XIAP may be part of the survival
molecular program underlying gastric-specific resistance to E-cadherin loss. Currently, we are further characterizing the molecular
relationship between these anti-apoptotic proteins and the gastric-specific S100P and CTSE proteins, and how they may interact and
contribute to cancer transformation.
Functional evaluation of new E-cadherin germline missense mutations
Four new E-cadherin germline missense mutations were found in gastric cancer patients, and reported to our laboratory in order to
evaluate their pathogenicity through in vitro assays and in silico bioinformatic analysis. For in vitro functional characterization, we
performed slow aggregation and matrigel invasion assays in cells transiently transfected the vectors encoding WT or the hE-cadherin
mutants. Bioinformatic prediction of the impact of each mutation was performed using SIFT, Polyphen-2 and FoldX programmes.
New bioimaging approach to distinguish WT from mutant forms of E-cadherin
We developed a bioinformatic tool that uses immunofluorescence images to quantify protein expression level and also to map the
protein at intra- and intercellular spaces. The output of the software can be analyzed by statistical methods or by constructing a
virtual cell that mimics the whole cell population examined. Using this approach, we were able to discriminate cells expressing wildtype E-cadherin from those expressing mutant forms of the protein. When compared to WT cells, we verify that mutant cells display
decreased fluorescence intensity at the membrane and present aberrant protein peaks corresponding to protein accumulation in the
perinuclear region.
DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro and in
vivo
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2013
HDGC-associated E-cadherin missense mutations can lead to folding defects and premature proteasome-dependent endoplasmic
reticulum-associated degradation (ERAD), but the molecular determinants for this fate were unidentified. Using a Drosophila-based
genetic screen, we found that Drosophila DnaJ-1 interacts with wild type (WT) and mutant human Ecad in vivo. DnaJ (Hsp40) homolog,
subfamily B, member 4 (DNAJB4), the human homolog of DnaJ-1, influences Ecad localization and stability even in the absence of Ecad
endogenous promoter, suggesting a post-transcriptional level of regulation. Increased expression of DNAJB4 leads to stabilization
of WT Ecad in the plasma membrane, while it induces premature degradation of unfolded HDGC mutants in the proteasome. The
interaction between DNAJB4 and Ecad is direct, and is increased in the context of the unfolded mutant E757K, especially when
proteasome degradation is inhibited, suggesting that DNAJB4 is a molecular mediator of ERAD. Post-translational regulation of
native Ecad by DNAJB4 molecular chaperone is sufficient to influence cell adhesion in vitro. Using a chick embryo chorioallantoic
membrane assay with gastric cancer derived cells, we demonstrate that DNAJB4 stimulates the anti-invasive function of WT Ecad
in vivo. Additionally, the expression of DNAJB4 and Ecad is concomitantly decreased in human gastric carcinomas. Altogether, we
demonstrated that DNAJB4 is a sensor of Ecad structural features that might contribute to gastric cancer progression (Simões-Correia
et al. Human Molecular Genetics 2013).
Genetic variants in the IL1A gene region contribute to gastric carcinoma susceptibility in European populations
In the context of sporadic gastric cancer, we have also analysed the influence of human genetic variation on disease risk. Although
evidence points to the IL1 region as a genetic susceptibility region involved in gastric carcinoma risk (e.g. polymorphisms in the IL1B and
IL1RN genes), definite functional variants reproducible across populations of different genetic background have not been discovered
so far. Therefore, a high density linkage disequilibrium (LD) map of the IL1 gene cluster was established using HapMap to identify
haplotype tagSNPs. 87 SNPs were genotyped in a Portuguese case-control study (358 cases, 1485 controls) for the discovery analysis.
A replication study, including a subset of those tagSNPs (n=43), was performed in an independent analysis containing individuals from
10 European countries (365 cases, 1284 controls). Single SNP and haplotype block associations were determined for overall gastric
carcinoma and anatomopathological subtypes.
The most robust association was observed for SNP rs17042407, which is located 16Kb upstream of the IL1A gene. Although several
other SNP associations were observed, only the inverse association of rs17042407 allele C with intestinal type gastric carcinoma was
observed in both studies, retaining significance after multiple testing correction (p=0.0042) in the combined analysis. The haplotype
analysis of the IL1A LD block in the combined dataset revealed the association between a common haplotype carrying the rs17042407
variant and gastric carcinoma, particularly of the intestinal type (p=3.1x10-5) and non-cardia localisation (p=4.6x10-3). These results
confirm the association of IL1 gene variants with gastric carcinoma and reveal a novel SNP and haplotypes in the IL1A region associated
with intestinal type gastric carcinoma in European populations (Durães et al. International Journal of Cancer, accepted for publication).
Strain-specific sequences at the Helicobacter pylori cagA promoter influence CagA expression and interleukin-8
secretion
CagA is an important H. pylori virulence factor and a major contributor for gastric carcinoma development. Sequence heterogeneity
in the promoter region of the cagA gene which was linked to high CagA expression and worse gastric histopathology has been recently
identified. We have characterized the H. pylori cagA promoter region, to assess the relationship between variation in this region and
CagA expression, and to investigate the influence of cagA promoter variation and CagA expression on interleukin (IL)-8 secretion in
Portuguese strains. We have confirmed that the cagA promoter showed high heterogeneity among strains, including variation in
sequence and copy number of several motifs. CagA protein expression was associated with a particular nucleotide sequence at the -10
motif (p=0.012) and with the presence of the +59 motif (p=0.003). Moreover, the levels of IL-8 secreted by gastric cells were correlated
with the levels of CagA expression (rp=0.391; p=0.009), as well as with the presence of the +59 motif in the cagA promoter (p<0.001).
Further studies are needed to confirm the usefulness of specific regions on the cagA promoter as markers to predict gastric carcinoma
risk (Ferreira et al. In preparation).
First-degree relatives of early-onset gastric cancer patients show a high risk for gastric cancer: phenotype and
genotype profile
First-degree relatives (FDR) of early-onset gastric cancer (EOGC; i.e. diagnosed before 45 years) are presumed to be a population with
a distinct molecular and phenotypic profile, regarding the prevalence of gastric premalignant conditions and the association with H.
pylori infection and host pro-inflammatory gene polymorphisms. A case-control study was conducted with FDR of EOGC patients and
age and gender matched controls. After upper endoscopy, the Operative Link on Gastritis Assessment (OLGA) system was used for
staging and the prevalence of H. pylori and of the bacterial virulence genotypes, as well as the host polymorphisms in inflammationrelated genes was determined.
In this setting, 70% of cases showed atrophy of which 19% presented high-stage gastritis (OLGA stage III or IV; p<0.001). Gastric
dysplasia was present in 7 cases (vs none in controls; p=0.007). H. pylori was present in 82% of the cases (vs 62% in controls; p=0.004)
and vacA s1/m1 strains were significantly associated with the presence of atrophy. Individuals homozygous for IL1B-511*T showed
a significantly higher risk for dysplasia. An increased global prevalence of IFNGR1-56*T/*T polymorphism (37% in cases vs 24% in
controls; p=0.03) was observed, but with no association with atrophic changes or dysplasia. These results show that FDR of EOGC
patients have an increased prevalence of high risk OLGA stages and dysplasia that may be associated with high-virulence H. pylori
strains and pro-inflammatory host genotypes. FDG of EOGC patients may deserve specific management and surveillance through
adequate endoscopic and histopathological assessment of the gastric mucosa (Marcos-Pinto et al Virchows Archiv 2013).
Helicobacter pylori infection affects mitochondrial function and DNA repair, thus, mediating genetic instability
in gastric cells
H. pylori infection is an important factor for the development of gastric cancer. However, the mechanisms that contribute for
carcinogenesis are not fully elucidated. We have previously shown that H. pylori induces genetic instability in both nuclear and
mitochondrial DNA of gastric epithelial cells. Further, we have shown that the mutagenic effect of H. pylori on nuclear DNA is a
consequence, in part, of a down-regulation of expression and activity of major DNA repair pathways. In this study, we demonstrated
that H. pylori infection of gastric adenocarcinoma cells causes mtDNA mutations and a decrease of mtDNA content. We also showed
a decrease of respiration coupled ATP turnover and respiratory capacity and, accordingly, a lower level and activity of complex I
of the electron transport chain. We also investigated if the increased mutational load in the mitochondrial genome was caused by
down-regulation of mitochondrial DNA repair pathways. Our results suggest the participation of APE-1 and YB-1, which are involved
in mitochondrial base excision repair and mismatch repair, in mtDNA repair during H. pylori infection. Furthermore, the results
demonstrated that multiple DNA repair activities are involved in protecting mtDNA during infection (Machado et al. Mechanisms of
Ageing and Development 2013).
Cholesterol uptake by Helicobacter pylori impacts resistance to docosahexaenoic acid
We have demonstrated that docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid known for its anti-inflammatory and
antitumor effects, directly inhibits H. pylori growth in vitro and in mice (Correia et al. PLOS One 2013). Nevertheless, the concentration
of DHA shown to reduce H. pylori mice gastric colonization was ineffective in vitro. Related to the auxotrophy of H. pylori for cholesterol,
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2013
we hypothesized that other mechanisms, in addition to the direct antibacterial effect of DHA, must be responsible for the reduction
of the infection burden. Therefore, we investigated if DHA affects also H. pylori growth by reducing the availability of membrane
cholesterol in the epithelial cell for H. pylori uptake. Levels of cholesterol in gastric epithelial cells and of cholesteryl glucosides in
H. pylori were determined by thin layer chromatography and gas chromatography. The consequences of cholesterol depletion in
epithelial cells on H. pylori growth were assessed in liquid cultures. We showed that H. pylori uptakes cholesterol from epithelial
cells. In addition, DHA lowered cholesterol levels in epithelial cells, decreased its de novo synthesis, leading to a lower synthesis of
cholesteryl glucosides by H. pylori. A previous exposition of H. pylori to cholesterol influenced the bacterium response to the direct
inhibitory effect of DHA. Overall, our results suggest that a direct effect of DHA on H. pylori survival is modulated by its access to
epithelial cell cholesterol, supporting the notion that cholesterol enhances the resistance of H. pylori. The cholesterol-dependent
resistance of H. pylori to antimicrobial compounds raises new important aspects for the development of new anti-bacterial strategies
(Correia et al. International Journal of Medical Microbiology 2013).
Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of
Helicobacter pylori clarithromycin resistance in gastric biopsy specimens
We have evaluated a peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for the
detection of H. pylori clarithromycin resistance in paraffin-embedded gastric biopsy specimens. For that, we conducted a retrospective
study as well as a prospective cohort study to evaluate the specificity and sensitivity of the PNA-FISH method in determining H. pylori
clarithromycin resistance. In the retrospective study, there was full agreement between PNA-FISH and PCR-sequencing. Compared
to the reference method (culture followed by the E-test), the specificity and sensitivity of PNA-FISH were 90.9% (95% CI, 57.1% to
99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort, 21 cases were positive by culture. For the patients
harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8%
(95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients
infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin
resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific
visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that different H. pylori
strains can subsist in very close proximity in the stomach (Cerqueira et al. Journal of Clinical Microbiology 2013).
Helicobacter pylori detection using advanced locked nucleic acid (LNA) probes
We assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization
(FIVH), for the detection of H. pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2’-O-methyl RNAs
(2’OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that
hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsy specimens. Furthermore,
the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be
replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides
an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo
applications either for this microorganism or for others (Fontenete et al PloS One 2013).
Characterization of the gastric microbiota in patients with chronic gastritis and gastric carcinoma
To determine the influence of the stomach microbiota in H. pylori-associated gastric carcinogenesis, we have characterized the
microbiota of 17 chronic gastritis and 12 gastric carcinoma patients, using the next generation sequencing platform Ion PGM. We
have found that the majority of the gastric microbiota was distributed in five main Phyla, Proteobacteria, Firmicutes, Actinobacteria,
Bacteroidetes, and Fusobacteria. The most representative Phylum in the stomach was the Proteobacteria with 75.6% and 88.9% of
reads in chronic gastritis and in gastric carcinoma, respectively. The remaining four Phyla were always less represented in patients
with gastric carcinoma than in patients with chronic gastritis. Interestingly, the abundance of Helicobacter sp. was significantly lower
in patients with gastric carcinoma (8.1%) than in patients with chronic gastritis (47.3%). Analysis of the phylogenetic diversity between
groups of individuals in a principal coordinate analysis showed distinct gastric microbiota profiles in the two groups of patients.
P-cadherin functional role is dependent on E-cadherin cellular context: a proof of concept using the breast
cancer model
P-cadherin overexpression is associated with worse breast cancer survival, being a poor prognostic marker as well as a putative
therapeutic target for the aggressive triple-negative and basal-like carcinomas (TNBCs). Previously, we have shown that P-cadherin
promotes breast cancer invasion of cells where membrane E-cadherin was maintained; however, it suppresses invasion in models
without endogenous cadherins, like melanomas. Thus, we hypothesized that P-cadherin expression would interfere with the normal
adhesion complex, constituting, in this way, a new mechanism by which E-cadherin invasive-suppressor function was disrupted. Using
breast TNBC models, we demonstrated, for the first time, that P-cadherin co-localizes with E-cadherin, promoting cell invasion due
to the disruption caused in the interaction between E-cadherin and cytoplasmic catenins. P-cadherin also induces cell migration and
survival, modifying the expression profile of cells expressing wild-type E-cadherin and contributing to alter their cellular behaviour.
Additionally, E- and P-cadherin co-expressing cells significantly enhanced in vivo tumour growth, compared with cells expressing only
E- or only P-cadherin. Finally, we still found that co-expression of both molecules was significantly correlated with high-grade breast
carcinomas, biologically aggressive, and with poor patient survival, being a strong prognostic factor in this disease. Our results show
a role for E- and P-cadherin co-expression in breast cancer progression and highlight the potential benefit of targeting P-cadherin in
the aggressive tumours expressing high levels of this protein (Ribeiro et al. Journal of Pathology 2013).
P-cadherin signals through the laminin receptor a6ß4 integrin to induce stem cell and invasive properties in
basal-like breast cancer cells
P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression,
being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem
cell and invasive properties to basal-like breast cancer cells. During 2013, we clarify the downstream signaling pathways that are
triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion
of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor
a6ß4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere
forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the a6
integrin subunit expression and directly interacts with the ß4 integrin subunit. We still showed that P-cadherin downstream signaling,
in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression
of P-cadherin, a6ß4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there
is a crosstalk between P-cadherin and the laminin receptor a6ß4 integrin signaling pathway, which link has never been previously
described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer
cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule
(Vieira et al. Oncotarget 2014).
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2013
The basal epithelial marker P-cadherin associates with breast cancer cell populations harbouring a hypoxiaresistant phenotype
Cancer stem cells are hypoxia-resistant due to their glycolytic metabolism. In fact, this association is very well found in triple-negative
basal-like breast carcinomas, which show increased expression of cancer stem cell markers, as well as hypoxia-resistant proteins.
Recently, we demonstrated that P-cadherin expression, which is a biomarker of basal-like breast cancer and a poor prognostic factor in
this disease, mediates stem-like properties, as well as resistance to radiation therapy. Thus, we aimed to evaluate if its expression was
associated to cell populations with an adapted phenotype to hypoxia, using breast cancer as a model. Using a large series of primary
human breast carcinomas, we could demonstrate that P-cadherin overexpression was significantly associated with the expression of
HIF-1¿, GLUT1, CAIX, MCT1 and CD147, independently of the molecular subtype. Interestingly, using the basal-like breast cancer cell
models BT20 and SUM149, we were able to validate some of these associations. We could show that the induction of HIF1¿ by CoCl2
was accompanied by a significant increase of P-cadherin expression at the cell membrane. By real-time PCR, we still demonstrated
that P-cadherin silencing leads to a decrease of the mRNA levels of GLUT1 and CAIX, not altering the transcripts of HIF1¿, MCT1 and
CD147. Moreover, we could also find that the cancer cell fractions harboring high levels of P-cadherin were the same exhibiting more
GLUT1 and CAIX expression. In conclusion, our results establish, for the first time, a link between aberrant P-cadherin expression and
the breast cancer cell populations harboring a hypoxia-resistant phenotype (Sousa et al. [in preparation]).
P-cadherin expression in breast metastatic lymph nodes is associated with poor prognosis
Breast cancer is a major cause of mortality and morbidity in women and its metastatic spread is the major reason behind the fatal
outcome. The aggressive behavior mediated by the breast cancer stem cell markers CD44, CD49f and P-cadherin is already well
documented. However, metastasis-related research about these molecules in breast cancer is underdeveloped when compared with
the abundant literature on primary tumors. Thus, we compared the profile of CD44, CD49f and P-cadherin between primary tumors
and synchronous axillary lymph node metastases from 135 patients with breast cancer. We found an association of P-cadherin with
the stem cell markers CD44 and CD49f in lymph node metastases, but not in the primary tumors. We also found that P-cadherin
expression in lymph node metastases was associated with poor overall and disease-free survival of patients. Significantly, when
we accessed the expression of P-cadherin, CD44 and CD49f markers in matched samples, we found that overall 8.2% of primary
tumors (11/135) gained P-cadherin expression in lymph node metastasis and these tumors presented the worst disease-free and
overall survival of the whole series. We also detected changes in expression (gain/loss) between the primary tumors and lymph node
metastasis for CD44 and CD49f, but these alterations did not impact in patient outcome. Significantly, the gain in P-cadherin and
CD49f expression in the metastatic lymph nodes was mainly found within the triple negative molecular subtype of breast carcinomas.
In conclusion, P-cadherin is an important predictor of disease outcome of breast tumors with lymph node involvement, being a
valuable marker for disease progression (Dionísio et al. [in preparation]).
CCAAT/enhancer binding protein ß (C/EBPß) isoforms are transcriptional regulators of the pro-invasive CDH3/Pcadherin gene in human breast cancer cells
P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated
cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently
overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis.
However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we described the existence
of several CCAAT/Enhancer Binding Protein ß (C/EBPß) transcription factor binding sites at the CDH3 promoter, we aimed to assess
if the distinct C/EBPß isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells.
We demonstrated that C/EBPß is co-expressed with P-cadherin in breast cancer cells and all the three isoforms can function as
transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional
activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3
is a newly defined transcriptional target gene of C/EBPß isoforms in breast cancer, and we also identified the binding sites that are
relevant for this activation (Albergaria et al. PLoS One 2013).
CDH3/P-cadherin is negatively regulated by TAp63 in a p53-dependent manner in breast cancer cells
P-cadherin is frequently overexpressed in high-grade breast carcinomas tumours, being a well-established indicator of poor patient
prognosis and an important inducer of cancer cell migration and invasion. P-cadherin also confers stem cell features to breast
tumorigenic cells that could be linked to the aggressive behavior of basal-like breast cancers. In fact, P-cadherin has been associated
with already described stem cell markers, such as p63, which was recently demonstrated to transcriptionally regulate CDH3 in a
context of the developmental biology. However, in cancer, the relationship between p63 and P-cadherin was only explored in a
pathological perspective. Thus, we demonstrated that TAp63 isoforms transcriptionally represses CDH3 promoter, downregulating
P-cadherin protein expression in breast cancer cells. This repression is functionally reflected on P-cadherin-induced breast cancer
cellular invasion and mammosphere-forming efficiency. Interestingly, we also observed that this effect was not replicated in cells
harboring p53 mutations, and that the induction of p53 hotspot mutations on p53 wild-type cells restored CDH3 promoter activation.
These results suggest that the repressive effect of TAP63g isoform onto CDH3 promoter is disabled by the p53 mutants. The validation
of these observations in human breast cancer samples revealed that that breast tumours expressing TAp63g isoform, but harboring
some type of known pathogenic p53 mutations were positive for P-cadherin expression, while the only case negative for P-cadherin
expression was the one where no p53 mutations were detected. Taken together, our data reveal previously unknown molecular
functions of TAp63g isoforms on CDH3/P-cadherin, where TAp63g is able to represses CDH3 promoter activity and P-cadherin
expression levels, being this regulation dependent of p53 mutational status (Albergaria et al. [in preparation]).
The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer
cell models
Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human
cancer cells relative to normal cells. We demonstrated that the invasive phenotype of P-cadherin-overexpressing breast cancer cell
models was significantly reduced by azurin. Azurin also caused a specific decrease on P-cadherin protein levels from 30-50% in MCF-7/
AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity
of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation
levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherinmediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis
breast carcinomas overexpressing P-cadherin in a wild-type E-cadherin context (Bernardes et al. PLoS One 2013).
High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets
for the anti-cancer bacterial protein azurin
Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherinoverexpressing models, azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression
profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein,
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endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes
associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of
cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins a6, ß4 and ß1 and interfered with
the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin
in P-cadherin overexpression breast cancer models (Bernardes et al. The International Journal of Biochemistry & Cell Biology 2014).
Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma are key events in the progression from an in
situ to an invasive breast carcinoma
The progression from in situ to invasive breast carcinoma is still an event poorly understood. However, it has been suggested that
interactions between the neoplastic cells and the tumor microenvironment may play an important role in this process. Thus, the
determination of differential tumor-stromal metabolic interactions could be an important step in invasiveness. The expression of
stromal Caveolin-1 (Cav-1) has already been implicated in the progression from ductal carcinoma in situ (DCIS) to invasive ductal
carcinoma (IDC). Additionally, stromal Cav-1 expression has been associated with the expression of stromal monocarboxylate
transporter 4 (MCT4) in invasive breast cancer. However, the role of stromal MCT4 in invasiveness has never been explored, neither
the association between Cav-1 and MCT4 in the transition from breast DCIS to IDC. Therefore, we aimed to investigate in a series of
breast cancer samples including matched in situ and invasive components, if there was a relationship between stromal Cav-1 and
MCT4 in the progression from in situ to invasive carcinoma. We found loss of stromal Cav-1 in the progression to IDC in 75% of the
cases. In contrast, MCT4 stromal expression was acquired in 87% of the IDCs. Interestingly, a concomitant loss of Cav-1 and gain of
MCT4 was observed in the stroma of 75% of the cases, when matched in situ and invasive carcinomas were compared. These results
suggest that alterations in Cav-1 and MCT4 may thus mark a critical point in the progression from in situ to invasive breast cancer
(Martins et al. Cell Cycle 2013).
The expression of cancer stem cells markers is largely dependent on breast cancer histological type
CD44, CD24 and ALDH1 are the most consistently used biomarkers to identify and characterize the breast cancer stem cell (CSC)
phenotype. However, most studies performed until now analyzed samples of invasive ductal carcinomas of no special type (IDC-NST).
Therefore, prevalence and clinical significance of these CSC markers in breast carcinomas of special histological types (SHT) is largely
unknown. For that reason, we determined the distribution of the breast CSC markers among a series of invasive breast carcinomas of
SHT, in comparison with a series of IDC-NST. We could find that the expression prevalence of the breast CSC markers differed between
special types and IDC-NST. Medullary, papillary and tubular carcinomas were enriched in the CSC phenotype CD44(+)/CD24(-/low).
Considering the ALDH1 cytoplasmic tumour expression, only medullary and metaplastic carcinomas displayed significant increase in
CD44(+)/CD24(-/low)/ALDH1(+) CSC phenotype frequency. Thus, we could conclude that the expression distribution of breast CSC
markers is largely dependent on histological type. Interestingly, within the distinct SHT, medullary and metaplastic carcinomas are the
two types highly associated with high-grade carcinomas, basal-like and claudin-low molecular subtypes, and to the CSC phenotype
CD44(+)/CD24(-/low)/ALDH1(+) (Beça et al. Journal of Clinical Pathology 2013).
Interactions between tumor and stromal cells that contribute to colorectal cancer progression
The maintenance of the architecture and homeostasis of the colonic epithelium results from a very controlled spatial organization
of signals emanating from a crosstalk between epithelial and mesenchymal cells residing in close proximity to the epithelium.
Deregulation of this fine-tuned crosstalk promotes tumor development.
We focused on how KRAS activation in colonic cells modulate epithelial-stromal crosstalk and creates a pro-tumoral microenvironment.
In a panel of colorectal cancer cell lines (DLD-1KRASG13D, HCT116KRASG13D, SW480KRASG12V, HT29KRASWT, RKOKRASWT,
Caco2KRASWT) we characterized the expression of proteins from intestinal homeostasis-related signaling pathways, namely sonic
hedgehog (Shh), BMP7, and TGFbeta1 proteins, all known to be pro-tumorigenic. Our preliminary results show that all the cell lines
express variable levels of the three molecules, independently of the presence of a KRAS mutation. However, only two cell lines, SW480
and Caco2, were found to express high levels of the three molecules simultaneously. Further studies will elucidate the importance of
these signaling pathways in colorectal cancer progression.
New Strategies for Colorectal Cancer Therapy: a MAPK- and PI3K-Targeted Approach
At present, the therapeutic option for patients with metastatic CRC (mCRC) involves the use of EGFR antibodies but only a small
percentage of patients benefit from such therapies. Thus, it is urgent to unravel new strategies for CRC therapy and explore putative
biomarkers predictive of therapy outcome. Interestingly, we verified that inhibition of MEK1/2 and PI3K modulated cell viability/
proliferation, cell cycle and apoptosis differentially in human colorectal cancer cells with distinct mutational status for KRAS, BRAF
and PI3K. Furthermore, the analysis of a multitude of proteins by the use of antibody arrays revealed specific alterations in a wide
spectrum of kinases and apoptosis-related proteins upon MEK1/2 and/or PI3K silencing. More importantly, our data demonstrates a
pivotal role for PI3K in the survival of CRC cells.
National Collaborations
Adelina Gama, UTAD, Vila Real
Ana Paula Pêgo, INEB, Porto
Arsénio Fialho, IBB-Instituto Superior Técnico, Lisboa
Fátima Baltazar, ICVS, University of Minho, Braga
Florence Janody, IGC, Oeiras
Isabel Amendoeira, HSJ, Porto
Isabel Palmeirim, Department of Medicine, University of Algarve, Faro
Isabel Rocha, CEB, University of Minho, Braga
João Miguel Sanches, ISR, Instituto Superior Técnico, Lisboa
Manuel A. Santos, CESAM, University of Aveiro, Aveiro
Manuel Coimbra, University of Aveiro, Aveiro
Manuela Lacerda, IPOFG-CROC, Coimbra
Maria João Vieira, CEB, University of Minho, Braga
Maria Oliveira, INEB, Porto
Mário Dinis Ribeiro, FMUP, Porto
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2013
Mário Barbosa, INEB, Porto
Nuno Azevedo, FEUP, Porto
Nuno C. Santos, IMM, FMUL, Lisboa
Paula Alves, IBET, Oeiras
Paulo Pereira, IBILI, Coimbra
Pedro Granja, INEB, Porto
Peter Jordan, INSA Ricardo Jorge, Lisboa
International Collaborations
Brazil - Célia Carlini, Universidade Federal do Rio Grande do Sul, Porto Alegre; Dulciene Queiroz, Universidade Federal de Minas
Gerais, Belo Horizonte
Canada - David Huntsman, British Columbia Cancer Agency, Vancouver
Costa Rica - Vanessa Ramirez, Calderón Guardia Hospital, San José
Denmark - Lene J Rasmussen, University of Copenhagen, Copenhagen
Finland - Lauri Aaltonen, University of Helsinki, Helsinki
France - Alex Duval, INSERM, Hôpital Saint-Antoine, Paris; Eliette Touati, Institute Pasteur, Paris - Françoise Bono, Sanofi-Aventis
Rechérche, Toulouse
Germany - Sebastian Suerbaum, Hannover Medical School, Hannover
Israel - Yosef Yarden, Weismann Institute, Rehovot
Italy - Elia Stupka, Milan; Franco Roviello, University of Siena, Siena; Remo Sanges, Cluster in Biomedicine, Trieste
Spain - Carlos Gonzalez, Catalan Institute of Oncology, Barcelona; Fernando Casares, Centro Andaluz de Biología del Desarrollo, Sevilla;
Jorge Cameselle-Teijeiro, Complexo Hospitalar Universitario, Vigo
Sweden - Ola Söderberg, University of Uppsala, Uppsala
The Netherlands - Leen-Jan van Doorn, DDL Diagnostic Laboratory, Voorburg; Marjolijn Ligtenberg, University Nijmegen Medical
Centre, Nijmegen; Robert Hofstra, University of Groningen, Groningen
UK - Göran Landberg, University of Manchester, Manchester; Ian Sanderson, Institute of Cell and Molecular Science, London; John
Atherton, University of Nottingham, Nottingham; Karen Vousden, Cancer Research UK Beatson Institute, Glasgow; Robert Clarke,
Paterson University of Manchester, Manchester
USA - David Mooney, Harvard University, Cambridge; Kevin Haigis, Massachusetts General Hospital, Boston; Jorge Reis-Filho,
Memorial Sloan-Kettering Cancer Center, New York; Judy Lieberman, Harvard Medical School, Boston; Patricia Steeg, Center for
Cancer Research, National Cancer Institute, Bethesda
Future Research
In the gastric cancer setting:
To identify key players involved in the cell-ECM crosstalk in the context of E-cadherin mutations associated to Hereditary Diffuse
Gastric Cancer (HDGC);
To identify ECM components/composition that modulate the expression of integrins or associated molecules;
To study the consequences of integrin switch and ECM modulation with respect to cellular behavior;
To disclose the molecular mechanisms underlying the mutant E-cadherin-integrin-ECM associated cellular effects;
To evaluate if E-cadherin loss induces basal extrusion from an epithelium;
To identify the tensional forces that induce basal extrusion of E-cadherin defective cells;
To understand the crosstalk between tensional forces, cytoskeleton, and nuclear position;
To validate the role of PON2, XIAP, the gastric-specific S100P and CTSE in E-cadherin-related survival/apoptosis in the stomach, using
chick embryonic tissues.
In the gastric cancer setting mediated by Helicobacter pylori infection:
To characterize the H. pylori dupA virulence-associated locus in strains infecting Portuguese patients, in order to assess the relationship
between dupA variation and risk for gastric carcinoma development;
To characterize host cell signaling pathways and to identify the bacterial virulence factors that lead to up-regulation of matrix
metalloproteinase 10 in gastric epithelial cells induced by H. pylori infection;
To dissect the signaling pathways and to characterize the bacterial components involved in receptor tyrosine kinase activation induced
by H. pylori and to investigate the respective functional consequences;
To extend the characterization of the stomach microbiota to patients at different stages of gastric carcinogenesis, in order to evaluate
the influence of bacteria other than H. pylori in gastric carcinoma development.
In the breast cancer setting:
To identify key signaling pathways induced by P-cadherin expression, namely its role in cancer cell metabolism, survival, invasion, as
well as miRNAs expression;
To study the role of P-cadherin in the seeding of metastasis in lymph nodes and distant sites;
To study the role of soluble P-cadherin in the disruption of the blood brain barrier;
To identify cancer stem cell markers with relevance in brain metastatic colonization;
To study the impact of P-cadherin expression in the DNA damage/repair signaling triggered by radio/chemotherapy in normal and
cancer cells;
To study the role of P-cadherin in cell polarity, namely in tight junction stability/assembly and in mitotic spindle orientation;
To find new therapeutic strategies to inhibit P-cadherin expression or associated signaling pathways;
To identify new mechanisms of CDH3 gene regulation in normal epithelial tissues and cancer;
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2013
To measure cell-cell adhesion mediated by both P-cadherin and E-cadherin by AFM.
In the colon cancer setting:
To determine if the survival of KRAS, BRAF and PI3K mutant CRC cells are all modulated by PI3K activation and validate the relevance
of PI3K activity and downstream targets in primary cultures of CRC tissues.
Raquel Seruca, Ceu Figueiredo, Joana Paredes, Fátima Carneiro, José Carlos Machado, Fernando Schmitt, Marina Leite, Patrícia
Carneiro, Cecília Durães. Programa Graduado em Áreas da Biologia Básica e Aplicada (GABBA), Universidade do Porto. Coordenação
e participação no módulo de Oncobiologia; Orientação de estudantes
Ceu Figueiredo, Joana Paredes, Raquel Seruca. International Doctoral Programme on Cellular and Molecular Biotechnology Applied
to Health Sciences (BiotechHealth)
Joana Paredes, Raquel Seruca. Doctoral Program in Experimental Biology and Biomedicine (BEB PhD Program), Center for Neuroscience
and Cell Biology (CNC), University of Coimbra
Fátima Carneiro. Programa de Doutoramento em Biomedicina (FMUP). Coordenação e participação no Módulo de Oncobiologia
Fátima Carneiro, Joana Paredes. Programa de Doutoramento em Medicina e Oncologia Molecular (FMUP). Coordenação e participação
no Módulo de Oncobiologia; Orientação de estudantes
Fátima Carneiro, Fernando Schmitt. Programa de Doutoramento em Patologia e Genética Molecular (ICBAS). Coordenação e
participação no Módulo de Oncobiologia; Orientação de estudantes
Ceu Figueiredo, Joana Paredes. Mestrado em Oncologia, Instituto de Ciências Biomédicas Abel Salazar. Orientação de estudantes
Fátima Carneiro, Ceu Figueiredo, Joana Paredes, José Carlos Machado. Mestrado em Medicina e Oncologia Molecular, Faculdade de
Medicina da Universidade do Porto. Coordenação e participação no Módulo de Oncobiologia; Orientação de estudantes
Joana Paredes. Master of Science in Oncobiology - Developmental Biology and Cancer. Faculty of Medicine of the University of Lisbon
Joana Paredes. Post-Graduation Course in Fundamentals of Genetics, Development and Neoplasia. Institute of Life and Health
Sciences (ICVS) – University of Minho
Joana Paredes. III Workshop on Cancer Research: Biological and Molecular Basis, IPATIMUP
Joana Paredes. Post-Graduation Course, Cancer Therapy: from basic research to clinic. Department of Biology - University of Minho
Ceu Figueiredo, Fatima Carneiro, Fernando Schmitt. Mestrado Integrado em Medicina, Faculdade de Medicina da Universidade do
Porto. Orientação de estudantes
Invited Talks
Ceu Figueiredo. Disruption of cell-cell adhesion by Helicobacter pylori. In session: Interactions between Helicobacter pylori and
epithelial cells. United European Gastroenterology Week UEGW2013. Berlin, Germany. 15/10/2013.
Ceu Figueiredo. Clinical relevance of Helicobacter pylori virulence in gastric carcinogenesis. Workshop Biologia das células epiteliais:
Mecanismos celulares e moleculares envolvidos na infecção por Helicobacter pylori e no Câncer.
Laboratório de Farmacologia Celular e Molecular, Departamento de Biologia Celular da Universidade do Estado do Rio de Janeiro
(UERJ). Rio de Janeiro, Brazil. 3/12/2013.
Marina Leite. Receptors tyrosine kinases as targets of Helicobacter pylori infection. Workshop Biologia das células epiteliais:
Mecanismos celulares e moleculares envolvidos na infecção por Helicobacter pylori e no Câncer
Laboratório de Farmacologia Celular e Molecular, Departamento de Biologia Celular da Universidade do Estado do Rio de Janeiro
(UERJ). Rio de Janeiro, Brazil. 03/12/2013.
Marina Leite. Receptors tyrosine kinases as targets of Helicobacter pylori infection. LAPROTOX- Center for Biotechnology of the
Federal University of Rio Grande do Sul (UFRGS). Porto Alegre, Brazil. 06/12/2013.
Joana Paredes. P-cadherin: a stem cell marker overexpressed in high-grade breast carcinomas. E-COST Meeting, COST Action CM1106.
1st Working Group Meeting, Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells. Porto, Portugal. 21/02/2013.
Joana Paredes. P-cadherin: a putative therapeutic target in breast cancer. Seminar at the Faculty of Medicine of the University of Porto
(FMUP). Porto, Portugal. 20/03/2013.
André Albergaria. Molecular Mechanisms of Resistance to Targeted Therapy in Breast Cancer. Encontros da Primavera – ONCOLOGIA.
Évora, Portugal. 19/04/2013.
Joana Paredes. Caderina-P: o seu papel na invasão/metastização do cancro da mama. IV Jornadas de Ciência e Medicina – Oncologia,
Núcleo de Estudantes de Medicina da Universidade do Minho (NEMUM), Escola de Ciências da Saúde, Universidade do Minho. Braga,
Portugal. 20/04/2013.
Joana Paredes. Stem Cells and Cancer: a challenging crosstalk for Regenerative Medicine. 8th International Meeting of the Portuguese
Society for Stem Cells and Cell Therapies (SPCE-TC), University of Algarve. Faro, Portugal. 9/05/2013.
Joana Paredes. Cadherins, Cancer Stem Cells and Metastasis. 25th European Congress of Pathology, Breast Pathology: Mechanisms of
metastasis, Centro de Congressos de Lisboa. Lisboa, Portugal. 3/09/2013.
Fátima Carneiro. Molecular and immunohistochemical markers of gastric carcinoma differentiation and progression. São Paulo School
of Advanced Sciences – Advances in molecular oncology: Translating molecular biology into cancer treatment. São Paulo, Brazil.
03/02/2013.
Fátima Carneiro. Impacto da informação molecular na abordagem global do cancro gastrico. 12ª Reunião Internacional de Cirurgia.
Lisboa, Portugal. 18/02/2013.
Fátima Carneiro. Molecular pathology of gastric cancer. Pathology Grand Rounds, Massachusetts General Hospital. Boston, USA.
28/02/2013.
Fátima Carneiro. “Outs” (Unknown Case) Conference. Massachusetts General Hospital. Boston, USA. 28/02/2013.
Fátima Carneiro. Gastrointestinal Pathology Specialty Conference on Early Neoplasia of the Gastrointestinal Tract: Diagnostic
Challenges and Clinical Implications (apresentação de um caso de carcinoma gastric hereditário de tipo difuso). USCAP Annual
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2013
Meeting 2013. Baltimore, USA. 06/03/2013.
Fátima Carneiro. What is new in premalignant lesions of the digestive system (WHO 2010). EScoP Course on Update in Gastro-Intestinal
Pathology. Belgrade, Serbia. 25/04/2013.
Fátima Carneiro. Classification of chronic gastritis, intestinal metaplasia and dysplasia. EScoP Course on Update in Gastro-Intestinal
Pathology. Belgrade, Serbia. 25/04/2013.
Fátima Carneiro. Slide Seminar on Chronic Gastritis (apresentação de 13 casos). EScoP Course on Update in Gastro-Intestinal Pathology.
Belgrade, Serbia. 25/04/2013.
Fátima Carneiro. Current understanding of the pathogenesis of gastric carcinogenesis. Spring Meeting in Pathology. Stockholm,
Sweden. 15/05/2013.
Fátima Carneiro. Current understanding of the pathogenesis of gastric cancer. Gastric Cancer Discovery Working Group Meeting,
organized by Astellas Oncology. London, UK . 17/05/2013.
Fátima Carneiro. Seminario de Patología “Salvados por la campana”. XXVI Congresso Nacional SEAP e IAP. Cadiz, Spain. 24/05/2013.
Fátima Carneiro. Patología molecular del cáncer de estómago. Curso de patologia Molecular II. XXVI Congresso Nacional SEAP e IAP.
Cadiz, Spain. 24/05/2013.
Fátima Carneiro. Pólipos gástricos. Curso de Patología Digestiva. XXVI Congresso Nacional SEAP e IAP. Cadiz, Spain. 24/05/2013.
Fátima Carneiro. Moderadora de Mesa-Redonda sobre “Pathogenesis: basic facts for basic sciences”. World Digestive Health day.
Porto, Portugal. 21/05/2013.
Fátima Carneiro. Prediction of high risk patients on gastric biopsy. 10th International Gastric Cancer Congress (IGCC 2013). Verona,
Italy. 20/06/2013.
Fátima Carneiro. Biobanking: The role of biobanking and translational research for the pathologist. The 3rd Global Cancer Genomics
Consortium Symposium (From Oncogenomics To Cancer Care). Lisbon Portugal. 20/09/2013.
Fátima Carneiro. Valor da histoquímica no atual panorama do diagnóstico anátomo-patológico. IV Simpósio Técnico de Anatomia
Patológica – Histopatologia e Qualidade. Porto, Portugal. 28/09/2013.
Fátima Carneiro. Pathology and carcinogenesis of gastric cancer. ESMO Preceptorship in Gastric Cancer on “Multidisciplinary
management, standards of care, therapeutic targets and future perspectives. Berlin, Germany. 12/10/2013.
Fátima Carneiro. Molecular events in gastric cancer. ESMO Preceptorship in Gastric Cancer on “Multidisciplinary management,
standards of care, therapeutic targets and future perspectives. Berlin, Germany. 12/10/2013.
Fátima Carneiro. Hereditary gastric cancer. Today’s Science: Tomorrow’s Medicine on Genetically predisposed GI cancers. UEGW
2013. Berlin, Germany. 15/10/2013.
Fátima Carneiro. Identifying and preventing hereditary gastric cancer (Lunch Session). UEGW 2013. Berlin, Germany. 15/10/2013.
Fátima Carneiro. Cancro gástrico – relevância dos aspectos anatomopatológicos. 9º Congresso Nacional Cancro Digestivo. Albufeira,
Portugal. 19/10/2013.
Fátima Carneiro. Case Presentation. Clinical Hepatology Perceptorship 2013. Porto, Portugal. 24/10/2013.
Fátima Carneiro. Inflamação e cancro. Congresso Nacional de Dermatologia. Porto, Portugal. 01/11/2013.
Fátima Carneiro. Doença oncológica: abordagem multidisciplinar. Jornadas do Internato Médico do Algarve. Faro, Portugal.
08/11/2013.
Fátima Carneiro. A Rede nacional de Bancos de Tumores. Sessão de Lançamento da Rede Nacional de Bancos de Tumores. Lisboa,
Portugal. 12/12/2013.
Fátima Carneiro. Pathology and carcinogenesis. ESMO Preceptorship in Gastric Cancer on “Multidisciplinary management, standards
of care, therapeutic targets and future perspectives. Singapore. 21/12/2013.
Fátima Carneiro. Molecular events in gastric cancer. ESMO Preceptorship in Gastric Cancer on “Multidisciplinary management,
standards of care, therapeutic targets and future perspectives. Singapore. 21/12/2013.
Fernando Schmitt. Triple-Negative Breast Cancer: an example for translational cancer research. SPSAS Advances in Molecular
Oncology. São Paulo, Brazil. 03/02/2013.
Fernando Schmitt. Pathology of triple negative breast tumors in 2012. XXII Porto Cancer Meeting & III Porto-Bordeaux Joint Meeting.
Porto, Portugal. 11/04/2013.
Fernando Schmitt. Molecular Cytopathology-main applications and technical issues. Workshop Pathology of the Breast: Possible
artefacts benefits of standardization, Institut Jules Bordet. Bruxelas, Bélgica. 20/04/2013.
Fernando Schmitt. Molecular Cytopathology on FNA specimens: technical issues and main applications. The Aasmund Berner
Cytopathological Symposium. Oslo, Noruega. 07/05/2013.
Fernando Schmitt. Molecular techniques in cytology. Varmote I Patologi. Estocolmo, Suécia. 16/05/2013.
Fernando Schmitt. Metastatic cancer: challenges and opportunities for molecular cytopathology. 18th International Congress of
Cytology. Paris, França. 26/05/2013.
Fernando Schmitt. Molecular Classification of Breast Cancer. Breast Cancer Workshop-Medical Informatics. Porto, Portugal.
19/06/2013.
Fernando Schmitt. Metastatic cancer: challenges and opportunities for molecular cytopathology. Grand Rounds St. Vicents Hospital.
Sidney, Austrália. 16/07/2013.
Fernando Schmitt. Molecular Cytopathology in Breast. Advanced Solutions for Advanced Pathology. Nice, França. 28/11/2013.
Fernando Schmitt. Dieta e Prevenção do Cancro: Que sabemos nós?. Fórum FNAC. Porto, Portugal. 27/04/2013.
Fernando Schmitt. Patologia Digital – Que Futuro?. XIV Congresso Técnico de Anatomia Patológica. Covilhã, Portugal. 18/05/2013.
Fernando Schmitt. THYROID FNAC. Toward a more personalized medicine. 18th International Congress of Cytology. Paris, França.
26/05/2013.
Fernando Schmitt. HER2: Atualidade e perspetivas futuras. 5ª Edição da reunião PHC (Personalised Healthcare in Oncology). Amadora,
Portugal. 06/06/2013.
Fernando Schmitt. EAPCP: The future of postgraduate education in Pathology in Europe. Subspecialty development: the dawn of
diaspora?. 25th European Congress of Pathology. Lisboa, Portugal. 31/08/2013.
Fernando Schmitt. Breast Pathology: Mechanisms of metastasis. Role of EGFR in breast cancer metastasis. 25th European Congress of
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2013
Pathology. Lisboa, Portugal. 03/09/2013.
Fernando Schmitt. Cytopathology: Cytology and molecular testing: what is effective? Molecular testing in cytology: overview and preanalytical issues. 25th European Congress of Pathology. Lisboa, Portugal. 31/08/2013.
Fernando Schmitt. Cytopathology: Cytology and molecular testing: what is effective? Molecular testing in cytology: overview and preanalytical issues. ASCP 2013 Annual Meeting. Chicago, USA. 18/09/2013.
Oral Presentations
Ferreira RM, Costa JL, Figueiredo C, Machado JC. Session: H. pylori and gastric cancer risk. Characterization of the gastric microbiota
in patients with chronic gastritis and gastric carcinoma. 21st United European Gastroenterology Week (UEGW 2013). Berlin, Germany.
15/10/2013.
Ferreira RM, Costa JL, Figueiredo C, Machado JC. Session: The metagenomic approach to GI disease. Characterization of the gastric
microbiota in patients with chronic gastritis and gastric carcinoma. 21st United European Gastroenterology Week (UEGW 2013).
Berlin, Germany. 14/10/2013.
Ferreira RM, Costa JL, Figueiredo C, Machado JC. Characterization of the gastric microbiota in patients with chronic gastritis and
gastric carcinoma. XXVIth International Workshop on Helicobacter and related bacteria in chronic digestive inflammation and gastric
cancer. Madrid, Spain. 15/09/2013.
Ana Rita Nobre. P-cadherin - A new predictive biomarker for Dasatinib therapeutic response in breast cancer. AstraZeneca Foundation
Innovate Competition - iMed 5.0 Conference. Reitoria da Universidade NOVA de Lisboa. Campolide, Portugal. 12/10/2013.
Prizes
Rui Ferreira, Jose L. Costa, Ceu Figueiredo, Jose C. Machado. Characterization of the gastric microbiota in patients with chronic
gastritis and gastric carcinoma.
Travel grant to Rui Ferreira - United European Gastroenterology Week (UEGW 2013). Berlim, Germany.
Ana Sofia Ribeiro, Ana Rita Nobre, André Vieira, André Albergaria, Bárbara Sousa, Rene Gerhard, Raquel Seruca, Fernando Schmitt,
Joana Paredes. P-cadherin activates Src-kinase: A new signaling pathway with implications in breast cancer progression.
TEMTIA Best Poster Award, VI International Epithelial-Mesenchymal Transition Meeting. Alicante, Spain.
Fátima Carneiro. O papel dos anticorpos IgA anti-endomisio no diagnóstico de doença celíaca numa amostra pediátrica.
Prémio do “melhor trabalho apresentado” na XXVI Reunião da Sociedade Portuguesa de Gastrenterologia e Nutrição Pediátrica
(2013). Vilamoura, Portugal
Fernando Schmitt. GOLDBLATT AWARD 2013
International Academy of Cytology. Paris, França, 2013
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2013
Objectives
The main objective of the group is to understand the molecular mechanisms involved in gastric carcinogenesis with emphasis on
the identification of transcriptional pathways responsible for cancer/pre-cancer transdifferentiation and the characterization of the
glycoproteome remodelling. We intend as well to extend our expertise to other cancer models (ex. ovary).
Main Achievements
Understanding the pathophysiology of gastric carcinogenesis
We have identified a novel CDX2 regulatory mechanism, at the post-transcriptional level, through the RNA-binding protein MEX3A.
This protein, in addition to repressing CDX2 expression, consequently impairing cellular differentiation, also impairs cellular adhesion
and polarity with potential implications in GI cancers (Pereira B, Nucleic Acids Res. 41:3986-99, 2013; Pereira B, Trends Biochem. Sci.
38:477-9, 2013; Bruno Pereira’s PhD thesis, November 2013).
We have characterized the expression of SOX2 in gastric premalignant lesions, intestinal metaplasia and dysplasia, showing that it is
expressed in normal gastric mucosa, in intestinal metaplasia of the incomplete type and is lost in most dysplastic lesions. Furthermore,
we demonstrated that SOX2 regulates CDX2 expression in Caco-2 cells. This study contributes to understand how intestinal metaplasia
appears and progresses (Camilo V, submitted).
We developed two quitosan-based nanoparticles to deliver siRNAs against CDX2 in vivo. We demonstrated, in vitro, their efficacy in
downregulating CDX2 expression and we assessed their behavior in the gastrointestinal mucus, concluding that they are able to cross
the gastric mucus barrier but not the intestinal one, suggesting this is a suitable strategy for gastric siRNAs delivery in vivo (Sadio A,
submitted).
We developed a transgenic mouse model with overexpression of the BMP pathway in the gastrointestinal tract. So far we did not
observe gastric phenotypic alterations but now we have a model that will allow to modulate the BMP pathway in vivo (Ana Luisa
Amaral Master thesis “A model of BMP pathway overexpression in the gastrointestinal tract – an in vivo approach”, December 2013,
ICBAS).
Glycoproteome remodeling and biomarker identification I
We demonstrated that infection of the gastric mucosa with CagA+ Helicobacter pylori strains, in non-secretor individuals, leads to the
expression of alpha1,2-fucosylated glycans, which allows the adhesion of the Norovirus GII. This observation shows that Helicobacter
pylori infection leads to a glycan remodeling that allows infection by a virus, that otherwise would not be able to infect and that is
responsible for great part of epidemic gastroenteritis(Ruvoën-Clouet N, in press).
We have applied a glycoproteomic approach to identify altered glycoproteins expressing the simple mucin-type carbohydrate antigens
T and STn in the serum of patients with gastritis, IM (complete and incomplete subtypes), and control healthy individuals. One of the
glycoproteins identified was plasminogen, a protein that has been reported to play a role in H. pylori chronic infection of the gastric
mucosa and is involved in extracellular matrix modeling and degradation. These results provide evidence of serum proteins displaying
abnormal O-glycosylation in patients with precursor lesions of gastric carcinoma and include a panel of putative targets for the noninvasive clinical diagnosis of individuals with gastritis and IM (Gomes C, J Proteome Res. 12:1454-66, 2013).
We have shown that CDX2 regulates the enzyme ST6GalNAc-I, that synthesizes the antigen Sialyl-Tn, potentially impacting on whole
glycoproteome remodelling and we have generated two CDX2 knock-out cell lines, using the zinc finger nuclease strategy, which
will constitute an invaluable tool to study the CDX2-dependent glycoproteome alterations (through glycoarray analysis) as well as
glycosyltransferase regulation.
Glycoproteome remodeling and biomarker identification II
We engineered OVCAR-3 cell line (ovarian cancer cell line), to knock-out cosmc, a chaperone protein of glycosyltransferases that
elongate glycan chains. This will lead to the biosynthesis of short glycans, potentially enriched in sialic acids, mimetizing what happens
in cancer (Steentoft C, EMBO J., 32:1478-88, 2013).
We developed a novel microarray-based platform for profiling specific aberrant glycoforms present in CA125 (MUC16) and CA153 (MUC1), which are biomarkers used to monitor ovarian tumors. The combined glycoform profile was able to distinguish benign
ovarian neoplasms from malignant serous ovarian carcinomas with a specificity of 61.1% at 90% sensitivity.These findings suggest
that glycoprofiling improves differential diagnosis and significantly reduces the number of patients elected for further testing (Chen
K, J Proteome Res. 12:1408-18, 2013).
We have collaborated in a robust study showing autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian,
lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection (Burford B,
Br J Cancer. 108:2045-55, 2013).
We identified the epitope specificity of two monoclonal antibodies against MUC16, used in the clinics. The predicted structure of
the human tandem repeat of MUC16 was obtained after extensive glycoproteome characterization and in collaboration with Pedro
Pereira (IBMC).This was not known and will be useful to define strategies to obtain novel cancer-specific antibodies (Marcos-Silva L,
submitted).
Other
We have collaborated in a study, performed in a Mozambican population, showing that infection with cutaneous HPV is associated
with squamous cell carcinoma of the conjuntiva (Carrilho C, Eur J Cancer Prev. 22:566-8, 2013).
INSERM, Nantes – Jacques Le Pendu - This collaboration has been critical for the prosecution of the study of Secretor and Lewis
phenotypes/genotypes, due to the unique expertise of Jacques Le Pendu in the field. We are currently folllowing the interesting
observation (published this year) of cooperation of Helicobacter pylori in glycoengeneering the host for certain Calicivirus strain
infections.
University of Gothemburg - Gunnar Hansen - This collaboration allowed us to study the behaviour of nanoparticles in the gastrointestinal
mucus in an ex vivo model.
Faculty of Health Sciences of the University of Copenhagen - Ulla Mandel and Henrik Clausen - Collaboration has been fundamental for
characterization of carbohydrate antigens and glycosyltransferases using unique monoclonal antibodies. In 2013 a new joint project
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2013
was started aiming at developing a CDX2 knock-out cell line (using the zinc-finger nuclease strategy) to study glycosyltransferases
regulation. One PhD student of the group (Lara Silva) is doing a joint PhD thesis and another PhD student (Rita Pinto) stayed at Henrik’s
lab for short periods. We regularly collaborate in pos-graduate teaching activities
Launching the Portuguese Association for Cancer Research - ASPIC - Two members of the group - Leonor David and Raquel Almeida were deeply involved, in conection to EACR, in launching the portuguese “branch” of this european cancer research network.
Faculty of Medicine, Hospital S. João, IPATIMUP - Fátima Carneiro - We are collaborating on the characterization of gastric premalignant
lesions and cancer.
INEB - Ana Paula Pêgo - This collaboration has been invaluable for the obtention and characterization of chitosan nanoparticles.
INSERM, Strasbourg – Jean-Noel Freund - This collaboration is essential for the study of CDX2 regulation in intestinal metaplasia using
animal models. A PhD thesis (Rita Barros) was completed between our two groups and joint projects are being developed.
University of Uppsala - Ola Soderberg - This collaboration has allowed the successful establishment of Proximity-Ligation assays for
identification of glycopeptide structures in situ.
INSERM, Grenoble - Marc Billaud - This collaboration is essential for the study of MEX3A function in gastrointestinal differentiation
and cancer. Two joint publications were obtained in 2013. New results have been obtained and pursued regarding the role of MEX3A
in cell polarity.
Instituto de Oncologia de Lisboa, Portugal - Paula Chaves - We are collaborating with the group in Lisbon using Barrett’s oesophagus
as a parallel model to intestinal metaplasia.
Future Research
Understanding the pathophysiology of gastric carcinogenesis I
We will study the interplay between CDX2 and SOX2 in gastric preneoplastic lesions and cancers. We have preliminary data showing
that SOX2 is expressed in 50% of gastric tumors and associates with invasion, metastases and poor survival, which is counterbalanced
by CDX2 expression. We will explore the relevance and interaction of these two transcription factors in gastric cancer biology.
Hypothesis is that the differentiation/stemness features are at stake and are determined by CDX2 and SOX2, respectively. We will
tackle this by performing phenotypic assays both in vitro and in vivo, using xenografts; we will address SOX2 regulation in gastric
cancers through epigenetics, miRNAs and gene amplification.
Understanding the pathophysiology of gastric carcinogenesis II
We will continue characterizing the cellular phenotype associated with MEX3A expression and CDX2 downregulation in normal and
pathological conditions of the GI tract, namely concerning differentiation, stemness and polarity. We have preliminary data suggesting
that MEX3A downregulates a variant form of the polarity determinant LKB1. We will explore this new research avenue by dissecting
the molecular mechanisms involved in this regulation, the impact in cellular phenotype and the biological contexts where it might
have an impact.
Understanding the pathophysiology of gastric carcinogenesis III
We will progress with the study of the BMP pathway in gastric preneoplastic and neoplastic lesions. We will expand this line to gastric
and esophageal cancers by studying BMP pathway expression and regulation and its impact in cancer biology. Moreover, we will
tackle a translational approach towards preventing gastric cancer. Statin use is beneficial in different types of tumors and one of the
mechanisms is through regulation of the BMP pathway. Hence, we will address the potential relevance of statins in impairing gastric
carcinogenesis, using in vitro and in vivo models.
Understanding the pathophysiology of gastric carcinogenesis IV
We will continue developing a surgical strategy to deliver siRNAs in vivo. We have preliminary data showing that it is possible to deliver
nanoparticles or drugs directly to intestine. We want to improve this model as well as develop methods to monitor the movement of
the nanoparticles in vivo and assess changes in cellular phenotype.
Glycoproteome remodeling and biomarker identification I
We will characterize the transcriptome of the CDX2 knock-out/knock-in cell lines, obtained using the zinc-finger strategy, as well as
its phenotypic characterization (proliferation, apoptosis, migration, invasion) and also promoter analysis. We will specifically look at
glycoproteome and glycosyltransferases alterations.
Glycoproteome remodeling and biomarker identification II
We will characterize two new monoclonal antibodies produced for MUC16 and use them to identify cancer-associated MUC16
glycoforms (using immunohistochemistry and Proximity Ligation Assay) and to assess the interaction of tumor cells with Mesothelin
(a critical step in many cancers invading serous cavities). Further, we will extend a preliminary study with mucinous carcinomas from
different locations in which a highly significant association between CDX2 expression and absence of MUC16 was observed.
Glycoproteome remodeling and biomarker identification III
The ovarian carcinoma cell line OVCAR3 - wild type and cosmc knock-out (with a cancer-associated glycan profile) will be phenotipically
characterized in in vitro assays and, most importantly, in vivo, by peritoneal injection in nude mice.
Raquel Almeida, Leonor David - Workshop on Cancer Research: biological and molecular basis Local: IPATIMUP Instituição: IPATIMUP/
ICBAS Âmbito: Curso de formação contínua
Raquel Almeida and Leonor David - Programa Doutoral de Medicina e Oncologia Molecular of the Medical Faculty of the University
of Porto
Raquel Almeida and Leonor David - Programa Doutoral em Patologia e Genetica Molecular do Instituto de Ciencias Biomedicas Abel
Salazar
Raquel Almeida and Leonor David - PhD program GABBA (Graduate program in areas of Basic and Applied Biology) of the University
of Porto
Raquel Almeida and Leonor David - Programa Doutoral em Biomedicina
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2013
Raquel Almeida and Leonor David - Mestrado em Oncologia Molecular do Instituto de Ciencias Biomedicas Abel Salazar
Invited Talks
Leonor David. Glycoprotein biomarkers in gastric cancer: from diagnosis to follow-up. XXII Porto Cancer Meeting on “Translational
Research in Cancer. Bringing Basic Science and Pathology to Clinical Oncology”. Porto, Portugal. 21/04/2013.
Leonor David. Glycopeptide identification as cancer biomarkers. Rudbeck Seminar. Uppsala, Suécia. 5/5/2013.
Leonor David. Estratégias para melhorar a sensibilidade e especificidade dos marcadores tumorais com utilidade clínica. . Conferências
em Oncobiologia 2013, da Faculdade de Farmácia da Universidade do Porto.. Porto, Portugal. 20/5/2013.
Leonor David. Reprogramming pathways and biomarkers in metaplasia. UEG/ESP Joint Symposium: Pathology meets endoscopy –
metaplasia in the gastrointestinal tract.. 25th European Congress of Pathology. Lisboa, Portugal. 31/08/2013.
Oral Presentations
Ana Sadio. Regulação da expressão de CDX2 e penetrabilidade no muco gastrointestinal usando nanopartículas de siRNA e quitosano/
trimetilquitosano. Semana digestiva 2013. Vilamoura, portugal. 05/06/2013.
Bruno Pereira. Regulation of CDX2 and intestinal differentiation in homeostasis and carcinogenesis: unveiling the role of the RNAbinding protein MEX3A.. 10th International Medical Postgraduate Conference. Hradec Králové, Republica Checa. 21/11/2013.
Prizes
Bruno Pereira. Regulation of CDX2 and intestinal differentiation in homeostasis and carcinogenesis: unveiling the role of the RNAbinding protein MEX3A. Best work presented in the Meeting “10th International Medical Postgraduate Conference”, Hradec Králové,
Republica Checa
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Objectives
The Expression Regulation in Cancer (ERiC) group was created in January 2013.
The main management objectives were consolidation of funding for consumables, services and human resources and the integration
in FCT-funded Networks of strategic interest.
The main scientific objective of the group in 2013 was to identify and understand somatic and germline genetic mechanisms involved
in gastric cancer, using next generation sequencing and bioinformatics, as well as molecular and cellular biology applied to patients’
cohorts and private cell line in vitro models.
The specific scientific objectives were:
1- Determine new genes involved in causing familial gastric cancer and explore novel diagnostic and therapeutic strategies for
Hereditary Diffuse Gastric Cancer (HDGC);
2-Identify new prognostic factors in gastric cancer and apply molecular characterization with these prognostic factors and targets of
therapy to stratify sporadic gastric cancer for whole genome seq, Methylseq and RNAseq;
3- Explore in vitro models of Epithelial-Mesenchymal-Epithelial transition to study tumour heterogeneity and cellular crosstalk
through secreted microvesicles;
4- Explore the role of RNA mistranslation in cancer.
A parallel objective of two group members (Carla Oliveira and Patrícia Oliveira) was the launching of a Spin-off from IPATIMUP,
together with GENETEST, another spin-off of IPATIMUP.
Main Achievements
Novel causative genes identified in a quarter of Gastric Cancer Families
In collaboration with D Huntsman (BCCA, Vancouver, CA) and K Bedard (Halifax, CA), we recently identified pathogenic germline
mutations in non-CDH1 genes in FGC cases using a multiplex panel sequencing approach. We developed a custom panel of 55 genes
for which germline or somatic aberrations have been implicated in familial upper gastrointestinal cancers or cancer susceptibility
syndromes. The list included well-established tumor suppressor genes and those associated with loss of expression in upper
gastrointestinal tumour samples. We screened germline DNA from 116 probands from families who met International Gastric
Cancer Linkage Consortium clinical criteria for HDGC (n=107, all tested negative for CDH1 mutations) or FIGC (n=9). Samples were
collected from BC Cancer Agency (Vancouver, CA), IPATIMUP, University of Siena (Siena, Italy) and Halifax, CA. Using a multiplexed
next generation sequencing assay, we identified pathogenic or likely pathogenic variants in 18% of cases (n=21) (17 HDGC and 4 FIGC
families). Variants included clearly pathogenic, novel truncating mutations in 5 unrelated families (two different mutations in CTNNA1,
two different mutations in BRCA2 and one novel mutation in MAP3K6). Mutations in SDHB (2 families) and STK11 (2 families) and 4
missense MAP3K6 (5 families) were also identified. Additional truncating mutations were identified in genes of moderate-penetrance
(risk-association): ATM (4 families), MSR1 (2 families) and PALB2 (1 family). Novel missense variants predicted to be damaging by in
silico methods were found in 32 cases (27.6%) in different genes within the panel.
(Hansford et al., “Genetic basis of familial gastric cancer: Beyond the CDH1-locus”. Manuscript under review in JAMA; and Gaston et
al., “Germline Mutations in MAP3K6 Predispose to Gastric Cancer.” Manuscript under review in PloS Genetics).
Rare CDH1 non-coding variants occur in a fraction of Hereditary Diffuse Gastric Cancer negative for CDH1
coding mutations
We hypothesize (Our lab in collaboration with D Huntsman’s lab) that HDGC families lacking CDH1-coding mutations may harbour
germline alterations in CDH1 non-coding regions, as of the 50% HDGC families that remain without molecular diagnosis, 2/3 present a
phenotype of monoallelic CDH1 downregulation and >90% display loss of E-cadherin protein expression in tumours.
We screened the complete CDH1 locus of 90 bonafide CDH1-negative HDGC probands by next generation sequencing. We filteredout 42 true rare heterozygous germline CDH1 non-coding variants, which may be disease causative for additional 39% of probands.
Importantly, 10/42 probands carrying these variants were assessed for CDH1 germline ASE, and 5/10 displayed massive CDH1 mRNA
monoallelic downregulation, supporting our hypothesis. Our bioinformatics query predicted that the presence of unique CDH1
non-coding variants often interfered with binding of several transcription factors, some of which are classical CDH1 expression
modulators. Moreover, several variants (and surrounding sequence) occur in highly conserved regions and locate within DNAseI-HSS,
widely accepted as regulatory regions.
Our data suggest that at least some CDH1 non-coding variants may cause germline CDH1 monoallelic downregulation if they occur within
regulatory sequences crucial for adequate E-cadherin expression control, namely at binding sites for CDH1 expression modulators.
Any novel pathogenic alteration identified in this cohort will warrant a complete redefinition of the screening methodology currently
applied to HDGC families, and will provide important insights for the understanding of CDH1 expression regulation.
(Pinheiro H, Oliveira P et al., Manuscript in preparation.
In vitro models to test tumor-targeted nanoparticles for early diagnosis of gastric cancer
Previous work by the group identified CD44v6 as an adequate molecular marker for gastric cancer and corresponding pre-malignant
lesions, with particular specific expression cells that loose E-cadherin expression from HDGC families. We aimed at identifying
molecular probes able to specifically target CD44v6 in gastric cancer invasive cells, which will then be grafted in nanoparticles
ultimately designed to attach to the stomach wall, cross the mucosal barrier epithelium, target CD44v6 positive cells and provide a
signal for diagnosis.
We were successful in establishing two gastric cancer cell lines, expressing de-novo either, the canonical form of CD44 (CD44 standard),
or the exon V6 containing CD44v6 proteins. We could complete functional characterization of both cell lines including invasion and
slow aggregation assays, apoptosis resistance, proliferation and expression of CD44 interactors/effectors. These cell lines were made
available to the project team members at INEB to be used as test tubes for development of tumor-targeted nanoparticles.
(Ferreira D et al, ongoing work)
We studied the physic-chemical characterization and stability of nanoparticles produced by chitosan chains electrostatically linked by
ionic interactions. Several factors affecting the stability of chitosan nanoparticles were examined in vitro. The results obtained in the
simulated gastric conditions proved that these nanoparticles meet the necessary requirements to be used in GC diagnostic strategy.
Chitosan nanoparticles have a positive charge to adhere to gastric wall (16-23 mV) and a particle size between 100-200 nm at a pH
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2013
range from 2.3 to 5.8 in order to be able to transpose the gastric mucosa. At a pH of 1.2 particle disintegration occurred, showing the
sensitivity of these nanoparticles to pH values under 2.3, which can be overcome in clinics.
(Lourenço B, et al, ongoing work).
Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA
We hypothesized that 20% of HDGC families which are caused by nonsense CDH1 mutations, leading to premature protein truncation
could benefit from readthrough therapies using suppressor-tRNAs that can replace premature stop codons with a cognate amino
acid, and generate full-length wild-type proteins. The use of Suppressor-tRNAs in HDGC patients could constitute a less invasive
therapeutic option for nonsense mutation-carriers, delaying the development of gastric cancer.
Our analysis revealed that 23/108 (21.3%) of E-cadherin mutant families carried nonsense mutations that could be potentially
corrected by 8 suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis,
we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin deficient cell lines were
transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, Westernblot, Immunofluorescence, Flow-cytometry and PLA-Proximity ligation assay were used to establish the model and monitor mRNA and
protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene carrying a nonsense mutation and
the suppressor-tRNA recovered full-length E-cadherin expression, and its correct localization and incorporation into the adhesioncomplex.
This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate-amino acid
replacement with suppressor-tRNAs. 21.3% of HDGC families are candidates for correction with suppressor-tRNAs to potentially delay
cancer onset.
(Bordeira-Carriço et al, Accepted for publication in Eur J Hum Genet, 2013).
Prognostic factors and targets of therapy allow molecular stratification of gastric cancer for whole genome seq,
Methylseq and RNAseq
We systematically determine the frequency of somatic CDH1 alterations in gastric cancer and analyzed their impact in the prognosis
and survival of patients with GC. We found that CDH1 somatic alterations exist in all clinical settings and histotypes of gastric cancer
and, most importantly, correlated with different survival rates. Patients with tumours carrying CDH1 structural alterations, particularly
those belonging to families with familial aggregation of intestinal gastric cancer, presented the worst survival rate among all cases.
This study disclosed that screening of CDH1 alterations at gastric cancer diagnosis may predict patient prognosis and is likely to
improve patients’ clinical management.
(Corso G and Carvalho J, et al, Journal of Clinical Oncology 2013).
We hypothesize that appropriate stratification of gastric cancer according to clear molecular features with impact in prognosis and
treatment would shed light into gastric cancer-specific molecular signatures crucial for the identification of therapy targets and the
development of new therapeutic options for personalized therapy. We extracted DNA under rigorous quality control standards from
a series of 100 cases (matched tumour and normal) for CDH1 deletion and HER2 overexpression/amplification as a bad prognosis
markers, and Microsatellite instability (MSI) as a good prognosis marker. We found that CDH1 deletion alone was present in 12% of
cases, HER overexpression/amplification alone was present in 9% of cases, and MSI was present in 29% of cases. A series of 50 cases
with these particular features was subjected to mapping of genetic variation at the genome level (Whole genome seq), the epigenetic
level (Methyl-seq) and expression level (RNA-seq)
(Carvalho J et al, Ongoing). The ERiC group, IPATIMUP Diagnostics team and Fátima Carneiro’s team were involved in this study in
collaboration with Coimbra Genomics and BGI.
In vitro models of Epithelial-Mesenchymal-Epithelial transition mimic tumour heterogeneity phenotypes and
highlight molecules secreted in microvesicles
Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are fundamental mechanisms during embryonic
development and cancer. We established two EMT-MET in vitro models by treating near-normal immortalized mammary cell lines
with/without TGF-ß1. We hypothesise that we could reproduce, in these transitions, features of cancer cells from transformation to
invasive and aggressive phenotypes, namely trans-differentiation into heterogeneous cell populations, self-renewal and tumorigenic
ability, activation of tumorigenic pathways, and crosstalk with neighbour cells through microvesicles.
We found that these cell lines, after undergoing EMT-MET, display: heterogeneous phenotypes that are nor epithelial nor mesenchymal;
increased self-renewal and increased ability to form tumours in the Chicken CAM and in nude mice, and; activation of tumorigenic
pathways
(Oliveira P et al, Manuscript in preparation).
We validated miR-22 as a direct modulator of CDH1/E-cadherin expression that lead to E-cadherin membrane expression loss
accompanied by miR-101 downregulation and consequent EZH2 overexpression. We found that overexpression of miR-22 seems to
induce EMT and that miR-22 was differentially expressed across EMT/MET. We started establishing and optimizing several techniques
related with extracellular vesicles’ isolation and characterization, that will allow the functional evaluation of the role of extracellular
vesicles in EMT/MET and cancer metastasis
(Carvalho J et al, Ongoing)
We also found that Dies1 is differentially expressed across EMT/MET and downregulated in primary gastric cancers and cell lines. We
are further exploring the role of this gene in gastric tumorigenesis by using human gastric cancer samples and studies in Zebrafish
(Oliveira P et al, Ongoing).
mRNA translational errors accelerate tumour progression through deregulation of protein synthesis
Misexpression and misprocessing of transfer RNAs, aminoacyl-tRNA synthetases, ribosomes and translational factors are common
in cancer, but their biomedical relevance is not understood. We have hypothesized that such deregulation would affect mRNA
decoding accuracy and could work either as a trigger to initiate cancer and/or confer advantageous features to malignant neoplastic
cell populations. Near-normal NIH3T3 cells engineered for mistranslation formed foci on soft agar, increased angiogenesis in a
chorioallantoic membrane (CAM) assay and accelerated tumor development in mice. These mistranslations also up-regulated the PP1
regulator GADD34, dephosphorylation of the eIF2a, and increased of protein synthesis rate. The overall data show that proteomes
of cancer cells are statistical and up-regulate translation initiation. We postulate, therefore, that mistranslation contributes to the
cellular heterogeneity of tumors and might be highly oncogenic.
(Pereira P and Santos Mafalda et al, Manuscript in preparation).
A bioinformatics data analysis company named Bioinf2Bio was created in August 2013
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Bioinf2Bio is a bioinformatics data analysis company aimed at researchers and clinicians from all biological backgrounds working with
high throughput technologies, such as microarrays and next-generation sequencing. The company aims at drawing relevant biological
meaning from medium and large scale genomic and transcriptomic approaches, namely microarrays and next-generation sequencing.
Such approaches may result from biological and clinical queries performed by the Researcher/Clinician (data analysis) or from
database-deposited data (data mining) relevant for the Researcher/Clinician.
Bioinf2Bio’s provides highly personalized and customizable analysis, combined with biological know-how, allowing Researchers and
Clinicians to define the relevant next wet-lab steps necessary for research thriving.
Website: http://www.frombioinformatics2biology.com/home.html
Collaborative partners for AIM 1. Genetics, diagnosis and therapy of Familial Gastric Cancer
International collaborators:
David Huntsman, British Columbia Cancer Agency, Vancouver Canada
Karen Bedard, Dalhousie University, Halifax, Canada
Franco Roviello, University of Siena, Italy
Rebecca Fitzgerald, University of Cambridge, UK
National collaborators:
Pedro Granja, INEB, Porto, Portugal
José Carlos Machado, IPATIMUP, Porto, Portugal
Raquel Seruca, IPATIMUP, Porto, Portugal
Fátima Carneiro, IPATIMUP and Medical faculty of Porto, Porto, Portugal
Gabriela Soares, Center of Medical Genetics Jacinto de Magalhães, Porto Hospital Center, Porto; Portugal
Teresa Almeida Santos, University Hospitals of Coimbra & Faculty of Medicine, University of Coimbra, Coimbra, Portugal
Isabel Claro, Instituto Português de Oncologia de Lisboa Francisco Gentil E.P.E., Lisbon, Portugal
Collaborative partners for AIM 2. Prognostic factors and molecular characterization/stratification of gastric
cancer
Beijing Genomics Institute, China
Angela Nieto, Instituto de Neurociencias de Alicante, Alicante, Spain
José Carlos Machado, IPATIMUP, Porto, Portugal
Fátima Carneiro, IPATIMUP and Medical faculty of Porto, Porto, Portugal
Collaborative partners for AIM 3. In vitro models of Epithelial-Mesenchymal-Epithelial transition to study
tumour heterogeneity and cellular crosstalk through secreted microvesicles
David Huntsman, British Columbia Cancer Agency, Vancouver, Canada
Cristina Barrias, INEB, Porto, Portugal
Susana Santos, INEB, Porto, Portugal
Raquel Almeida, IPATIMUP, Porto, Portugal
Celso Reis, IPATIMUP, Porto, Portugal
Joana Paredes, IPATIMUP, Porto, Portugal
João Taborda Barata, IMM, FMUL, Lisboa, Portugal
Nuno Bonito, IPO, Coimbra, Portugal
Organization of International Meetings:
Gabriela Almeida was a co-Organizer of the XXII Porto Cancer Meeting on “Translational Research in Cancer – Bringing basic science
and pathology to clinical oncology”, 11-12 April 2013, Porto, Portugal.
Carla Oliveira was a co-Organizer and Scientific Program Committee Member of the European Society of Human Genetics Meeting,
08-11 June 2013, Paris, France.
Networking within European COST Action I:
Carla Oliveira and Joana Carvalho are Management Committee member (C. Oliveira) and Management Committee substitute member
(J Carvalho) of the BMBS COST Action BM1202 “European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD)”
(http://www.cost.eu/domains_actions/bmbs/Actions/BM1202?management). This network currently has partners from 20 different
countries. Within the scope of this COST Action, CO and JC have actively participated in two meetings: 1) Work group and Management
Committee Meeting, May 16th - 17th, 2013, Dublin, Ireland; 2) 2nd Working Group Meeting, 06 – 07 October 2013 Budapest, Hungary.
JC also participated in the course on “Extracellular vesicles: implications in Biomedicine” held in Valencia, Spain.
Networking within European COST Action II:
Gabriela Almeida is a Management Committee member, and the Short Term Scientific Missions Coordinator, of European COST Action
CM1106, on “Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells” (http://www.cost.eu/domains_actions/cmst/
Actions/CM1106?management) that currently has partners from 65 different laboratories from 33 different countries. Within the
scope of this COST Action, G Almeida has actively participated in two meetings since joining the ERiC Group in April 2013: 1) Coregroup Meeting, April 16th - 17th, 2013, Milano – Università degli Studi di Milano, Italy; 2) 2nd Working Group Meeting, 19 – 20
September 2013 Warsaw – Warsaw University of Life, Poland.
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Future Research
Aim 1. Molecular determinants of familial gastric cancer
A large cohort of gastric cancer families (n=500), negative for germline mutations in CDH1, will be analyzed (Research application
submitted to “No Stomach for Cancer”, D. Huntsman and C. Oliveira co-PIs. Clinical history and tumor histopathology will be revisited
and probands will be tested by NGS under a targeted-based approach (gene panels and intronic portion of CDH1). Findings from these
analyses will be validated on a hypothesis-driven perspective, using in vitro and in vivo (chick embryo/CAM and mouse models) tools
to assess functional significance of variants found.
This work will be conducted in collaboration with D Huntsman and several members of the International Gastric Cancer Linkage
Consortium (IGCLC), as well as with our medical partners in Portuguese hospitals.
Diagnostic tools for early detection of gastric cancer based on nanoparticles and immobilized antibodies will also be developed in
collaboration with P Granja’s team at INEB, Porto, Portugal.
Aim 2. Intra-tumoral molecular landscapes and mediators of cellular crosstalk during gastric cancer initiation
and progression
Using Bioinformatics and molecular biology, we will analyze gastric cancer cases stratified according to selected prognostic markers/
therapy targets status, and submitted to WGS, RNAseq and Bissulphite sequencing, to obtain and validate specific molecular signatures.
We will use in vitro cell line models and high-throughput genomics, transcriptomics and proteomics approaches to disclose factors
from the gastric cancer secretome (exosomes and microvesicles), that have functional autocrine/paracrine effects and perpetuate
aggressive cancer phenotypes. This work will be conducted in collaboration with BGI, Coimbra Genomics, Fátima Carneiro, José Carlos
Machado, Nuno Bonito and Manuel Santos.
Aim 3. Determine whether gastric cancer-specific molecular signatures impact patient’s survival and therapy
We wish to understand whether gastric cancers, displaying particular gastric cancer-associated molecular signatures and/or particular
profiles of secreted biomarkers, display different survival rates and/or respond to combinations of conventional chemotherapy with
targeted therapies designed for those signatures. To assess impact in survival, we will run retrospective analysis in discovery and
validation series. To test therapy design, we will select/produce cell line models mimicking the molecular signatures that will be
xenografted in mouse /CAM models and then submitted to conventional chemotherapy and targeted. This work will be conducted in
collaboration with Fátima Carneiro, Ola S¿derberg (Upsala, Sweden) and Nuno Bonito.
Carla Oliveira: Member of the scientific committee, Supervisor and Lecturer of the FCT-funded PhD Programs: Graduate Program in
Areas of Basic and Applied Biology (GABBA); Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences
(BiotechHealth)
Patrícia Oliveira, Gabriela Almeida and Carla Oliveira: Graduate Program in Areas of Basic and Applied Biology (GABBA). Oncobiology
Module.
Carla Oliveira: Programa de Doutoramento em Medicina e Oncologia Molecular, Faculdade de Medicina da Universidade do Porto.
Oncobiology Module.
Carla Oliveira: Programa de Doutoramento em Ciências da Saúde, Faculdade de Medicina da Universidade de Coimbra.
Carla Oliveira: Examiner and Jury member at PhD Thesis: Invited Jury member (examiner): Ph.D. Dissertation of Pedro Costa. Univ.
Coimbra, Portugal; 2013; Member of the Ph.D. Dissertation Committee of Bruno Pereira, FMUP, Univ. Porto, Portugal; 2013; Invited
Jury member (examiner): Ph.D. Dissertation of Cristovão Sousa. L’Université Pierre et Marie Curie. Paris, France; 2013 ;Member of the
Ph.D. Dissertation Committee of Renata Bordeira-Carriço. FMUP, Univ. Porto, Portugal; 2013
Carla Oliveira: Mestrado em Medicina e Oncologia Molecular, FMUP. Módulo de Oncobiologia
Carla Oliveira: Mestrado em Biologia da Universidade de Aveiro. Módulo de Biologia do Genoma
Carla Oliveira: Examiner at Masters Thesis Juries: Invited Jury member (examiner): Masters Dissertation of Marta Cardoso. ICBAS,
Univ. Porto, Portugal; 2013
Invited Talks
Carla Oliveira. “Dissecting the role of cadherins with OMICS technologies”. Course: “New frontiers of OMICs in Medicine - from the lab
to a clinical setting”. IBILI Coimbra, Portugal. 18/01/2013.
Carla Oliveira. “From disease to mechanisms: old and new stories about E-cadherin”. IMMS - Instituto Mexicano de Seguro Social .
Guadalajara, Mexico. 22/01/2013.
Carla Oliveira. “Cancer Metastization: Examples from the wet lab”. 3rd Workshop on Cancer Research, IPATIMUP. Porto, Portugal .
16/05/2013.
Carla Oliveira. “Genetics of hereditary diffuse gastric cancer – HDGC”. Funderburg Symposium - “Current Approach to Hereditary
Diffuse Gastric Cancer (HDGC) syndrome”. DDW 2013 Digestive Disease Week . Orlando, USA. 19/05/2013.
Carla Oliveira. “HDGC: Updated guidelines for clinical management” . IGCC 2013, 10th International Gastric Cancer Congress. Verona,
Italy. 20/06/2013.
Carla Oliveira. “Molecular mechanisms modulating the expression and function of the invasion suppressor protein E-cadherin”.
Institut Curie . Paris, France. 11/07/2013.
Carla Oliveira. “Cancro Gástrico e seu tratamento”.. Workshop Universidade do Minho “A terapêutica do doente oncológico: o papel
dos profissionais de saúde” . Braga, Portugal. 19/07/2013.
Carla Oliveira. “Modulation of E-cadherin expression and function in gastric cancer - clinical and molecular features”. 19TH GI CANCER
FORUM - The Martin Cohen Lecture Theatre, CRUK CRI - Li Ka Shing Centre. Cambridge, UK. 26/09/2013.
Carla Oliveira. “Germline and somatic determinants in gastric cancer - impact in diagnosis, prognosis and treatment”. Monday
Seminar Medical Faculty University of Porto. Porto, Portugal. 16/10/2013.
Carla Oliveira. “Metastastic pathways in gastric cancer: is there a role of Extracellular Vesicles?” . 2nd Meeting COST Action_BM1202
ME-HAD. Budapest, Hungary. 07/10/2013.
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Carla Oliveira. “Modulation of cell-cell adhesion in cancer and epithelial-mesenchymal-epithelial transition?”. IMM Monday lectures.
Lisbon Portugal. 04/11/2013.
Carla Oliveira. “Using molecular stratification and NGS to Understand Structural and Regulatory Variation in Gastric Cancer” . 17th
SPGH Annual Meeting. Coimbra Portugal. 22/11/2013.
Carla Oliveira. “Using NGS and in Vitro Models to Understand Structural and Regulatory Variation in Gastric Cancer” . Bio-IT World and
Cambridge Healthtech Institute’s Second Annual, Clinical Genomics and Genome Informatics Europe, Deciphering Disease through
Sequencing Data. Lisbon Portugal. 06/12/2013.
Carla Oliveira. “Using Next Generation Sequencing to Uncover Structural and Regulatory Variation in Gastric Cancer”. Lançamento da
Rede Nacional de Bancos de Tumores . Lisbon, Portugal. 12/12/2013.
Joana Carvalho. Modulating the expression and function of E-cadherin in Gastric Cancer: classical and alternative mechanisms. “I Ciclo
de Conferências – Biomédicas à Tarde” organized by students from Biomedical Sciences, Faculty of Health Sciences, University of Beira
Interior. Covilhã, Portugal. 7/05/2013.
Patrícia Oliveira. “(Re)-creating a dynamic EMT/MET in vitro model: from cells to RNA and maybe to novel biomarkers”. IV Simpósio
Técnico de Anatomia Patológica . Porto, Portugal. 27/09/2013.
Oral Presentations
Joana Carvalho. Extracellular Vesicles: shuttles of Epithelial and Mesenchymal transitions and Cancer Metastasis. ME-HAD COST
Meeting. Budapest, Hungary. 06/10/2013.
Patrícia Oliveira. “A dynamic Epithelial to Mesenchymal to Epithelial transition in vitro model to analyze drug response”. 2nd Working
Group Meeting COST Action CM1106. Warsaw, Poland. 20/09/2013.
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Genetic Diversity
Objectives
The group aims to establish a bridge between human population and clinical genetics. We study evolution, genetic drift, migration,
expansion, bottleneck and selection, which are the major forces designing the genetic diversity across the world. Then we apply the
evolutionary based studies to the identification of candidate genes/variants which model human adaptation and health.
Till recently, both population and complex-disease genetics were largely focused on the mitochondrial DNA (mtDNA). We were
successful in establishing international collaborations that enabled us to study populations that cover Eurasia, Africa and East Asia.
We have been investigating the main human migrations, responsible for setting up the worldwide genetic distribution, as the out-ofAfrica, the settlement of Europe and East Asia, and the trans-Atlantic slave trade. We are also showing that this knowledge is essential
to disentangle between neutral and pathologic variants, estimate times of emergence of polymorphisms, describe geographic
dispersions, and applying that knowledge in the clinical field, namely in the cancer area.
Accompanying the advances of the genomics era, we are upgrading to the screening of genome-wide chips (GWAS) containing millions
of single nucleotide polymorphisms (SNPs). These allow unbiased overall screens of the genome and reliable evaluations of candidate
genes and population structure. We are pursuing these screenings to study human population diversity and to evaluate the European
genetic susceptibility to dengue infection.
By the end of 2013, the group included the PI Susana Seixas and her students, who study proteolysis genes. We are improving the
genetic diagnosis and characterisation of deficient SERPINA1 genotypes, which underlie a common Mendelian disease in Europeans.
We are also analysing 3 gene clusters (SERPINB, WFDC and KLK) as potential targets of natural selection through their involvement in
reproduction and response against pathogens.
Main Achievements
Evaluating purifying selection in the mitochondrial DNA of various mammalian species
Mitochondrial DNA (mtDNA), the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under
the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of
nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by
phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of
predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection
in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of
other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of
purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher
pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors
of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this
selection is fundamental to evolutionary studies and to the identification of deleterious mutations. [Soares et al. (2013) PLoS One 8:
e58993].
A substantial prehistoric European ancestry amongst Ashkenazi maternal lineages
The origins of Ashkenazi Jews remain highly controversial. Like Judaism, mitochondrial DNA is passed along the maternal line. Its
variation in the Ashkenazim is highly distinctive, with four major and numerous minor founders. However, due to their rarity in the
general population, these founders have been difficult to trace to a source. Here we show that all four major founders, B40% of
Ashkenazi mtDNA variation, have ancestry in prehistoric Europe, rather than the Near East or Caucasus. Furthermore, most of the
remaining minor founders share a similar deep European ancestry. Thus the great majority of Ashkenazi maternal lineages were not
brought from the Levant, as commonly supposed, nor recruited in the Caucasus, as sometimes suggested, but assimilated within
Europe. These results point to a significant role for the conversion of women in the formation of Ashkenazi communities, and provide
the foundation for a detailed reconstruction of Ashkenazi genealogical history. [Costa et al. (2013) Nat. Commun. 4: 2543].
The first modern human dispersals across Africa
The emergence of more refined chronologies for climate change and archaeology in prehistoric Africa, and for the evolution of human
mitochondrial DNA (mtDNA), now make it feasible to test more sophisticated models of early modern human dispersals suggested
by mtDNA distributions. Here we have generated 42 novel whole-mtDNA genomes belonging to haplogroup L0, the most divergent
clade in the maternal line of descent, and analysed them alongside the growing database of African lineages belonging to L0’s sister
clade, L1’6. We propose that the last common ancestor of modern human mtDNAs (carried by “mitochondrial Eve”) possibly arose in
central Africa ~180 ka, at a time of low population size. By ~130 ka two distinct groups of anatomically modern humans co-existed in
Africa: broadly, the ancestors of many modern-day Khoe and San populations in the south and a second central/eastern African group
that includes the ancestors of most extant worldwide populations. Early modern human dispersals correlate with climate changes,
particularly the tropical African “megadroughts” of MIS 5 (marine isotope stage 5, 135–75 ka) which paradoxically may have facilitated
expansions in central and eastern Africa, ultimately triggering the dispersal out of Africa of people carrying haplogroup L3 ~60 ka.
Two south to east migrations are discernible within haplogroup L0. One, between 120 and 75 ka, represents the first unambiguous
long-range modern human dispersal detected by mtDNA and might have allowed the dispersal of several markers of modernity. A
second one, within the last 20 ka signalled by L0d, may have been responsible for the spread of southern click consonant languages
to eastern Africa, contrary to the view that these eastern examples constitute relicts of an ancient, much wider distribution. [Rito et
al. (2013) PlosOne. 8: e58993].
The genetic impact of the Lake Chad Basin population in North Africa as documented by mitochondrial diversity
and internal variation of the L3e5 haplogroup
The presence of sub-Saharan L-type mtDNA sequences in North Africa has traditionally been explained by the recent slave trade.
However, gene flow between sub-Saharan and northern African populations would also have been made possible earlier through the
greening of the Sahara resulting from Early Holocene climatic improvement. In this article, we examine human dispersals across the
Sahara through the analysis of the sub-Saharan mtDNA haplogroup L3e5, which is not only commonly found in the Lake Chad Basin
(~17%), but which also attains nonnegligible frequencies (~10%) in some Northwestern African populations. Age estimates point to its
origin ~10 ka, probably directly in the Lake Chad Basin, where the clade occurs across linguistic boundaries. The virtual absence of this
specific haplogroup in Daza from Northern Chad and all West African populations suggests that its migration took place elsewhere,
perhaps through Northern Niger. Interestingly, independent confirmation of Early Holocene contacts between North Africa and the
Lake Chad Basin have been provided by craniofacial data from Central Niger, supporting our suggestion that the Early Holocene
offered a suitable climatic window for genetic exchanges between North and sub-Saharan Africa. In view of its younger founder age in
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North Africa, the discontinuous distribution of L3e5 was probably caused by the Middle Holocene re-expansion of the Sahara desert,
disrupting the clade’s original continuous spread. [Podgorna et al. (2013) Ann. Hum. Genet. 77: 513-523].
A founder SDHB mutation in Portuguese paraganglioma patients
We report a genetic screening of SDHB, SDHC, SDHD and SDHAF2 genes (hereafter abbreviated to SDHx) in 37 patients with
paragangliomas (PGL) and phaeochromocytomas (PCC) from northern Portugal, diagnosed between 2009 and 2012, of which three
were familial cases and 34 were sporadic. Sporadic cases were defined as those having no familial history of PGL or PCC in the parental
and grandparental generations and disease was considered to be inherited when at least two first-degree relatives or two second
degree relatives were affected by these tumours. Data showed that the majority of PGL in northern Portugal patients develop as a
consequence of germline SDHx mutations, in particular a founder mutation in the SDHB gene (15 678 bp deletion). Our results support
the suggestion by Hensen et al. (2012) that population specificities, regarding the presence of founder germline SDHx mutations,
should be taken into account whenever deciding about the genetic testing in PGL/PCC patients. [Martins et al. (2013) Endocr Relat
Cancer. 20:L23-26].
Genetic and archaeological perspectives on the initial modern human colonization of southern Asia
It has been argued recently that the initial dispersal of anatomically modern humans from Africa to southern Asia occurred before
the volcanic “supereruption” of the Mount Toba volcano (Sumatra) at ~74,000 y before present (B.P.)-possibly as early as 120,000 y
B.P. We show here that this “pre-Toba” dispersal model is in serious conflict with both the most recent genetic evidence from both
Africa and Asia and the archaeological evidence from South Asian sites. We present an alternative model based on a combination of
genetic analyses and recent archaeological evidence from South Asia and Africa. These data support a coastally oriented dispersal of
modern humans from eastern Africa to southern Asia ~60–50 thousand years ago (ka). This was associated with distinctively African
microlithic and “backed-segment” technologies analogous to the African “Howiesons Poort” and related technologies, together with
a range of distinctively “modern” cultural and symbolic features (highly shaped bone tools, personal ornaments, abstract artistic
motifs, microblade technology, etc.), similar to those that accompanied the replacement of “archaic” Neanderthal by anatomically
modern human populations in other regions of western Eurasia at a broadly similar date. [Mellars et al. (2013) Proc Natl Acad Sci USA.
110:10699-10704].
Anavaj Sakuntabhai, Institut Pasteur, Paris, France and colleagues of the DENFREE EU project - Genetic susceptibility in infectious
diseases – dengue fever
Martin Richards, University of Huddersfield, UK - Genetic diversity of European and East Asian populations
Viktor Cerny, Academy of Sciences of the Czech Republic, Prague - Genetic diversity of Arabian Peninsula and African populations
David Samuels, Vanderbilt University Medical Center, Nashville, TN, USA - Statistics applied to mtDNA clinical genetics
Vincent Macaulay, University of Glasgow, UK - Statistics applied to genetics
João Zilhão, University of Barcelona, Spain - Dating of archaeological samples from the Mediterranean Neolithic
Future Research
Genome-wide screening in African Sahel
We are screening the Illumina chip for 2.5 million SNPs in 170 individuals from several populations. Preliminary results testify that
Sahel was the main corridor for west-east migrations, and for migrations towards north and south, and is revealing interesting genetic
patterns in the nomadic Fulani. This geographical region was important for the emergence of certain phenotypes, such as malaria
susceptibility, food adaptations and potentially dengue infection resistance – we evaluate the selection of these traits across Sahel.
This is part of the Marie Curie project EUROTAST.
Dengue infection and the European susceptibility
In the context of the 5-years EU-funded project DENFREE we will perform genome-wide association studies in Thailand, Cuba, Brazil
and Nicaragua. The Latin American samples are very interesting due to the high level of admixture between African (resistant) and
European (susceptible) gene pools. The admixture increases the statistical power to detect associations, as we have confirmed in a
preliminary analysis in Cuba. By studying these populations we will identify candidate genes/SNPs conferring susceptibility to dengue
in people with a main European background. We will also check the frequency distribution of these SNPs worldwide, by TaqMan assay,
with an emphasis in Europe: we will screen around 100 individuals from 30 worldwide populations and add information from the 1000
genomes project.
mtDNA diversity in Southeast Asia
This work was funded in the last round of FCT projects (beginning June 2013) and has a PhD student dedicated to it. If there is a region
of the globe for which we need more information about the genetic diversity this is Southeast Asia. We will dedicate a big effort to the
characterisation of mtDNA complete sequences and genome-wide diversity to unravel the past of human migrations in Asia.
Somatic mutations in mitochondrial and nuclear encoded genes, responsible for the oncocytic phenotype in
thyroid tumors
We will assemble the mtDNA for all thyroid tumor and paired normal samples available in The Cancer Genome Atlas (TCGA). We
will identify somatic mtDNA changes (fixed and heteroplasmic) in the tumor and assess its pathogenicity. This will also be done for
the nuclear DNA genes acting in the oxidative phosphorylation. The genetic evaluation will be coupled with a careful revision of
the oncocytic phenotype in thyroid tumors performed by expert pathologists in the TCGA slides. The results gathered on the TCGA
analyses will allow to formulate hypothesis that we will test in lab experiments. This work will be performed in collaboration with the
Cancer Biology group and David C. Samuels.
Evolution of the proteolysis-related genes and role in fertility and immunity
We will continue studying the WFDC and KLK clusters, now to uncover in which extent the genetic variation of these genes underlie
phenotypic variation in male reproductive function in both healthy and infertile individuals; while simultaneously contributing to
better characterization of these genes with combined roles in fertility and immunity. We anticipate a follow-up of this research line
centred in other genes possibly affecting human reproductive fitness.
Proteolysis and association to pulmonary disorders
We intend to explore the association of SERPINA1 to a complex autoimmune disorder characterized by the inflammation of the
respiratory tract and in SERPINA1 deficient patients we aim to identify additional genetic modifiers of pulmonary disease. Furthermore,
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we expect to investigate the role of proteolysis in Chronic Obstructive Pulmonary Disease (COPD) and lung cancer progression using
high-throughput approaches (by target sequencing of candidate genes identified in proteomic surveys). Our ultimate goal is to uncover
unsuspected genetic susceptibility to COPD and/or lung cancer, and to discriminate potentially selective advantageous mutations in
primary tumors and metastasis.
Luisa PereiraSeminar in the Programa Doutoral e Mestrado de Investigação Biomédica, Faculty of Medicine University of Coimbra
Luisa Pereira - co-supervisorMarta Daniela Araújo Costa - Genetic characterisation of the Early Upper Palaeolithic settlement of
Europe by modern humans - University of Leeds, UK
Luisa Pereira - co-supervisorJoana Isabel Barbosa Pereira - Genetic characterisation of modern human dispersals in the Greater
Mediterranean - University of Leeds, UK
Oral Presentations
TRISKA P, CERNY V, SOARES P, PEREIRA L. Genome-wide analysis across the African Sahel corridor. Bioinformatics Open Days. University
of Minho. Braga. 15/03/2013.
TRISKA P, CERNY V, SOARES P, PEREIRA L. Population Structure of the African Sahel inferred from Genome-Wide SNP Screening. XXXVIII
Portuguese Genetics Conference. ICBAS. Porto. 04/06/2013.
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Objectives
The main research line of the group “Glycobiology in Cancer” addresses the role that glycosylation plays in cancer.
The group has developed two main research projects:
1 - Characterization of the molecular mechanisms underlying the glycan-mediated adhesion of Helicobacter pylori and the
understanding of the importance of the host-pathogen crosstalk for the chronic infection.
2 - Evaluation of the role of glycans in cancer addressing the molecular mechanisms controlling glycosylation of key proteins involved
in cancer development and progression, and identification of novel biomarkers with clinical application.
Main Achievements
Main Achievement I
We have characterized glycosylation changes in circulating serum proteins in patients with precursor lesions of gastric cancer. Applying
a glycoproteomic approach we have identified altered glycoproteins expressing the simple mucin-type carbohydrate antigens T
and STn in the serum of patients with gastritis, IM (complete and incomplete subtypes), and control healthy individuals. One of the
glycoproteins identified was plasminogen, which was further characterized at the structural level and showed to carry STn antigens in
patients with gastritis and IM. These results provide evidence of serum proteins displaying abnormal O-glycosylation in patients with
precursor lesions of gastric carcinoma (Gomes et al., J Proteome Res, 2013)
Main Achievement II
We have demonstrated that E-cadherin is regulated by glycosylation controlled by specific glycosyltransferases (GnT-III and GnT-V)
in a gastric cancer context. We have showed that GnT-III-mediated glycosylation has a stabilizer effect on E-cadherin whereas GnTV-mediated glycosylation has a destabilizer effect. This supports the role of GnT-III on Ecadherin-mediated tumor suppression, and
GnT-V on E-cadherin-mediated tumor invasion. These novel regulatory mechanisms of E-cadherin functions were further validated
in human gastric carcinoma clinical samples (Pinho SS et al. Biochim Biophys Acta. 2013; Pinho SS et al. Trends in Molecular Medicine
2013).
We have shown that Insulin/IGF-I signaling pathways enhances tumor cell invasion through bisecting GlcNAc N-glycans modulation,
demonstrating an interplay with E-Cadherin. (de-Freitas-Junior JC Pinho SS et al. PLoS One. 2013)
Main Achievement III
We have characterized the role of SLe(x) expression in gastric carcinoma cells. We have shown that the expression of ST3GAL4 leads to
SLe(x) expression and induces c-Met activation as well as downstream signaling pathways modulating an invasive phenotype in gastric
carcinoma cells. (Gomes et al., PLoS One. 2013)
We have collaborated on the characterization of the role of other Sialylated glycans in cancer, including Sialyl-Tn (Ferreira et al., Mol
Oncol. 2013; Almeida et al., Electrophoresis 2013)).
Main Achievement IV
We have established innovative mass spectrometry methods for studying glycosylation in cancer (Osório and Reis; Methods Mol Biol.
2013) and described methods for evaluation of specific glycosyltransferases expression in gastric tissues (Gomes and Reis, Methods
Mol Biol. 2013).
Main Achievement V
In collaboration with INEB we have contributed for the development and characterization of bioengineered surfaces that promote
specific protein-glycan mediated binding of the gastric pathogen Helicobacter pylori (Parreira et al., Acta Biomater., 2013) and
characterized the.bacterial-binding chitosan microspheres for gastric infection treatment and prevention (Gonçalves et al., Acta
Biomater., 2013). This work resulted in a patent (US Patent Application No. 61/642,587 - Chitosan microspheres).
Main Achievement VI
Salomé Pinho, Celso Reis, et al: inventors of provisional patent application nº 20131000040844 (submitted to Instituto Nacional
de Propriedade Industrial) “N-GLICOSILAÇÃO DO RECETOR DAS CÉLULAS T: UM NOVO BIOMARCADOR NA DOENÇA INFLAMATÓRIA
INTESTINAL” 2013
Main Achievement VII
Celso Reis, Hugo Osorio, Catarina Gomes Inventors of a provisional patent application nº 20131000048433 (submitted to Instituto
Nacional de Propriedade Industrial) “CO-EXPRESSÃO DE CEA E GLICANOS SLEX COMO BIOMARCADOR NO CANCRO GÁSTRICO”
We have established an European network in the context of an EU Marie Curie Training Network - We have established an European
network in the context of an EU Marie Curie Training Network for the development of identification of glycoprotein biomarkers in
gastric cancer. This network has the participation of Prof. Pauline Rudd (Dublin University); Dr Niclas Karlsson (Gotenburg University),
Daniel Kolarich (Max Planck Institute of Colloids and Interfaces, Germany).
Faculty of Health Sciences of the University of Copenhagen - Ulla Mandel and Henrik Clausen - This collaboration has been
fundamental for characterization of carbohydrate antigens and glycosyltransferases using unique monoclonal antibodies. We have
been collaborating with this group for the characterization of the O-glycoproteome of gastric cells. A PhD student of the group (Diana
Campos) is doing a joint PhD thesis on this topic.
Umea University, Sweden – Thomas Borén - This collaboration has contributed for the study of H.pylori adhesion to gastric mucosa,
developing binding assays, and evaluating the novel glycoengineering strategies for treatment of H.pylori infection .
University of Lille, France . Anne Harduin-Lepers - This collaboration has been important in the characterization of sialyltranferases
in human cells.
Institut Pasteur, France - Eliette Touati - This collaboration is essential for the study of Helicobacter pylori using animal models. This
collaboration is essential for the study of Helicobacter pylori using animal models.
Jose Andres Morgado-Diaz – Instituto Nacional do Cancro, INCA, Brazil - This collaboration has contributed to address the signaling
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pathways involved in E-cadherin glycosylation in a cancer context. In collaboration with INCA, we are starting to evaluate the role of
glycosylation in the radio/chimio resistance of cancer cells.
Future Research
Role of glycans for H. pylori adhesion/infection
The group will continue to characterize the role of glycans for H. pylori adhesion/infection and to develop novel anti-adhesion
therapeutic strategies based on carbohydrates/analogs.
The following approaches will be used:
We will evaluate in gastric biopsies of individuals with H. pylori infection the glycosylation alterations induced by the bacteria and
characterize the glycosyltransferases repertoire responsible for such glycophenotype modifications. This evaluation will allow the
validation of our in vitro findings previously published in the Journal of Clinical Investigation 118:2325-36 (2008). These analyses will be
combined with the detailed characterization of the adhesins of the H pylori strains infecting each individual. The analysis of adhesins
will be performed by protein detection in Western Blots using specific antibodies, and in functional assays using labelled glycans.
This comprehensive study of both the H. pylori adhesins expression, the glycosyltransferases expression and the glycophenotype will
provide insights into mechanisms contributing for the H. pylori chronic infection of the gastric mucosa.
We will continue the development of novel strategies for inhibition of H. pylori adhesion and infection. This approach is being
developed within an on-going collaboration with Dr. Cristina Martins from INEB. In this project we are developing and testing
chitosan microspheres modified with synthetic carbohydrates receptors for inhibition of H. pylori adhesion, using cell line models
expressing specific glycan receptors, gastric mucosa of genetically modified mice (previously published by our group Magalhaes A et
al, Glycobiology 2009), and human gastric mucosas.
The group will continue to evaluate the role of glycans in disease and particularly on the cancer cell behaviour
addressing the molecular mechanisms controlling glycosylation of key proteins.
Various lines of research are going to be developed:
We will continue to analyse the role of E-cadherin glycosylation in gastric cancer cell behaviour. We plan to map the structurefunction of E-cadherin N-glycans in a gastric cancer context envisioning potential clinical applications. We will characterize the
different N-glycans structures attached to E-cadherin; we will evaluate the biological role of each E-cadherin N-glycan structure in
gastric cancer by transfecting cancer cell lines with mutant forms of E-cadherin for potential N-glycosylation sites and evaluate their
consequences on cancer cell behaviour (in vitro and in vivo). We will further translate and validate our results in a set of human gastric
carcinoma cases displaying E-cadherin dysfunction not explained by genomic alterations.
We will continue to evaluate the role of glycosylation in the immune system network by addressing the impact of glycosylation
modification on T Cell Receptor (TCR) functions in Inflammatory Bowel Disease (a pre-malignant condition of colorectal cancer).
The group will continue to analyse the role that sialyltransferases play in the terminal glycosylation of proteins important for the
cancer cell behaviour. We will use previously established gastric cell lines expressing sialyltransferases and displaying specific glycan
phenotypes. We aim to identify protein carriers of these sialylated glycans by proteomic analysis and perform in vitro and cell biology
assays and in vivo experiments to characterise the functional role of these glycans.
The group will continue to pursue the mechanisms underlying the dynamic changes in glycans and galectins which recognize them
during the metastatic process. We will use in vitro and in vivo models in order to understand the relationship between sialidase
inhibition and beta-galactosides availability for galectin recognition.
The group will continue to develop glycoproteomic strategies for identification of biomarkers that can be used
for cancer detection.
The group will perform glycoproteomic strategies for characterization of the glycoproteome of cancer patients both in tumour cells
and serum in order to detect novel targets that can be applied in cancer diagnosis tests. The following approaches will be used: a)
Development of Glycoengeneering gastric cells for the characterization of O-glycosylation; b) Validation of the identified glycoproteins
bearing cancer-associated carbohydrate antigens will be performed by complementary assays using clinical samples.
Celso Reis, Salome Pinho, Ana Magalhães, Hugo Osório - PhD program GABBA (Graduate program in areas of Basic and Applied
Biology) of the University of Porto
Celso ReisPD BiotechHealth
Celso Reis - Programa Doutoral de Medicina e Oncologia Molecular of the Medical Faculty of the University of Porto.
Celso Reis, Fatima Gartner - Programa Doutoral em Patologia e Genetica Molecular do Instituto de Ciencias Biomedicas Abel Salazar
Invited Talks
Reis CA. . Alterations of glycosylation in gastric carcinogenesis. Invited Keynote lecture . 22nd International Symposium on
Glycoconjugates. Dalian, China, 23-28 June 2013.. Dalian, China,. 23/06/2013.
Reis CA. . Gomes C, Osório H, Teixeira-Pinto M, Campos D, Reis CA. The expression of SLeX glycan antigen induced by ST3Gal4 leads to
an invasive gastric cancer cell behavior through activation of c-Met.. Mucins in Health and Disease; 12nd International Workshop on
Carcinoma-associated Mucins. . Cambridge, United Kingdom. 31/07/2013.
Reis CA. . Alterations of glycosylation in cancer. . Curso AMSUD-PASTEUR. Montevideo, Uruguai. . 01/12/2013.
Osorio H. Glycoprotein characterization by mass spectrometry in cancer and in congenital disorders of glycosylation. SPDM Portuguese Society of Inherited Metabolic Diseases Conference. Porto, Portugal. 06/12/2013.
Reis CA. Protein glycosylation
2º workshop on Carbohydrates and mass spectrometry; Universidade de Aveiro . Aveiro, Portugal. 15/05/2013.
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Population Genetics
Objectives
The group aims at understanding the origin and evolution of genetic diversity, their consequences and applications, both normal and
pathological, using autosomal, X and Y linked, and mtDNA markers.
In order to achieve the genetic characterisation of normal populations, their origins, phylogeny and evolution, and disease susceptibility
profiles, we develop descriptive and analytical formal tools and techniques adequate to specific genomic segments.
The applied research areas in which we concentrate our efforts are molecular diagnostics and forensics. Some of specific lines currently
pursued are: (a) the history, conservation and management of domesticates and laboratory animals (b) food quality assessment, and
(c) identification and diagnostic tools for humans, their commensal/pathogenic species, domesticates and laboratory animals.
Main Achievements
Clarification of the origins of native American dog breeds by the use of worldwide mtDNA sequences
Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/
street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and
South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic
analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua,
xoloitzcuintli and perro sín pelo del Peru, by comparing to extensive samples of East Asian (n = 984) and European dogs (n = 639), and
previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan
Malamute for which results were ambiguous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland
dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European
dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and
ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying
pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the
Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin..
Molecular characterization of Tunisian patients with Maple Syrup Urine Disease
Maple Syrup Urine Disease (MSUD) is a rare disorder of branched-chain amino acids metabolism caused by the defective function of
branched-chain a-ketoacid dehydrogenase complex. The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes
encoding respectively the E1a, E1ß and E2 subunits of the complex.Two novel putative mutations have been identified: the alteration
c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene.
The evolutionary constraints in the ß-globin cluster: the paradox of the conservation of the delta-globin locus
The ß-globin gene cluster comprises one pseudogene and five genes whose expression undergoes two critical switches: the
embryonic-to-fetal and fetal-to-adult transition. HBD encodes the d-globin chain of the minor adult hemoglobin (HbA2), which is
assumed to be physiologically irrelevant. Paradoxically, reduced diversity levels have been reported for this gene. We have shown that
HBD and its neighbor pseudogene HBBP1 have mainly evolved under purifying selection, suggesting that their roles are essential and
nonredundant. Moreover, in the light of recent studies on chromatin conformation, we present evidence sustaining that the strong
functional constraints underlying the decreased contemporary diversity at these two regions were not driven by protein function but
instead are likely due to a regulatory role in ontogenic switches of gene expression.
Linguistic and religious isolates in northeastern Portugal: new insights from Genetics and Demography
In Miranda do Douro, NE Portugal, two different languages, Portuguese and Mirandese (an Astur-Leonese dialect) coexist. We
characterized the population from Miranda through the analysis of maternal lineages (the entire control region of mitochondrial DNA)
in 121 individuals. Haplogroup composition was usual for a Western European population (63.6% belonging to macro-haplogroup
R0). Lineages ascribed to an African (L2a and L1b) origin, were detected, at the amount commonly found in Portugal. However a few
haplogroups typically found in Jewish populations, while rarely observed in other Iberian populations were also present. The finding
can be explained by gene flow with crypto-Jew communities that since long are known to be established in the same region.
Genetic determinants of male fertility
We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of
male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. Assaying genomewide
SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each
rare autosomal deletion detected in our study multiplicatively changes a man’s risk of disease by 10%, rare X-linked CNVs by 29%, and
rare Y-linked duplications by 88%.. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous
to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we
identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional
4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and
potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls).
The evolutionary portrait of metazoan NAD salvage pathway
Nicotinamide Adenine Dinucleotide (NAD) levels are essential for cellular homeostasis and survival. Main sources of intracellular
NAD are the salvage pathways from nicotinamide, where Nicotinamide phosphoribosyltransferases (NAMPTs) and Nicotinamidases
(PNCs) have a key role. NAMPTs and PNCs are important in aging, infection and disease conditions such as diabetes and cancer. These
enzymes have been considered redundant since either one or the other exists in each individual genome. The co-occurrence of
NAMPT and PNC was only recently detected in invertebrates though no structural or functional characterization exists for them. Here,
using expression and evolutionary analysis combined with homology modeling and protein-ligand docking, we show that both genes
are expressed simultaneously in key species of major invertebrate branches and emphasize sequence and structural conservation
patterns in metazoan NAMPT and PNC homologues. The results anticipate that NAMPTs and PNCs are simultaneously active, raising
the possibility that NAD salvage pathways are not redundant as both are maintained to fulfill the requirement for NAD production in
some species.
The role of recombination in the origin and evolution of Alu subfamilies.
Alus are the most abundant short interspersed nuclear elements found in primate genomes (about 10% of the human genome,
although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they
evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. We have
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addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences
of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome
searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These
results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination.
Development of molecular methods for detection, identification and genotyping of pathogens in clinical
specimens.
Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable
for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of
Aspergillus fumigatus in a single reaction combining 20 markers in a multiplex reaction followed by mini-sequencing reaction which
accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar
lavage.
The same methodology was successfully applied to Pseudomonas aeruginosa. This cost-effective technique allowed the analysis of
a Portuguese set of isolates (n=111) collected from three distinct hospitals and the genotyping data could be obtained in less than
six hours. Point mutations were shown to be the most frequent event responsible for diversification of the Portuguese population
sample. Corroborating the epidemic hypothesis for P. aeruginosa population.
Genetic history of South American populations
We have shown a continent-wide decoupling of Y-chromosomal genetic variation from language and geography in native South
Americans. Colombia’s admixed population of European, Amerindian, and African descent was studied through Y-chromosome
markers, defining paternal lineages. A high diversity of was found along with substantial regional variation of European, African, and
Native American contributions.
In Brazil, Acre was the last state to be inhabited by non-indigenous individuals. We profiled a sample of 503 unrelated individuals
living in Acre for 15 autosomal STR loci. Population comparisons between sub-samples from the five regions in Acre did not reveal the
presence of population substructure.
We have also revisited the genetic ancestry of Brazilians using autosomal AIM-Indels.
Population genetics of 38 autosomal InDels in San Basilio de Palenque, and Embera-Chami Amerindian community (Colombia) and on
10 X-STRs in a population sample from Lima, Perú were also reported.
Comparative behaviour of autosomal and X-chromosomal paternity exclusion power in standard and deficient
cases with inbreeding
We show that extending the standard estimation of the autosomal coancestry to X-chromosomal transmission can be a powerful
approach to bring into light distant genealogies and to discern specific pedigrees, especially if combined with the estimation of the
autosomal counterpart.
Aiming to provide a basis for designing strategies for selection of X-haploblocks defined by single nucleotide polymorphisms (SNPs)
using next generation sequencing approach we have shown that, given the size of the X chromosome, only four haploblocks could be
selected in order to guarantee their mutual independence.
The mitochondrial genome of the pinewood nematode (Bursaphelenchus xylophilus) lineage introduced in
Europe and new insights into the phylogeny and worldwide dispersion of two closely related nematode species,
Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.
The pinewood nematode (PWN) Bursaphelenchus xylophilus is the causative agent of pine wilt disease and the greatest biological
threat to conifer forests worldwide. We describe the near-complete mitochondrial DNA (mtDNA) sequence (12,945 bp) of the PWN
lineage recently introduced in Europe. The absence of polymorphisms across the mtDNA of three Portuguese isolates suggests that a
single mtDNA lineage was introduced in SW Europe and provides a basis for studying the European dispersion of this invasive species.
The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect
vectors, and similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus.
Using both nuclear and mtdnaDNA markers, we showed that B. xylophilus and B. mucronatus form distinct phylogenetic groups with
contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity, as expected for a species that
evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised
areas, such as in SW Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in
both mtDNA and nuclear phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests
intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of
the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their
morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for
understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.
Optimization of a pentaplex panel for microsatellite instability analysis
We have optimized a pentaplex panel for MSI analysis in a Brazilian population and describe accurate methodology to assess MSI
status without matched-normal DNA and independently of the ethnicity.
Comparative analysis of two indel-based ancestry informative multiplex PCR typing kits
Two different insertion/deletion (indel) multiplexes have been used to analyse a subset of the CEPH Human Genome Diversity Panel
as well as several additional populations collected locally in order to compare the effectiveness of the marker sets in differentiating
the populations on a continental level. We show that both marker sets by themselves are able to achieve full continental population
differentiation and combining all 67 markers leads to considerably higher accuracy of classification. While differentiation of the
European and Middle Eastern population groups remains impossible, surprisingly high accuracy is achieved in assigning Central South
Asian samples.
Frequency and pattern of heteroplasmy in the complete human mitochondrial genome
A set of primer pairs designed to avoid co-amplification of nuclear DNA (nDNA) sequences of mitochondrial origin (NUMTs) was used
to amplify the mitochondrial genome in 101 individuals. The analysed individuals represent a collection with a balanced representation
of genders and mtDNA haplogroup distribution, similar to that of a Western European population. The results show that the frequency
of heteroplasmic individuals exceeds 61%. The frequency of point heteroplasmy is 28.7%, with a widespread distribution across the
entire mtDNA. In addition, an excess of transitions in heteroplasmy were detected, suggesting that genetic drift and/or selection may
be acting to reduce its frequency at population level. In fact, heteroplasmy at highly stable positions might have a greater impact on
the viability of mitochondria, suggesting that purifying selection must be operating to prevent their fixation within individuals. This
study analyses the frequency of heteroplasmy in a healthy population, carrying out an evolutionary analysis of the detected changes
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2013
and providing a new perspective with important consequences in medical, evolutionary and forensic fields.
High-throughput sequencing of a South American Amerindian.
The genome of an individual from a South American tribe was completely sequenced. A total of 36.8 giga base pairs were sequenced
and aligned with the human genome. These Gbp corresponded to 95.92% of the human genome with an estimated miscall rate of
0.0035 per sequenced bp. The data obtained from the alignment were used for SNP (single-nucleotide) and INDEL (insertion-deletion)
calling, which resulted in the identification of 502,017 polymorphisms, of which 32,275 were potentially new high-confidence SNPs
and 33,795 new INDELs, specific of South Native American populations. The authenticity of the sample as a member of the South
Native American populations was confirmed through the analysis of the uniparental (maternal and paternal) lineages. The autosomal
comparison distinguished the investigated sample from others continental populations and revealed a close relation to the Eastern
Asian populations and Aboriginal Australian. Although, the findings did not discard the classical model of America settlement; it
brought new insides to the understanding of the human population history.
Characterization of the oral fungal microbiota
The study aims to assess the effect of smoking in the biodiversity of oral fungal microbiota of healthy young subjects, using an
improved culture method that assesses both total and pathogenic viable fungi. Tobacco smoking may alter the oral mycobiota and
facilitate the colonization of the oral cavity by yeasts and pathogenic molds. The effect of chronic fungal colonization on the oral
health of tobacco smokers cannot be neglected.
The oral fungal community presented a high inter-individual variability. Also, the frequency and quantification of each fungal taxon
were constant over the 30-weeks observation period, showing a consistent intra-individual stability over time. The intra-individual
stability as opposed to inter-individual variability may suggest a common and a variable group of fungi in the oral cavity.
Detection and discrimination of common bovine mastitis-causing streptococci
A total of 12 DNA markers were validated with a set of 50 reference strains and isolates, representative of the Streptococcus genus,
of closely related species and of microorganisms with matching habitats. This work suggests that the combined use of these novel
taxa-specific markers coupled with discriminatory functional markers presents a reliable approach for the rapid and cost-effective
detection and infrasubspecific discrimination of bovine mastitis-causing pathogens, which will contribute to an improved treatment
and control of this disease.
Mutation rates at Y chromosome microsatellites
We address the simplest model of transmission: the Y chromosome specific STRs. Within this model, and under an explicit set of
definitions and involved assumptions, we developed the theoretical framework required for the study of allelic transitions in
gametogenesis, identifying the required parameters and associated probabilities. We conclude that (i) for forensic casework the
relevant parameter for incorporation in a likelihood ratio is biallelic specific (i.e. the mutation rate estimate corresponds to the
probability of the specific allelic transition observed) and (ii) for these estimates as well as in order to provide data for testing mutation
models the absolute frequency of mutated and non-mutated transmissions per allele, along with the description of the observed
mutations should be reported.
Novel TTC19 mutation in a family with severe psychiatric manifestations and complex III deficiency.
We investigated a consanguineous Portuguese family where four siblings had reduced enzymatic activity of CIII in muscle and
harbored a novel homozygous mutation in TTC19. The clinical phenotype in the four sibs was consistent with severe olivo-pontocerebellar atrophy, although their age at onset differed slightly. Interestingly, three patients also presented progressive psychosis.
The mutation resulted in almost complete absence of TTC19 protein, defective assembly of CIII in muscle, and enhanced production
of reactive oxygen species in cultured skin fibroblasts. Our findings add to the array of mutations in TTC19, corroborate the notion
of genotype/phenotype variability in mitochondrial encephalomyopathies even within a single family, and indicate that psychiatric
manifestations are a further presentation of low CIII.
Mitochondrial DNA rearrangements in health and disease.
A meta-analysis of mtDNA deletions (n = 730) and partial duplications (n = 37) using information from more than 300 studies revealed
that both classes of mtDNA rearrangements are unequally distributed among disorders and their breakpoints have different genomic
locations. We also demonstrate that 100% of cases with sporadic mtDNA deletions and 97.3% with duplications have no breakpoints
in the 16,071 breakage hotspot site, in contrast with deletions from healthy and aged tissues. Notably, most deletions removing a
section of the D-loop are found in tumors. Deleted mtDNA molecules lacking the origin of L-strand replication (O(L)) represent only
9.5% of all reported cases, whereas extra origins of replication occur in all duplications. As previously shown for deletions, imperfect
stretches of homology are common in duplication breakpoints. A dedicated Website with detailed information on deleted/duplicated
mtDNA regions to facilitate the design of efficient methods for identification and screening of rearranged mitochondrial genomes was
made publicly available.
We further organized a mitochondrial DNA breakpoints database as free, web-accessible comprehensive list of breakpoints from
three classes of somatic mtDNA rearrangements: circular deleted (deletions), circular partially duplicated (duplications) and linear
mtDNAs from seven species and their associated phenotypic information.
A novel tool for genotyping Burkholderia cenocepacia genotyping
The strategy used for the SNaPBcen assay is based on targeting single nucleotide polymorphisms (SNPs) located in MLST genes instead
of sequencing full MLST sequences. Nonpolymorphic and redundant MLST positions were eliminated, and a set of 24 polymorphisms
included in the SNaPBcen assay ensures a high-resolution genomic characterization. These polymorphisms were identified based
on the comparative analysis of 137 B. cenocepacia MLST profiles available online (http://pubmlst.org/bcc/). The group of 81 clinical
isolates of B. cenocepacia examined in this study using the SNaPBcen assay revealed 51 distinct profiles, and a final discriminatory
power of 0.9997 compared with MLST was determined. The SNaPBcen assay was able to reveal isolates with microvariations and the
presence of multiple clonal variants in patients chronically colonized with B. cenocepacia.
Identification of human NLZ1/ZNF703conserved domains essential for proper subcellular localization and
transcriptional repression.
We characterize human NLZ1/ZNF703 (NocA-like zinc finger 1/Zinc finger 703), one of the two human NET family member genes.
We show that the gene is ubiquitously expressed in adult human and mouse tissues, that three mRNA species with the same coding
sequence are generated by alternative polyadenylation, and that the encoded protein contains six evolutionarily conserved domains,
three of which are specific to NET proteins. Finally, we present functional evidence that these domains are necessary for proper
subcellular distribution of and transcription repression by the NLZ1 protein, but not for its interaction with Groucho family corepressors.
43
2013
Trimethylaminuria (fish odor syndrome): genotype characterization among Portuguese patients.
TMAu is a metabolic disorder characterized by the inability to convert malodorous dietarily-derived trimethylamine (TMA) to odorless
TMA N-oxide by the flavin-containing monooxygenase 3 (FMO3). Affected individuals unable to complete this reaction exude a “fishy”
body odor due to the secretion of TMA in their corporal fluids leading to a variety of psychosocial problems. Interindividual variability
in the expression of FMO3 gene may affect drug and foreign chemical metabolism in the liver and other tissues. 25 Portuguese patients
with phenotype suggestive of TMAu were evaluated for molecular screening of the FMO3 gene. Herein, we found 16 variants in the
FMO3 coding region, some of which had not been previously documented (Gly38Trp, Asp232Val, Thr307Pro, Ser310Leu). Whenever
common variants (Glu158Lys, Glu308Gly) were considered in combination a distinct pattern between the control population and
patients was observed, mainly in what concerns the presence of Lys158 and Gly308 in homozygous state. Further studies are
necessary to clarify the pathogenicity of novel variants identified in this study, as well as the effect of the common single nucleotide
polymorphisms, which may play an important role in disease presentation and/or protective mechanism to xenobiotics drugs or
environment.
44
University of Évora - Dr Luis Costa: Phylogenetic analysis of the agricultural pests Batrocera oleae and Prays spp.
REQUIMTE - Rede de Química e Tecnologia, Faculty of Sciences, University of Porto, Porto, Portugal (Irina S. Moreira) - Theoretical
Chemistry
Instituto Gulbenkian de Ciência (IGC), Oeiras, Portugal - Dr. Lounès Chikhi: Evolutionary patterns of human chromosomal inversions
and development of inferential statistical methods. Study of candidate genes for speciation in non-human primates.
Compensatory mutations at the Human Gene Mutation Database (HGMD) - Prof David Cooper: Institute of Cancer & Genetics, Cardiff
University School of Medicine
IBMC - Jorge Sequeiros - Epidemiological genetics of Machado-Joseph disease
Royal Institute of Techonogy (KTH), Stockholm, Sweden - Prof. Peter Savolainen: Research on the origins and genetic diversity of dogs
in Continental Africa and Madagascar
Wildlife Institute of India (WII), Dehadrun, India - Analysis of forensic wildlife samples (elephant and reindeer): Phylogeography and
pig domestication in India
Research and Development Unit, Department of Genetics, CGMJM, INSA (Sandra Alves) - Exploring splicing therapeutics for patients
affected by lysosomal storage disorders
Cintia Alves - Member of the Executive Committee of the Spanish and Portuguese Speaking Working Group of the International Society
for Forensic Genetics (GHEP-ISFG)
Section of Forensic Genetics, Univ. Copenhagen, Denmark (Morling N, Tomas C) - X chromosome; population genetics and forensics
Pontificia Universidad Javeriana, Instituto de Genética Humana, Facultad de Medicina , Bogotá , Colombia (Noguera MC) - Population
genetics of Colombia: Y chromosome
DNA Diagnostic Laboratory - Institute of Biology, State University of Rio de Janeiro, UERJ, Brazil (Carvalho EF) - Population profiling,
ancestry markers and admixture
Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria (Michaela Lackner) - icrobiology;
molecular genetics of fungi
Center for Human Genetic Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA (Samuels DC) - Mitochondrial
DNA
Unitat d’Antropologia Biològica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autònoma de Barcelona,
Cerdanyola del Vallès, Barcelona, Spain.(Aluja MP) - Mitochondrial DNA
REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto,
Porto, Portugal (Lucília Saraiva ) - Bacterial genetics
Serviço de Pneumologia, Hospitais Universidade Coimbra-CHUC, EPE, Coimbra, Portugal (Fernanda Gamboa) - microbiological
genetics
Department of Pneumology, Faculty of Medicine of the University of Porto and Hospital S. João, Porto, Portugal (Adelina A. Amorim)
- Microbilogical genetics
Interdisciplinary Centre for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory, University of Porto, Porto,
Portugal (L. Filipe C. Castro) - Sequence motif analysis
CMUP - Centro de Matemática da Universidade do Porto, Porto, Portugal (Paulo Aguiar) - Bioinformatics
Faculty of Medicine of Monastir, Monastir, Tunisia (Jaafar N) - Molecular genetics of inborn errors of metabolism
REQUIMTE - Rede de Química e Tecnologia, Faculty of Sciences, University of Porto, Porto, Portugal (Irina S Moreira, Maria João
Ramos) - Theoretical Chemistry
Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal (Filipa Carvalho, Susana Fernandes, Alberto Barros)
- genetics of male infertility
Department of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal (João Gonçalves) - genetics of male
infertility
Genomics Medicine Group, National Genotyping Center, University of Santiago de Compostela, Santiago de Compostela, Spain (Inés
Quintela, Angel Carracedo) - genetics of male infertility
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA(Donald F. Conrad) genetics of male infertility
Centro Hospitalar e Universitário de Coimbra, Serviço de Hematologia, Portugal (C Bento) - hemoglobinopathies
IMAR-CMA, Department of Life Sciences, University of Coimbra, Coimbra, Portugal (Luís Fonseca, Isabel Abrantes) - Nematode
genetics
ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Evora, Portugal (Manuel
Mota) - Nematode genetics
Norwegian University of Life Sciences, IKBM, Aas, Norway (Egeland T) - Biostatistics and forensics
Departamento de Toxicología y Legislación Sanitaria, Facultad de Medicina, Universidad Complutense de Madrid, Spain (Arroyo-Pardo
E) - Y chromosome, ancient DNA
2013
Leonor Gusmão - Member of the Executive Committee of the International Society for Forensic Genetics (ISFG)
CISA – Centro de Investigação em Saúde de Angola (Miguel Brito) - Epidemiology of hemoglobinopathies and erythrocyte enzymes in
Bengo, Angola
Future Research
Population genetics of domestic species and insect agricultural pests
A comprehensive study of dog and wolf mtDNA will allow for the clarification of the origins and dispersal of dogs in Africa and
Madagascar. This collaboration has already produced an important paper in the field that demonstrates the Asian origin of Native
American dog breeds (van Asch et al. Proc R Soc B 2013, 280:20131142).
I will analyse mtDNA sequences from India to clarify the history of pigs in Asia. Preliminary results indicate an independent
domestication event in India. The results have implications in the spread of the Neolithic technologies in Asia.
We develop collaborative work on Bactrocera olea and Prays spp. with the University of Évora for clarification of the origins and
dispersal patterns of the species in the Mediterranean, with potential applications in pest control and management. Comprehensive
collections of samples from Europe, North Africa and the Near East will allow for deep characterization of mtDNA.
Understanding the phenotypic variability associated to lysosomal storage diseases (LDS)
Besides contributing to enrich the spectrum of mutations that causes LDS, we intend to provide new insights into the structural
requirements for localization and activity of the enzyme that underlies Mucolipidosis II (MLII), helping to explain the clinical phenotype
of MLII patients.
Genetics of Male Infertility
In a follow up of our recent landmark work on the genomics of male infertility (Lopes et al., PLoS Genet 9(3): e1003349) we will analyse
the genetic variation in key regulators of sexual development in patients with severe spermatogenic failure and in fertile controls.
Compensatory mutations
Understanding the role of compensatory mutations in phenotype-genotype associations
Novel genes with a de novo origin
Biological characterization of lineage-specific genes with origin in ancestrally non-coding regions.
Diversity in the androgen receptor CAG repeat has been shaped by a multistep mutational mechanism.
We aimed at clarifying the mechanisms on the origin of newly CAG sized alleles through a SNP-based haplotype strategy, crucial in
future association studies.
Machado-Joseph disease in a Nigerian family: mutational origin and review of the literature.
We report a Nigerian family with MJD from Calabar, once settled by Portuguese slave traders, and assessed its mutational origin. This
family bears the Joseph haplotype, which has a founder effect in the island of Flores, in the Azores, but is also the most common in
non-Portuguese populations worldwide.
Modifiers of (CAG)n instability in Machado-Joseph disease (MJD/SCA3) transmissions: an association study with
DNA replication, repair and recombination genes.
By analyzing postzygotic instability, we found a variant in ERCC6 associated with an expansion bias of MJD alleles. When using a genegene interaction model, the allele combination of RPA3-CDK7 is also associated with MJD instability in a direction-dependent manner.
Pharmacogenetics
Development of a panel to simultaneously genotype 10 polymorphisms in genes known to have pharmacogenetic implications,
namely TPMT, CYP2C9, CYP2C19, NAT2 and VKORC1.
Characterization of a Portuguese Gypsy community at a Pharmacogenetics and Forensic Toxicology level.
Metabolic Diseases
Contribution to the molecular spectrum of Maple Syrup Urine disease: demonstration of the pathogenic role of putative splicing
variations identified in BCKDHA gene using a minigene strategy.
Exploring the relationship between lifestyles, diets and genetic adaptations in humans
Population genetic tools will be used to address whether genes involved in metabolism and tasting of food compounds present signs
of adaptations to dietary shifts underwent by human populations
MLST@SNaP software
MLST@SNaP: user-friendly software for simplification of multilocus sequence typing and dissemination of microbial population
analyses
Mutation rates
Development of theoretical framework, models and sample design for the estimation of mutation rates
Forensic Genetics
Development of pilot quality control programs for non-human samples; validation of new genotyping systems
Chromosomal Inversions
We will analyze the recombination patterns inside and around inversion polymorphisms in humans and their variation across
populations.
Epidemiology of hemoglobinopathies: genetic variation of hemoglobin and erythrocyte enzymes in Bengo,
Angola
Estimation of the prevalence of main hemoglobinopathies and Glucose-6-phosphate dehydrogenase (G6PD) deficiency in children
born in Hospital Geral do Bengo
Determination of the proportion of malaria infection in children homozygous for hemoglobinopathies, heterozygous children, children
without detected mutations in globin genes, and children with G6PD deficiency, during the two first years of life.
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2013
Beta-globin cluster
Elucidation of the evolutionary history of the cluster and implications in ontogeny.
Genetics of populations of Jewish origin
Analysis of maternal lineages through complete mtDNA genomes and genome-wide autosomal investigation.
Population genetics of linguistic isolates.
Revision and extension of the work on maternal lineages to Y-chromosome and autosomal markers.
Barbara van Asch GABBA (Graduate Program in Areas of Basic and Applied Biology)
Alexandra M LopesGABBA (Graduate Program in Areas of Basic and Applied Biology)
Luisa Azevedo GABBA (Graduate Program in Areas of Basic and Applied Biology)
Martins S.GABBA (Graduate Program in Areas of Basic and Applied Biology)
Sofia QuentalGABBA (Graduate Program in Areas of Basic and Applied Biology)
Maria João PrataGABBA
Amorim A.GABBA (Graduate Program in Areas of Basic and Applied Biology)
Barbara van Asch Forensic Genetics, Faculty of Sciences, University of Porto
Alexandra M LopesMolecular Diagnostics, Faculty of Sciences, University of Porto
Luisa Azevedo Master’ s degree in Forensic Genetics, Faculty of Sciences, University of Porto
Amorim A. Forensic Genetics, Faculty of Sciences, University of Porto
Martins S. Molecular Diagnostics, Faculty of Sciences, University of Porto
Sofia QuentalMolecular Diagnostics, Faculty of Sciences, University of Porto
Maria João PrataMaster’s degree in Cell and Molecular Biology , Faculty of Sciences, University of Porto
Maria João PrataMaster’ s degree in Forensic Genetics, Faculty of Sciences, University of Porto
Cíntia Alves. Master’ s degree in Forensic Genetics, Faculty of Sciences, University of Porto
Invited Talks
Amorim A. Bases de dados e arquivos genéticos. . Conferência Bases de Dados Genéticos – a Ética, o Direito e a Investigação Criminal
. Braga, Portugal. 08/03/2013.
Amorim A. . Diagnóstico Genético - situação presente e perspetivas. . XIV Jornadas Biologia Aplicada 2013. Braga, Portugal. 01/03/2013.
Amorim A. Diversidade genética humana. . Ciência por quem a faz e por quem a ensina. Aguas Santas, Portugal. 06/09/2013.
Amorim A. Genética Forense. V Jornadas Nacionais de Genética e Biotecnologia. Vila Real, Portugal. 20/02/2013.
Amorim A. Profiling in Forensic Genetics. I International Symposium on Profiling. Caparica, Portugal. 02/09/2013.
Amorim A. A origem das espécies. Conferências no âmbito da exposição “Terra em Transformação” - Histórias da Terra e da Vida: do
Big Bang ao Homem. Reitoria Universidade do Porto. Porto, Portugal. 12/12/2013.
Amorim A. Chair: Session 3 (Solidarity and biosecurity) . Brocher Foundation Symposium “Solidarity: towards new solutions in the
bioethics of biobanking; biosecurity; and health inequalities”. Geneva, Switzerland. 22/03/2013.
Barbara van Asch. DNA Canino: aplicaciones de interés forense. Seminario AND no Humano, GENFOREN, Departamento de Toxicología
y Legislación Sanitaria Facultad de Medicina, Universidade Complutense Madrid (UCM), Madrid, Espanha.. Madrid. 26/11/2013.
Araujo R.. Microbiologia y virologia forense. Seminario AND no Humano, GENFOREN. Departamento de Toxicología y Legislación
Sanitaria Facultad de Medicina, Universidade Complutense Madrid (UCM), Madrid, Espanha. 26/11/2013.
Araujo R. Micologia forense: especies, complejos y diversidad genetica. Seminario AND no Humano, GENFOREN. Departamento de
Toxicología y Legislación Sanitaria Facultad de Medicina, Universidade Complutense Madrid (UCM), Madrid, Espanha. 27/11/2013.
Oral Presentations
Raquel M Silva. An evolutionary perspective on metazoan NAD salvage pathways. XXXVIII Jornadas Portuguesas de Genética. Porto,
Portugal. 04/06/2013.
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2013
Proteolysis in diseases
Objectives
The main goal of our research is to increase the understanding of the evolutionary history of proteolysis-related genes, having a
particular focus on genes with functions in male reproduction and innate immunity and with potential implications for current
patterns of human health and disease. More specifically, we aim to continue our research focused on the study of the human WFDC
cluster located at chromosome 20q13 and of the kallikrein (KLK) cluster located in chromosome 19q13.4 and to address the extent of
the contribution of their variants in human adaptations and in male infertility.
Mechanistic insights into anti-cancer drugs by MS-based drug profiling or pharmacoproteomics MS-based drug profiling or
pharmacoproteomics holds great potential for elucidating drug function and mechanisms.
Pharmacoproteomics, in contrast to pharmacogenomics, provide essential information about post translation modifications. For
example, many anticancer drugs work by inhibiting enzymes (e.g. kinases, histone deacetyltransferases, etc) that add or remove post
translational modifications thereby directly affecting the level of post translational modifications. Furthermore, pharmacoproteomics
based toxicology studies on human cell lines models delivers information about tissue specific toxicity which otherwise requires
unreliable animal testing systems. High throughput MS-based proteomics nowadays routinely identifies and quantify >12000 proteins
and 30000-50000 peptides together with their associated post translational modifications from raw cell extracts. Three great benefits
of MS-based pharmacoproteomics are: provide information about post translational information, the effect of toxicity is more evident
than using genomics or transcriptomics technologies and it profiles directly the sample content in contrast to array technology that
needs targets predefined which may turn out not even to be present in the sample.
Main Achievements
Natural Selection in the Human WFDC Locus
The whey acidic protein four-disulfide core domain (WFDC) locus located on human chromosome 20q13 spans 19 genes with WAP
and/or Kunitz domains. These genes participate in antimicrobial, immune, and tissue homoeostasis activities. Neighboring SEMG
genes encode seminal proteins Semenogelin 1 and 2 (SEMG1 and SEMG2). To better understand the selection pressures acting on
WFDC genes in human populations, we resequenced 18 genes and 54 noncoding segments in European, African, and Asian individuals.
We confirmed a signature of short-term balancing selection on WFDC8 in Europeans and a signature of positive selection spanning
genes PI3, SEMG1, SEMG2, and SLPI. Associated with the latter signal, we identified an unusually homogeneous-derived 100-kb
haplotype with a frequency of 88% in Asian populations. A putative candidate variant targeted by selection is Thr56Ser in SEMG1,
which may alter the proteolytic profile of SEMG1 and antimicrobial activities of semen. All the well-characterized genes residing in
the WDFC locus encode proteins that appear to have a role in immunity and/or fertility, two processes that are often associated with
adaptive evolution. We provided further evidence that the WFDC and SEMG loci have been under strong adaptive pressure within the
short timescale of modern humans.
Ferreira et al.(2013) Mol Biol Evol
Sequence diversity of Pan troglodytes subspecies and signatures of natural selection at the WFDC locus
Recent efforts have attempted to describe the population structure of common chimpanzee, focusing on four subspecies: Pan
troglodytes verus, P. t. ellioti, P. t. troglodytes, and P. t. schweinfurthii. However, few studies have pursued the effects of natural
selection in shaping their response to pathogens and reproduction. WFDC genes and neighboring semenogelin genes encode proteins
with combined roles in immunity and fertility. They display a strikingly high rate of amino acid replacement (dN/dS), indicative
of adaptive pressures during primate evolution. To evaluate the patterns of genomic variation and selection at the WFDC locus
in chimpanzees, we sequenced 17 WFDCgenes and 47autosomal pseudogenes in 68 wild caught chimpanzees. We found a clear
differentiation of P. t. verus and estimated the divergence of P. t. troglodytes and P. t. ellioti subspecies in 0.173 Myr; further, at the
WFDC locus we identified a signature of strong selective constraints common to the three subspecies in WFDC6. Chimpanzees and
humans do not display similar footprints of selection across the WFDC locus, possibly due to different selective pressures between the
two species related to immune response and reproductive biology.
Ferreira et al (2013) Gen Biol Evol
Evolutionary Constraints in the beta-Globin Cluster
Human hemoglobins are composed by two alpha-like and two beta-like globin monomers. The beta-globin gene cluster located at
11p15.5 comprises one pseudogene and five genes. HBD encodes the delta-globin and it is assumed to be physiologically irrelevant.
Paradoxically, reduced diversity levels have been reported for this gene. We sought a detailed portrait of the genetic variation within
the beta-globin cluster in a large human population panel from different geographic backgrounds. Comprehensive analyses, based on
classic neutrality tests, empirical and haplotype-based studies, revealed that HBD and its neighbor pseudogene HBBP1 have mainly
evolved under purifying selection, suggesting that their roles are essential and non-redundant. Moreover, in the light of recent studies
on the chromatin conformation of the beta-globin cluster, we presented evidence for strong functional constraints at these two
regions driven by a regulatory role instead of protein function.
Moleirinho et al (2013) Gen Biol Evol (in collaboration with Population Genetics group)
SERPINA2 Is a Novel Gene with a Divergent Function from SERPINA1
Serine protease inhibitors (SERPINs) are a superfamily of highly conserved proteins that play a key role in controlling the activity of
proteases in diverse biological processes. The SERPIN cluster located at the 14q32.1 region includes the gene coding for SERPINA1, and
a highly homologous sequence, SERPINA2, which was originally thought to be a pseudogene. To investigate the function of SERPINA2,
we used HeLa cells stably transduced with the different variants of SERPINA2 and SERPINA1 (M1, S and Z). We identified SERPINA2
as a 52 kDa intracellular glycoprotein, which is localized at the endoplasmic reticulum (ER), independently of the variant analyzed.
SERPINA2 is not significantly regulated by proteasome, proposing that ER localization is not due to misfolding. Using phylogenetic
analysis, we propose an origin of SERPINA2 in the crown of primates, and we unveiled the overall conservation of SERPINA2 and A1.
Nonetheless, few SERPINA2 residues seem to have evolved faster, contributing to the emergence of a new advantageous function,
possibly as a chymotrypsin-like SERPIN. We showed that SERPINA2 is an active gene, coding for an ER-resident protein, which may act
as substrate or adjuvant of ER-chaperones.
Marques et al (2013) Plos One
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2013
Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex
chromosomes, including the gene DMRT1
Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful
mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic
architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal
spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and
more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man’s risk
of disease by 10% (OR 1.10 [1.04-1.16], p<2 × 10(-3)), rare X-linked CNVs by 29%, (OR 1.29 [1.11-1.50], p<1 × 10(-3)), and rare Y-linked
duplications by 88% (OR 1.88 [1.13-3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets
from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic
impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two
patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW
chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic
azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined
results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure
(frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2 × 10(-5)). Our study identifies other recurrent CNVs as potential
causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and
IVF outcomes.
Lopes et al (2013) Plos Genetics (in collaboration with Population Genetics group)
Methods and algorithms for quantitative proteomics by mass spectrometry
Protein quantitation by mass spectrometry (MS) is attractive since it is possible to obtain both identification and quantitative values of
proteins and their posttranslational modifications in a single experiment. In contrast, protein arrays only provide quantitative values
of targeted proteins and their modifications. There are an overwhelming number of quantitative MS methods for protein and peptide
quantitation. The aim here is to provide an overview of the most common MS methods and algorithms used in quantitative proteomics
and discuss the computational algorithms needed to reliably quantitate proteins, peptides, and their posttranslational modifications.
One of the main challenges in data analysis of many experimental projects is to pipe together a number of software solutions that
are either commercial or freely available. The aim of this chapter is to provide a good set of algorithms, ideas, and resources that can
easily be implemented in scripting language like R, Python, or Perl. By understanding the algorithmic ideas presented here, data from
any instrument or modified experimental protocol can be analyzed and is therefore in the authors’ opinion more valuable than a black
box concept.
MS-based quantitation of peptide and proteins (Update chapter), Carvalho AS and Matthiesen R , (2013) Methods Mol Biol
Interpretation of tandem mass spectra of posttranslationally modified peptides.
Tandem mass spectrometry provides a sensitive means of analyzing the amino acid sequence of peptides and modified peptides by
providing accurate mass measurements of precursor and fragment ions. Modern mass spectrometry instrumentation is capable of
rapidly generating many thousands of tandem mass spectra and protein database search engines have been developed to match
the experimental data to peptide candidates. In most studies there is a schism between discarding perfectly valid data and including
nonsensical peptide identifications-this is currently a major bottleneck in data-analysis and it calls for an understanding of tandem
mass spectrometry data. Manual evaluation of the data and perhaps experimental cross-checking of the MS data can save many
months of experimental work trying to do biological follow-ups based on erroneous identifications. Especially for posttranslationally
modified peptides there is a need for manual validation of the data because search algorithms seldom have been optimized for the
identification of modified peptides and because there are many pitfalls for the unwary. This chapter describes some of the issues that
should be considered when interpreting and validating tandem mass spectra and gives some useful tables to aid this process.
Interpretation of Collision-Induced Fragmentation Tandem Mass Spectra of Posttranslationally Modified Peptides (Update from first
edition) Bunkenborg J and Matthiesen R, (2013) Methods Mol Biol.
Algorithms for database-dependent search of MS/MS data.
The frequent used bottom-up strategy for identification of proteins and their associated modifications generate nowadays typically
thousands of MS/MS spectra that normally are matched automatically against a protein sequence database. Search engines that take
as input MS/MS spectra and a protein sequence database are referred as database-dependent search engines. Many programs both
commercial and freely available exist for database-dependent search of MS/MS spectra and most of the programs have excellent user
documentation. The aim here is therefore to outline the algorithm strategy behind different search engines rather than providing
software user manuals. The process of database-dependent search can be divided into search strategy, peptide scoring, protein
scoring, and finally protein inference. Most efforts in the literature have been put in to comparing results from different software
rather than discussing the underlining algorithms. Such practical comparisons can be cluttered by suboptimal implementation and the
observed differences are frequently caused by software parameters settings which have not been set proper to allow even comparison.
In other words an algorithmic idea can still be worth considering even if the software implementation has been demonstrated to be
suboptimal. The aim in this chapter is therefore to split the algorithms for database-dependent searching of MS/MS data into the
above steps so that the different algorithmic ideas become more transparent and comparable. Most search engines provide good
implementations of the first three data analysis steps mentioned above, whereas the final step of protein inference are much less
developed for most search engines and is in many cases performed by an external software. The final part of this chapter illustrates
how protein inference is built into the VEMS search engine and discusses a stand-alone program SIR for protein inference that can
import a Mascot search result.
Matthiesen R, (2013) Met
LC-MS spectra processing
Peak extraction from raw data is the first step in LC-MS data analysis. The quality of this procedure is important since it affects the
quality and accuracy of all subsequent analysis such as database searches and peak quantitation. The most important and most
accurately measured physical entity provided by mass spectrometers is m/z values which need to be extracted by state of art
algorithms and scrutinized thoroughly. The aim of this chapter is to provide a discussion of peak processing methods and furthermore
discuss some of the yet unresolved or neglected issues. A few novel concepts are also proposed for analysis and visualization. The final
section of this chapter provides a note on possible software for spectra processing
Matthiesen R, (2013) Methods Mol Biol.
Introduction to mass spectrometry-based proteomics
Mass spectrometry has been widely applied to study biomolecules and one rapidly developing field is the global analysis of proteins,
48
2013
proteomics. Understanding and handling mass spectrometry data is a multifaceted task that requires many decisions to be made to
get the most comprehensive information from an experiment. Later chapters in this book deal in-depth with various aspects of the
process and how different tools can be applied to the many analytical challenges. This introductory chapter is intended as a basic
introduction to mass spectrometry (MS)-based proteomics to set the scene for newcomers and give pointers to reference material.
There are many applications of mass spectrometry in proteomics and each application is associated with some analytical choices,
instrumental limitations and data processing steps that depend on the aim of the study and means of conducting it. Different aspects
of the proteome can be explored by choosing the right combination of sample preparation, MS instrumentation and data processing.
This chapter gives an outline for some of these commonly used setups and some of the key concepts, many of which are explored in
greater depth in later chapters.
MS-based proteomics (Update from first edition), Matthiesen R and Bunkenborg J (2013) Methods Mol Biol
Tools for protein posttranslational modifications analysis: FAK, a case study
Recent advances in mass spectrometry have resulted in an exponential increase in annotation of posttranslational modifications
(PTMs). Just in the Swiss-Prot Knowledgebase, there are 89,931 of a total of 27 characterized PTM types reported experimentally. A
single protein can be dynamically modified during its lifetime for regulation of its function. Considering a PTM can occur at different
levels and the number of different PTMs described, the number of possibilities for a single protein is unthinkable. Narrowing the study
to a single PTM can be rather unmerited considering that most proteins are heavily modified. Currently crosstalk between PTMs is
plentifully reported in the literature. The example of amino acids serine and threonine on one hand and lysine on the other hand, as
targets of different modifications, demand a more global analysis approach of a protein. Besides the direct competition for the same
amino acid, a PTM can directly or indirectly influence other PTMs in the same protein molecule by for example steric hindrance due
to close proximity between the modifications or creation of a binding site such as an SH2 binding domain for protein recruitment
and further modifications. Given the complexity of PTMs a number of tools have been developed to archive, analyze, and visualize
modifications. VISUALPROT is presented here to demonstrate the usefulness of visualizing all annotated protein features such as
amino acid content, domains, amino acid modification sites and single amino acid polymorphisms in a single image. VISUALPROT
application is demonstrated for the protein focal adhesion kinase (FAK) as an example. FAK is a highly phosphorylated cytoplasmatic
tyrosine kinase comprising different domains and regions. FAK is crucial for integrating signals from integrins and receptor tyrosine
kinases in processes such as cell survival, proliferation, and motility.
Voabil P, Fonseca C, Carvalho AS and Matthiesen R, (2013) Methods Mol Biol.
An evolutionary perspective into the role of Kallikreins (KLKs) in male reproductive biology - Collaboration with Victor Quesada and
Carlos López-OtÍn from 2Department of Biochemistry and Molecular Biology-IUOPA, University of Oviedo, Oviedo, Spain
Ubiquitin traps - Collaboration with Dr. Manuel S. Rodriguez, inbiomed, 20009 San Sebastián – Gipuzkoa – Spain
Studying Genetic Variation and Positive selection at the WFDC locus in Hominids - Collaboration with Belen Hurle from National Human
Genome Research Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA and Andrew G. Clark from Department of
Molecular Biology and Genetics, Cornell University, USA.
Proteomics of P.aeruginosa biofilms: from establishment of the components, their spatiotemporal relationships towards inhibitors of
the biofilm formation. - Collaboration with two French groups on combating microbial biofilms was initiated. The French groups are
experts in biofilms and computational structural biology. Although we address medical problems of microbial biofilms in our project,
biofilm constitute problems in many other areas as well such as agriculture. In fact biofilms are one of the major topics in Horizon
2020 program.
Rune Matthiesen GABBA PhD program
Invited Talks
Rune Matthiesen . Global MS- and transcriptomics array drug profiling provides novel insight into glucosamine-induced ER stress.
Waters day. Lisbon. 10/2013.
Rune Matthiesen. Computational methods in high throughput MS-based proteomics,. CRBM. Montpellier, France. 02/213.
Rune Matthiesen . Computational methods in high throughput MS-based proteomics.. ITQB. Lisbon, Portugal. 02/2013.
49
50
2013
2013
Post-Graduation
51
2013
Post-Graduation Unit
Overview
The Post-Graduation activities were done as planned.
The Unit organized the participation on the GABBA program, The workshop on Cancer Research, as well as contributed on other
activities of Post-Graduation.
Highlights
GABBA
Programa Graduado em Biologia Básica e Aplicada da UP - Módulo de Oncobiologia
Workshop on Cancer Research 2013 - Organization of the Workshop on Cancer Research 2013
Seminar IPATIMUP
Seminários de Quartas feiras, 9h
Jornal Club IPATIMUP 2013
JC temáticos.
52
2013
Outreach Activities
53
54
2013
2013
Science Diffusion
Overview
The Science Diffusion Unit aims to promote scientific culture, interacting whether with schools or with the community. Thus, during
2013 the Science Diffusion Unit has developed/been involved in the following activities/projects:
Laboratório Aberto; Porto de Crianças; Mostra UP ; Ciência Viva no Verão; IPATIMUP’s Day ; Noite dos Investigadores; Escola das
Ciências da Vida e Saúde; School meetings; Traz um amigo também; Segunda há Ciência; Escola Científica; Auto Laboratório
Highlights
Laboratório Aberto
In 2013 the objectives of the Laboratório Aberto have been met. The Laboratório Aberto has received about 5,000 students from
around the country that performed hands-on practical activities.
Porto de Crianças
In 2013, the protocol with the City Council of Porto, which was about to end, was maintained meeting the project objectives.
Four schools benefited from this project which contemplated 64 sessions in the classroom, with the participation of 83 students in
4th grade.
Ciência Viva no Verão
The project received two more students than last year (31 total) and there was also an increase in funding.
Escola Científica
This project aims twinning two laborat’rios in two schools, Esc. Sec. Cinfães and Esc. Sec. Aurélia de Sousa in collaboration / scientific
support IPATIMUP. This project is supported by the Agência Ciência Viva.
Auto Laboratório
Funded project ON.2 / CCRN which is based experimental activities lead to schools, conducting activities in the context of the
classroom. To this end, via a vehicle moves schools without guaranteeing the quality of experimental teaching and reducing the costs
associated with the students.
Activity Statistics
Visits to Laboratório Aberto in 2013 per district and per month
Mês
Nº Alunos
Responsáveis
pelas Visitas
Total
Distrito
Nº Alunos
Responsáveis
pelas Visitas
Total
1
650
68
718
Aveiro
541
36
577
2
561
46
607
3
359
42
401
Braga
108
6
114
4
647
57
704
Guarda
16
2
18
5
686
68
754
Leiria
141
12
153
6
200
24
224
7
143
27
170
Lisboa
60
6
66
8
0
0
0
Porto
3854
408
4262
9
89
9
98
Santarém
53
4
57
10
531
43
574
11
603
55
658
Setúbal
62
4
66
12
376
40
416
Viseu
10
1
11
TOTAL
4845
479
5324
TOTAL
4845
479
5324
Quality Control
Laboratório Aberto
At the end of each visit, students and teachers filled out a satisfaction survey of the activities, with the following topics:
1. Satisfaction of the activities presented;
2. Appropriate scientific language;
3. Scientific rigor;
4. Would you return to Laboratório Aberto?
Nearly 100% of people have given maximum rating on the topics asked.
55
2013
Overview
During 2013 the Public Awareness of Cancer Unit (PACU) developed an extensive activity focused on Cancer Prevention Education.
Besides reinforcing the team and expanding national and international collaborations, the PACU participated in several public events
promoting new strategies of cancer prevention. The results of the research work were presented in national and international
meetings and publications. Several grant applications (individual and projects) were prepared and submitted.
Highlights
Publications
Freitas DP, Teixeira CA, Santos-Silva F, Vasconcelos MH, Almeida GM. Therapy-induced enrichment of putative lung cancer stem-like
cells. Int J Cancer. Epub 2013 Oct 21.
Coelho S, Rocha S, Juzenas P, Sampaio P, Almeida GM, Santos-Silva F, Pereira M, Coelho M. Gold nanoparticle delivery-enhanced
proteasome inhibitor effect in adenocarcinoma cells. Expert Opinion on Drug Delivery (Impact Factor: 4.87). 08/2013; DOI:10.1517/1
7425247.2013.827659
Barros A, Moreira L, Santos H, Carvalho L, Ribeiro N, Santos-Silva F. “Cancer – Educate to Prevent” – High-school teachers, the new
promoters of cancer prevention education campaigns. (review re-submitted) Plos One.
Communications
Barros A, Marcos NT, Ribeiro N (equal authors), “”Prevenir o Cancro da Mama””, Hospital de Braga, Out.2013, no âmbito do Dia
Nacional da Prevenção do Cancro da Mama, promovido pelo Movimento Vencer e Viver da Liga Portuguesa contra o Cancro.
Barros A, Ribeiro N, Santos-Silva F. Horizon 2020 Flash Presentation about the projects developed in Public Awareness of Cancer Unit
at Sessão Oportunidades & Desafios I&I em Saúde, Alterações Demográficas e Bem-estar, FCUP, 7TH November 2013, promoted by
FCT.
Ribeiro N, Almeida AM, Silva FS (2013) Happy: Health Awareness and Prevention Personalized for You in Conference “SaudeCUF2013”,
21 Junho, Estoril.
Lamas S, Santos-Silva F. Website - Cancronafamilia.com. Update, dissemination and preliminary results. Program Harvard Medical
School Portugal Annual Retreat. Dezembro 2013.
Marcos NT, “”Beyond the Lab: New Strategies for Cancer Prevention””, IBMC, Jan. 2013, 17ª edição do Programa Doutoral GABBA.”
Abstracts
Abstract accepted: “Cancer Educate to Prevent – Bringing Teachers to a Brave New Word” for The Fourth International Conference on
Health, Wellness and Society, 14-15th March 2014, Vancouver, Canada. Dr. Luis Filipe Santos-Silva, Dr. Ana Barros, Dr. Luís M. Moreira,
Dr. Helena Santos, Dr. Nuno Ribeiro, Dr. Luis Carvalho. (Oral communication).
Abstract accepted : “”Cancro, Educar para Prevenir”” - apresentação de um estudo de prevenção primária for the VIII Congresso
Português de Sociologia, 14-16 April 2014, Universidade de Évora, Portugal.Authors: Ana Barros, Helena Santos, Luís Moreira, Filipe
Santos-Silva.
Grants Preparation
FCT - Call for Individual Grants – Doctoral Grant (student Nuno Ribeiro): Title: Happy: Health Awareness and Prevention Personalized
for You; Supervisors: Margarida Almeida; Filipe Santos Silva. AWARDED
FCT - Call for Individual Grants – Doctoral Grant (student Ana Barros); Title: Cancer Prevention Education: Building the Future;
through Schools; Supervisors: Filipe Santos-Silva; Helena Santos.
Happy: Health Awareness and Prevention Personalized for You. Set. 2013, Fundação Calouste Gulbenkian - Health Literacy Program
Call.
Happy: Health Awareness and Prevention Personalized for You. Dez. 2013, Fundação Portugal Telecom.
VITAL.MoBi - Virtual Training Advanced Lab in Molecular Biology, Jul 2013, FCT Exploratory Projects Call, in collaboration with
researchers from Health Sciences Section from University of Aveiro and Superior School of Health Jean Piaget.
Prevenção de Cancro: novas tácticas contra um problema antigo, Set. 2013, Fundação Calouste Gulbenkian - Health Literacy
Program Call.
PinCancer – Retrato das representações sociais do cancro na comunidade escolar e suas implicações no planeamento de campanhas
de prevenção.Set. 2013, Fundação Calouste Gulbenkian - Health Literacy Program Call.
Other Activities
Main organizer of: COST Action CM1106 1st Working Group Meeting on “Chemical approaches to targeting drug resistance in cancer
stem cells”, 21-22 February 2013, IPATIMUP, Porto, Portugal.
Media and ICT co-organizer. Porto Cancer Meeting 2013.
Dissemination Coordinator of European COST ACTION BM1204 - An integrated European platform for pancreas cancer research: from
basic science to clinical and public health interventions.
National Jury for Biomedical area of “Concurso Jovens Investigadores”.
International Jury: Member of Scientific Review Committee (SRC) of Intel ISEF 2014.
Participation Public Events: Dissemination of Cancer Prevention Education at “”Corrida da Mulher””. Maio 2013.
56
2013
IPATIMUP
Diagnostics
57
58
2013
2013
Overview
In 2013, IPATIMUP Diagnostics maintained the high quality standards successfully achieved in the previous years through CAP
accreditation, after CAP inspection held in Februrary 2013. Also, the ISO 9001:2008 certification was maintained through the followup audit held in June 2013.
Other goals for 2013, such as maintaining the unit as a professional training center, maintaining the number of tests from the
previous year, achieving better client satisfaction with our services and participating in research projects with health institutions and
pharmaceutical companies, and inclusion in international networks, were totally accomplished.
Highlights
- CAP accreditation and ISO 9001:2008 certification was successfully maintained by IPATIMUP Diagnostics;
- Participation in international networks – maintained for EUROFORGEN and began our inclusion in EUROGENETEST;
- Client satisfaction increased relatively to the previous year;
- Training increased considerably in relation to previous years. IPATIMUP has reinforced its position as a professional training centre
through its nomination by the Portuguese Physician’s Association (Ordem dos Médicos) as a reference centre in molecular pathology
of the Anatomical Pathology internship;
- Participation in research projects with health institutions and pharmaceutical companies, as well as the establishment of specific
national and international service contracts with hospitals and other health institutions, increased and diversified in 2013.
Activity Statistics
EXAMS
LAP
Total
17314
Histology
1263
Cytology (includes 2852 cases of FNA cytology)
10673
Molecular Pathology
723
Consultations
225
Telepathology
4226
LDG
4901
Total
Tumour mutation screening
2131
Genetic Diagnosis
2770
LPIG
Total
278
Genetic Characterizations (including lineage markers)
82
Parentage investigations (individuals) and genetic profile
comparisons (sample/individual)
196
CONSULTATIONS
Brazil
United Kingdom
16 Ireland
9 Norway
Portugal
58 Italy
Spain
10 Belgium
France
7 CAP
9 Turkey
1 USA
2
7 Germany
2 Slovenia
1
2 Switzerland
10 The Netherlands
67
13 Angola
1
1 Algeria
9
Total
225
59
2013
Quality Control
1. INTERNAL QUALITY CONTROL
A) LAP
In 2013 the “turn-around time” (TAT) was the following:
- Histological Exams: 49.53% of reports are sent in 2 working days.
- Cytological Exams: 96.89% of reports are sent in 2 working days.
- Screening cytologies: 97% of reports are sent in 7 working days.
The main results for the Gynecological Cytology quality control analysis were:
2011
2012
2013
Total cases
6977
6862
7108
Reviewed cases
1281
1349
1391
Concordance between pathologist
and cytotechnician
2011
2012
2013
1204
(93.99%)
1245
(92.3%)
1326
(95.3%)
77
(6.01%)
104
(7.7%)
65
(4.7%)
Discordance between pathologist and
cytotechnician
The total amount of unsatisfactory cases for analysis was 1.69% (1.43% in 2012, 0.85% in 2011, 0.59% in 2010).
ASCUS/ACG were diagnosed in 303 cases with a ASCUS/Lesion reason of 2.61 (1.84 in 2012, 2.23 in 2011, 2.57 in 2010).
B) LDG
The diagnostic genetics laboratory has an internal quality control system that includes proficiency testing.
Internal proficiency testing is performed twice a year using samples that represent diseases not covered in the external quality control
exercises. In 2013 we accomplished a 100% concordance rate in the internal quality control exercise.
C) LPIG
The main features comprising internal quality control are measured according to PR.QUA.10. The main finding concerns the overall
turn-around time (Tat), where better results were obtained for 2013 relatively to 2012, especially for more routine cases (exams with
simple reference samples):
Type of exam
Tat
% exams where Tat was accomplished
2011
2012
2013
1
Exams with simple
reference samples
10 working days
80,75%
82,26%
87.34%
2
Exams with complex
reference samples
20 working days
62,75%
100,00%
100.00%
3
Exams with limit samples
30 working days
87,50%
96,43%
83.33%*
All the other quality control measures are within the goals established as acceptable.
2. EXTERNAL QUALITY CONTROL
In the following tables is a summary of the results obtained for all proficiency tests and quality assurance programs in which IPATIMUP
Diagnostics participated in 2013.
60
2013
1. CAP - Proficiency Tests
Evaluation of Cervicovaginal Cytology (PAPM)
Evaluation of Non-gynecological Cytology (NGC)
Program for performance improvement in Surgical
Pathology (PIP)
FISH for Paraffin-Embedded Tissue
Evaluation of Immunohistochemistry (MK) Immunohistochemistry HER-2- (HER2) Hybrid capture for HPV (CHPVJ)
ER and PgR Immunohistochemistry (PM2) ISH HER2 (ISH2) Molecular Genetics (MGL1 e MGL3) – CAP/ACMG
Parentage/Relationship-Filter Paper (PARF)
Molecular Pathology
Program
% Concordance
PAPM-A
100%
PAPM-B
100%
NGC-A
NR
NGC-B
NR
NGC-C
NR
NGC-D
NR
PIP-A
NR
PIP-B
NR
PIP-C
NR
PIP-D
NR
CYP-A
Good
MK-A
84,6%
MK-B
100%
HER2-A
94,7%
HER2-B
100%
CHPVJ A
100%
CHPVJ B
100%
CHPVJ C
80%
PM2-A
100%
PM2-B
100%
ISH-A
100%
ISH-B
100%
MGL
98%
PARF-A
100%
PARF-B
100%
PARF-C
100%
EGFR, MSI, KIT,
KRAS
98%
2. Other Proficiency Tests
UK NEQAS for Immunohistochemistry
Quality Control Program of the European Society
of Pathology
Quality Control Program of the GHEP-ISFG
NR - Results not received, as of 14th March 2014.
GP
Breast
In House
16,50
13,00
UK-NEQAS
16,50
14,00
Program
KRAS
100%
Basic Parentage
100%
Advanced
Parentage
Basic Forensic
Advanced
Forensic
Not evaluated
100%
92.95%
61
2013
Other Activities
TRAINING
In total, there were 23 people who underwent training at IPATIMUP Diagnostics during 2013, according to the following table:
NAME
COUNTRY
START DATE
END DATE
Adriana Furtado Honório Dornelas
Brazil
13.04.01
13.05.31
Ana Isabel Moreira Paulino
Portugal
13.05.08
13.11.08
António José Polónia Rodrigues de Oliveira
Portugal
13.11.14
14.01.31
ARTHUR CONELIAN GENTILI
Brazil
13.04.01
13.05.31
Bernardo Gago da Câmara Dias Pereira
Portugal
13.01.01
13.03.31
Carlos Guilherme Santos de Oliveira
Portugal
13.02.25
13.09.30
Catarina Callé Lucas Mendes
Portugal
13.04.02
13.07.15
Daniela Marisa Pais de Sousa Almeida
Portugal
13.02.04
13.11.30
Deise Reis de Paula Mendes
Brazil
13.02.04
13.04.29
Edenir Inêz Palmero
Brazil
13.02.04
13.02.26
Elisabete Maria de Oliveira Couto
Portugal
13.03.12
13.05.31
Evalina Cândida de Almeida Ventura
Angola
13.03.18
13.05.30
Filipe Barbosa Araújo
Portugal
13.03.13
13.05.31
Inês Nascimento Mendonça
Portugal
13.02.18
13.03.04
Janaina Siqueira de Sá Barreto
Brazil
13.06.01
13.07.31
Márcia Daniela Lemos Baixia
Portugal
13.04.15
13.07.07
Mariana da Costa Dias
Portugal
13.09.23
13.11.22
Marta Filipa de Almeida Gonçalves Ferreira
Portugal
13.05.01
13.07.31
Nelson Fuentes Martínez
Spain
13.02.01
13.03.31
Nuria Vanessa de Sousa Borges Camacho
Portugal
13.03.18
13.05.30
Priscila Mendes Maia
Brazil
13.04.01
13.05.31
Rafaela Isabel Magalhães Leal Silva
Portugal
13.02.18
13.11.30
Sara Isabel Ribeiro de Meireles
Portugal
13.07.01
13.12.31
PUBLICATIONS
•
•
•
•
•
•
•
•
•
•
•
•
62
Badior M, Trigo F, Eloy C, Guimarães JE. Mucor infection: difficult diagnosis. Clin Drug Investig. 2013;33 (Suppl 1):S19-21.
Costa JL, Sousa S, Justino A, Kay T, Fernandes S, Cirnes L, Schmitt F, Machado JC. Non Optical Massive Parallel DNA Sequencing of
BRCA1 and BRCA2 Genes in a Diagnostic Setting. Hum Mutat 2013;34:629-3.
Durães C, Machado JC, Portela F, Rodrigues S, Lago P, Cravo M, Ministro P, Marques M, Cremers I, Freitas J, Cotter J, Tavares
L, Matos L, Medeiros I, Sousa R, Ramos J, Deus J, Caldeira P, Chagas C, Duarte MA, Gonçalves R, Loureiro R, Barros L, Bastos I,
Cancela E, Moraes MC, Moreira MJ, Vieira AI, Magro F. Phenotype-genotype profiles in Crohn’s disease predicted by genetic
markers in autophagy-related genes (GOIA study II). Inflamm Bowel Dis 2013;19:230-9.
Eloy C, Cameselle-Teijeiro JM, Rousseau E, Sobrinho-Simões M. Rare Flowers in the Thyroid Gland: The Case of Small Cell Tumors.
Int J Surg Pathol. 2013; [Epub ahead of print].
Eloy C, Cruz J, Vieira J, Teixeira MR, Cameselle-Teijeiro J, Sobrinho-Simões M. Carcinoma of the Thyroid With Ewing Family Tumor
Elements: A Tumor With Unknown Histogenesis. Int J Surg Pathol. 2013; [Epub ahead of print].
Eloy C, Oliveira M, Vieira J, Teixeira MR, Cruz J, Sobrinho-Simões M. Carcinoma of the Thyroid With Ewing Family Tumor Elements
and Favorable Prognosis: Report of a Second Case. Int J Surg Pathol. 2013; [Epub ahead of print].
Fernandes F, Eloy C, Carimo A, Pinto P, Graves S, Simões J, Carrilho C, Lopes JM. Simultaneous presentation of Kaposi sarcoma
and HHV8-associated large B-cell lymphoma in the same lymph node: A rare diagnosis in an HIV-negative patient. Am J Case Rep.
2013;14:263-6.
Monteiro D, Horta R, Eloy C, Silva P, Silva Á. Kaposi’s sarcoma arising in a burn scar mimicking Marjolin’s ulcer. Burns. 2013;39:e258.
Nakazawa T, Celestino R, Machado JC, Cameselle-Teijeiro JM, Vinagre J, Eloy C, Benserai F, Lameche S, Soares P, SobrinhoSimões M. Cribriform-morular variant of papillary thyroid carcinoma displaying poorly differentiated features. Int J Surg Pathol.
2013;21:379-89.
Pinto N, Magalhães M, Conde-Sousa E, Gomes C, Pereira R, Alves C, Gusmão L, Amorim A. Assessing paternities with inconclusive
STR results: The suitability of bi-allelic markers. Forensic Sci Int Genet. 2013;7:16-21.
Prieto L, Alves C, Zimmermann B, Tagliabracci A, Prieto V, Montesino M, Whittle MR, Anjos MJ, Cardoso S, Heinrichs B, Hernandez
A, López-Parra AM, Sala A, Saragoni VG, Burgos G, Marino M, Paredes M, Mora-Torres CA, Angulo R, Chemale G, Vullo C, SánchezSimón M, Comas D, Puente J, López-Cubría CM, Modesti N, Aler M, Merigioli S, Betancor E, Pedrosa S, Plaza G, Masciovecchio
MV, Schneider PM, Parson W. GHEP-ISFG proficiency test 2011: Paper challenge on evaluation of mitochondrial DNA results.
Forensic Sci Int Genet. 2013;7:10-15.
Ribeiro C, Campelos S, Moura CS, Machado JC, Justino A, Parente B. Well-differentiated papillary mesothelioma: clustering in a
Portuguese family with a germline BAP1 mutation. Ann Oncol 2013;24:2147-50.
2013
PROJECTS
Project with Sanofi Pasteur MSD – Epidemiological study for evaluation of anogenital condilomas in a population that undergoes
dermatology and/or DST counselling in Portugal.
Project with Politécnico Viseu – Prevalence and relationship of Helicobacter pylori with other microorganisms from the oral flora.
QREN Projects:
- Anti-EGFR – Development of a diagnostic solution for mutations associated with colorectal carcinoma for commercialization.
- DoIt – Optimization of an algorithm for therapy decision in metastatic colorectal carcinoma.
- MuSa – Detection of mutations and epigenetic alterations in blood circulating tumoral DNA.
FCT Project – Portuguese study on Familial Dilated Cardiomyopathy.
Projects with Life Technologies:
- Ion Torrent Ampliseq – RASopathy Panel, a partnership with “RASopathy Ion Torrent Network”, Life technologies
- Lung Fusion transcripts – RNA Ion Ampliseq Cancer Panel, a partnership with “OncoNetwork - Fusions”, Life technologies
63
64
2013
2013
Innovation
&
Translation
65
66
2013
2013
Ipatimup Innovation
Overview
There is an increasing social demand that R&D comes to constitute the basis for an added value economy that generates knowledgebased services or products and opens pathways for scientific employment. As such, R&D Institutes such as IPATIMUP face the need
to supply examples of how national R&D can generate economic revenue. The Innovation Unit’s mission is to create value from the
commercial exploitation of intellectual property (I.P.) and from stimulating the creation and growth of spin-off companies based at
IPATIMUP. Through consulting and coaching, the Innovation Unit helped researchers achieve the different steps in the innovation
cycle of services and products derived from their core research activities. In 2013, the Innovation Unit undertook four main lines
of action: 1) Registration of I.P. and Licensing to established companies; 2) Application to Innovation funding programs/awards; 3)
Launching of new spin-off companies; 4) Direct presentation to investors and Venture Capitals.
Highlights
Portfolio of innovation projects
The Innovation unit took a pro-active role in prospecting knowledge and research that can constitute the basis for innovative products
or services which have highly differentiated, cutting-edge, science-based character and, most importantly, a potential market that is
economically interesting. In 2013 seventeen projects/technology/materials with some potential for innovation and commercialization
came forward to be supported by the Innovation Unit. The 2013 portfolio of innovative solutions includes: formulations of
nanobiomaterials for eradication of Helicobacter pylori, original sources of anticancer compounds (microalgae), new cell models for
HCV replication, chiken-egg in vivo assays, in vitro assays to determine potential antitumor effects, genomic bioinformatics services,
informatics algorithm to quantify protein expression at intercellular interfaces, proprietary hybridomas to produce diagnostic and
therapy selection antibodies, serum biomarkers associated with gastric cancer and it’s precursor lesions, bladder cancer biomarkers
in urine, multiplex system for human identification and digital cytology for oral cancer.
The Innovation unit undertook several types of actions, established in a case by case basis, in order to take each of the above
mentioned projects further in the innovation cycle aiming to obtaining economic revenue from promotion of their commercialization.
Registration of IP and Licensing
In terms of patent submission, overall, in 2013 the Innovation Unit made a total of six new IP registrations in which IPATIMUP is
applicant, with entitled ownership. These corresponded to 4 provisional patent applications and 2 international stage applications.
In 2013 a total of 14 proposals were sent to companies offering a license to commercialize antibody-producing hybridomas that have
been synthetized by IPATIMUP researchers. As a result, one licensing agreement was accomplished with DAKO, a leading supplier of
immunohistochemistry reagents.
In collaboration with the University of Santiago de Compostela, a second licensing agreement was achieved for the commercialization
of an indels-based multiplex system for human identification. Licensee was a Spanish company, GENÓMICA.
Application to Innovation funding programs/awards
In 2013 the Innovation Unit promoted eighteen applications to innovation funding projects, programs or awards. One or more
applications were sent to the following calls: “Concurso InovPortugal”; Portugal Venture’s Call for Entrepreneurship; QREN (several
calls); “Building Global Innovators; Universal Biotech Innovation Prize; “BES Inovação”, “Arrisca C” and “Bluepharma Inovação”.
Up to date, a total of 16 applications were evaluated and 2 are still waiting final assessment. Overall 7 applications achieved the final
stages of assessment process and 4 applications achieved funding.
Launching of new spin-off companies
In 2013 the Innovation Unite launched 2 new spin-off companies devoted to providing services for researchers and Pharma companies.
Bioinf2Bio will provide bioinformatics analysis to next generation sequencing platforms. Expertus will perform in vivo assays for
fast and affordable drug screening and prediction of response to therapy. Knowledge-transfer agreements were negotiated so that
IPATIMUP can benefit from royalties over services or sales provided by these spin-offs.
Direct presentation to Investors and Venture Capitals
As a means to attract investment to our portfolio of projects, the Innovation Unit directly contacted investors and venture capitals
(VCs). Projects were presented to 10 Investors/Venture Capitals. Out of these, 4 projects raised interest and were called for board
meetings and 1 project achieved a positive decision for investment. Currently the investment agreement is under negotiation.
Main Achievements during 2013
•
•
•
•
•
•
•
•
•
7 new additions to the innovation portfolio
6 new patent applications (2 International stages; 4 new registries)
2 Licencing deals
6 partnerships for Co-development/Marketing with companies
4 projects financed in Innovation Programs: QREN (3) and Arrisca C
1 External service
2 New spin-offs
1 Participation in equity
1 VC Investment
Main challenges for 2014
•
•
•
Extract H2020 funding available for SMEs and for Innovation Vouchers,
Help new spin-offs structure their companies and generate earnings,
Integrate the new spin-offs in Cooperative Networks: P-Bio; HCP Oncology Sub-cluster, other,
67
•
•
•
•
•
•
•
2013
Extract funding from the Portugal 2020 Program (the new QREN),
Increase projects co-promoted between IPATIMUP and Companies in the Portugal 2020 Program,
Partner with a VC fund to launch an yearly; national award for innovation in personalized cancer medicine,
Increase maturation of some technologies to potentiate chances of licencing deals,
Increase direct contact with Venture Capitals and Investors to fund proof-of-concept projects,
Improve pitch competences to increase success in final stages of innovation awards,
Keep launching new spin-offs and maintain close relationships between IPATIMUP and spin-offs operating in the field of cancer.
Activity Statistics (tables only)
Table summarizing activity
IP: New applications
IP: Licencing Proposals
IP: Licencing
Agreements
IP Licensing Sucssess
rate
2012
6
0
0
0%
- €
2013
6
14
2
14%
23.383,30 €
Projects/Prizes:
Applications Submitted
Projects/Prizes:
Applications already
Evaluated
Projects/Prizes:
Applications still under
evaluation
Projects/Prizes:
Applications Approved
Projects/Prizes:
Success rate
2012
2
2
0
0
0%
- €
2013
18
16
2
4
25%
85.000,00 €
Spin-Offs: New
Companies
Spin-Offs: Overall
Royalties
Venture Capital:
Presentations
Venture Capital: Board
meetings
Venture Capital:
Investment
Venture Capital:
Success rate
0
0%
1
10%
2012
0
- €
0
2013
2
- €
10
4
IP: Overall Fees and
Royalties
Projects/Prizes: Overall
raised funds
Other Activities
Technical work (agreements, application forms, business plans, etc.)
In the setting of the above mentioned activities, the Innovation Unit performed a body of technical work, which could be summarized
in: IP sharing agreements (3); IP registries (6); Innovation Award Applications (18); Company Business Plans (4); Pitch Presentations
(12); Video pitch presentations (4); Licensing Proposals (14); Licensing agreements (2) and VC Investment Plans (1).
Aside from the above mentioned activities, the innovation Unit has:
•
•
•
•
•
•
•
•
•
•
68
Negotiated preferential access to UPTEC for IPATIMUP spin-offs
Received and negotiated technology offers from UPIN
Integrated spin-offs in open innovation platforms such as Biotechzone
Contributed the COTEC Academia-Industry cooperation award in which U Porto achieved to 2nd place
Contributed to Institutional QREN applications (Re-equipment)
Established relationships with the UP Innovation Ecosystem (interlocution with UPIN, incubation at UPTEC)
Integrated cooperative networks: POP; HCP sub-cluster; ANJE
Planned an I3S Knowledge Transfer Platform
Hosted visits from other Innovation experts
Contributed to create and maintain connections between IPATIMUP and several spin-off companies (Genetest, Expertus;
Bioinf2bio; TargeTalent; Biomode; Infogene; Coimbra Genomics).
2013
Ipatimup Translational Research
Overview
In a global environment of budget cuts, including investment in scientific research, industry/academia R&D cooperative strategies are
crucial to leverage resources and expertise.
The IPATIMUP Translational Research Unit (TRU) is at the interface between the industrial communities, academic research teams and
clinicians, potentiating the Institutes’ scientific knowledge as a means of delivering innovative clinical and translational research in
cancer. By furnishing the appropriate environment to share ideas and discuss how IPATIMUP’s best research can impact unmet clinical
and scientific needs of the industry, the IPATIMUP Translational Research Unit contributes to narrow the gap between the cancer
research discoveries and the development of new therapies to help cancer patients.
Between February 2012 (the date of inception of the unit) and the beginning of 2013 the TRU was fully dedicated to the process of its
own implementation and especially to the establishment of formal contacts with Global R&D company departments in the Pharma
and Biotech, namely through the proposal of innovative research projects at an international level. As consequence of this strategic
effort, during the course of 2013 the TRU reached important achievements in terms of recognition, confidence and commitment,
which was materialized by signing of contract research agreements.
During 2013, the TRU worked on eight new research projects, which were analysed for a potential interest for submission to Pharma
or Biotech companies. From these, TRU submitted six research projects to Pharma/biotech companies, from which four are currently
under negotiations or appreciation by the R&D Department company counterparts. Most importantly, from these projects submitted
and discussed, TRU signed two Investigator-initiated Research Contracts Agreements with two world-level Biotech/Pharma companies.
Favouring the collaborative involvement with health care and biotech companies, and also looking forward to the improvement
of international diffusion of IPATIMUP and TRU among these types of companies, we attended the AACR-NCI-EORTC International
Conference on Molecular Targets and Cancer Therapeutics, in Boston. Being hosted by the American Association for Cancer Research,
the National Cancer Institute, and the European Organisation for Research and Treatment of Cancer, this scientific conference is
devoted to stimulate scientific partnerships between academia/cancer research institutions and pharma companies. At the national
level, and with the aim of create de appropriate environment to share expertise and unique technologies, TRU participated in the first
Meeting Academia-Industries organized by P-BIO.
2013 was the year of official implementation of the Porto Cancer Platform, a structure that aims to position Porto strategically as a
region of reference for Cancer research, by catalyzing national collaborations and international partnerships (including in clinical trials)
and improving the local capacity to attract investment. During 2013, several meeting were held with Executive Director of this Platform
in order to start putting in place several thematic meetings involving clinicians and cancer scientists, envisioning the discussion and
structuring of pure translational research projects. Having in mind the importance of global networking for translational research, in
2013, the TRU have concluded the process of inclusion of IPATIMUP into the Portuguese network that will constitute the European
Infrastructure for Translational Medicine (EATRIS), a non-profit organization comprising European academic centers of excellence in
translational research.
The IPATIMUP translational research team organized the XXII Porto Cancer Meeting, specifically devoted to translational research in
cancer under the theme “Translational Research in Cancer: Bringing Basic Science and Pathology to Clinical Oncology”. In contrast
with previous meetings, this edition of the Porto Cancer Meeting will focus on the discussion of the latest developments in oncologyrelated translational research. Introducing an innovative approach, the TRU will bring scientists from pharma industry, together with
oncologists and cancer researchers, with the goal of generating a multidisciplinary and informal atmosphere for exciting discussions
on Translation Research in Cancer.
Overall Appreciation and Directions
During the second year the TRU actively promoted IPATIMUP internationally among industries, organized and participated events
focused on translational research and work for the integration of networks that bring together good science and industrial players.
Most importantly, TRU generated Contracted Research Agreements for joint R&D projects between IPATIMUP researchers and major
companies. Being the establishment of these types of contracts with health care industries the main goal of the TRU, this strategic
line will be maintained and reinforced in 2014. In addition to this, a special effort will be given to the preparation of international
collaborative research projects for IMI initiative calls.
Highlights
IPATIMUP-Industry Projects Management & Contracted Research Performance (TRU)
•
•
•
•
Management of 8 research projects, which were analyzed for a potential interest for submission to Pharma or Biotech companies.
Six new projects submitted.
Four new Projects ongoing and in advanced negotiations or under appreciation with the following companies: Abbvie, Roche,
Ipsen, Pfizer.
Signature of Investigator-initiated Research Contracts Agreements with two world-level Biotech/Pharma companies, namely
one research contract with and consortia formed by the multinational Beijing Genomic Institute (BGI) and Coimbra Genomics
S.A and another research contract with Merck Serono (KGaA).
International Networking at the Translational Research Setting
•
•
•
Active participation on the structuring and inception of the formal started Porto Cancer Platform, a structure that aims to foster
translational research projects that bridge basic science with pre-clinical and clinical studies by ensuring that basic research
institutions, faculties and hospitals work in tight coordination.
Promotion of the integration in the translation medicine network ptATRIS (Portuguese Infrastructure for Translational Medicine),
which will be integrated in the European Group, designated as EATRIS.
Registration, expertise profiling and diffusion of IPATIMUP in the IMI (The Innovative Medicines Initiative) networking database.
This action is of outstanding importance for the Institute since The Innovative Medicines Initiative is the Europe's largest publicprivate initiative aiming to speed up the development of better and safer medicines for patients. IMI supports collaborative
research projects and builds networks of industrial and academic experts in order to boost pharmaceutical innovation in Europe.
IMI is a joint undertaking between the European Union and the pharmaceutical industry association EFPIA (European Federation
of Pharma Industry and Associations).
69
2013
External Diffusion Activities
•
•
Set up of project discussion meetings with Portuguese and International Big Pharma/Biotech companies such as Novartis,
Hoffman- La Roche, Coimbra Genomics, Abbvie, Beijing Genomic Institute, Bristol-Myers Squibb, Pfizer, Ipsen, Amgen, Celgene
and Pharmamar.
Participation in AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, in Boston (October
2013). Hosted by the American Association for Cancer Research, the National Cancer Institute, and the European Organisation
for Research and Treatment of Cancer, the 2013 Molecular Targets and Cancer Therapeutics conference brought together an
estimated 3,000 academics, scientists, and pharmaceutical industry representatives from across the globe to discuss innovations
in drug development, target selection, and the impact of new discoveries in molecular biology.
Invited Talks or Presentations
•
•
Invited talk on the GABBA Program entitled “How can I Translate Research into Business”, held at IBMC on May 2013.
Invited talk on the Conference entitled “A Montante e a Jusante das Empresas de Biotecnologia Encontro no Luso P-BIO,
organized by P-BIO and held at Hotel da Curia on February 2013.
Translational Research Promoting Events
•
Organization and realization of the XXII Porto Cancer Meeting & III Porto-Bordeux Joint Meeting, devoted to translational
research in cancer, under the theme: Translational Research in Cancer: Bringing Basic Science and Pathology to Clinical Oncology.
This year, and for the first time, the Porto Cancer Meeting brought together the perspectives from pharma industry scientists,
oncologists, cancer researchers and pathologists. The multidisciplinary and informal atmosphere of the meeting provided
the excellent conditions for discussing the latest developments in oncology translational research with the ultimate goal of
narrowing the gap between basic research and clinical practice.
Activity Statistics
Ratio TRU/Total IPATIMUP Research Funding 2013
The graph represents the percentage of funding contratctualized by the Translational Reseach Unit in 2013 over the total of research
funding contractualized by IPATIMUP in 2013.
Other Activities
Projection of Future Strategical Actions
In the end of 2013, the TRU, together with the Innovation Unit and with the knowledge transfer INEB, started the projection of a
future I3S knowledge Transfer Platform, a unit that will foster the presentation of early stage technologies to venture capital investors,
the application for innovation prizes and funding for collaborative research with companies, promoting the translation of IP and the
establishment of strong links with industry. The merging of the 3 units is envisaged into a common Platform, dedicated to academiaindustry partnerships, contracted research and to the end-stages of the innovation cycle. Synergies will generate further cooperative
strategies in R&D, setting up I3S as a partner for innovation and development of cutting-edge projects with hospitals and international
health sciences companies.
70
2013
Internal Services
71
72
2013
2013
Animal Model Facility
Overview
Since July 2012 IPATIMUP’s Animal Experimentation Unit is located at the CIM-FMUP Animal House under a protocol established
between the two Institutions. Until December 2013 IPATIMUP’s Unit occupied four rooms, one in the “SPF-like” area for breeding and
stock of our immunodeficient mouse strain (N: NIH(s) II-nu/nu) and three at the conventional area, one of them exclusively used for
maintenance and procedures involved in tumor xenograft research to allow the routine surveillance and monitoring of the animals
by all the members directly involved in the experimental process. In December 2013 we decided to move the breeding and stock of
immunodeficient mice to a room in the conventional area with very restricted permits in terms of access. This was decided because
of the need to better control our animals. Since then, our Unit is distributed in three rooms, all at the conventional area, two of them
dedicated to nude mice and a bigger room for the conventional strains. Animal maintenance areas are kept under controlled conditions
of light and humidity. Conventional mice strains allocated in the Animal Facility were received by our Researchers under collaboratin
with external groups/Institutions. These include wild-type strains and transgenic models. They are maintained and expanded to fulfill
Researchers needs. At our Institution all in vivo research, is regulated by the European and National Law that standardize the use
of animals in research. The 3Rs (that is the replacement, refinement and reduction of the use of animals in research) are implicit in
the Directive 2010/63/EU of the European Parliament and of the Council of 22nd September 2010 on the protection of animals used
for scientific purposes. Any researcher planning to use animals in their project research has to first demonstrate why there is no
alternative method, justify the number of animals plan to be used and prove that any suffering or distress will be kept to a minimum.
All the research protocols are made under direct supervision.
In terms of personnel IPATIMUP’s Animal Experimentation Unit include a Veterinarian doctor (Director), a Research technician
(Responsible technician/Coordinator) and 2 Animal Handlers. All personnel are certified by DGAV (Direção Geral de Alimentação e
Veterinária), the National Regulator Entity) under National and European Laws.
Highlights
Tumor Biology Research - Immunodeficient and Conventional Models
Tumor biology research – tumorigenesis, invasion, metastization and response to stimuli
NIH(s) II-nu/nu mice are breeded in our Unit in SPF-like conditions. Breeding is maintained in a reasonable level in order to maintain
(1) an outbred colony of genetically-variable composition mice, (2) allow the periodic substitution of active reproductive couples
and (3) improve the reproduction level when there is the need to perform experiments. During the year 2013 we started 10 in vivo
assays – 3 were internal and 7 were performed in collaboration with two external Institutions. One assay that started in the end of
December 2012 and kept in the first months of 2013 is taken into account. In terms of type of experiment, 3 were tumorigenesis assays
and 7 were tumorigenesis and metastization assays associated to response to therapy (one of them was exclusively a toxicologic
assay). In all of these cases, all the procedures, invasive (examples: inoculation of tumor cells, surgical procedures) or not (examples:
monitorization of animal health status, anthropometric parameters registry), were done under the directives that regulate the use and
care of laboratory animals and were performed by the experiment Coordinator or its Director aided by a well trained Animal Handler
or by the certified researcher. Is relevant to mention that 2013 was a year in which the cooperation with an External Institution
became stronger, highlighting the quality of our research experience. Number of animals, days of experimentations and procedures
will be presented in the point “Activity statistics”.
Conventional strains - maintenance and expansion of transgenic and/or knockout mice
In 2013 we had in expansion and maintenance 14 colonies of wild-type and transgenic models. All the manipulations performed were
done by the personnel from the Facility accompanied by the certified researchers, under supervision. Number of cages maintained
per month will be presented in the point “Activit
Activity Statistics
TABLE 1 : N: NIH(s) II-nu/nu reproduction maintained per month - year 2013
Active
couples
N (cages)*
jan13
fev13
mar13
abr13
mai13
jun13
jul-13
ago13
set-13
out13
nov13
dez13
Total
30
56
44
30
34
36
22
13
11
17
20
15
328
1594
1572
1870
1731
1654
1894
1815
1493
987
1153
1434
1578
18775
* total number of cages (maintenance and research)
TABLE 2 : Weaning NIH(s) II-nu/nu - males and females - and CBA/N - females - maintained per day per month year 2013
jan-13
fev-13
mar-13
abr-13
mai-13
jun-13
jul-13
ago-13
set-13
out-13
nov-13
dez-13
nude males
12
15
9
28
36
19
20
21
18
16
9
22
nude females
3
13
5
32
36
14
12
18
11
13
21
15
CBA/N females
2
15
5
24
40
8
50
26
12
20
12
12
Total*
17
43
19
84
112
41
82
65
41
49
42
49
* total number refers to the number of weaned animals maintained per month. Excludes CBA/N males
73
2013
TABLE 3 : Number of NIH(s) II-nu/nu used in research, duration and procedures according to Internal /External
Research Groups - year 2013
Na
Nb
Nc
Nd
Ne
Nf
Internal
71
64
502
49
484
3
External
110
61
385
37
570
7
181
125
887
86
1054
10
Total
a) N, number of animals (includes animals used in toxicologic studies)
b) N, number of necropsies
b) N, total number of maintenance days
c) N, Number of surgical procedures (primary tumours removal/heterotransplantation procedures/orthotopic inoculations)
e) N, total number of injection of drugs
f) N, number of assays
TABLE 4: Conventional strains cages - WT and transgenic- maintained per month - year 2013
Research Group
jan-13
fev-13
mar-13
abr-13
mai-13
jun-13
jul-13
ago-13
set-13
out-13
nov-13
dez-13
TOTAL
Glicobiology in
Cancer
1106
772
746
706
938
1050
1201
1355
1668
2133
1907
1668
15250
Cancer Biology
746
982
1357
1550
1776
1599
1610
1790
1780
1888
1827
1864
18769
Differentiation
and Cancer
370
381
486
412
479
509
539
530
540
613
977
915
6751
Cancer Genetics
11
30
77
90
129
337
2222
2135
2589
2668
3193
3158
3350
3686
4018
4711
4801
4576
41107
TOTAL
Other Activities
Animal Model Unit gives support to researchers in all phases of the licensing process which have to be submitted to the National
Regulator Entity (DGAV) in accordance with National Law.
74
2013
Cell Lines Bank
Overview
IPATIMUP’s Cell Line Bank (BLC) main goal is the maintenance of stocks of parental cells characterized for their genetic profile (genetic
identity) and microbiological status, in particular infection by bacteria of the genus Mycoplasma. BLC is composed by a collection of
parental cell lines, mostly tumors of various histological topographies, provided by the research groups of IPATIMUP and representing
an unquestionable heritage of the Institute. BLC cell lines were commercially obtained and others were established at the Institute or
provided by external institutions under scientific collaboration.
MAIN OBJECTIVES
During the year 2013 IPATIMUP’s BLC continued the permanent task of maintaining stocks of cell lines already characterized within the
quality parameters as well as receiving and starting new cultures provided by internal research groups. This is a continuous task and
new cell lines are always arriving to fulfill researchers and research needs. In 2013, services provided by the Unit were maintained in
a reasonable level. These include (1) provision of parental cell lines genotypically identified and tested for contaminants (specifically
bacteria of the genus Mycoplasma), provided in frozen aliquots or in culture and (2) Mycoplasma testing to cell samples donated by
internal research groups and (3) support in acquisition of new commercially available cell lines. BLC Technician also gave support in
any case of cell culture problems, recommendations for the use of consumables (for example, FBS testing control and acquisition)
and any other question considered relevant for the improvement of in vitro quality performance. In addition, during the year 2013
were fully characterized 3 new cell lines and decontaminated all the cell lines that were found to bee contaminated with Mycoplasma
strains. The updating at IPATIMUP’s website of the considered relevant information, including pictures, about each particular cell line,
available internally to all researchers was kept in a minimal level, due to reduction of personnel.
BLC TASKS / ACTIVITIES
Table 1: Numbers of stock cells and cultured cell lines per topography - year 2013
Cell line Topography
Na
Nb
Gastic Ca.
54
2
Colorectal Ca.
21
1
Breast Ca.
45
3
Thyroid Ca.
12
1
Ovarian Ca.
24
1
156
8
Total
a) N, number of frozen vials in 2012
b) N, number of cultured or maintained cell lines in 2012
Table 2: Services executed by IPATIMUP’s Cell Line Bank - year 2013
Internal
BLC
Total
N (a)
24
24
N (b)
22
21*
43
N (c)
2
3**
5
a) N, number of Mycoplasma tests executed by the service, includes collection, DNA extration and controls[* this number is for BLC internal quality control]
a) N, number of Mycoplasma tests executed by the service, includes collection, DNA extration and controls[* this number is for BLC internal quality control]
c) N, number of new cell lines acquired with BLC support [** number of cell lines donated to BLC]
Other Activities
Continuous testing of new cell culture media and reagents in order to obtain the best results in terms of cost-effectiveness.
Quality Control
IPATIMUP’s BLC Technician perform an internal control for microbiological status, specifically in the case of Mycoplasma infection,
using a commercial kit based on high specific PCR techniques. All the controls, positive, negative and internal (to test the performance
of the reaction itself) are included. We also microscopically check all the BLC cell lines in culture and perform bacterial and fungal
examinations when there is the suspicion of other possible infections. The genetic identification of our cell lines is made by IPATIMUP's
Diagnostic Service, which is accredited by CAP.
75
2013
In vivo CAM Assays
Overview
The chick embryo is a well-known animal model extensively used in cancer studies. The chick embryo presents several advantages
to mammalian models: it is naturally immunoincompetent, simple to manipulate, inexpensive, it involves short experimental times
and there is no need for official authorization from animal experimentation committees (as long as experiments end before hatching;
Directive 2010/63/EU). The CAM is an extraembryonic membrane, connected to the embryonic circulation and highly vascularised. It
provides a perfect environment to grow mammalian grafts and therefore evaluate features such as tumour mass formation, tumour
induced angiogenesis, cell migration, intravasion, extravasion and metastization. The 3Rs (that is the replacement, refinement and
reduction of the use of animals in research) are implicit in the Directive 2010/63/EU of the European Parliament and of the Council of
22nd September 2010 on the protection of animals used for scientific purposes. The use of the chick embryo as an animal model meets
this directive given that it will allow validation of in vitro results without resorting to rodent animal models or, at least, by refining
experimental conditions in order to reduce the number of mice used in a given experiment.
IPATIMUP’s “In vivo CAM assays Unit” is functioning as an internal IPATIMUP’s service unit since June 2012. In 2013, we continued to
perform services based on the established knowledge (please see “activities” and “highlights”), but we have also done a parallel work
to develop and explore the innovative and business potential of CAM assays (please see “other activities).
Highlights
Expression of ST3GAL4 leads to SLex expression and induces c-Met activation and an invasive phenotype in
gastric carcinoma cells
Sialyl-Lewis X (SLe(x)) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated
with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the
expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize
the SLe(x) antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe(x) overexpression in cancer cell behavior both
in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their
downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells
leads to the synthesis of SLe(x) antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis
of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream
molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins
and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive
phenotype. In conclusion, the expression of ST3GAL4 leads to SLe(x) antigen expression in gastric cancer cells which in turn induces
an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases
activation.
Gomes, C., H. Osório, M. Pinto,… C. Reis (2013). PLoS One Jun 14;8 (6):e66737.
DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro and in
vivo.
E-cadherin (Ecad) is a well-known invasion suppressor and its loss of expression is common in invasive carcinomas. Germline Ecad
mutations are the only known genetic cause of hereditary diffuse gastric cancer (HDGC), demonstrating the causative role of Ecad
impairment in gastric cancer. HDGC-associated Ecad missense mutations can lead to folding defects and premature proteasomedependent endoplasmic reticulum-associated degradation (ERAD), but the molecular determinants for this fate were unidentified.
Using a Drosophila-based genetic screen, we found that Drosophila DnaJ-1 interacts with wild type (WT) and mutant human Ecad
in vivo. DnaJ (Hsp40) homolog, subfamily B, member 4 (DNAJB4), the human homolog of DnaJ-1, influences Ecad localization and
stability even in the absence of Ecad endogenous promoter, suggesting a post-transcriptional level of regulation. Increased expression
of DNAJB4 leads to stabilization of WT Ecad in the plasma membrane, while it induces premature degradation of unfolded HDGC
mutants in the proteasome. The interaction between DNAJB4 and Ecad is direct, and is increased in the context of the unfolded
mutant E757K, especially when proteasome degradation is inhibited, suggesting that DNAJB4 is a molecular mediator of ERAD. Posttranslational regulation of native Ecad by DNAJB4 molecular chaperone is sufficient to influence cell adhesion in vitro.
Using a chick embryo chorioallantoic membrane assay with gastric cancer derived cells, we demonstrate that DNAJB4 stimulates
the anti-invasive function of WT Ecad in vivo. Additionally, the expression of DNAJB4 and Ecad is concomitantly decreased in human
gastric carcinomas. Altogether, we demonstrate that DNAJB4 is a sensor of Ecad structural features that might contribute to gastric
cancer progression.
Silva DI, Melo S, Figueiredo J, Caldeira J, Pinto MT…… Simões-Correia J. Human Molecular Genetics, 2013 1–12.
Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y, c-Src, Erk1/2 and Akt
phosphorylation and smallGTPase activity.
We used the CAM assay to evaluate the Angiogenic response of gastric AGS cell suspended in conditioned medium (CM) from different
populations of macrophages namely, M0, M1 and M2. AGS cells subjected to CMM0 and CMM1 had a reduced angiogenic response
while cells with CMM2 presented a significant increased angiogenic response
Cardoso AP, Pinto ML, Pinto AT, Oliveira MI, Pinto MT, …Olivera MJ. Oncogene, 2013 May 6. doi: 10.1038/onc.2013.154. [Epub ahead
of print]
Activity Statistics
Services performed
During 2013, 450 eggs were manipulated, 330 were inoculated in a total of 8 requisitions from IPATIMUP researchers but also from
others, including INEB, FEP and Coimbra University.
Other Activities
Creation of a spin-off
In September 2013 the company - "Expertus, CAM assays" was created.
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2013
FUNDING
In collaboration IPATIMUP’s Innovation Unit, and in the company context, two applications for funding - Vales Inovação e
Empreededorismo - were approved in October 2013, and in February 2014, respectively.
SEMI-FINALIST
Expertus, CAM assays was one of the top 20 semi-finalists, to the contest "BGI - Building Global Innovators", ISCTE-IUL MIT venture
competition (May to November 2013).
AWARD
Expertus, CAM assays was award with IDDNET-Technology Network award at the “Arrisca C, Concurso de Provas de Conceito”
(application in October 2013, received in February 2014).
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2013
Proteomics
Overview
The IPATIMUP Proteomics Unit offers to the scientific community analysis of protein samples from solutions, bands or protein spots
from 1D or 2D SDS PAGE gels.
The following proteomics approaches were performed in 2013:
1. Mass Analysis of Intact Proteins, Peptides and Metabolites by mass spectrometry.
2. Protein identification and characterization by Peptide Mass Fingerprint -PMF (MALDI-TOF).
3. Protein identification and characterization by PMF following peptide sequencing/fragmentation (MALDI-TOF/TOF, PMF+MS/MS).
4. Protein separation by 1D or 2D gel electrophoresis.
The provided services included: protein separation; enzymatic digestion and peptide extraction of isolated proteins from 1D or
2D SDS-PAGE gels or purified proteins in solution; peptide analysis by mass spectrometry (MALDI-TOF) with the option of peptide
sequencing (MALDI-TOF/TOF); protein identification using UniProt and/or NCBI protein sequence databases; data analysis; and
delivery of a report with the identified protein(s). Specific and “personalized” analysis were also performed, including: analysis of
amino acids sequences, gene identification, determination of protein molecular weight and isoelectric point, search for relevant
proteins and enzymatic characterization studies.
The overall income in 2013 was 39 078 euros. The expenses in lab materials, reagents, and missions were 10 961.70 euros. Equipment
maintenance costs were 14 244 euros. Staff costs were 39 464 euros (supported by QREN since May 2013). The MALDI mass
spectrometer was replaced by a new instrument in May 2013 with an investment cost of 104 550 euros.
In addition, the IPATIMUP proteomics unit has participated in 5 research projects, 10 scientific publications, 3 patents, and has been
involved in the organization of the 4th Proteomics Workshop.
Highlights
IPATIMUP Proteomics Unit – Highlights 2013
The IPATIMUP Proteomics Unit developed its activity in 2013, similarly to the previous years, by performing research-based activities
and providing services to the inside and outside scientific community.
The average income per month increased from 947.90 euros (2007-2012) to 3256.56 euros (2013). 65% of the generated income
was from analysis performed to external institutions. These results were the outcome of the following approaches: 1) progressive
replacement of the collaboration prices by the standard costs of the analyses; 2) increase in the number and variety of the analyses
performed; 3) promotion of networking activities with research groups developing projects from different scientific backgrounds and
participation in multidisciplinary studies; and 4) stimulation of scientific collaborations leading to the publication of scientific papers,
submission of research projects and patent applications.
In May the MALDI mass spectrometer was replaced (trade-in) by a new instrument. The novel, state of the art, equipment has better
sensitivity, higher throughput, and enhanced capacity of peptide sequencing. The new instrument was installed, calibrated and has
successfully performed the vendor certification test and also the IPATIMUP “real-life” tests.
Major activities
The IPATIMUP Proteomics Unit has performed a total of 63 independent analysis divided by the following activities:
•
Molecular weight determination of protein and peptides: 377 analyses
•
Protein identification by Peptide Mass Fingerprint (PMF): 178 analyses
•
Protein identification by PMF and MS/MS peptide sequencing / characterization of Protein post-translational modifications: 263
analyses
•
Protein separation by 1D and 2D gel electrophoresis: 41 analyses
Proteomic analysis were performed to the following institutions:
•
IPATIMUP: Cancer Genetics, Cancer Biology, Population Genetics, and Glycobiology in Cancer research groups
•
IBMC
•
INEB
•
CIIMAR
•
University of Porto: FCUP, FEUP and ICBAS
•
University of Minho
•
University of Coimbra
•
University of Algarve
•
Polytechnic Institute of Porto
•
Technical University of Lisbon: Institute of Agronomy (ISA)
•
Portuguese Catholic University: Faculty of Biotechnology
•
Bioalvo (Biotechnology company)
Participation in the following research projects:
•
1. Gastric GlycoExplorer, EU Grant 316929, FP7-PEOPLE-2012-ITN; PI: Celso Reis
•
2. Zinc-finger nuclease-glycoengineered SimpleCell lines for characterization of the O-glycoproteome of cancer cells, PTDC/
BBB-EBI/0786/2012; PI: Celso Reis
•
3. Glycomic approach to unravel the role of glyco structures in gastric carcinogenesis, Merieux Research Grant; PI: Celso Reis
•
4. Unravelling a new Helicobacter pylori virulence factor involved in tight junction dysfunction, PTDC/BIA MIC/116890/2010; PI:
Céu Figueiredo
•
5. Mucin MUC16 - CA125 cancer biomarker – biological functions and development of new biomarker assays, PTDC/SAUONC/117216/2010; PI: Leonor David
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2013
Scientific publications (Part1)
1. Gomes C, Almeida A, Ferreira JA, Silva L, Santos-Sousa H, Pinto-de-Sousa J, Santos LL, Amado F, Schwientek T, Levery SB, Mandel U,
Clausen H, David L, Reis CA, Osório H. Glycoproteomic analysis of serum from patients with gastric precancerous lesions. J Proteome
Res. 2013 Mar 1;12(3):1454-66. doi: 10.1021/pr301112x.
2. Malagolini N, Catera M, Osório H, Reis CA, Chiricolo M, Dall'Olio F. Apoptotic cells selectively uptake minor glycoforms of vitronectin
from serum. Apoptosis. 2013 Apr;18(4):373-84. doi: 10.1007/s10495-013-0812-z.
3. Correia M, Michel V, Osório H, El Ghachi M, Bonis M, Boneca IG, De Reuse H, Matos AA, Lenormand P, Seruca R, Figueiredo C,
Machado JC, Touati E. Crosstalk between Helicobacter pylori and gastric epithelial cells is impaired by docosahexaenoic acid. PLoS
One. 2013;8(4):e60657. doi: 10.1371/journal.pone.0060657. Epub 2013 Apr 5.
4. Burford B, Gentry-Maharaj A, Graham R, Allen D, Pedersen JW, Nudelman AS, Blixt O, Fourkala EO, Bueti D, Dawnay A, Ford J, Desai
R, David L, Trinder P, Acres B, Schwientek T, Gammerman A, Reis CA, Silva L, Osório H, Hallett R, Wandall HH, Mandel U, Hollingsworth
MA, Jacobs I, Fentiman I, Clausen H, Taylor-Papadimitriou J, Menon U, Burchell JM. Autoantibodies to MUC1 glycopeptides cannot be
used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer. Br J Cancer. 2013 May 28;108(10):2045-55.
doi: 10.1038/bjc.2013.
5. Gomes C, Osório H, Pinto MT, Campos D, Oliveira MJ, Reis CA. Expression of ST3GAL4 Leads to SLe(x) Expression and Induces
c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells. PLoS One. 2013 Jun 14;8(6):e66737. doi: 10.1371/journal.
pone.0066737.
6. Neves J, Campos A, Osório H, Antunes A, Vasconcelos V. Conopeptides from Cape Verde Conus crotchii. Mar Drugs. 2013 Jun
19;11(6):2203-15. doi: 10.3390/md11062203.
7. Almeida A, Ferreira JA, Teixeira F, Gomes C, Cordeiro MN, Osório H, Santos LL, Reis CA, Vitorino R, Amado F (2013). Challenging
the limits of detection of sialylated Thomsen-Friedenreich antigens by in-gel deglycosylation and nano-LC-MALDI-TOF-MS.
Electrophoresis, 34, 2337-41. doi: 10.1002/elps.201300148
8. Campos A, Araújo P, Pinheiro C, Azevedo J, Osório H, Vasconcelos V (2013). Effects on growth, antioxidant enzyme activity and
levels of extracellular proteins in the green alga Chlorella vulgaris exposed to crude cyanobacterial extracts and pure microcystin and
cylindrospermopsin. Ecotoxicol Environ Saf, 94, 45-53. doi: 10.1016/j.ecoenv.2013.04.019
9. Azevedo CC, Azevedo J, Osório H, Vasconcelos V, Campos A (2013). Early physiological and biochemical responses of rice seedlings
to low concentration of microcystin-LR. Ecotoxicology. 2013 Dec 10 in press. doi: 10.1007/s10646-013-1156-8
Book chapters
1. Osório H, Reis CA.
Mass spectrometry methods for studying glycosylation in cancer. Methods Mol Biol. 2013;1007:301-16. doi: 10.1007/978-1-62703392-3_13.
Patent applications
1. Gomes C, Reis CA, Osório H. Biomarcadores séricos para diagnóstico do cancro do estômago, Portuguese patent reference:
20131000002498, 2013/01/10.
2. Gomes C, Osório H, Reis CA. Co-expressão de CEA e glicanos SLeX como biomarcador no cancro gástrico. Portuguese patent
reference: 20131000048433, 2013/06/27.
3. Thompson G, Silva E, Marques S, Osório H, Carvalheira J. HCV Homolog Fragments, cell-lines and applications thereof. International
patent reference: PCT/IB2013/052636, WO2013150450 A1. International publication date: 10/10/2013.
Activity Statistics
Facturação serviços
internos
13.733,60 €
externos
25.345,18 €
Total
39.078,78 €
Other Activities
Participation in graduation and training programs:
GABBA graduated program from University of Porto
III Workshop on Cancer Research
Organization of the Workshop: “Proteomics course: gel based protein separation by two- dimensional gel electrophoresis and protein
characterization by MALDI-TOF/TOF mass spectrometry”. This workshop was attended by 17 students from different countries and
institutions.
Post-graduate education program of the Portuguese Society of Inherited Metabolic Diseases
Networking
Full integration at RNEM – Rede Nacional de Espectrometria de Massa / Portuguese Mass Spectrometry Network (September 2013)
Submitted Projects
Application submitted to the FCT Research Infrastructure (RI) call: Portuguese Mass Spectrometry Network (October 2013)
Communications in scientific meetings:
Poster presentation at the “61st Conference on Mass Spectrometry and Allied Topics – ASMS, Minneapolis”, MN, USA (June 2013).
Presentation title: MALDI TOF/TOF determination of serum plasminogen sialylation profile from patients with gastric precancerous
lesions”
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2013
Sequencing Service
Overview
IPATIMUP’s sequencing service was initiated in 2006 to serve investigators and technicians who develop their work there. The
sequencing service performs the sequencing reaction of plasmids and PCR products and automated analysis of genotyping and
sequencing products by capillary electrophoresis.
Nowadays, IPATIMUP’s sequencing service is also open to the general scientific community.
Highlights
Improvement of Sequencing Service - I
The Sequencing Service for external scientific community now includes the possibility of performing sequencing reactions. Since
March, the occupancy rate by the external service was about 6% (both automatic electrophoresis only and sequencing reaction
included) mainly by IBMC.
Improvement of Sequencing Service - II
The Sequencing Service has an extra 4-capillary Genetic Analyzer which allows for a reduced turn-around time and an increased
sequencing results output.
Activity Statistics
Number of samples processed per month in 2013
Number of samples processed per research group in 2013
32.421
Population Genetics
12.317
Cancer Biology
9.951
Genetic Diversity
6.132
Proteolysis in Diseases
4.181
External Service (automatic electrophoresis only)
3.823
1.012
Cancer Genetics
936
111
Internal/External Service (sequencing reaction included)
89
69
2
Other Activities
Real-Time Service
The Sequencing Service is now in charge of operating and maintaining the Real-Time Service.
80
2013
81
82
2013
2013
Core Services
83
84
2013
2013
Informatics
Overview
The main goal of this service it’s to give access to the Institute Researchers all necessary tools available to register, promote and
publish their work.
Our Unit it’s divided in 2 areas, the Networking and the Information System.
The networking staff is responsible for the maintenance and configuration of all informatics infrastructure and gives helpdesk support
for all informatics equipment.
In other hand the Information System staff is responsible for the creation and maintenance of all web applications mainly created in
the Outsystems Platform and Oracle Application Express.
Highlights
File Module [Development]
In the IPATIMUP's site was made available a screen (My Org Unit Documents) that allows members of a group see the associated files
to your group. Thus, even if it are not available on the site public or internally it still are visible within the group.
Web Docs Manager Module [Development]
Change our API, data model and interface widgets from Issuu publish service to Calameo publish service. Besides uploading a
document to Calameo, now is possible to update and delete an existing document on Calameo.
It was necessary to create a script to upload all the files to Calameo Server which were already on Issuu, handles the response and
update our database with the links to documents.
Business Process Manager Module [Development]
Since the creation and implementation of Workflows, it was felt the need to have an interface where the user can see all his workflows
and tasks.
So, this interface was created and someone with special permissions can also see all the workflows.
VARS/AM - Variable Acquisition and Reporting System / Alarm Management [Development]
This module is important for IPATIMUP Diagnostics Service, it allows to monitor temperatures and humidity of devices such as
refrigerators, freezers, etc..., and is also a requirement for the certification of the service.
It was created a script to get temperatures and humidity values from Poseidon device via Http/Xml on each minute and store them in
the database and it was created a script to repair database gaps with Poseidon device internal log.
For users to consult, it was developed an interface in APEX platform where users can view charts with alarms, temperatures and
humidity of all sensors.
This interface shows a hierarchical disposition of two locations and the sensor. When user selects a sensor, view an alarm chart since
previous week, temperature/humidity chart of current day and current week and the respective histogram. Is also possible to print,
export to CSV file and generate a chart for each week of the year.
This module was extended to monitor duration of open-close doors of some freezers of the institute.
It was made a development of a C# service called SNMP Manager. This service is a network service which is listening on 162 UDP Port
for SNMP traps sent by Damocles Device.
It processes this traps and inserts into the Database. All information gathered from Damocles is also possible to view on the interface
as charts.
Site Module [Development]
The appearance of our site www.ipatimup.pt was changed. It was created a new home page where the announcements are the main
content and is possible to filter by category. All pages were modified in accordance with this new look.
It was developed a bar menu widget for the new vertical and horizontal menus. Is possible now to search for content, the results
appear on a new page separated by tabs and subsections. The search text is highlighted on each page and there is a navigation toolbar to navigate through all matched words.
The site has also now a Sitemap page where the content is dynamic depending if the user is inside the facility or not.
Tracking of visits to our website was implemented using Piwik.
Person Module [Development]
Sending emails to warn anyone who has to make an appointment of occupational medicine, new people or that the link with the
IPATIMUP has changed so that it has necessary to make an appointment of occupational medicine.
Event Registration Manager Module [Development]
Enable to generate and print all cards from every person which is registered on an event.
Given an Excel file with a list of people with Name and Institution, allow to generate and print the respective cards.
Requisition Module [Development]
Added an authorization to requisitions with amount superior to 5000 €.
Send requisitions for suppliers by email
External Requisitions [Development]
Added the option to make requesitions of service packages pre-paid.
Form Generator Module [Development]
Added the functionality to create forms sequential.
Absence Notification Module [Development]
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2013
Now users can schedule absences on days where already exist other absences. Because of this, the interface warns the user and who
is going to approve if has more than one absence on same day.
In Person Calendar is also possible to view and edit more than one absence on a specific day.
It is possible now to schedule vacations before the respective "vacation year". It was also created a new permission to reopen missions.
Allowances Module [Development]
Now is possible to submit expenses to reimburse as soon as the mission starts, never allowing to insert with date higher than the
current date. The secretary can reimburse and pay the user several times before closing the mission.
It was included the account item in the form when user inserts allowances, besides the account, the user can also select the item on
which the allowance will take effect. All interfaces were changed in order reflect this new requirement.
When allowances need to be approved by the account responsible, they are grouped all together and send on single email. It was
created an interface where the responsible for the account can approve or reject each allowance individually and manages concurrent
allowances approval by different responsible of the account.
In order to agile the work of the secretary, it was included a check box to select or not all allowances at once.
The allowance page has also a warnings when the user has already other absences on the mission interval and also warns when the
date of the expense to be reimbursed has already other absences.
Update action which interacts with www.xe.com site and gets the exchange rate for a given date and currency.
Who Is In Manager Module [Development]
When an person force checkout of other persons, the system send an email to him with a list of persons that he forced checkout.
Person Admission Module [Development]
The Visiting Admission Workflow was changed in order when the Organization Unit is not from the category Research, the user should
specify the account instead of projects.
Meeting Manager Module [Development]
Update of this module in order to have validations to prevent insertion of blank fields on database, insertion of incoherent and
contradictory data, unable more than one meeting being opened with same meeting type, unable a specific scanner being active on
more than one meeting.
Also when closing a meeting, include chapters, topics and attendees in order to edit them for the next meeting.
Add new features when meeting is open such as edit chapters and topics and enable to edit decisions even when exists other meetings
of the same type created after.
Changing of Data Model and optimization of querys.
Kioskes (Risk Management) [Development]
Added two new screens:
Safety Rules: with our oficial documents with safety rules.
Devices: with GLPI devices page with all documents associated, instructions manuals, maintenance reports, etc...
Scientific Data Module [Development]
Module for storing scientific data to be used by research groups.
[Networking]
All software maintenance is performed by our department, like upgrading versions or changing licenses. When asked by the users, we
install specific software on the computers of the Institution or in the personal laptops, if there is a valid license to perform this task.
The update of the operating system, from Windows XP to Windows 7, is still a work in progress. Some of the machines were not
updated because their hardware doesn't support Windows 7.
We have finished replacing the computers in the library. We found out that the computers are working well with the new operating
system (Windows 7).
Our department is expected to perform some operations in the telephone network, like create new telephone extensions, unlock
telephone for external calling and resolution of other minor problems.
The Windows 8.1 Operating System is available. We've started to perform tests with this operating system in some specific machines.
We are responsable for the maintenance of specific Linux servers that perform specific data analisys operations.
We have replaced most of the local printers to network printers. The new Canon printers needed specific software for accounting,
named Uniflow, we needed to replace the printer server in order to install and configure this specific software.
We changed our SPAM filter machine from physical to virtual and with this change the service performance was improved.
This year we updated several times the anti-virus server due major releases from the anti-virus company. We also upgraded our
ticketing service and the domain upgrade was performed.
Activity Statistics
Informatics > Computer
86
Number of tickets
Number of resolved tickets
Average resolution delay
89
85
7 Day(s) 22 Hour(s) 48 Min(s)
Informatics > Computer > Hardware
27
28
Informatics > Computer > Software
120
121
Informatics > Internet
33
34
Informatics > Printer
154
154
Helpdesk Team [Networking]
569
573
2013
Other Activities
Future Objectives [Development]
Provide all applications for new versions of IE, and Chrome.
Management of sending warning emails for alarms in module VARS.
Future Objectives [Networking]
We need to improve our policies to get better performance from the system.
And the migration/substitution of our mail server is still on the roadmap to be performed.
Programs Office
Overview
During the year 2013, the office announced to the researchers of I3S hundreds of funding opportunities such as projects, awards,
fellowships and job positions; it supported the submission of over 150 research project proposals (77% to national funding programs
of which 67% to FCT (in its majority to the 2013 call for proposals to exploratory projects in all scientific domains) and 23% to
international agencies with 14% to European Commission programs, the majority to FP7) and also supported other scientific research
related programs, which are included in the regular activities of the institutes.
Like in previous years, the responsible for the office attended information sessions about research funding programs such as Horizon
2020 (European Commission’s Research & Development Funding Program) among others. The programs’ office also had a relevant
role in the coordination of the relations between the 3 institutes, concerning the I3S Seminars and other information and diffusion
activities.
Risk Management System
Overview
The IPATIMUP undertakes to develop a systematic approach whose objective is to minimize the various professional risk factors for
their colaborators, risks to users and all stakeholders through:
• Comply with the rules of safety and health at work in force;
• Implement a continuous process of identifying, assessing and monitoring risks;
• Promote and ensure a safe and healthy workplace for all facility users;
• Promote training activities so that all employees are involved in the area of safety and health;
• Review and improve the system of health and safety;
• Provide this policy to the general public.
Highlights
TRAINING
- “Risk Management Systems Workshop: Organizational and legal aspects”, Mário Seixas, March 2013
- “Risk Management Systems Workshop: Accident & Incidents Management”, Ana Sofia Silva, March 2013
- “Risk Management Systems Workshop: Safety rules & risk assessment”, Filipa Valente, March 2013
- “Internal Security Plan”, Manuel Carvalho, May 2013
- “Internal Security Plan”, Manuel Carvalho, July 2013
- “Pallet jack: Risks and preventive measures for use”, Filipa Valente, September 2013
- “Revision to Standard 18001”, David Sanders, October 2013
- “Analysis of the simulacrum 2013 and opportunities for improvement”, Manuel Carvalho, November 2013
- “Good Lab Practices & Chemical Spill Kits“, Filipa Valente, December 2013
- “Reagents internal MSDS sheetsdatabase”, Filipa Valente, December 2013
- “Residues: the classes and safety discard”, Filipa Valente, December 2013
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2013
The contents of training are available for consultation on the IPATIMUP intranet in the Risk Management System area.
INTERNAL AUDITS
In 2103 eight internal audits were performed and, seven of which were first made by the IPATIMUP Local Safety Contacts:
- Audit to Cancer Biology Laboratory, 2013.10.03
- Audit to Cancer Drug Resistance Laboratory, 2013.10.03
- Audit to Cancer Genetics/ Gen. Diversity/ Exp. Reg. in Cancer Laboratory, 2013.10.04
- Audit to IPATIMUP Diagnostics Laboratories, 2013.10.04
- Audit to INEB Laboratory, 2013.10.03
- Audit to Population Genetics Laboratory, 2013.10.04
- Audit to Glycobioloy inCancer/Differentiation in Cancer Laboratory, 2013.10.04
- Audit to IPATIMUP by Always the Best, 2013.10.15
The reports resulting from audits are available in in the IPATIMUP intranet, in the Risk Management System documents.
SIMULACRUM 2013
At 17.07.2013 was performed the simulacrum and were involved:
- All the IPATIMUP collaborators, Porto Firefighters, Public Security Police & Nacional Medical Emergency (INEM);
PERIODIC MEETINGS
As part of the risk management system several working groups met periodically throughout the year to discuss and analyze issues of
health and safety and their risks and propose corrective and /or preventive actions.
The working groups are constituted by:
- Comission Risk;
- Local Safety Contacts, Mario Seixas, Filipa Valente;
- Ana Paula Teixeira, David Sanders, Mario Seixas, Filipa Valente e Ana Sofia Silva;
DOCUMENTS
In order to maintain the OSHAS 18001:2007 certification the Risk Management System produced several documents. Some examples
are:
- IPATIMUP Risk Assessment 2013;
- System Review Acta;
- IPATIMUP Health & Safety Plan for 2013;
- Action Planning / 2013;
- Analysis reports to the submitted accident/incident reports;
- Periodic checklists completing.
Quality Control
ENERGY CERTIFICATION
During this year IPATIMUP accomplished the Energy Certification, an legal requirement getting the energetic rating B+. This certification
also included the indoor air quality measurement where IPATIMUP complies the legislation in force.
The Energy Certification report is available for consult in the Risk Man. System Documents
Secretary General
Overview
The General Secretary carries out the general administrative tasks of IPATIMUP:
- Treasury and accounting;
- Projects management, comprising all the tasks of financial reporting and providing assistance to the Principal Investigators in
administrative and legal related issues;
- Human Resources management;
- Organization of events;
- Control of purchase orders and Contratação Pública;
- Validation of contracts with general suppliers;
- Updating data on the information system
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2013
Highlights
General highlights
A controlling table for infrastructural expenditure has been monthly updated . In 2013, we have achieve a reduction of €87.000 on
general costs (facilities and maintenance).
The management software developed in-house has been improved following specific needs.
The accounting is made in accordance with the SNC – Standard Accounting System, using a certified software (SAGE).
All assets are labelled.
All requisitions of goods and services and equipment maintenance are administratively verified.
The General Secretary supports the maintenance and renewal of the IPATIMUP’s library.
The administrative services receives and instructs all requests for human resources movements - entries, renewals or voluntary
trainings - through workflows created for this purpose.
The General Secretary takes charge of all organization tasks related to international meetings and seminars.
There are specific workflows for control of accidents, for monitoring of occupational health and for updating a database of applicable
laws.
Technical Body
Overview
The Technical Body is an infrastructural service and is responsible for the management of the IPATIMUP’s common research resources
crossing the different areas and research groups:
- Looking to keep the equipments in a good maintenance status;
- Planning and monitoring the use of those equipments by the different researchers;
- Establishing, gather with the Board of Directors, procedures and good work practices;
- Proposing to the Board of Directors technical solutions to problems related with the laboratories management;
- Keeping work records;
- Training new users;
Technical Body has the operational responsibility of the Sequencing , Real Time PCR, Cell Line Bank and QIAxcel internal services and
in 2013 it was composed by Thecnical Supervisor Filipa Valente as the coordinator, by Ana Mafalda Rocha as the Sequencing Service
Responsible, Nuno Mendes as the Cell Lines Bank Service Responsible, and by Maria José Ferreira (Washing Room Responsible) and
Mário Ramos as maintenance assistants.
Highlights
TRAINING
- "Risk Management Systems Workshop: Safety rules & risk assessment", March 2013
- "ThermalCyclers":Safety use and maintenance", July 2013
- "Porta paletes Riscos e medidas preventivas de utilização", September 2013
- "Good Lab Practices & Chemical Spill Kits“, December 2013
- "Reagents internal MSDS sheetsdatabase”, December 2013
- “Residues: the classes and safety discard", December 2013
COMMON WORKING ROOMS MAINTENANCE
Technical body is responsible for the daily maintenance of several rooms ( eg. Cell culture rooms 1 and 2, Pré PCR room, Advanced
microscopy room, Dark room, HPLC and Image acquisition room, Centrifuges and incubators
room, -80ºC and 4ºC Cold rooms).
Technical Body permanently ensure that, in these rooms, the equipments are in operating conditions, the correct use of the facilities
and equipments, give specific formation, check the users access to the rooms, and provide logbooks, Standart Operating Procedures
(SOP) for the equipments and the Safety Rules of each room.
In fact, during 2013, the Safety Rules and MSDS access for all was simplified by the use of the "computers stations" existent in the
intitute corridor (floor 1).
EQUIPMENT ACQUISITION
Equipment aquired during 2013 by the institute:
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2013
- Thermalcyclers, 5 units
- Freezers -20ºC, 3 units
- Time lapse microscope, 1 unit
- Ultracentrifuge, 1 unit
- Rotor 70Ti & Rotor SW32
- Ion Proton
DATABASES SUPERVISION & MAINTENANCE
- "Reagents MSDS" database was continuously updated (by Ana Mafalda Rocha and Filipa Valente).
- “Device Inventory” database was continuously updated with insertion of new equipment acquired data, instruction manuals,
technical assistance and maintenance/inspection reports, Conformity Declarations, etc. (by F. Valente).
- "Tickets" from Helpdesk database was daily coordinated/maintained. This is an important work because is through this tickets that
people report equipment or infrastructure problems, needs, etc. (by Filipa Valente)
Activity Statistics
Total of tickets submitted in 2013 in the building, maintenance and equipment categories.
Other Activities
RISK MANAGEMENT SYSTEM (RMS)
Technical body was fully involved in the Risk Management Project, namely Filipa Valente as member of risk comission and responsible
for health and safety at IPATIMUP and Ana M. Rocha also as member of the risk comission.
In the aim of this project Filipa Valente produced several Safety Rules/SOP's for equipments and rooms with specific requirements;
- 2013 IPATIMUP risk evaluation table;
- Training;
- New Checklists development for verification of equipments/facilities and improvement of the Checklists already existent;
- Maintenance of the RMS Database (insertion and actualization of documentation);
- Elaboration of meetings oficial records (p.e. commission risk);
- Coordination of the accident/incident reports submitted;
- Elaboration of the Technical Supervisor reports related to the accident/incident reports submitted;
- Participation, as the Institute responsible for Safety and Hygiene, in all the 2013 audits in the scope of OSHAS 18001:2007 certification
;
OTHER ACTIVITIES
- Maintenance of the 2013 Pricelist for the most used consumables in IPATIMUP (by F. Valente);
- Management of the "Equipment Acquisition Forms"
- Elaboration of specifications/requisits necessary during equipment acquisitions processes;
- Coordination of the Dark room reformulation in other space;
- Coordination of the Gel and Cell Counter Room and Radioimunoassay rooms elimination with the re-instalation os equipments in
other spaces;
Quality Control
QUALITY/SAFETY CONTROL
Preventive Maintenance/Verification Contracts establishedfor the laboratorial equipments:
- Microtome, Thermo Electron Corporation Shandon Finesse 325, S/N: FI50850603
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2013
- Cooling plate Kunz CP-4, S/N: 2006-1262
- Histologic Bath Kunz Hir-3, S/N: 2006-2041
- Centrifuge SHANDON Cytospin 3, S/N: MA170403R
- Genetic Analyser Applied Biosystems, 3130-4, S/N: 19336-040
- Genetic Analyser 310, Applied Biosystems, S/N: 1205-017
- Real Time PCR Applied Biosystems, 7500, S/N: 275012980
- Autoclave AJC, S/N: 100
- Dishwasher MIELE, S/N: 16/18332096
- Dishwasher MIELE, S/N: 16/18332095
- Drying Incubator Memmert 800, Int. Ref.: 120008/1996
- Sterilizer Machine MIELE, Int. Ref.: 1200048/1997 94
- Hottes
Preventive Maintenance/Verification Contracts establishedfor the insfrastructural equipments:
- Elevator;
- AVAC equipments;
- Plague control units;
- Personal hygiene units;
Preventive Maintenance/Verification Contracts establishedfor the insfrastructural services:
- Collection and treatment of waste (Types III and IV);
- Gas cylinders;
- Emergency units (Automatic system of fire detection);
- Extinguisher;
- Garden;
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2013
Annexes
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2013
Recent PhD’s
Vânia Alves e Silva Pereira
03-01-2013, FCUP
A Study on Patterns of X-chromosomal diversity
Supervisor: Maria João Prata
Renata Bordeira Carriço
18-03-2013, FMUP
Suppressor-tRNAs as therapeutic tools for cancer associated syndromes: HDGC as a model system
Supervisor: Carla Oliveira
Ricardo Jorge Pinto
23-04-2013, ICBAS
Characterization of a population with increased risk of gstric cancer: first degree relatives of early onset gastric carcinoma patients
Supervisor: Fátima Carneiro
Luís Pedro Resende
07-05-2013, ICBAS
The role of the headcase gene in the Drosophila melanogaster testis and intestinal stem cells niches
Supervisor: Leonor David
Joana Isabel Barbosa Pereira
03-06-2013, Universidade de Leeds
Genetic characterisation of modern human dispersals in the Greater Mediterranean
Supervisor: Luísa Pereira
Marta Daniela Araújo Costa
03-06-2013, Universidade de Leeds
Genetic characterisation of the Early Upper Palaeolithic settlement of Europe by modern humans
Supervisor: Luísa Pereira
João Miguel Sotto Maior Faria Carneiro
09-07-2013, FCUP
The Role of Non-coding DNA Structual Information in Phylogeny, Evolution ad Disease
Supervisor: António Amorim
Maria Francisca de Lima Magriço Coutinho
29-07-2013, FCUP
Molecular, Biochemical and Functional Studies in Genes Determining Missorting of Lysosomal Proteins
Supervisor: Maria João Prata
Zélia Alexandra Mota Quintas Ferreira
06-09-2013, FCUP
The Human Whey-acidic-protein Four-Disulfide Core—domain (WFDC) cluster on 20q13 region: evolutionary history and role in
human health and disease
Supervisor: Susana Seixas
Bruno Miguel Correia Pereira
18-11-2013, FMUP
Regulation of CDX2 and intestinal differentiation in homeostasis and carcinogenesis: emphasis on the role of MEX3A
Supervisor: Raquel Almeida
Diana Raquel Fernandes Martins
02-12-2013, ICBAS
Transition from In Situ to Invasive Breast carcinoma
Supervisor: Fernando Schmitt
Catarina de Sena Bastos Gomes
13-12-2013, FMUP
Discovery of novel biomarkers in gastric cancer based on post-translational modifications of glycoproteins
Supervisor: Celso Reis
Hugo Seca Teixeira
19-12-2013, FFUP
Increasing sensitivity to drugs in leukemias by modulation of microRNA expression
Supervisor: Helena Vasconcelos
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2013
Research Projects
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Fellowship para apoio ao Internato de Anatomia Patológica no Hospital de S. João [FCG]
Molecular and nanotecchnology-based approaches to improve the antitumor activity of small molecules [FCT]
Desenvolvimento de um sistema de informação médica sobre Cancro Hereditário da Mama e Colorectal [FCT]
Characterization of a population with increased risk of Gastric Cancer: First Degree Relatives of Early Onset Gastric Carcinoma
Patients [HSA]
Mutant E-Cadherin and Novel Interactors in Cancer (MECANIC). The role of mutant E-cadherin-integrin-ECM crosstalk in gastric
cancer [FCT]
Combining siRNA and AAV therapy approaches to target human basal-like breast cancer: from vector development to anti-tumor
efficacy evaluation [FCT]
O Nemátode-Da-Madeira-Do-Pinheiro (NMP), Bursaphelenchus Xyliophilus [IMAR]
Transcriptional and post-transcriptional mechanisms of LRP1B inactivation in sporadic and familial non-medullary thyroid cancer
[FCT]
Biomarcadores para avaliação de efectividade terapêutica no carcinoma colorectal [ADI - Programa IDEIA]
Portuguese Wild Mushrooms: Chemical characterization and functional study of antiproliferative and proapoptotic properties
in cancer cell lines [FCT]
Genetic and chronological characterisation of the European settlement by modern humans in the Upper Palaeolithic [FCT]
Characterization of voriconazole susceptibility in relevant clinical molds [Pfizer]
Telepathological Assessment of histopathological and cytological techniques [UE]
Disorders on the Glycolytic and Pentose Phosphate Pathaways of the Red Blood Cell - how do they affect Plasmodium infection?
[IHMT]
Helicobacter pylori genetic variation: An opportunity to identify individuals at increased risk for disease [FCT]
MUC1 mucin transcriptional regulation by the maternal hormones progesterone and oestrogen and by the embryo in bovine
endometrium cells - a crucial process on implantation [FCT]
Zinc-finger nuclease-glycoengineered SimpleCell lines for characterization of the O-glycoproteome of cancer cells. [FCT]
Estudo da via AKT-mtor em carcinomas das glândulas salivares [Novartis Farma]
The role of sialylation in modulating galectin-3 functions in the metastatic process [FCT]
Conhecer a doença: Os doentes em primeiro lugar [FCG]
Dengue research framework for resisting epidemics in Europe [UE]
Development of siRNA-loaded nanoparticles to circumvent chemoresistance in cancer stem cells [FCT]
Dissection of the molecular role of O-GLcNAc in the multinucleation phenotype of the neoplasic cells in Hodgkin´s lymphoma
[FCT]
Urease de helicobacter pylori: propriedades não enzimáticas que potencialmente contribuem para gastrite e cancro gástrico
[FCT]
Mapeamento de genes de susceptibilidade para tumores familiares da tireóide [FCT]
Modulation of CDX2 expression in gastric intestinal metaplasia using na siRNA delivery system in vivo [SPG]
Linguistic and religious isolates in northeastern Portugal: new insights from Genetic and Demography [FCT]
Insulin Growth Factor and PI3K pathway ROLe in Luminal Breast Cancer survival [FCT]
Tumour spectrum in hereditary Diffuse Gastric Cancer [FCT]
Glycomic Approach to unravel the role of Glycostructures in Gastric Carcinogenesis [Institut Mérieux]
Unravelling a new Helicobacter pylori virulence factor involved in tight junction dysfunction [FCT]
Mucin MUC16 - CA125 cancer biomarker - biological functions and development of new biomarker assays [FCT]
Alternative oncogenic signalling pathways in medullary thyroid cancer [UIT]
Microenvironment, metabolism and cancer [ON]
Acções de Colaboração com instituições norte-americanas durante o ano de 2013 [FLAD]
PYLORICIDAL - Engineered biomaterials with Helicobacter PYLORI bacteriCIDAL effect [FCT]
Computational disease prediction systems based on molecular markers [FCT]
The Role of Protein Quality Control in the regulation of E-cadherin and its relevance in cancer [FCT]
Desenvolvimento de um novo método de detecção de mutações com relevância preditiva na resposta de doentes com carcinoma
colo rectal ao tratamento com cetuximab [ADI - Programa IDEIA]
New E-cadherin RNAs: the dark side of a tumour suppressor gene [FCT]
Functional, molecular and pharmacological studies of p53 family proteins: from yeast to human cells [FCT]
Adhesion, Differentiation and Invasion: different processes, common cadherin players [FCT]
Identification of genetic markers for prediction of the clinical course and development of complications in Portuguese Crohn's
Disease patients. [GEDII]
Glycosylation of the T cell receptor: a new mechanism underlying IBD pathogenesis Icuplications for therapy stratification [GEDII]
Initial Training Network - Systems Glycobiology of Gastric Cancer. [Seventh Framework Programme (FP7)]
Genetic history and population structure in Southeast Asia [FCT]
TUMORTAG - Tumor-targeted nanoparticles for early diagnosis of gastric cancer [FCT]
Portuguese study of familial dilated cardiomyopathies [FCT]
Using NGS to uncover structural and regulatory variation in Gastric Cancer [Coimbra Genomics S.A]
Search for genomic structural variants in azoopermia:a study in the Portuguese population [FCT]
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Programa de Acções Integradas Luso-Francesas PAULIF2011 [CRUP]
GRIM-19, a novel protein involved in cell apoptosis: structure-function characterization [FCT]
Study of mTOR pathway in GISTs [Novartis Farma]
L and U6 mtDNA lineages in Southern and Western Iberia: migratory links between Africa and Europe [CRUP]
Project support by FCG - Financial Fee [FCG]
CONtaminant-driven GENEtic ERosion: consequences on the viability of Amphibia populations [FCT]
Projecto financiado pelo Ciência Viva Escola Científica Ciência Viva [Agência Nacional Ciência Viva]
Molecular mechanisms underlying cancer cell invasion in two cancer disease models, gastric and breast [ON]
Glicosilação e cancro: da glicómia funcional à aplicação clínica [Convénio FCT/CAPES 2013/2014]
T cell receptor N-glycosylation as a new mechanism underlying Inflammatory Bowel Disease pathogenesis. Implications for
therapy. [FCT]
Wich is the role of both Cancer Stem Cells and Neural Stem Cells to the establishment of HER2+Breast Cancer Brain Metastases?
[Novartis Farma]
HERICA - Histological and Endoscopic Evaluation of Remission induced by Infliximab in moderately to severely active ulcerative
Colitis Patients [GEDII]
miRNAs como alvos moleculares em leucemia humana [FCG]
HeliSysBio - Molecular Systems Biology Helicobacter pylori [FCT]
Alterações metabólicas e cancro: GRIM-19 um gene do metabolismo celular e um gene supressor tumoral, que poderá ser um
target terapêutico interssante [Merck]
Auto Laboratório [ON]
Phylogeography and population structure of the powdery mildew fungus, Erysiphe necator, from diverse Vitis vinifera cultivars
grown in Portugal [Reitoria Universidade do Porto]
Helicobacter pylori diversity in pathogenesis, antibiotic resistance, and evasion from natural and vaccine-induced immune
responses (HELDIVPAT) [FCT]
Co-Evolutionary Study of Nad Biochemical Networks [FCT]
Bacterial protein azurin as a new candidate drug to trat poor-prognosis breast cancer [FCT]
The causes and consequences of mitochondrial DNA deletions in animals cells [FCT]
E-cadherin Post-Translational Modifications by N-glycosylation: a potencial new mechanism of E-cadherin deregulation in cancer
[FCT]
Effect of radio-and chemotherapy on cancer cell invasion: the role of macrophages [FCT]
Cooperação Portugal/França Programa Pessoa 2011-2012 [FCT]
Evaluating the role of proteolysis in the male reproductive system through the study of KLK (19q13.4) and WFDC (20q.13) gene
clusters [FCT]
Differentiation in cancer with emphasis on glycoproteome alterations [ON]
Cancro-Educar para prevenir [ACS]
A European Initial Training Network on the History, Archaeology, and New Genetics of the Trans-Atlantic Slave Trade [UE]
Glycosylation of cellular receptors in Cancer. [Industry]
97
2013
Scientific Papers
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Durães C, Machado JC, Portela F, Rodrigues S, Lago P, Cravo M, Ministro P, Marques M, Cremers I, Freitas J, Cotter J, Tavares
L, Matos L, Medeiros I, Sousa R, Ramos J, Deus J, Caldeira P, Chagas C, Duarte MA, Gonçalves R, Loureiro R, Barros L, Bastos I,
Cancela E, Moraes MC, Moreira MJ, Vieira AI, Magro F. Phenotype-Genotype Profiles in Crohn’s Disease Predicted by Genetic
Markers in Autophagy-Related Genes (GOIA Study II). Inflammatory bowel diseases; 19: 230-239. 2013. [Article] DOI: 10.1002/
ibd.23007 PMID: 22573572 IF=5,119
Pinto N, Magalhães M, Conde-Sousa E, Gomes C, Pereira R, Alves C, Gusmão L, Amorim A. Assessing paternities with inconclusive
STR results: The suitability of bi-allelic markers. Forensic science international. Genetics; 7: 16-21. 2013. [Article] DOI: 10.1016/j.
fsigen.2012.05.002 PMID: 22658886 IF=3,861
Di Lorito A, Rosini S, Falò E, Gustapane S, Gomes M, Costa JL, Schmitt FC. Molecular alterations in endometrial archived liquidbased cytology. Diagnostic cytopathology; 41: 492-6. 2013. [Article] DOI: 10.1002/dc.22869 PMID: 22807394 IF=1,487
Garziera M, De Re V, Geremia S, Seruca R, Figueiredo J, Melo S, Simões-Correia J, Caggiari L, De Zorzi M, Canzonieri V, Cannizzaro
R, Toffoli G. A novel CDH1 germline missense mutation in a sporadic gastric cancer patient in north-east of Italy. Clinical and
experimental medicine; 13: 149-57. 2013. [Article] DOI: 10.1007/s10238-012-0184-7 PMID: 22543498 IF=2,397
Figueiredo J, Söderberg O, Simões-Correia J, Grannas K, Suriano G, Seruca R. The importance of E-cadherin binding partners to
evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC. European journal of human genetics : EJHG;
21: 301-9. 2013. [Article] DOI: 10.1038/ejhg.2012.159 PMID: 22850631 IF=4,319
Fernandes MS, Carneiro F, Oliveira C, Seruca R. Colorectal cancer and RASSF family-A special emphasis on RASSF1A. International
journal of cancer. Journal international du cancer; 132: 251-8. 2013. [Review] DOI: 10.1002/ijc.27696 PMID: 22733432 IF=6,198
Celestino R, Lima J, Faustino A, Vinagre J, Máximo V, Gouveia A, Soares P, Lopes JM. Molecular alterations and expression of
succinate dehydrogenase complex in wild-type KIT/PDGFRA/BRAF gastrointestinal stromal tumors. European journal of human
genetics : EJHG; 21: 503-10. 2013. [Article] DOI: 10.1038/ejhg.2012.205 PMID: 22948025 IF=4,319
Beirão F, Araujo R. State of the art diagnostic of mold diseases: a practical guide for clinicians. European journal of clinical
microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology; 32: 3-9. 2013. [Review]
DOI: 10.1007/s10096-012-1722-7 PMID: 22903167 IF=3,024
González M, Gomes V, López-Parra AM, Amorim A, Carracedo A, Sánchez-Diz P, Arroyo-Pardo E, Gusmão L. The genetic landscape
of Equatorial Guinea and the origin and migration routes of the Y chromosome haplogroup R-V88. European journal of human
genetics : EJHG; 21: 324-31. 2013. [Article] DOI: 10.1038/ejhg.2012.167 PMID: 22892526 IF=4,319
Sousa E, Palmeira A, Cordeiro AS, Sarmento B, Ferreira D, Lima RT, Vasconcelos MH, Pinto M. Bioactive xanthones with effect
on P-glycoprotein and prediction of intestinal absorption. Medicinal Chemistry Research; 22: 2115-2123. 2013. [Article] DOI:
10.1007/s00044-012-0203-y PMID: 0 IF=1,612
Gil A, Castillo A, Rodríguez F, Amorim A, Gusmão L. Linkage between HPRTB STR alleles and Lesch-Nyhan syndrome inside
a family: Implications in forensic casework. Forensic science international. Genetics; 7: e5-6. 2013. [Letter] DOI: 10.1016/j.
fsigen.2012.09.005 PMID: 23063963 IF=3,861
Beça FF, Caetano P, Gerhard R, Alvarenga CA, Gomes M, Paredes J, Schmitt F. Cancer stem cells markers CD44, CD24 and
ALDH1 in breast cancer special histological types. Journal of clinical pathology; 66: 187-91. 2013. [Article] DOI: 10.1136/
jclinpath-2012-201169 PMID: 23112116 IF=2,439
Lima RT, Barron GA, Grabowska JA, Bermano G, Kaur S, Roy N, Helena Vasconcelos M, Lin PK. Cytotoxicity and Cell Death
Mechanisms Induced by a Novel Bisnaphthalimidopropyl Derivative against the NCI-H460 non-small Lung Cancer Cell Line. Anticancer agents in medicinal chemistry; 13: 414-21. 2013. [Article] PMID: 23092269 IF=2,61
Pereira-Castro I, Costa AM, Oliveira MJ, Barbosa I, Rocha AS, Azevedo L, da Costa LT. Characterization of human NLZ1/ZNF703
identifies conserved domains essential for proper subcellular localization and transcriptional repression. Journal of cellular
biochemistry; 114: 120-33. 2013. [Article] DOI: 10.1002/jcb.24309 PMID: 22886885 IF=3,062
Almeida LA, Araujo R. Highlights on molecular identification of closely related species. Infection, genetics and evolution :
journal of molecular epidemiology and evolutionary genetics in infectious diseases; 13: 67-75. 2013. [Review] DOI: 10.1016/j.
meegid.2012.08.011 PMID: 22982158 IF=2,768
Marinšek ZP, Nolde N, Kardum-Skelin I, Nizzoli R, Onal B, Rezanko T, Tani E, Ostovic KT, Vielh P, Schmitt F, Kocjan G. Multinational
study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates. Cytopathology
: official journal of the British Society for Clinical Cytology; 24: 7-20. 2013. [Article] DOI: 10.1111/cyt.12024 PMID: 23082931
IF=1,714
Vogelaar IP, Figueiredo J, van Rooij IA, Simões-Correia J, van der Post RS, Melo S, Seruca R, Carels CE, Ligtenberg MJ, Hoogerbrugge
N. Identification of germline mutations in the cancer predisposing gene CDH1 in patients with orofacial clefts. Human molecular
genetics; 22: 919-26. 2013. [Article] DOI: 10.1093/hmg/dds497 PMID: 23197654 IF=7,692
Ribeiro AS, Sousa B, Carreto L, Mendes N, Nobre AR, Ricardo S, Albergaria A, Cameselle-Teijeiro JF, Gerhard R, Söderberg O,
Seruca R, Santos MA, Schmitt F, Paredes J. P-cadherin functional role is dependent on E-cadherin cellular context: a proof of
concept using the breast cancer model. The Journal of pathology; 229: 705-18. 2013. [Article] DOI: 10.1002/path.4143 PMID:
23180380 IF=7,585
Manta FS, Pereira R, Caiafa A, Silva DA, Gusmão L, Carvalho EF. Analysis of genetic ancestry in the admixed Brazilian population
from Rio de Janeiro using 46 autosomal ancestry-informative indel markers. Annals of human biology; 40: 94-8. 2013. [Article]
DOI: 10.3109/03014460.2012.742138 PMID: 23151124 IF=1,484
Alvelos MI, Vinagre J, Fonseca E, Barbosa E, Teixeira-Gomes J, Sobrinho-Simões M, Soares P. MEN1 intragenic deletions may
represent the most prevalent somatic event in sporadic primary hyperparathyroidism. European journal of endocrinology /
European Federation of Endocrine Societies; 168: 119-28. 2013. [Article] DOI: 10.1530/EJE-12-0327 PMID: 23093699 IF=3,136
Nobre AR, Albergaria A, Schmitt F. p40: a p63 isoform useful for lung cancer diagnosis - a review of the physiological and
pathological role of p63. Acta cytologica; 57: 1-8. 2013. [Review] DOI: 10.1159/000345245 PMID: 23221041 IF=,693
Amaro HM, Barros R, Guedes AC, Sousa-Pinto I, Malcata FX. Microalgal compounds modulate carcinogenesis in the gastrointestinal
tract. Trends in Biotechnology; 31: 92-8. 2013. [Review] DOI: 10.1016/j.tibtech.2012.11.004 PMID: 23260440 IF=9,66
Pinto N, Gusmão L, Egeland T, Amorim A. Paternity exclusion power: comparative behaviour of autosomal and X-chromosomal
markers in standard and deficient cases with inbreeding. Forensic science international. Genetics; 7: 290-5. 2013. [Article] DOI:
10.1016/j.fsigen.2012.12.002 PMID: 23312390 IF=3,861
2013
24. van Krieken JH, Normanno N, Blackhall F, Boone E, Botti G, Carneiro F, Celik I, Ciardiello F, Cree IA, Deans ZC, Edsjö A, Groenen PJ,
Kamarainen O, Kreipe HH, Ligtenberg MJ, Marchetti A, Murray S, Opdam FJ, Patterson SD, Patton S, Pinto C, Rouleau E, Schuuring
E, Sterck S, Taron M, Tejpar S, Timens W, Thunnissen E, van de Ven PM, Siebers AG, Dequeker E. Guideline on the requirements
of external quality assessment programs in molecular pathology. Virchows Archiv : an international journal of pathology; 462:
27-37. 2013. [Article] DOI: 10.1007/s00428-012-1354-4 PMID: 23250354 IF=2,676
25. Albuquerque A, Cardoso H, Lopes J, Cipriano A, Carneiro F, Macedo G. Familial occurrence of nodular regenerative hyperplasia
of the liver. The American journal of gastroenterology; 108: 150-1. 2013. [Letter] DOI: 10.1038/ajg.2012.370 PMID: 23287953
IF=7,553
26. Ferreira Z, Seixas S, Andrés AM, Kretzschmar WW, Mullikin JC, Cherukuri PF, Cruz P, Swanson WJ, , Clark AG, Green ED, Hurle B.
Reproduction and Immunity-Driven Natural Selection in the Human WFDC Locus. Molecular biology and evolution; 30: 938-50.
2013. [Article] DOI: 10.1093/molbev/mss329 PMID: 23292442 IF=10,353
27. Costa AM, Leite M, Seruca R, Figueiredo C. Adherens junctions as targets of microorganisms: A focus on Helicobacter pylori. FEBS
letters; 587: 259-65. 2013. [Review] DOI: 10.1016/j.febslet.2012.12.008 PMID: 23262219 IF=3,582
28. Jaafar N, Moleirinho A, Kerkeni E, Monastiri K, Seboui H, Amorim A, Prata MJ, Quental S. Molecular characterization of maple
syrup urine disease patients from Tunisia. Gene; 517: 116-9. 2013. [Article] DOI: 10.1016/j.gene.2012.12.097 PMID: 23313820
IF=2,196
29. Gomes C, Almeida A, Ferreira JA, Silva L, Santos-Sousa H, Pinto-de-Sousa J, Santos LL, Amado F, Schwientek T, Levery SB, Mandel
U, Clausen H, David L, Reis CA, Osório H. Glycoproteomic analysis of serum from patients with gastric precancerous lesions.
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30. Corso G, Carvalho J, Marrelli D, Vindigni C, Carvalho B, Seruca R, Roviello F, Oliveira C. Somatic mutations and deletions of the
e-cadherin gene predict poor survival of patients with gastric cancer. Journal of clinical oncology : official journal of the American
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31. Moleirinho A, Seixas S, Lopes AM, Bento C, Prata MJ, Amorim A. Evolutionary Constraints in the ß-Globin Cluster: The Signature of
Purifying Selection at the d-Globin (HBD) Locus and Its Role in Developmental Gene Regulation. Genome biology and evolution;
5: 559-71. 2013. [Article] DOI: 10.1093/gbe/evt029 PMID: 23431002 IF=4,759
32. Malagolini N, Catera M, Osorio H, Reis CA, Chiricolo M, Dall’olio F. Apoptotic cells selectively uptake minor glycoforms of
vitronectin from serum. Apoptosis : an international journal on programmed cell death; 18: 373-84. 2013. [Article] DOI: 10.1007/
s10495-013-0812-z PMID: 23381642 IF=3,949
33. Pereira B, Sousa S, Barros R, Carreto L, Oliveira P, Oliveira C, Chartier NT, Plateroti M, Rouault JP, Freund JN, Billaud M, Almeida R.
CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness. Nucleic acids research;
41: 3986-99. 2013. [Article] DOI: 10.1093/nar/gkt087 PMID: 23408853 IF=8,278
34. Pereira F, Moreira C, Fonseca L, van Asch B, Mota M, Abrantes I, Amorim A. New Insights into the Phylogeny and Worldwide
Dispersion of Two Closely Related Nematode Species, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. PloS one;
8: e56288. 2013. [Article] DOI: 10.1371/journal.pone.0056288 PMID: 23409167 IF=3,73
35. Lima RT, Seca H, Palmeira A, Fernandes MX, Castro F, Correia-da-Silva M, Nascimento MS, Sousa E, Pinto M, Vasconcelos MH.
Sulfated small molecules targeting eBV in Burkitt lymphoma: from in silico screening to the evidence of in vitro effect on viral
episomal DNA. Chemical biology & drug design; 81: 631-44. 2013. [Article] DOI: 10.1111/cbdd.12109 PMID: 23350710 IF=2,469
36. Carracedo A, Butler JM, Gusmão L, Linacre A, Parson W, Roewer L, Schneider PM. New guidelines for the publication of genetic
population data. Forensic science international. Genetics; 7: 217-20. 2013. [Editorial Material] PMID: 23375886 IF=3,861
37. Monteiro-da-Silva F, Sampaio-Maia B, Pereira Mde L, Araujo R. Characterization of the oral fungal microbiota in smokers and
non-smokers. European journal of oral sciences; 121: 132-5. 2013. [Article] DOI: 10.1111/eos.12030 PMID: 23489903 IF=1,42
38. Gerhard R, Teixeira S, Gaspar da Rocha A, Schmitt F. Thyroid fine-needle aspiration cytology: Is there a place to virtual cytology?.
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39. Oliveira C, Seruca R, Hoogerbrugge N, Ligtenberg M, Carneiro F. Clinical utility gene card for: Hereditary diffuse gastric cancer
(HDGC). European journal of human genetics : EJHG; 21: .. 2013. [Editorial Material] DOI: 10.1038/ejhg.2012.247 PMID: 23443028
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40. Corso G, Roncalli F, Marrelli D, Carneiro F, Roviello F. History, Pathogenesis, and Management of Familial Gastric Cancer: Original
Study of John XXIII’s Family. BioMed Research International; 2013: 385132. 2013. [Article] DOI: 10.1155/2013/385132 PMID:
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41. Flor-de-Lima F, Gonçalves D, Marques R, Silva C, Lopes J, Silva R, Tavares M, Trindade E, Carneiro F, Amil-Dias J. Phlegmonous
Gastritis: A Rare Cause of Abdominal Pain. Journal of pediatric gastroenterology and nutrition; Epub ahead of print. 2013. [Epub
ahead of print] DOI: 10.1097/MPG.0b013e318291ff1f PMID: 23518491 IF=2,196
42. Oliveira C, Pinheiro H, Figueiredo J, Seruca R, Carneiro F. E-cadherin alterations in hereditary disorders with emphasis on
hereditary diffuse gastric cancer. Progress in molecular biology and translational science; 116: 337-59. 2013. [Review; Book
Chapter] DOI: 10.1016/B978-0-12-394311-8.00015-7 PMID: 23481202 IF=2,322
43. Cazal CM, Choosang K, Severino VG, Fernandes JB, da Silva MF, Vieira PC, Nascimento MS, Almeida GM, Vasconcelos MH, Pakkong
P, Pinto MM. Natural compounds with cell growth inhibitory activity in human tumor cell lines. Anti-cancer agents in medicinal
chemistry; 13: 1582-9. 2013. [Article] PMID: 23438826 IF=2,61
44. Almeida A, Albuquerque P, Araujo R, Ribeiro N, Tavares F. Detection and discrimination of common bovine mastitis-causing
streptococci. Veterinary microbiology; 164: 370-7. 2013. [Article] DOI: 10.1016/j.vetmic.2013.03.011 PMID: 23578710 IF=3,127
45. Correia A L, Mori H, Chen E I, Schmitt F. The hemopexin domain of MMP3 is responsible for mammary epithelial invasion and
morphogenesis through extracellular interaction with HSP90ß. Genes & Development; 805-817. 2013. [Article] PMID: 23592797
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46. Correia M, Michel V, Osório H, El Ghachi M, Bonis M, Boneca IG, De Reuse H, Matos AA, Lenormand P, Seruca R, Figueiredo C,
Machado JC, Touati E. Crosstalk between Helicobacter pylori and gastric epithelial cells is impaired by docosahexaenoic acid.
PloS one; 8: e60657. 2013. [Article] DOI: 10.1371/journal.pone.0060657 PMID: 23577140 IF=3,73
47. Albergaria A, Resende C, Nobre AR, Ribeiro AS, Sousa B, Machado JC, Seruca R, Paredes J, Schmitt F. CCAAT/enhancer binding
protein ß (C/EBPß) isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer cells.
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48. Soares P, Abrantes D, Rito T, Thomson N, Radivojac P, Li B, Macaulay V, Samuels DC, Pereira L. Evaluating purifying selection in
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49. Lopes AM, Aston KI, Thompson E, Carvalho F, Gonçalves J, Huang N, Matthiesen R, Noordam MJ, Quintela I, Ramu A, Seabra
C, Wilfert AB, Dai J, Downie JM, Fernandes S, Guo X, Sha J, Amorim A, Barros A, Carracedo A, Hu Z, Hurles ME, Moskovtsev S,
Ober C, Paduch DA, Schiffman JD, Schlegel PN, Sousa M, Carrell DT, Conrad DF. Human spermatogenic failure purges deleterious
mutation load from the autosomes and both sex chromosomes, including the gene DMRT1. PLoS genetics; 9: e1003349. 2013.
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50. Cerqueira L, Fernandes RM, Ferreira RM, Oleastro M, Carneiro F, Brandão C, Pimentel-Nunes P, Dinis-Ribeiro M, Figueiredo C,
Keevil CW, Vieira MJ, Azevedo NF. Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes
for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens. Journal of clinical microbiology; 51:
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51. Azevedo CM, Afonso CM, Sousa D, Lima RT, Helena Vasconcelos M, Pedro M, Barbosa J, Corrêa AG, Reis S, Pinto MM.
Multidimensional optimization of promising antitumor xanthone derivatives. Bioorganic & medicinal chemistry; 21: 2941-59.
2013. [Article] DOI: http://dx.doi.org/10.1016/j.bmc.2013.03.079 PMID: 23623253 IF=2,903
52. Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT, Lavrsen K, Dabelsteen S, Pedersen NB,
Marcos-Silva L, Gupta R, Bennett EP, Mandel U, Brunak S, Wandall HH, Levery SB, Clausen H. Precision mapping of the human
O-GalNAc glycoproteome through SimpleCell technology. The EMBO journal; 32: 1478-88. 2013. [Article] DOI: 10.1038/
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53. Amorim A, alves C. Genética: uma introdução à sua aplicação na investigação de parentescos. Testes Paternidade Ciência, ética
e sociedade; I: 13-24. 2013. [Chapters of Books] PMID: 0
54. Osório H, Reis CA. Mass spectrometry methods for studying glycosylation in cancer. Methods in molecular biology (Clifton, N.J.);
1007: 301-16. 2013. DOI: 10.1007/978-1-62703-392-3_13 PMID: 23666732
55. Squire JM, Guerreiro MJ, Sidebotham RL, Reis CA, Wiseman J, Luther PK. Quantitative MUC5AC and MUC6 mucin estimations
in gastric mucus by a least-squares minimization method. Analytical biochemistry; 439: 204-11. 2013. [Article] DOI: 10.1016/j.
ab.2013.04.013 PMID: 23643621 IF=2,582
56. Pinho SS, Figueiredo J, Cabral J, Carvalho S, Dourado J, Magalhães A, Gärtner F, Mendonfa AM, Isaji T, Gu J, Carneiro F,
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glycosyltransferases involving N-glycan branching biosynthesis, N-acetylglucosaminyltransferases III and V. Biochimica et
biophysica acta; 1830: 2690-700. 2013. PMID: 23671930 IF=3,848
57. Ferreira Z, Hurle B, Andrés AM, Kretzschmar WW, Mullikin JC, Cherukuri PF, Cruz P, Gonder MK, Stone AC, Tishkoff S, Swanson WJ,
, Green ED, Clark AG, Seixas S. Sequence Diversity of Pan troglodytes Subspecies and the Impact of WFDC6 Selective Constraints
in Reproductive Immunity. Genome biology and evolution; 5: 2512-23. 2013. [Article] DOI: 10.1093/gbe/evt198 PMID: 24356879
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58. Gomes J, Reis CA. Immunodetection of glycosyltransferases in gastrointestinal tissues. Methods in molecular biology (Clifton,
N.J.); 1022: 349-56. 2013. DOI: 10.1007/978-1-62703-465-4_26 PMID: 23765674
59. Lima Raquel T., Nascimento M . S. José, Vasconcelos M. Helena. EBV-associated cancers: Strategies for targeting the virus.
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60. Carneiro F, Oliveira C, Seruca R. Hereditary diffuse gastric cancer and other gastric cancers associated with hereditary
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61. Carneiro F, Lauwers G. Epithelial tumours of the stomach. Morson and Dawson’s: Gastrointestinal Pathology, Fifth Edition.
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62. Carneiro F. Pathology of Hereditary Gastric Cancer. Spotlight on Familial and Hereditary Gastric Cancer. Corso G and Roviello F
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63. Simões-Correia J, Silva DI, Melo S, Figueiredo J, Caldeira J, Pinto MT, Girão H, Pereira P, Seruca R. DNAJB4 molecular chaperone
distinguishes WT from mutant E-cadherin, determining their fate in vitro and in vivo. Human molecular genetics; Epub ahead of
print. 2013. [Epub ahead of print] DOI: 10.1093/hmg/ddt602 PMID: 24293545 IF=7,692
64. Machado AM, Desler C, Bøggild S, Strickertsson JA, Friis-Hansen L, Figueiredo C, Seruca R, Rasmussen LJ. Helicobacter pylori
infection affects mitochondrial function and DNA repair, thus, mediating genetic instability in gastric cells. Mechanisms of ageing
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65. Carneiro P, Figueiredo J, Bordeira-Carriço R, Fernandes MS, Carvalho J, Oliveira C, Seruca R. Therapeutic targets associated
to E-cadherin dysfunction in gastric cancer. Expert opinion on therapeutic targets; 17: 1187-201. 2013. [Review] DOI:
10.1517/14728222.2013.827174 PMID: 23957294 IF=4,13
66. Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos RG, Arraiano CM, Seruca R, Paredes J, Fialho AM. The bacterial protein azurin
impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models. PloS one; 8: e69023. 2013.
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67. van der Werf CS, Hsiao NH, Conroy S, Paredes J, Ribeiro AS, Sribudiani Y, Seruca R, Hofstra RM, Westers H, van Ijzendoorn SC.
CLMP is essential for intestinal development, but does not play a key role in cellular processes involved in intestinal epithelial
development. PloS one; 8: e54649. 2013. [Article] DOI: 10.1371/journal.pone.0054649 PMID: 23460781 IF=3,73
68. Portela A, Vasconcelos M, Fernandes MH, Garcia M, Silva A, Gabriel J, Gartner F, Amorim I, Cavalheiro J. Highly focalised
thermotherapy using a ferrimagnetic cement in the treatment of a melanoma mouse model by low temperature hyperthermia.
International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American
Hyperthermia Group; 29: 121-32. 2013. [Article] DOI: 10.3109/02656736.2013.767478 PMID: 23418916 IF=2,591
69. Santos AA, Lopes CC, Ribeiro JR, Martins LR, Santos JC, Amorim IF, Gärtner F, Matos AJ. Identification of prognostic factors
in canine mammary malignant tumours: a multivariable survival study. BMC veterinary research; 9: 1. 2013. [Article] DOI:
10.1186/1746-6148-9-1 PMID: 23289974 IF=1,861
70. Taulescu MA, Amorim I, Gärtner F, Farcas L, Mircean M, Catoi C. Gastric Smooth Muscle Hamartomas Mimicking Polyps in a Dog:
A Case Description and a Review of the Literature. Case Reports in Veterinary Medicine; .. 2013. PMID: 0
71. Moniz S, Martinho O, Pinto F, Sousa B, Loureiro C, Oliveira MJ, Moita LF, Honavar M, Pinheiro C, Pires M, Lopes JM, Jones
C, Costello JF, Paredes J, Reis RM, Jordan P. Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas
and triggers Rac1-mediated tumour cell invasiveness. Human molecular genetics; 22: 84-95. 2013. [Article] DOI: 10.1093/hmg/
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72. Costa JL, Sousa S, Justino A, Kay T, Fernandes S, Cirnes L, Schmitt F, Machado JC. Nonoptical massive parallel DNA sequencing of
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73. Fontenete S, Guimarães N, Leite M, Figueiredo C, Wengel J, Filipe Azevedo N. Hybridization-Based Detection of Helicobacter
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74. Figueiredo C, Garcia-Gonzalez MA, Machado JC. Molecular pathogenesis of gastric cancer. Helicobacter; 18 Suppl 1: 28-33. 2013.
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75. Correia M, Casal S, Vinagre J, Seruca R, Figueiredo C, Touati E, Machado JC. Helicobacter pylori’s cholesterol uptake impacts
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77. Marques M, Cardoso H, Lopes J, Carneiro F, Macedo G. Colangiocarcinoma em doente com hemocromatose hereditária. Jornal
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78. Lima L, Severino PF, Silva M, Miranda A, Tavares A, Pereira S, Fernandes E, Cruz R, Amaro T, Reis CA, Dall’olio F, Amado F, Videira
PA, Santos L, Ferreira JA. Response of high-risk of recurrence/progression bladder tumours expressing sialyl-Tn and sialyl-6-T to
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79. Costa MD, Pereira JB, Pala M, Fernandes V, Olivieri A, Achilli A, Perego UA, Rychkov S, Naumova O, Hatina J, Woodward SR,
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maternal lineages. Nature communications; 4: 2543. 2013. [Article] DOI: 10.1038/ncomms3543 PMID: 24104924 IF=10,015
80. Pereira F, Amorim A, van Asch B. Genetic and DNA-Based Techniques. Elsevier Science; 195-220. 2013. [Chapters of Books] DOI:
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81. Almeida A L, Boaventura M P, Soares P. Etiopathogenic factors of thyroid cancer. Clinical Management of Thyroid Cancer; 46-62.
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82. Saloum de Neves Manta F, Pereira R, Vianna R, Rodolfo Beuttenmüller de Araújo A, Leite Góes Gitaí D, Aparecida da Silva D, de
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83. Mellars P, Gori KC, Carr M, Soares PA, Richards MB. Genetic and archaeological perspectives on the initial modern human
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84. Santos T, Oliveira M, Sousa D, Lima R T, Martins A, Ferreira I, Vasconcelos M H. Suillus luteus methanolic extract inhibits
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85. Melo M, Costa G, Ribeiro C, Carrilho F, Martins MJ, da Rocha AG, Sobrinho-Simões M, Carvalheiro M, Soares P. Stimulated
Thyroglobulin at Recombinant Human TSH-Aided Ablation Predicts Disease-free Status One Year Later. The Journal of clinical
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86. Damas J, Carneiro J, Amorim A, Pereira F. MitoBreak: the mitochondrial DNA breakpoints database. Nucleic acids research; 1.
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87. Noguera MC, Schwegler A, Gomes V, Briceño I, Alvarez L, Uricoechea D, Amorim A, Benavides E, Silvera C, Charris M, Bernal
JE, Gusmão L. Colombia’s racial crucible: Y chromosome evidence from six admixed communities in the Department of Bolivar.
Annals of human biology; Epub ahead of print. 2013. [Epub ahead of print] DOI: 10.3109/03014460.2013.852244 PMID: 24215508
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88. van Asch B, Zhang AB, Oskarsson MC, Klütsch CF, Amorim A, Savolainen P. Pre-Columbian origins of Native American dog breeds,
with only limited replacement by European dogs, confirmed by mtDNA analysis. Proceedings of The Royal Society B-Biological
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89. Mairal Q, Santos C, Silva M, Marques SL, Ramos A, Aluja MP, Amorim A, Prata MJ, Alvarez L. Linguistic isolates in Portugal:
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fsigen.2013.08.009 PMID: 24041913 IF=3,861
90. Rito T, Richards MB, Fernandes V, Alshamali F, Cerny V, Pereira L, Soares P. The First Modern Human Dispersals across Africa. PloS
one; 8: e80031. 2013. [Article] DOI: 10.1371/journal.pone.0080031 PMID: 24236171 IF=3,73
91. Amorim A. Produção e interpretação da prova genética. A Ciência na Luta contra o crime; 1. 2013. [Chapters of Books] DOI: 3
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92. Pereira F, Amorim A. Evolution: Viruses. Brenner’s Encyclopedia of Genetics; 407-411. 2013. [Chapters of Books] DOI: 4 PMID: 0
93. Amorim A. Population Genetics. Brenner’s Encyclopedia of Genetics; 407-411. 2013. [Chapters of Books] DOI: 1 PMID: 0
94. Campanella NC, Berardinelli GN, Scapulatempo-Neto C, Viana D, Palmero EI, Pereira R, Reis RM. Optimization of a pentaplex
panel for MSI analysis without control DNA in a Brazilian population: correlation with ancestry markers. European journal of
human genetics : EJHG; Epub ahead of print. 2013. [Epub ahead of print] DOI: 10.1038/ejhg.2013.256 PMID: 24193342 IF=4,319
95. Martins RG, Nunes JB, Máximo V, Soares P, Peixoto J, Catarino T, Rito T, Soares P, Pereira L, Sobrinho-Simões M, Santos AP,
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96. Podgorna E, Soares P, Pereira L, Cerny V. The Genetic Impact of the Lake Chad Basin Population in North Africa as Documented by
Mitochondrial Diversity and Internal Variation of the L3e5 Haplogroup. Annals of human genetics; 513-523. 2013. DOI: 10.1111/
ahg.12040 PMID: 0 IF=2,215
97. Caramalho R, Gusmão L, Lackner M, Amorim A, Araujo R. SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for
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98. Ramos A, Santos C, Mateiu L, Gonzalez Mdel M, Alvarez L, Azevedo L, Amorim A, Aluja MP. Frequency and pattern of heteroplasmy
in the complete human mitochondrial genome. PloS one; 8: e74636. 2013. [Article] DOI: 10.1371/journal.pone.0074636 PMID:
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99. de-Freitas-Junior JC, Carvalho S, Dias AM, Oliveira P, Cabral J, Seruca R, Oliveira C, Morgado-Díaz JA, Reis CA, Pinho SS. Insulin/
IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with
E-Cadherin. PloS one; 8: e81579. 2013. [Article] DOI: 10.1371/journal.pone.0081579 PMID: 24282611 IF=3,73
100. Roewer L, Nothnagel M, Gusmão L, Gomes V, González M, Corach D, Sala A, Alechine E, Palha T, Santos N, Ribeiro-Dos-Santos A,
Geppert M, Willuweit S, Nagy M, Zweynert S, Baeta M, Núñez C, Martínez-Jarreta B, González-Andrade F, Fagundes de Carvalho
E, da Silva DA, Builes JJ, Turbón D, Lopez Parra AM, Arroyo-Pardo E, Toscanini U, Borjas L, Barletta C, Ewart E, Santos S, Krawczak
M. Continent-wide decoupling of Y-chromosomal genetic variation from language and geography in native South Americans.
PLoS genetics; 9: e1003460. 2013. [Article] DOI: 10.1371/journal.pgen.1003460 PMID: 23593040 IF=8,517
101. Ferreira JA, Videira PA, Lima L, Pereira S, Silva M, Carrascal M, Severino PF, Fernandes E, Almeida A, Costa C, Vitorino R, Amaro
T, Oliveira MJ, Reis CA, Dall’Olio F, Amado F, Santos LL. Overexpression of tumour-associated carbohydrate antigen sialyl-Tn in
advanced bladder tumours. Molecular oncology; 7: 719-31. 2013. [Article] DOI: 10.1016/j.molonc.2013.03.001 PMID: 23567325
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102. Gerhard R, Carvalho A, Carneiro V, Bento RS, Uemura G, Gomes M, Albergaria A, Schmitt F. Clinicopathological significance of
ERCC1 expression in breast cancer. Pathology, research and practice; 209: 331-6. 2013. [Article] DOI: 10.1016/j.prp.2013.02.009
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103. Carneiro J, Duarte-Pereira S, Azevedo L, Castro LF, Aguiar P, Moreira IS, Amorim A, Silva RM. The Evolutionary Portrait of
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104. Burford B, Gentry-Maharaj A, Graham R, Allen D, Pedersen JW, Nudelman AS, Blixt O, Fourkala EO, Bueti D, Dawnay A, Ford
J, Desai R, David L, Trinder P, Acres B, Schwientek T, Gammerman A, Reis CA, Silva L, Osório H, Hallett R, Wandall HH, Mandel
U, Hollingsworth MA, Jacobs I, Fentiman I, Clausen H, Taylor-Papadimitriou J, Menon U, Burchell JM. Autoantibodies to MUC1
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106. Eusebio N, Pinheiro T, Amorim AA, Gamboa F, Saraiva L, Gusmão L, Amorim A, Araujo R. SNaPaer: a practical single nucleotide
polymorphism multiplex assay for genotyping of Pseudomonas aeruginosa. PloS one; 8: e66083. 2013. [Article] DOI: 10.1371/
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107. Marques PI, Ferreira Z, Martins M, Figueiredo J, Silva DI, Castro P, Morales-Hojas R, Simões-Correia J, Seixas S. SERPINA2 Is a
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108. Santos T, Tavares C, Sousa D, Vaz J, Calhelha R C, Martins A, Almeida G M, Ferreira I, Vasconcelos M H. Suillus luteus methanolic
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110. Moreira C, van Asch B, Fonseca L, Pereira-Castro I, Silva R, Azevedo L, Mota M, Abrantes I, Amorim A, Pereira F. The mitochondrial
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112. Seca H, Lima RT, Lopes-Rodrigues V, Guimaraes JE, Almeida GM, Vasconcelos MH. Targeting miR-21 Induces Autophagy and
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113. Rodriguez-Paredes M, Martinez de Paz A, Simó-Riudalbas L, Sayols S, Moutinho C, Moran S, Villanueva A, Vázquez-Cedeira
M, Lazo PA, Carneiro F, Moura CS, Vieira J, Teixeira MR, Esteller M. Gene amplification of the histone methyltransferase
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114. Vinagre J, Almeida A, Pópulo H, Batista R, Lyra J, Pinto V, Coelho R, Celestino R, Prazeres H, Lima L, Melo M, Rocha AG, Preto A,
Castro P, Castro L, Pardal F, Lopes JM, Santos LL, Reis RM, Cameselle-Teijeiro J, Sobrinho-Simões M, Lima J, Máximo V, Soares
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115. Ibarra A, Martinez M, Freire-Aradas A, Fondevila M, Carracedo A, Porras L, Gusmão L. Using STR, MiniSTR and SNP markers to
solve complex cases of kinship analysis Forensic Science International. Forensic science international. Genetics; 4: e91-e92. 2013.
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116. Ribeiro-Dos-Santos AM, de Souza JE, Almeida R, Alencar DO, Barbosa MS, Gusmão L, Silva WA, de Souza SJ, Silva A, Ribeiro-DosSantos A, Darnet S, Santos S. High-throughput sequencing of a South american amerindian. PloS one; 8: e83340. 2013. [Article]
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117. Carneiro F. Promoting Pathology. Pan European Networks: Science & Technology; .. 2013. PMID: 0
118. Birolo L, Amorim A, Alves C, Mesquita AGG, Gusmão L. Analysis of 15 autosomal STR loci in the population of the State of Acre,
Brazilian Amazonia. Forensic science international. Genetics; 4: e11-e12. 2013. PMID: 0 IF=3,861
119. Pereira V, Tomas C, Pietroni C, Andersen JD, Fordyce SL, Pinto N, Mikkelsen M, Borsting C, Amorim A, Gusmão L, Prata MJ,
Morling N. Assessing the potential application of X-chromosomal haploblocks in population genetics and forensic studies.
Forensic science international. Genetics; 4: e9-e10. 2013. PMID: 0 IF=3,861
120. Pinto N, Gusmão L, Egeland T, Amorim A. Estimating relatedness with no prior specification of any genealogy: The role of the
X-chromosome. Forensic science international. Genetics; 4: e252-e253. 2013. PMID: 0 IF=3,861
121. Martínez B, Builes JJ, Gaviria A, Burgos G, Manrique A, Aguirre DP, Mendonza L, Bravo MLI, Pereira R, Gusmão L, Marrugo J.
Population genetic data of 38 autosomal InDels in San Basilio de Palenque, the first free town in America. Forensic science
international. Genetics; 4: e73-e74. 2013. DOI: 10.1016/j.fsigss.2013.10.037 PMID: 0 IF=3,861
122. Builes JJ, Castro JF, Velilla CM, Manrique A, Aguirre DP, Mendoza L, Bravo MLI, Gusmão L. Results of Colombian exercise
interlaboratory quality control 2012. Forensic science international. Genetics; 4: e158-e159. 2013. DOI: 10.1016/j.
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123. Builes JJ, Ospino JM, Manrique A, Aguirre DP, Mendoza L, Bravo MLI, Pereira R, Gusmão L. Genetic population data of 38
autosomal InDels for the Amerindian community Embera-Chami of Lapo, Antioquia-Colombia. Forensic science international.
Genetics; 4: e170-e171. 2013. DOI: 10.1016/j.fsigss.2013.10.088 PMID: 0 IF=3,861
124. Barletta C, Builles JJ, Bravo MLI, Aguirre D, Descailleaux J, Gusmão L. Genetic data of 10 X-STRs in a population sample from Lima,
Perú. Forensic science international. Genetics; 4: e168-e169. 2013. DOI: 10.1016/j.fsigss.2013.10.087 PMID: 0 IF=3,861
125. Alves JM, Lopes AM. Population Genomics: Macro vs Microevolution. Brenner’s Encyclopedia of Genetics; 412-414. 2013. PMID:
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126. Melo M, Costa G, Carrilho F, Soares P. Radiotherapy: radioiodine and external beam irradiation treatment of differentiated
thyroid carcinomas. Clinical Management of Thyroid Cancer; 78-92. 2013. [Chapters of Books] PMID: 0
127. Ziros PG, Manolakou SD, Habeos IG, Lilis I, Chartoumpekis DV, Koika V, Soares P, Kyriazopoulou VE, Scopa CD, Papachristou DJ,
Sykiotis GP. Nrf2 is commonly activated in papillary thyroid carcinoma, and it controls antioxidant transcriptional responses and
viability of cancer cells. The Journal of clinical endocrinology and metabolism; 98: E1422-7. 2013. [Article] DOI: 10.1210/jc.20131510 PMID: 23766517 IF=6,43
128. Nakazawa T, Celestino R, Machado JC, Cameselle-Teijeiro JM, Vinagre J, Eloy C, Benserai F, Lameche S, Soares P, Sobrinho-Simões
M. Cribriform-morular variant of papillary thyroid carcinoma displaying poorly differentiated features. International journal of
surgical pathology; 21: 379-89. 2013. [Article] DOI: 10.1177/1066896912473355 PMID: 23349472 IF=,756
129. Eloy C, Cameselle-Teijeiro JM, Rousseau E, Sobrinho-Simões M. Rare Flowers in the Thyroid Gland: The Case of Small Cell Tumors.
International journal of surgical pathology; Epub ahead of print. 2013. [Epub ahead of print] DOI: 10.1177/1066896913510029
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130. Barroca H, Souto Moura C, Lopes JM, Lisboa S, Teixeira MR, Damasceno M, Bastos P, Sobrinho Simões M. Ewing’s Sarcoma/
Primitive Neuroectodermal Tumor With Neuroendocrine Differentiation: Report of an Unusual Lung Tumor. International journal
of surgical pathology; Epub ahead of print. 2013. [Epub ahead of print] DOI: 10.1177/1066896913502227 PMID: 24141023 IF=,756
131. Bialas M, Okon K, Dyduch G, Ciesielska-Milian K, Buziak M, Hubalewska-Dydejczyk A, Sobrinho-Simoes M. Neuroendocrine
markers and sustentacular cell count in benign and malignant pheochromocytomas - a comparative study. Polish journal of
pathology : official journal of the Polish Society of Pathologists; 64: 129-35. 2013. [Article] PMID: 23900871 IF=,49
132. Carrilho C, Gouveia P, Yokohama H, Lopes JM, Lunet N, Ferro J, Ismail M, Walboomers J, Sobrinho-Simões M, David L. Human
papillomaviruses in intraepithelial neoplasia and squamous cell carcinoma of the conjunctiva: a study from Mozambique.
European journal of cancer prevention : the official journal of the European Cancer Prevention Organisation (ECP); 22: 566-8.
2013. [Article] DOI: 10.1097/CEJ.0b013e328363005d PMID: 23752127 IF=2,974
133. Boos LA, Dettmer M, Schmitt A, Rudolph T, Steinert H, Moch H, Sobrinho-Simões M, Komminoth P, Perren A. Diagnostic and
prognostic implications of the PAX8-PPAR¿ translocation in thyroid carcinomas-a TMA-based study of 226 cases. Histopathology;
63: 234-41. 2013. [Article] DOI: 10.1111/his.12150 PMID: 23738683 IF=2,857
134. Vilallonga R, Zafon C, Ruiz-Marcellan C, Obiols G, Fort JM, Baena JA, Villanueva B, Garcia A, Sobrinho-Simões M. Malignant
thyroid teratoma: report of an aggressive tumor in a 64-year-old man. Endocrine pathology; 24: 132-5. 2013. [Article] DOI:
10.1007/s12022-013-9250-2 PMID: 23702575 IF=1,6
135. Eloy C, Cruz J, Vieira J, Teixeira MR, Cameselle-Teijeiro J, Sobrinho-Simões M. Carcinoma of the Thyroid With Ewing Family Tumor
Elements: A Tumor With Unknown Histogenesis. International journal of surgical pathology; Epub ahead of print. 2013. [Epub
ahead of print] DOI: 10.1177/1066896913486697 PMID: 23637257 IF=,756
136. Eloy C, Oliveira M, Vieira J, Teixeira MR, Cruz J, Sobrinho-Simões M. Carcinoma of the Thyroid With Ewing Family Tumor Elements
and Favorable Prognosis: Report of a Second Case. International journal of surgical pathology; Epub ahead of print. 2013. [Epub
ahead of print] DOI: 10.1177/1066896913486696 PMID: 23637256 IF=,756
137. Sigstad E, Paus E, Bjøro T, Berner A, Grøholt KK, Jørgensen LH, Sobrinho-Simões M, Holm R, Warren DJ. Reply to ‘The new
molecular markers DDIT3, STT3A, ARG2 and FAM129A are not useful in diagnosing thyroid follicular tumors’. Modern pathology
: an official journal of the United States and Canadian Academy of Pathology, Inc; 26: 613-5. 2013. [Letter] DOI: 10.1038/
modpathol.2013.39 PMID: 23542526 IF=5,253
138. Prazeres H, Salgado C, Duarte C, Soares P. LRP1B (low density lipoprotein receptor-related protein 1B). Atlas Genet Cytogenet
Oncol Haematol; .. 2013. PMID: 0
139. Zaumsegel D, Pereira R, Rothschil MA, Phillips C, Gusmão L, Schneider PM, EUROFORGEN-NoE Consortium. Comparative Analysis
of Two Indel-based Ancestry Informative Multiplex PCR Typing Kits. Forensic science international. Genetics; 4: e21-e22. 2013.
DOI: 10.1016/j.fsigss.2013.10.010 PMID: 0 IF=3,861
140. Campos A, Araújo P, Pinheiro C, Azevedo J, Osório H, Vasconcelos V. Effects on growth, antioxidant enzyme activity and levels
of extracellular proteins in the green alga Chlorella vulgaris exposed to crude cyanobacterial extracts and pure microcystin
and cylindrospermopsin. Ecotoxicology and environmental safety; 94: 45-53. 2013. [Article] DOI: 10.1016/j.ecoenv.2013.04.019
PMID: 23726538 IF=2,203
141. Almeida A, Ferreira JA, Teixeira F, Gomes C, Cordeiro MN, Osório H, Santos LL, Reis CA, Vitorino R, Amado F. Challenging the limits
of detection of sialylated Thomsen-Friedenreich antigens by in-gel deglycosylation and nano-LC-MALDI-TOF-MS. Electrophoresis;
34: 2337-41. 2013. [Article] DOI: 10.1002/elps.201300148 PMID: 23775548 IF=3,261
142. Neves J, Campos A, Osório H, Antunes A, Vasconcelos V. Conopeptides from Cape Verde Conus crotchii. Marine drugs; 11: 220315. 2013. [Article] DOI: 10.3390/md11062203 PMID: 23783403 IF=3,978
143. Gomes C, Osório H, Pinto MT, Campos D, Oliveira MJ, Reis CA. Expression of ST3GAL4 leads to SLe(x) expression and induces
c-Met activation and an invasive phenotype in gastric carcinoma cells. PloS one; 8: e66737. 2013. [Article] DOI: 10.1371/journal.
pone.0066737 PMID: 23799130 IF=3,73
144. Marcos-Pinto R, Dinis-Ribeiro M, Carneiro F, Wen X, Lopes C, Figueiredo C, Machado JC, Ferreira RM, Reis CA, Canedo P, Durães
C, Ferreira J, Pedroto I, Areias J. First-degree relatives of early-onset gastric cancer patients show a high risk for gastric cancer:
phenotype and genotype profile. Virchows Archiv : an international journal of pathology; 463: 391-399. 2013. [Article] DOI:
10.1007/s00428-013-1458-5 PMID: 23887584 IF=2,676
145. Boaventura P, Pereira D, Celestino R, Mendes A, Nakasawa T, Teixeira-Gomes J, Sobrinho-Simões M, Soares P. Genetic alterations
in thyroid tumors from patients irradiated in childhood for tinea capitis treatment. European journal of endocrinology / European
Federation of Endocrine Societies; 169: 673-679. 2013. [Article] DOI: 10.1530/EJE-13-0543 PMID: 23966419 IF=3,136
146. Nogueira C, Barros J, Sá MJ, Azevedo L, Taipa R, Torraco A, Meschini MC, Verrigni D, Nesti C, Rizza T, Teixeira J, Carrozzo R,
Pires MM, Vilarinho L, Santorelli FM. Novel TTC19 mutation in a family with severe psychiatric manifestations and complex III
deficiency. Neurogenetics; 14: 153-60. 2013. [Article] DOI: 10.1007/s10048-013-0361-1 PMID: 23532514 IF=3,575
103
2013
147. Ferreira F, Esteves S, Almeida LS, Gaspar A, da Costa CD, Janeiro P, Bandeira A, Martins E, Teles EL, Garcia P, Azevedo L, Vilarinho
L. Trimethylaminuria (fish odor syndrome): Genotype characterization among Portuguese patients. Gene; 527: 366-70. 2013.
[Article] DOI: 10.1016/j.gene.2013.05.025 PMID: 23791655 IF=2,196
148. Pópulo H, Tavares S, Faustino A, Nunes JB, Lopes JM, Soares P. GNAQ and BRAF mutations show differential activation of the
mTOR pathway in human transformed cells. PeerJ; 1: e104. 2013. DOI: 10.7717/peerj.104 PMID: 23904987
149. Coelho SC, Rocha S, Juzenas P, Sampaio P, Almeida GM, Silva FS, Pereira MC, Coelho MA. Gold nanoparticle delivery-enhanced
proteasome inhibitor effect in adenocarcinoma cells. Expert opinion on drug delivery; 10: 1345-52. 2013. [Article] DOI:
10.1517/17425247.2013.827659 PMID: 23937147 IF=4,869
150. Martins D, Beça FF, Sousa B, Baltazar F, Paredes J, Schmitt F. Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma:
Key events in the progression from an in situ to an invasive breast carcinoma. Cell cycle (Georgetown, Tex.); 12: 2684-90. 2013.
[Article] DOI: 10.4161/cc.25794 PMID: 23907124 IF=5,243
151. Eusebio N, Coutinho CP, Sá-Correia I, Araujo R. SNaPBcen: a Novel and Practical Tool for Genotyping Burkholderia cenocepacia.
Journal of clinical microbiology; 51: 2646-53. 2013. [Article] DOI: 10.1128/JCM.01019-13 PMID: 23761147 IF=4,068
152. de Beça FF, Rasteiro C, Correia A, Costa S, Amendoeira I. Improved malignancy prediction by B3 breast lesions subclassification.
Annals of diagnostic pathology; 17: 434-6. 2013. [Article] DOI: 10.1016/j.anndiagpath.2013.05.003 PMID: 23773891 IF=,975
153. Cardoso AP, Pinto ML, Pinto AT, Oliveira MI, Pinto MT, Gonçalves R, Relvas JB, Figueiredo C, Seruca R, Mantovani A, Mareel M,
Barbosa MA, Oliveira MJ. Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2
and Akt phosphorylation and smallGTPase activity. Oncogene; Epub ahead of print. 2013. [Epub ahead of print] DOI: 10.1038/
onc.2013.154 PMID: 23644655 IF=7,357
154. Dabbs DJ, Schnitt SJ, Geyer FC, Weigelt B, Baehner FL, Decker T, Eusebi V, Fox SB, Ichihara S, Lakhani SR, Palacios J, Rakha E,
Richardson AL, Schmitt FC, Tan PH, Tse GM, Vincent-Salomon A, Ellis IO, Badve S, Reis-Filho JS. Lobular neoplasia of the breast
revisited with emphasis on the role of E-cadherin immunohistochemistry. The American journal of surgical pathology; 37: e1-11.
2013. [Article] DOI: 10.1097/PAS.0b013e3182918a2b PMID: 23759937 IF=4,868
155. Parreira P, Magalhães A, Reis CA, Borén T, Leckband D, Martins MC. Bioengineered surfaces promote specific protein-glycan
mediated binding of the gastric pathogen Helicobacter pylori. Acta biomaterialia; 9: 8885-8893. 2013. [Article] DOI: 10.1016/j.
actbio.2013.06.042 PMID: 23831721 IF=5,093
156. Gonçalves IC, Magalhães A, Fernandes M, Rodrigues IV, Reis CA, Martins MC. Bacterial-binding chitosan microspheres for gastric
infection treatment and prevention. Acta biomaterialia; 9: 9370-8. 2013. [Article] DOI: 10.1016/j.actbio.2013.07.034 PMID:
23920152 IF=5,093
157. de Beça FF, Lopes J, Maçoas F, Carneiro F, Lopes JM. Tactoid Body Features in Colon Mucosal Schwann Cell Hamartoma.
International journal of surgical pathology; Epub ahead of print. 2013. [Epub ahead of print] DOI: 10.1177/1066896913501384
PMID: 23994879 IF=,756
158. Pinho SS, Carvalho S, Marcos-Pinto R, Magalhães A, Oliveira C, Gu J, Dinis-Ribeiro M, Carneiro F, Seruca R, Reis CA. Gastric
cancer: adding glycosylation to the equation. Trends in molecular medicine; 19: 664-76. 2013. [Review] DOI: 10.1016/j.
molmed.2013.07.003 PMID: 23932995 IF=9,571
159. Pereira B, Le Borgne M, Chartier NT, Billaud M, Almeida R. MEX-3 proteins: recent insights on novel post-transcriptional
regulators. Trends in biochemical sciences; 38: 477-9. 2013. [Editorial Material] DOI: 10.1016/j.tibs.2013.08.004 PMID: 23999169
IF=13,076
160. Lopes-Rodrigues V, Seca H, Sousa D, Sousa E, Lima RT, Vasconcelos MH. The network of P-glycoprotein and microRNAs
interactions. International journal of cancer. Journal international du cancer; Epub ahead of print. 2013. [Epub ahead of print]
DOI: 10.1002/ijc.28500 PMID: 24122334 IF=6,198
161. Queiroz MJ, Peixoto D, Calhelha RC, Soares P, Dos Santos T, Lima RT, Campos JF, Abreu RM, Ferreira IC, Vasconcelos MH. New
di(hetero)arylethers and di(hetero)arylamines in the thieno[3,2-b]pyridine series: Synthesis, growth inhibitory activity on
human tumor cell lines and non-tumor cells, effects on cell cycle and on programmed cell death. European journal of medicinal
chemistry; 69: 855-62. 2013. DOI: 10.1016/j.ejmech.2013.09.023 PMID: 24121236 IF=3,499
162. Azevedo CM, Afonso CM, Soares JX, Reis S, Sousa D, Lima RT, Vasconcelos MH, Pedro M, Barbosa J, Gales L, Pinto MM.
Pyranoxanthones: Synthesis, growth inhibitory activity on human tumor cell lines and determination of their lipophilicity in two
membrane models. European journal of medicinal chemistry; 69: 798-816. 2013. [Article] DOI: 10.1016/j.ejmech.2013.09.012
PMID: 24113365 IF=3,499
163. Vranic S, Schmitt F, Sapino A, Costa JL, Reddy S, Castro M, Gatalica Z. Apocrine carcinoma of the breast: A comprehensive review.
Histology and histopathology; 28: 1393-1409. 2013. [Review] PMID: 23771415 IF=2,281
104
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Members of IPATIMUP at Editorial Boards
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American Journal of Cancer Therapy and Pharmacology
Current Diagnostic Pathology
Dataset Papers in Biology
Dataset Papers in Oncology
European Journal of Cancer Prevention
European Journal of Human Genetics
Forensic Science International: Genetics
Frontiers in Genomic Assay Technology
Histopathology
ISRN Genomics
Journal of Integrated OMICS
Journal of Pathology
Open Pathology Journal
Pathology, research and practice
Patologia
PloS one
Research in Cancer and Tumor
Seminars in diagnostic pathology
The Open Forensic Science Journal
The Scientific World Journal
Ultrastructural pathology
World Journal of Biological Chemistry
World Journal of Clinical Infectious Diseases
World journal of gastroenterology: WJG
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