RELATÓRIO de ACTIVIDADE

RELATÓRIO
de
ACTIVIDADE
2012
Activity Report
2012
INDEX
Introdução
4
Produção Científica
6
Research Groups
9
Cancer Genetics
11
Population Genetics
23
Cancer Biology
31
Carcinogenesis
37
Cancer Drug Resistance
42
Proteolysis in diseases
46
Genetic Diversity
55
Outreach Activities
59
Science Diffusion
61
Public Awareness of Cancer
63
IPATIMUP Diagnostics
65
Innovation & Translation
73
IPATIMUP Innovation
75
IPATIMUP Translation
80
Internal Services
83
Sequencing Service
85
Proteomics Service
86
Animal Model Service
88
Cell Lines Bank
90
In vivo CAM Assays
92
Slides Preparation Service
93
Core Services
Technical Body
95
97
Informatics
100
Secretary General
103
Programs Office
104
Risk Management System
104
ANNEXES
2
51
Post-Graduation Unit
105
Recent PhDs
106
Research Projects
108
Scientific Papers
111
Members of IPATIMUP at Editorial Boards
119
Activity Report
2012
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Introdução
Nos últimos cinco anos, de 2008 a 2012, o IPATIMUP publicou 743 artigos científicos, correspondendo a 14% dos artigos publicados pelas
69 Unidades de Investigação da Universidade do Porto.
Entre o período acima mencionado, no ano de 2012 o IPATIMUP atingiu o número de 177 artigos publicados em revistas. Dos 177 artigos,
foram publicados 29 em revistas com FI superior a 6; 71 em revistas com FI entre 3 a 6; 65 em revistas com FI entre 1 e 3.
Assim o ano de 2012 poderá ser considerado como um dos melhores dos 24 anos do IPATIMUP. Os resultados científicos foram excelentes, apesar das condições financeiras muito adversas.
Em 2012, doutoraram-se 20 elementos do IPATIMUP, um número muito elevado tendo em conta o número de investigadores séniores
doutorados que trabalham no IPATIMUP.
Pela primeira vez, o IPATIMUP registou 8 patentes. Estes resultados refletem o investimento que o IPATIMUP fez ao criar a Unidade de
Inovação dirigida por um elemento Doutorado no IPATIMUP e com forte visão empresarial.
A Unidade IPATIMUP Inovação também assinou um Memorandum of Understanding com a Portugal Capital Ventures, com o objectivo
de promover o acesso de projetos de base tecnológica em fases de proof of concept, seed e early stage a investimento de capital de risco,
agilizando e sistematizando os processos de deal flow, acompanhamento e exit. Assim esta Unidade assegura no IPATIMUP a protecção
de Propriedade Intelectual com valor comercial, a exploração desse valor e estímulo à criação de empresas “spin-off”.
Foi também formalmente criada, em Fevereiro de 2012, a Unidade IPATIMUP Translação, com os objectivos de angariação de fundos externos, na forma de investigação contratada, integração em redes internacionais de translação na área da oncologia e estabelecimento
de parcerias estratégicas com a indústria. Esta Unidade terá uma actividade determinante no futuro do IPATIMUP, que procura diminuir
a sua dependência face ao Estado e à FCT. Até agora foram já contactadas mais de 50 empresas nacionais e internacionais e 4 projectos
encontram-se em fase de negociação avançada. Esta Unidade assegura no ano de 2013 a organização do Porto Cancer Meeting sobre
o tema Translational Research in Cancer onde participarão não só cientistas e clínicos mas também profissionais da indústria dedicados
ao desenvolvimento e aplicação de novas drogas para o cancro.
A Unidade IPATIMUP Diagnósticos obteve a renovação da acreditação pelo College of American Pathologists e a renovação da certificação em qualidade pela norma NP EN ISO 9001: 2008, Sistemas de Gestão da Qualidade.
O IPATIMUP foi oficialmente certificado como fornecedor de serviços para a plataforma de sequenciação de nova geração da empresa
Ion Torrent. O IPATIMUP é a primeira instituição em Portugal a utilizar esta tecnologia inovadora que acelera o tempo de diagnóstico de
doenças genéticas e hereditárias, como alguns tipos de cancro.
O IPATIMUP obteve a certificação em higiene e segurança pela OSHAS 18001: 2007 Occupational Health and Safety Assessment Service, assegurando que o IPATIMUP segue padrões de segurança no trabalho estando assim preparado para competir internacionalmente, como
instituição de acolhimento de recursos humanos envolvidos na realização de projetos de investigação com altos níveis de exigência.
Para redefinição da estratégia científica do IPATIMUP e desenho das linhas na área de investigação do cancro para os próximos anos,
foi criada uma task force composta por group leaders e investigadores seniores. Esta task force elencou as linhas de investigação mais
competitivas no IPATIMUP na área do cancro. Esta analise baseou-se nos seguintes critérios: capacidade de atrair financiamento competitivo nacional e internacional, publicação de resultados em revista internacionais indexadas e visibilidade internacional em sociedades cientificas.
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Foram criados três novos grupos de investigação, respondendo à evolução verificada de autonomização de campos de investigação por
parte de investigadores seniores:
•
Grupo Differentiation and Cancer
•
Grupo Glycobiology in Cancer
•
Grupo Expression Regulation in Cancer
Os grupos Carcinogenesis e Tumour Molecular Models foram extintos.
O grupo de investigação do INEB NEW Therapies mantém-se nas instalações do IPATIMUP, ocupando um laboratório de 50m2 onde foi
instalado um Centro de Bioimagem que servirá o I3S e outras instituições ligadas ao sistema de saúde. O IPATIMUP considera este acolhimento físico fundamental para o estímulo da colaboração inter-institucional e multidisciplinar, nomeadamente na interface de I&D
Cancro e Medicina Regenerativa.
O IPATIMUP foi instituição de acolhimento para a Associação Portuguesa de Investigação em Cancro- ASPIC. Esta associação teve como
promotora a Profª Leonor David que dedicou todo o seu empenho científico no desenvolvimento desta rede cientifica nacional ligada à
instituição europeia com maior visibilidade na área do cancro, a EACR (European Association for Cancer Research).
O grupo de Genética Populacional do IPATIMUP foi distinguido em 2012, pelo jornal «Ciência Hoje», na categoria «Seed of Science Especial», um reconhecimento baseado na análise de citações por referência de artigos publicados e factor h.
Iniciaram-se em 2012, 21 projectos de investigação dos quais onze são financiados pela FCT, dois financiados pela indústria farmacêutica,
um financiado pela Agência de Inovação e um pelo 7º Programa Quadro.
No concurso 2012 em todos os domínios científicos da FCT foram aprovados nove projetos de investigação. Foram aprovados 2 candidaturas de investigadores do IPATIMUP no âmbito do concurso para Investigador da FCT e que estavam anteriormente contratados pelo
IPATIMUP com financiamento do programa da FCT Ciência 2007.
Em 2012 realizou-se a edição XXI do Porto Cancer Meeting com o titulo «Metabolism and cancer - From etiopathogenesis to therapy»
e com organização cientifica de Paula Soares, Valdemar Máximo, Jorge Lima e Manuel Sobrinho Simões e contou com mais de 200
inscritos de diversas nacionalidades. Manteve-se a reunião anual do Portugaliae Genetica com o tema ‘Iberian Peninsula: at the gate
between the Mediterranean and the Atlantic worlds’e com a organização científica assegurada pelo grupo da Genética Populacional
sob a coordenação do António Amorim.
O III Scientific Retreat do I3S realizou-se na Póvoa de Varzim, em 10 e 11 de Maio de 2012, com uma forte participação dos investigadores
do IPATIMUP. Duas das investigadoras do IPATIMUP (Raquel Seruca, Vice-Presidente da Direcção e Luisa Pereira, Group Leader), fizeram
parte da comissão científica deste encontro.
O IPATIMUP manteve 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 celebrou um protocolo com a Faculdade de Medicina do Porto para utilização do biotério desta Faculdade abandonando-se
as antigas as instalações no Hospital de S. João. O IPATIMUP assegura neste novo protocolo os recursos humanos necessários para o
manuseamento dos animais de experiência e a Faculdade de Medicina assegura a infraestrutura.
A Universidade Federal do Rio de Janeiro apresentou formalmente o seu interesse em ser admitida como Associada Efectiva do
IPATIMUP e em celebrar contratos-programa para actividades de investigação e de ensino.
O IPATIMUP promoveu, em Março de 2012, a site visit anual regular do seu External Advisory Board.
Iniciaram-se as obras de construção do edifício decorrente da aprovação da candidatura apresentada pela Reitoria da Universidade do
Porto ao QREN, para o I3S - Instituto de Investigação e Inovação em Saúde da U.Porto. Foi também proposto, pela U. Porto e votado
favoravelmente pelo Conselho Geral, a constituição de um consórcio externo entre a UP e IBMC, INEB e IPATIMUP.
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Produção Científica
No ano de 2012, o IPATIMUP atingiu o número de 177 artigos publicados em revistas internacionais indexadas. Dos 177 artigos, foram
publicados 29 em revistas com FI superior a 6; 71 em revistas com FI entre 3 a 6; 65 em revistas com FI entre 1 e 3.
O gráfico seguinte ilustra a evolução do número de artigos publicados desde 2002, distribuídos por factor de impacto das revistas:
O gráfico seguinte ilustra a evolução do número de citações por ano, distribuídos por factor de impacto das revistas:
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A taxa de citações de artigos científicos do IPATIMUP mantem-se muito elevada, quer em “Clinical Medicine”, quer em “All Fields”:
Fonte:
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RESEARCH
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Cancer Genetics
Objectives
The Cancer Genetics group is focused on the genetics of three common types of epithelial cancer: gastric, breast and colorectal.
The group aims at:
1. identifying individuals at increased risk for cancer;
2. identifying clinicopathological features and molecular markers occurring in the setting of familial and sporadic carcinoma;
3. identifying signaling pathways mediated by genetic and environmental factors pivotal for tumor development. A special interest is on
the identification of the environmental and epi/genetic factors underlying cancer cell invasion, a topic of crucial importance in cancer
control.
Specifically, the Cancer Genetics team aims at developing new strategies for improving the quality of detection and treatment of poorprognosis cancer. To design efficient methods for detection of invasive tumor cells and new therapeutic strategies targeting invasion,
it is mandatory to identify key molecular/cell/ECM players in cancer. Therefore, the Group has been focusing on the identification of the
environmental and epi/genetic factors and associated signaling pathways underlying loss of cell-cell adhesion, increased cell-ECM interaction, cancer cell invasion and survival. A strong area of research of the Group aims at identifying the molecular mechanisms that regulate P-cadherin and E-cadherin expression in highly invasive cancer (breast and gastric, respectively), their associated cellular effects
and dependent signaling (upstream and downstream) pathways. Furthermore, the Group aims at unraveling how Helicobacter pylori
pathogenicity-associated factors interact with gastric cells and activates signaling pathways important for gastric cancer development.
In colon, we focus on the role on oncogenic mutations: K-Ras, N-Ras, BRAF, and PI3K.
Main Achievements
Somatic mutations and deletions of the E-cadherin gene predict poor survival of gastric cancer patients
We demonstrated that the presence of CDH1 somatic alterations in gastric cancers (~30% of all GC cases) associate with different patient
survival rates. In particular and rather surprisingly, patients with familial history of intestinal type gastric cancer (FIGC), who carry CDH1
somatic structural alterations, present the worst overall survival among all gastric cancer patients. The screening of such alterations
at diagnosis may predict patient prognosis and is likely to improve management of gastric cancer patients, particularly those with FIGC
(Corso and Carvalho et al, JCO, 2012).
Lack of microRNA-101 causes E-cadherin functional deregulation through EZH2 upregulation in intestinal gastric
cancer
We demonstrated that intestinal type gastric cancers often present E-cadherin dysfunction due to genomic deletion of miR-101 loci and
consequent upregulation of EZH2 that induce E-cadherin delocalization to the cytoplasm (Carvalho et al, J Pathol, 2012).
Characterization of the intronic portion of cadherin superfamily members, common cancer orchestrators and
identification of novel tissue-specific transcripts arising from E-cadherin/CDH1 intron2
We systematically characterized the intronic portion of cadherin superfamily members and identified intronic regions (MIR and MaLR
elements located in introns 2 and 3 of most human cadherin genes) constituting putative targets/triggers of regulation, using a bioinformatics approach and biological data mining (Oliveira et al, EJHG, 2012). Furthermore, we demonstrated, for the first time, the existence
of novel transcripts starting within CDH1 intron 2 and discovered a new mechanism by which a novel E-cadherin isoform impairs the
canonical E-cadherin function. This novel protein isoform of E-cadherin increases gastric cancer cell invasion and angiogenesis (Pinheiro
et al, HMG, 2012).
Development of a gene therapy approach that allows efficient recovery of E-cadherin expression in HDGC-associated truncating mutations
Using a CDH1 mini-gene encoding and a suppressor-tRNA based on a stop-codon readthrough strategy we were able to recover the normal expression (membrane) and function of E-cadherin (cell-cell adhesion and invasion supression) in epithelial cancer cells harbouring
truncating CDH1 mutations (Carriço et al, Trend in Molecular Medicine, 2012, made the cover of the journal and unpublished).
Development of a new method to classify E-cadherin missense mutants (pathogenic versus non-pathogenic)
based on their ability to interact with protein trafficking regulators
Using as model system the E-cadherin negative cells stably transfected with different E-cadherin germline missense mutations, we
tested by proximity ligation assays (PLA) if and how the E-cadherin mutations interfere with key trafficking-related partners, leading to
abnormal E-cadherin expression, localization and function, thus supporting their pathogenic relevance using. We verified that mutants
impairing concomitantly the binding of p120, ß-catenin and PIPKI¿, had the strongest impact on adhesive properties, presenting a close-
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to-isolated phenotype. In contrast, those mutants that only affected the interplay with one binding partner presented a mild effect on
cell aggregation, emphasizing the importance of these partners for E-cadherin-mediated cell-cell adhesion. This allowed us to propose
PLA as a screening method for identifying E-cadherin pathogenic mutants (Figueiredo et al, EJHG, 2012).
Pathogenic significance of eleven new germline E-cadherin missense mutations found in HDGC patients
As the International reference centre for studying the functional relevance of E-cadherin missense mutations, eleven new mutants were
reported to our laboratory 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 with empty vector
pIRES-GFP (Mock) and 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. Ten of the 11 mutations were functionally relevant and in all cases the
results were reported to the respective patient genetic counselors.
E-cadherin missense mutations associated to strong structural alterations lead to earlier gastric cancer development
We have previously found a new mechanism of E-cadherin regulation at the level of the Endoplasmic Reticulum, known to triage the
quality of protein structures soon after their synthesis. This protein quality control mechanism recognizes improper proteins for disclosure, leading to their elimination in the proteasome as is commonly referred as ERAD (Endoplasmic Reticulum Associated Degradation).
We previously shown that some E-cadherin missense mutations found in HDGC are efficiently recognized by ERAD as unfolded and degraded in the proteasome, leading to their loss of expression and function. We hypothesized that E-cadherin mutants unfolding could
account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. We
showed that strong structural alterations are found in approximately half of the HDGC-associated missense mutations. Interestingly,
HDGC patients harbouring germline E-cadherin destabilizing mutations are characterized by a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. Moreover, we showed that, in vitro,
unfolded mutant E-cadherin variants exhibit a shorter half-life, dependent on proteasomal degradation. For a set of new mutations analysed, we found a perfect correlation between loss of native-state stability and loss of function. In this context, we created structural
models that allow us to predict, in silico, the structural impact of most missense mutations, and this has been included in the workflow
for the prediction of pathogenicity of newly found E-cadherin missense mutations.
In the Drosophila model system, E-cadherin negative cells invade the neighboring tissue by expressing ectopically metalloproteinases and laminin
Our main goal was to try to recapitulate in vivo, how loss E-cadherin can trigger cancer cells to invade as an initiating event as verified in
Hereditary Diffuse Gastric Cancer (HDGC). By using a hairpin-type dsRNA for E-cadherin together with a tissue-specific driver construct,
we found that E-cadherin negative cells in a tissue context disappear from the epithelium with time due to cell death or invading the
neighbouring compartment. Further, we verified that some of the invading cells started expressing ectopically MMP1 and laminin A at
the interface between wild-type and E-cadherin negative cells. To further validate our results with human cells we demonstrated that
when E-cadherin transfected AGS cells come into contact with AGS parental cells (negative for E-cadherin), MMP2 is overexpressed in
E-cadherin negative AGS cells in comparison to E-cadherin positive, with a stronger expression at the border. The same holds true for
laminin gama2 and alfa5 (unpublished).
The tumor suppressor potential of DNAJB4/HLJ1 depends on its role as a molecular chaperone of E-cadherin
Using a genetic functional screen in Drosophila, we found that Drosophila DNAJ-1 interacts with human Ecad, suggesting that its human
homolog DNAJB4 is a new Ecad regulator. To dissect the role of DNAJB4 in the regulation of Ecad, we manipulated its expression in WT
and mutant Ecad genetic backgrounds and found that it directly interacts with Ecad, especially in the mutant context. DNAJB4 stabilizes
WT Ecad, but reduces the half-life of a misfolded mutant, presumably by increasing its degradation in the proteasome, indicating that it
acts as a molecular chaperone of Ecad. DNAJB4 forms a multimeric complex with Hsp90, and synergizes with the chemical chaperone
DMSO, exacerbating Ecad expression. In our models, DNAJB4 influences cell adhesion and motility in a cadherin-dependent manner,
indicating that the anti-tumorigenic role of DNAJB4 depends on its function as a molecular chaperone of Ecad. This work allowed us the
identification of the first molecular chaperone of Ecad, and to propose that the previously described tumor suppressor role of DNAJB4
is cadherin-dependent.
A specific gastric expression program that may allow gastric cells to overcome cell death mediated by E-cadherin loss in stomach
We aim to determine the tissue specific molecular determinants behind the predominant increased risk for stomach cancer in HDGC
despite that E-cadherin is expressed in all epithelial tissues. Our working hypothesis is that CDH1 biallelic inactivation is only tolerated in
gastric epithelium and not in other epithelia due to a favourable gastric-specific expression program that allows cell-autonomous survival which thus becomes more prone to acquire de novo alterations, increasing its risk of transformation. A stomach-specific gene list was
previously compiled based on data mining analysis of 4 datasets of expression arrays of normal tissues by assessing which genes were
present in at least two of those datasets. We obtained 25 common stomach-specific genes, 15 of which have been reported as displaying
a cell survival/apoptosis relationship with gastric cancer. We have validated the bioinformatic analysis by RT-PCR for 10 selected genes
and confirmed their expression to be confined to stomach tissue RNA. A set of genes displayed retained expression in gastric cancer cell
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lines (diffuse type), namely MUC1, S100P and CTSE. We have subsequently performed RNAi assays for MUC1, S100P and CTSE in gastric
cancer cell lines displaying WT or absent E-cadherin expression and we are currently performing apoptosis inductions assays using Paclitaxel and Staurosporine for functional characterization. Further, we determined the expression profile of apoptosis-related proteins to
identify putative pathways underlying cell survival of stomach epithelial cells upon E-cadherin loss. We performed a proteomic profiler
approach using a “Human Apoptosis Array” from R&D Systems in gastric cancer cells expressing WT E-cadherin vs siE-cadherin. Data
analysis unraveled the most differentially expressed proteins in the experimental settings, which are now being validated in biological
replicas as well as in the settings of apoptosis inductions (Carneiro et al, FEBS Letters, 2012 and unpublished)
Gastric cells with loss of E-cadherin and activation of the non-canonical Notch pathway do not induce angiogenesis in vivo but instead show a switch to a glycolytic phenotype
Two cells lines negative for E-cadherin (AGS and MDA435) were stably transfected with CDH1. Using the chorialantoic membrane (CAM)
we evaluated whether AGS cells overexpressing E-cadherin showed increased angiogenic response. We verified that cells overexpressing E-cadherin appear to preferentially activate an angiogenic response to address their metabolic needs while E-cadherin negative cells
use the glycolytic pathway. MDA435 E-cadherin negative cells had higher GLUT1 expression and increased lactate production/glucose
consumption. Furthermore, the inverse relation between E-Cadherin and Notch- 1 expression, previously described in vitro, was confirmed in vivo. The relationship between Notch and angiogenesis remains to be addressed (unpublished).
A small percentage of patients with orofacial clefts (OFC) harbor germline mutations in the CDH1 gene
We previously described an association between non-syndromic OFC and CDH1 germline mutations and determined that during embryonic development, the cell adhesion molecule E-cadherin is highly expressed in the median edge epithelium of the palate. In this study
we found that 4 out of 81 (5%) patients with non-syndromic OFC had functionally relevant CDH1 germline missense mutations (Vogelaar
and Figueiredo , et al, HMG, 2012).
Mgat3/GnT-III-mediated E-cadherin N-glycosylation is a driver mechanism of EMT/MET in an in vitro model of
TGFß1-induced Epithelial-Mesenchymal Transition and Mesenchymal-Epithelial Transition (EMT/MET)
We generated a database that encompasses all differentially expressed genes and pathways that change along EMT/MET, using whole
transcriptome sequencing (RNAseq). A deep bioinformatics analysis of this database as well as RNA and protein expression validations
have shown a large set of differentially expressed genes and biological pathways, many related with cancer and metastases pathways.
Moreover, we found that during EMT/MET, different metabolic circuitries are used as sources of energy production (unpublished). Taking advantage of the model, we identified a novel mechanism that regulates E-cadherin function during EMT/MET, and depends on the
E-cadherin glycosylation with bisecting GlcNAc structures catalyzed by the GnT-III enzyme (encoded by Mgat3 gene), which expression
is controlled by CpG island promoter methylation (Pinho and Oliveira et al, PLOS One, 2012).
Inter-individual variation in chronic inflammatory response leads to increased risk of gastric carcinoma
We have shown before that genetic variation in genes that regulate chronic inflammatory response to Helicobacter pylori infection lead
to an increased risk of development of gastric carcinoma. We have now shown that IL1B expression leads to increased expression of the
transcription factors CREB and C/EBPbeta, which in turn lead to an increased survival capacity of gastric epithelial cells. Both CREB and
C/EBPbeta had been previously shown to play a role in tumourigenesis in several cancer models. Our own results have demonstrated
that C/EBPbeta expression is correlated with loss of differentiation in the gastric epithelium. Our current results add to the model that
in individuals with a “pro-inflammatory” genetic make-up, chronic inflammation triggered by H. pylori infection, leads to a mucosal
environment of increased epithelial cell survival. This, in turn, increases the likelihood of fixation of genetic mutations with oncogenic
potential (unpublished).
A novel method for genotyping the intermediate (i)-region of Helicobacter pylori vacA virulence factor
We have developed and optimized a new genotyping method for characterizing the virulence-associated vacA i-region in archive material. The genotypes determined using the method were completely concordant with those of sequence analysis and of functional
vacuolation activity. The method was further validated directly in gastric biopsy specimens of 386 H. pylori-positive cases, and effective
characterization of the vacA i-region was obtained in 99.5% of frozen and in 95.9% of formalin-fixed paraffin-embedded gastric biopsy
specimens, respectively (Ferreira et al, J Clin Microbiol 2012).
Helicobacter pylori vacA i-region genotypes and gastric carcinoma development
The novel vacA i-region genotyping method was used to address the relationship between H. pylori vacA i-region genotypes and gastric
disease development in the Portuguese population. Patients infected with vacA i1 strains showed an increased risk for gastric atrophy
and for gastric carcinoma with odds ratio of 8.0 (95%, CI 2.3-27) and of 22 (95%, CI 7.9-63), respectively. The results suggest that the characterization of the vacA i-region may be useful to identify patients at higher risk of gastric carcinoma development (Ferreira et al, J Clin
Microbiol 2012).
Helicobacter pylori vacA i-region genotypes and the progression of premalignant lesions of the stomach
The novel vacA i-region genotyping method was also used to characterize this virulence-associated locus in a Spanish population of a
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region with a high-risk of gastric carcinoma. This was part of a follow-up study in which we had previously demonstrated that the risk
of progression of gastric preneoplastic lesions was higher in patients infected with the most virulent H. pylori cagA and vacA s- and
m-region genotypes than in patients infected with the least virulent strains. After a mean of 12.8 years of follow-up, and in multivariate
analysis adjusted for age, sex, histological diagnosis, smoking, family history of gastric cancer, and consumption of non-steroidal antiinflammatory drugs, infection with vacA i1 strains was associated with lesion progression with OR of 3.4 (95%, CI 1.4-8.1). After fitting
different models, for each bacterial virulence-associated locus and for loci combinations, the model that best explained the results is
the one that combines the vacA s- and m-regions and cagA, suggesting that genotyping of the vacA i-region in this population does not
improve the prediction of progression given by the other H. pylori virulence-associated loci (Ferreira et al, Am J Gastroenterology, 2012).
A method for short-term culture of human gastric epithelial cells to study the effects of Helicobacter pylori
In vitro studies of H. pylori pathogenesis mostly rely on the use of tumor-derived cell lines. Although invaluable, tumor cell lines are not
representative of the normal cell physiology. Thus, the use of primary gastric epithelial cell cultures provides an important tool for investigating the mechanisms underlying H. pylori infection, as well as for validating the in vitro findings obtained with tumor-derived cell
line models. We described a method for isolation and short-term culture of human primary gastric epithelial cells obtained from gastric
biopsy specimens, and the use of these cells to evaluate the effect of H. pylori on the junctional adhesion molecule-A protein (Leite and
Figueiredo, Methods Mol Biol, 2012).
Docosahexaenoic Acid Inhibits Helicobacter pylori Growth in vitro and Colonization of the Mouse Gastric Mucosa
H. pylori drug-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. Since for some bacterial species it has been demonstrated that fatty acids have a growth inhibitory effect, we decided to assess the ability of docosahexaenoic acid (DHA) to inhibit H. pylori growth. Our data demonstrate that DHA decreases H. pylori growth in vitro in a dose-dependent manner. Furthermore, DHA is able to inhibit H. pylori gastric colonization in vivo as well as to decrease mouse gastric mucosa inflammation.
Addition of DHA to standard antibiotic therapy (ST) was also associated with lower H. pylori infection recurrence in the mouse model. In
conclusion, we have shown that DHA is an inhibitor of H. pylori growth. DHA treatment is also associated with a lower recurrence of H.
pylori infection in combination with ST. These observations may pave the way to consider DHA as an adjunct agent in H. pylori eradication treatment (Correia et al, PLOS One, in press).
P-Cadherin expression mediates stem cell properties in basal-like breast cancer
Using a series of breast cancer cell lines and primary tumors, we showed that P-cadherin was directly associated with the expression of
the breast stem markers CD44, CD49f and ALDH1 in the basal subtype. Moreover, cell populations enriched for P-cadherin expression
comprised increased in vitro mammosphere forming efficiency and capacity to grow colonies in 3D cultures, as well as greater tumourigenicity. Importantly, an association was found with stem/progenitor-like phenotypes of the breast, including the luminal progenitor
population, CD49f+CD24+. Additionally, P-cadherin expression conferred resistance to X-ray induced cell death, sustaining a role for this
molecule in another stem cell property. In summary, we demonstrated, for the first time, that P-cadherin mediates stem cell properties,
which could be explored in order to better define the CSC phenotype of basal-like breast tumors and the cell-of-origin of this malignancy
(Vieira et al. Stem Cells 2012).
P-cadherin intracellular signalling is dependent on a6ß4 integrin activation to induce breast cancer stem cell
and invasive properties
We showed that P-cadherin is essential for the cell adhesion to the extracellular matrix substrates laminin, vitronectin and fibronectin.
The a6ß4 integrin heterodimer was implicated in the downstream signaling of P-cadherin in response to laminin, as well as in the invasive capacity and the stem cell activity of cancer cells. The activation of FAK and Src signaling was also dependent on P-cadherin expression. Moreover, P-cadherin was shown to control the levels of the a6 integrin subunit mRNA and to directly interact with the ß4 integrin
subunit. In conclusion, we showed, for the first time, that there is a crosstalk between P-cadherin and the a6ß4 integrin receptor, with
both molecules having a central role in tumor progression. The identification of this signaling crosstalk is of major importance since
P-cadherin, as a breast cancer target, would have implications in the invasive and stem cell properties that are dependent on integrin
signaling (unpublished)
P-cadherin functional role is dependent on E-cadherin cellular context
We aimed to investigate if P-cadherin expression would interfere with the normal adhesion complex and which were the cellular/molecular consequences, constituting, in this way, a new mechanism by which E-cadherin invasive-suppressor function was disrupted in
cancer. 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
behavior. Additionally, E- and P-cadherin co-expressing cells significantly enhanced in vivo tumor 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 tumors expressing high levels of this protein (Ribeiro et al. J Pathol 2013).
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C/EBPß isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer
cells
Since we recently described the existence of several CCAAT/Enhancer Binding Protein ß (C/EBPß) transcription factor binding sites at the
CDH3 promoter, the aim of this study was to assess if the distinct C/EBPß isoforms were directly involved in the transcriptional activation
of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been
performed. We demonstrated that C/EBPß is co-expressed with P-cadherin in breast cancer cells and all the three isoforms 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).
Aberrant P-cadherin expression is associated with hypoxic/glycolytic and acid resistant phenotype in breast
cancer
We aimed to evaluate if P-cadherin expression was associated with the adaptive response of breast cancer cells to hypoxic tumor conditions, the acquisition of a glycolytic metabolism and with the extracellular acidification of the tumor microenvironment. We found
that P-cadherin overexpression was significantly associated with the expression of HIF1a, GLUT1, CAIX, MCT1 and CD147. Accordingly,
we still found that P-cadherin was expressed in the same cell population as GLUT1 and CAIX in triple negative and basal-like breast cancer cell lines. Furthermore, P-cadherin silencing was able to induce a significant downregulation of GLUT1 and CAIX mRNA. Thus, we
established, for the first time, a link between aberrant P-cadherin expression and the hypoxic, glycolytic and acid-resistant phenotype
in breast cancer. This link needs to be further explored in order to reveal how P-cadherin expression regulates breast cancer cell metabolism (unpublished).
The bacterial protein azurin impairs P-cadherin-dependent invasion of breast cancer cells via decreased FAK/
Src signaling
Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity towards human cancer cells, after its preferential penetration rather than normal cells. We found that azurin caused a specific decrease on P-cadherin protein levels from 30-50%
in breast cancer cell lines, but the levels of E-cadherin remain unaltered. The invasive phenotype of these breast cancer cells was significantly reduced by azurin, which also led to a decrease in the phosphorylation levels of both FAK and Src proteins. Also, the levels of
sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells. Our data show that azurin specifically
targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be
considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin (unpublished).
1Alpha,25-dihydroxyvitamin D3 induces de novo E-cadherin expression in triple-negative breast cancer cells by
CDH1-promoter demethylation
The triple-negative subgroup of breast cancer includes a cluster of tumors exhibiting low E-cadherin expression (metaplastic carcinomas). In several cancer models, 1alpha,25-dihydroxyvitamin D3 (1a,25(OH)2D3) induces differentiation by increasing E-cadherin expression. The Vitamin D receptor (VDR) was evaluated as a possible therapeutic target for metaplastic carcinomas and 1a,25(OH)2D3 effects
as a differentiating agent in triple-negative breast cancer cells were assessed. We found that most of the metaplastic carcinomas were
positive for VDR expression. Furthermore, 1a,25(OH)2D3 promoted differentiation of MDA-MB-231 cells by inducing de novo E-cadherin
expression, an effect that was time- and dose-dependent. Also, E-cadherin expression was due to promoter demethylation. Thus, metaplastic carcinomas may respond to 1a,25(OH)2D3, since they express VDR and 1a,25(OH)2D3 induces de novo E-cadherin expression in
breast cancer cells by promoter demethylation (Lopes et al. Anticancer Research, 2012).
CLDN3, CLDN4 and CLDN7 expression is insufficient to identify the CLDN-low molecular subtype of breast carcinomas
The recently characterized CLDN-low molecular subgroup of breast tumours increased the interest in these molecules. Our aim was to
explore the pattern of expression of CLDNs among a large series of invasive breast carcinomas and analysed the correlation between
the combinatorial expression of CLDN3/CLDN4 and classical prognostic factors and biological markers. In addition, we also compared
the characteristics of tumours with low expression of CLDN3, CLDN4 and CLDN7, assessed by immunohistochemistry (IHC), and the ones
from CLDN-low subgroup of tumours previously defined by genomic assays. The combinatorial analysis of the expression of CLDN3/
CLDN4 showed a significant association between high CLDN3/CLDN4 levels and triple-negative tumours, as well as with a worse patient
outcome. This combined analysis may provide useful information for breast carcinomas, since these two CLDN members are putative
therapeutic targets. Comparing tumours with low expression of CLDN3, CLDN4 and CLDN7 with tumours previously referred as CLDNlow by genomic assays, we demonstrated that the evaluation of these three specific CLDNs is insufficient to identify the CLDN-low
molecular subtype of breast tumours. The analysis of several other molecular markers, such as EMT markers, should be probably added
to improve the identification of this subgroup of tumours by IHC, which seems be most likely enriched in carcinomas with metaplastic
differentiation (Ricardo et al. Histol & Histopathol, 2012).
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Activity Report
2012
Metaplastic breast carcinomas have similar features to the ones included in the claudin-low molecular subtype
The claudin-low molecular subtype of breast cancer includes triple negative invasive carcinomas, with a high frequency of metaplastic
and medullary features. The aim of this study was to evaluate the immunohistochemistry expression of claudins in a series of metaplastic breast carcinomas. We also assessed other claudin-low features, such as the cancer stem cell-like and epithelial-to-mesenchymal
transition phenotypes. The negative/low expression of claudins and E-cadherin, high levels of vimentin, and the breast cancer stem
cell phenotype suggests that metaplastic breast carcinomas have similar features to the ones included in the claudin-low molecular
subtype, specially their mesenchymal components (Gerhard et al. The Breast, 2012).
Cancer stem cells markers CD44, CD24 and ALDH1 in breast cancer special histological types
The prevalence and clinical significance of CD44, CD24 and ALDH! CSC markers in breast carcinomas of special histological types (SHT) is
largely unknown. For that reason, we aimed to determine their distribution among a series of invasive breast carcinomas of SHT, in comparison with a series of IDC-NST. 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(+) (de Beça et al. J Clin Pathol, 2013).
Immunohistochemical profiling of special histological types (HST) of breast carcinomas
The purpose of the present study was to apply the recently used immunohistochemical profiling of HST breast carcinomas (BC), as a
surrogate for the molecular subtyping, what could be relevant for therapeutic purposes. In summary, tubular, mucinous and papillary
types were all categorised as luminal-like A and B. The medullary and metaplastic types corresponded largely to the basal-like tumours.
Cases of the micropapillary type were luminal A, luminal B and HER2 overexpressing, whereas the apocrine carcinomas presented a
heterogeneous profile. The proliferation rate (Ki-67) varied among the types, being the medullary carcinoma subtype with higher proliferation. Comparing the current data with those based on molecular studies, there was good agreement in the classification of the
tubular, mucinous and papillary types. Only a partial concordance was achieved for the other types, which may be due to sampling, and
to the relatively low frequency of such cases. The present work supports the clinical usage of immunohistochemistry as a surrogate to
molecular classification of special types of BC (Alvarenga et al. J Clin Pathol 2012).
MAPK- and PI3K-targeted approaches as a new strategy for colorectal cancer therapy
We aim to understand if inhibition of molecules of the MAPK and PI3K pathways could provide an alternative therapeutic approach to
patients with metastatic colorectal cancer (CRC) and to pursue the identification of novel putative biomarkers able to predict therapy
sensitivity and to help in the stratification of CRC patients for therapy. We verified that in distinct CRC cell lines harbouring activating
mutations in KRAS, BRAF and/or PIK3CA, siRNA-mediated targeted depletion of MEK1/2 and/or PIK3CA differentially affected cell survival
and death. Moreover, the results demonstrate a pivotal role for PI3K in all cell lines. To identify the molecular mechanisms underlying the
effects of MAPK and/or PI3K inhibition, a human phospho-kinase array was evaluated and validation on the levels of phosphorylation is
being performed for several candidate proteins (unpublished).
Mutant N-Ras activates Stat3 in colorectal cancer and correlates with a less favorable clinical outcome of the
patients
Little is known about how the N-Ras mutant protein contributes to the onset and progression of colorectal cancer. Using genetically
engineered mice, we find that mutant N-Ras strongly promotes tumorigenesis in the context of inflammation. The protumorigenic
nature of mutant N-Ras is related to its antiapoptotic function, which is mediated by activation of a noncanonical mitogen-activated
protein kinase pathway that signals through Stat3. As a result, inhibition of MAP–ERK kinase selectively induces apoptosis in autochthonous colonic tumors expressing mutant N-Ras. The translational significance of this finding is highlighted by our observation that
NRAS mutation correlates with a less favorable clinical outcome for patients with colorectal cancer. These data show for the first time
the important role that N-Ras plays in colorectal cancer (Wang and Velho, et al, Cancer Discov, 2012).
Internationalization/Networking
National collaborations
Ana Paula Pêgo, INEB, Porto
Arsénio Fialho, IBB-Instituto Superior Técnico, Lisboa
Cristina Barrias, INEB, Porto
Fátima Baltazar, ICVS, University of Minho, Braga
Fernando Magro, FMUP, 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
João Taborda Barata, IMM, FMUL, Lisboa
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Activity Report
2012
Manuel A. Santos, CESAM, University of Aveiro, Aveiro
Manuel Coimbra, University of Aveiro, Aveiro
Maria João Vieira, CEB, University of Minho, Braga
Maria Oliveira, INEB, Porto
Mário Dinis Ribeiro, FMUP, Porto
Mário Barbosa, INEB, Porto
Nuno Azevedo, FEUP, Porto
Nuno C. Santos, IMM, FMUL, Lisboa
Nuno Lunet, FMUP, Porto
Paulo Pereira, IBILI, Coimbra
Pedro Granja, INEB, Porto
Peter Jordan, INSA Ricardo Jorge, Lisboa
International collaborations
Australia
Amanda Charlton, Department of Pathology, the Children’s Hospital at Westmead, Sydney
Georgia Chenevix-Trench, The Queensland Institute of Medical Research, Brisbane
Belgium
Marc Mareel, Laboratory of Experimental Cancer Research, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, Ghent
Marc Bracke, Laboratory of Experimental Cancer Research, Ghent University Hospital, Ghent
Brazil
Célia Carlini, Universidade Federal do Rio Grande do Sul, Porto Alegre
Dulciene Queiroz, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte
Rozany Dufloth, Faculdade de Medicina de Botucatu-UNESP
Canada
David Huntsman, British Columbia Cancer Agency, Vancouver
Chile
Paul Harris, Pontificia Universidad Católica de Chile, Santiago
Costa Rica
Vanessa Ramirez, Calderón Guardia Hospital, San José
Denmark
Lene J Rasmussen, University of Copenhagen, Copenhagen
Finland
Lauri A Aaltonen, Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki
France
Alex Duval, INSERM, Hôpital Saint-Antoine, Paris
Eliette Touati, Institute Pasteur, Paris
Germany
Elisa Izaurralde, Max Plank Institute for Developmental Research, Tuebingen
Federico Canzian, German Cancer Research Center (DKFZ), Heidelberg
Sebastian Suerbaum, Hannover Medical School, Institute of Medical Microbiology and Hospital Epidemiology, Hannover
Thomas Meyer, Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin
Thomas Schulz, Institute of Virology, Medical School Hannover
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Activity Report
2012
Ireland
Dermot Kelleher, Department of Clinical Medicine, Trinity Centre for Health Sciences, St. James’s Hospital, Dublin
Israel
Yosef Yarden, Weismann Institute, Rehovot
Italy
Elia Stupka, Milan, Italy
Franco Roviello, Dept Surgery and Oncology, University of Siena, Siena
Remo Sanges, Cluster in Biomedicine, Trieste
Peru
Robert Gilman, Department of Microbiology, Infection Disease Laboratory, Universidad Peruana Cayetano Heredia, Rimac, Lima
Spain
Carlos Gonzalez, Unit of Nutrition, Environment and Cancer, Catalan Institute of Oncology (IDIBELL-ICO), Barcelona
Fernando Casares, Centro Andaluz de Biología del Desarrollo, Sevilla
Gabriel Capellá, Catalan Institute of Oncology (IDIBELL-ICO), Barcelona
Gema Moreno-Bueno, Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM), Instituto de Investigaciones Biomédicas
‘Alberto Sols’ CSIC-UAM, Madrid
Jorge F. Cameselle-Teijeiro, Department of Pathology, Complexo Hospitalar Universitario de Vigo (CHUVI), Vigo
José Luis Skarmeta, Centro Andaluz de Biología del Desarrollo, Sevilla
José Palacios, Servicio de Anatomía Patológica, Hospital Virgen del Rocío, Sevilla
Luis Serrano, Systems Biology, CRG, Barcelona
Manel Esteller, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona
Sweden
Ola Söderberg, Department of Genetics and Pathology, University of Uppsala, Uppsala
The Netherlands
Bauke Ylstra, Microarray Core Facility (MAF) of the VU University Medical Center/ Cancer Center Amsterdam
Beatriz Carvalho, Dept Pathology, VU Univ. Medical Center, Amsterdam
Leen-Jan van Doorn, DDL Diagnostic Laboratory, Voorburg
Marjolijn Ligtenberg and Han van Krieken, Radboud University Nijmegen Medical Centre, Nijmegen
Nicole C. van Grieken, VuMC, Amsterdam
Robert Hofstra, Department of Medical Genetics, University of Groningen, Groningen
United Kingdom
Carlos Caldas, Cambridge Research Institute Li Ka Shing Centre, Cambridge
Göran Landberg, Paterson Institute for Cancer Research (PICR), University of Manchester, Manchester
Ian Sanderson, Institute of Cell and Molecular Science, Barts and The London, London
Jean Crabtree, St James’ University Hospital, Leeds
John Atherton, University of Nottingham, Nottingham
Jorge Sérgio Reis-Filho, Institute of Cancer Research, London
Robert B. Clarke, Paterson University of Manchester, Manchester
USA
David Mooney, Lab of Cell and Tissue Engineering, School of Engineering and Applied Sciences (SEAS), Harvard University, Cambridge
Gregory Lauwers, Department of Pathology, General Hospital, Boston
Kevin Haigis, Molecular Pathology Unit of the Massachusetts General Hospital, Boston
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Activity Report
2012
Future Research
In the gastric cancer settings:
To develop a bioimaging method to discriminate WT E-cadherin from mutated forms based on the quantification of its expression at the
membrane and its cellular radial distribution profile;
To characterize the inter-nuclear distance between contiguous cells in WT and mutated E-cadherin cells in order to understand their
impact on cell morphology;
To identify key players involved in the cell-ECM crosstalk in the context of E-cadherin mutations associated to HDGC and identify the ECM
components/compositions that can modulate the expression of integrins or associated molecules;
To identify the mechanism by which E-cadherin loss induces MMP overexpression to induce cancer cell invasion;
To perform proteomic analysis of the molecular interactors of E-cadherin involved in its degradation by the proteasome or stabilization
by chemical chaperones, and consequent modulation of their expression in attempt to rescue expression and function of E-cadherin
mutants;
To determine the mechanisms of protein quality control sensing E-cadherin stability at the plasma membrane, and identify the stimuli
that trigger their activation;
To identify the gastric-specific molecular program underlying the ability of gastric cells to overcome E-cadherin mediated apoptosis and
functionally characterize the putative candidates and their relevance in E-cadherin-related survival/apoptosis in the stomach vs colon
and breast;
To characterize the promoter region of the H. pylori cagA virulence gene in strains infecting Portuguese patients, in order to assess the
relationship between cagA promoter variation, CagA expression, and capacity to elicit inflammation in the host;
To evaluate the genetic and functional diversity of the H. pylori virulence factor HtrA, a secreted serine protease that cleaves the extracellular domain of E-cadherin;
To identify the bacterial virulence factors and the host cell signaling pathways leading to up-regulation of MMP-10 in gastric epithelial
cells induced by H. pylori infection.
In the breast cancer setting:
To identify the key signaling pathways induced by P-cadherin expression in breast cancer, namely its role in the cell-ECM crosstalk, in
cancer cell metabolism and in survival/invasion capacity;
To study P-cadherin’s role in the seeding of breast cancer cell metastasis;
To identify the molecular mechanism by which P-cadherin expression influences cell polarity, misoriented cell division and genetic instability in breast cancer cells;
To identify specific biomarkers for the transition in situ/invasive breast cancer.
In the colon cancer setting:
To identify key signaling molecules underlying the effects of MAPK and/or PI3K inhibition in colorectal cancer cells with distinct mutational status for KRAS, BRAF and PI3K and validate the results obtained in vitro in primary CRC specimens;
To determine the role of mutant N-Ras in the development of colorectal cancer in the context of inflammatory bowel disease.
Note: Carla Oliveira started a new Group called Expression Regulation in Cancer in January 2013. This new research group aims to study
the: 1) regulation of cancer-associated genes and implication for gastric cancer diagnosis, prognosis and management, and; 2) mechanisms involved in Epithelial-Mesenchymal-Epithelial transitions (EMT/MET) associated to metastasis and chemotherapy-induced resistance.
Participation in PhD Programs
Céu Figueiredo, Fátima Carneiro (Coordenação e participação), Fernando Schmitt, Joana Paredes, José C. Machado, Marina Leite,
Raquel Seruca, Patrícia Oliveira, Patrícia Carneiro. Programa Graduado em Áreas da Biologia Básica e Aplicada (GABBA) da Universidade
do Porto. Oncobiology Module.
Fátima Carneiro, Fernando Schmitt, Joana Paredes, José C. Machado, Sofia Fernandes, Raquel Seruca (Coordenação e participação).
Doctoral Programme for Physicians (Fundações Calouste Gulbenkian e Champalimaud). Oncobiology Module.
Fátima Carneiro (Coordenação e participação), Fernando Schmitt, Joana Paredes, José C. Machado, Raquel Seruca. Programa de Doutoramento em Medicina e Oncologia Molecular, Faculdade de Medicina da Universidade do Porto. Oncobiology Module.
Fátima Carneiro, Fernando Schmitt, Joana Paredes, José C. Machado, Raquel Seruca. Programa Doutoral em Patologia e Genética Molecular, Instituto de Ciências Biomédicas Abel Salazar, Porto. Oncobiology Module.
Joana Paredes. Doctoral Program in Health Sciences, Faculty of Medicine of Coimbra University.
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Activity Report
2012
Participation in Master Programs
Fátima Carneiro. Mestrado em Medicina e Oncologia Molecular, FMUP. Módulo de Oncobiologia (Coordenação e participação).
Raquel Seruca, Joana Paredes. Advanced Course on Oncobiology, Universidade de Coimbra.
Joana Paredes. Course on Fundamentals of Genetics, Development and Neoplasia, ICVS, Minho University.
Joana Paredes. Mestrado Integrado em Medicina, Universidade do Algarve.
Joana Paredes. Master in Molecular Genetics, Biology Departament of Minho University.
Prizes
A.F. Vieira, A.S. Ribeiro, M.R. Dionísio, B. Sousa, A.R. Nobre, A. Albergaria, A. Santiago-Gómez, F. Schmitt, R.B. Clarke, J. Paredes. Pcadherin intracellular signalling is dependent on a6b4 integrin activation to induce breast cancer stem cell and invasive properties.
First best poster in the Oncobiology Area in Third I3S Scientific Retreat. Póvoa do Varzim, Portugal. May 2012.
P. Oliveira, J. Carvalho, M. Azevedo, D. Ferreira, A. Moussavi, J. Vinagre, N. Mendes, F. Carneiro, D. Huntsman, R. Seruca, C. Oliveira. An
in vitro model of Epithelial-Mesenchymal-Epithelial transitions as a novel tool to study cancer progression.
Second best poster in the Oncobiology Area in Third I3S Scientific Retreat. Póvoa do Varzim, Portugal. May 2012.
B. Sousa, A. S. Ribeiro, N. Lopes, D. Martins, A. Albergaria, S. Ricardo, C. Pinheiro, R. Gerhard, F. Schmitt, F. Baltazar, J. Paredes. Aberrant
P-cadherin expression is associated with glycolytic and acid-resistant phenotype in invasive breast carcinoma.
Third best poster in the Oncobiology Area in Third I3S Scientific Retreat. Póvoa do Varzim, Portugal. May 2012.
Martins DF, Sousa B, Paredes J, Schmitt F. Mtor expression in basal-like cancer and the ability of everolimus to inhibit the invasion cancer
cell capacity. Annals of Oncology 23: (Suppl 9) doi:10.1093, 2012.
«Best Poster Award”na categoria Breast Cancer no 37º Congresso da ESMO, Viena, Áustria, 2012.
Fernando Schmitt. Recebeu o Prémio Especial ADICAM, Espanha, 2012.
Invited Talks
Raquel Seruca . E-cadherin dysfunction in gastric cancer--cellular consequences, clinical applications and open questions. FEBS Congress. Sevilha, Spain. 2012.
Raquel Seruca. The hallmarks of cancer explained by a single molecule: E-cadherin. IMM. Lisbon, Portugal. 2012.
Raquel Seruca. Molecular biomarkers in gastric cancer. Highlights of the EORTC St. Gallen International Expert Consensus on the primary therapy of gastric, gastroesophageal and oesophageal cancer - differential treatment strategies for subtypes of early gastroesophageal cancer. St. Gallen. 2012.
Raquel Seruca. E-cadherin associated signalling and cancer. IGC. Lisbon, Portugal. 2012.
Carla Oliveira. Dissecting the role of cadherins with genome-wide technologies. User Meeting Genómica. IBILI. Coimbra, Portugal. 2012.
Carla Oliveira. Novel molecular mechanisms modulating the function of the invasion suppressor protein E-cadherin. 2nd IBEC-INEB Advanced Summer School. IBEC.. Barcelona, Spain. 2012.
Carla Oliveira. From disease to mechanisms: old and new stories about E-cadherin. CDR Saint-Antoine, Paris. Paris, France. 2012.
Carla Oliveira. Epigenetic Changes in Cancer and Epithelial to Mesenchymal Transitions . Mini-symposium on Epigenetics and Cancer.
IBMC. Porto, Portugal. 2012.
Patrícia Pereira. MicroRNAs in Human Cancer. User Meeting Genómica. IBILI. Coimbra, Portugal. 2012.
Patrícia Pereira. MicroRNAs in Human Cancer. Workshop in Microarrays Applications in Biomedicine. Biocant . Cantanhede, Portugal.
2012.
Fátima Carneiro. Anatomia Patológica hoje. Sessão de divulgação das Especialidades do Centro Hospitalar de São João. Porto, Portugal.
2012.
Fátima Carneiro. O envelhecimento é um processo patológico? Trata-se?. Sessão “do médico para os médicos – especificidades que interessam à generalidade” organizada pela Secção Regional do Norte da Ordem dos Médicos.. Porto, Portugal. 2012.
Fátima Carneiro. Cancro gástrico: Oncogénese na doença esporádica e familiar. Jornadas Internacionais de Cirurgia. Coimbra, Portugal.
2012.
Fátima Carneiro. Diagnóstico anatomopatológico na doença hepática. Curso Teórico-Prático de Contrastes, Fibroscan, Elastografia
Dinâmica e Biopsia na Patologia Hepática. Porto, Portugal. 2012.
Fátima Carneiro. Quality assurance in gastrointestinal molecular pathology. 2nd Pannonia Congress of Pathology. Siófok, Hungary. 2012.
Fátima Carneiro. Anatomia Patológica. 1º Curso Teórico-Prático sobre Co-infecção VIH/vírus das hepatites. Porto, Portugal. 2012.
Fátima Carneiro. Hereditary gastric cancer. 96th Annual Meeting of the “Deutsche Gesellschaft für Pathologie. Berlin, Germany. 2012.
Fátima Carneiro. Gastric cancer: what’s new?. 13th Panhellenic Congress of Pathology. Kalamata, Greece. 2012.
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Activity Report
2012
Fátima Carneiro. Carcinogenesis in gastric cancer. ESMO 14th World Congress on Gastrointestinal Cancer. Barcelona, Portugal. 2012.
Fátima Carneiro. Novos horizontes no tratamento médico do cancro gástrico. Semana Digestiva 2012. Porto, Portugal. 2012.
Fátima Carneiro. Lesões gástricas displásicas: visão anátomo-patológica. Semana Digestiva. Porto, Portugal. 2012.
Fátima Carneiro. Special pathological techniques in the management of gastric cancer. XXIX Congress of the International Academy of
Pathology. Cape Town, South Africa. 2012.
Fátima Carneiro. Inflammatory pathology and its consequences in the gut (chairperson). XXIX Congress of the International Academy of
Pathology. Cape Town, South Africa. 2012.
Fátima Carneiro. Molecular pathogenesis of gastric carcinoma (European Society of Pathology Symposium). XXIX Congress of the International Academy of Pathology. Cape Town, South Africa. 2012.
Fátima Carneiro. What is new in hereditary gastric cancer?. XXX Saltyrow Memorial Meeting. Zagreb, Croatia. 2012.
Fátima Carneiro. Tumour banks in Portugal. A One Day Symposium with Carlos Caldas sponsored by EACR. Porto, Portugal. 2012.
Fátima Carneiro. GAPPS syndrome: a new hereditary gastric cancer syndrome. Hereditary Cancer of the Digestive System in the VIIIth
International Course on Updated Surgical Pathology. Madrid, Spain. 2012.
Fátima Carneiro. Gastric dysplasia: immunophenotypic classification. Bertinoro, a meeting point for high grade dysplasia and early gastric cancer between East and West toward the 10th IGCC. Bertinoro, Italy. 2012.
Fátima Carneiro. Molecular pathology of gastric cancer. Bertinoro, a meeting point for high grade dysplasia and early gastric cancer
between East and West toward the 10th IGCC. Bertinoro, Italy. 2012.
Fátima Carneiro. Pathology of gastric cancer. First ESMO Preceptorship meeting on the multidisciplinary management of gastric cancer,
standards of care, targets and future perspectives. Berlin, Germany. 2012.
Fátima Carneiro. Gastric carcinogenesis. First ESMO Preceptorship meeting on the multidisciplinary management of gastric cancer,
standards of care, targets and future perspectives. Berlin, Germany. 2012.
Fátima Carneiro. Molecular events in gastric cancer. First ESMO Preceptorship meeting on the multidisciplinary management of gastric
cancer, standards of care, targets and future perspectives. Berlin, Germany. 2012.
Joana Paredes. Alvos moleculares – Dos mitos à realidade em cancro da mama. Actualizações em Oncologia 2012. Auditório dos Hospitais da Universidade de Coimbra. Coimbra, Portugal. 2012.
Joana Paredes. P-cadherin in Breast Cancer: a prognostic factor, an inducer of cancer cell invasion and metastasis and a therapeutic
target. II Workshop on Cancer Research: biological and molecular basis. IPATIMUP.. Porto, Portugal. 2012.
Joana Paredes. Tamanho do tumor: relevância biológica para a progressão e prognóstico do cancro da mama. VIII Congresso Nacional
de Senologia. Porto, Portugal. 2012.
José C. Machado. O impacto da biologia molecular na clínica. Jornadas Internacionais de Cirurgia. Lisboa, Portugal. 2012.
José C. Machado. Instabilidade de microssatélites e principais vias de sinalização: implicações para a terapêutic. Encontros da primavera
em Oncologia. Évora, Portugal. 2012.
José C. Machado. How does understanding cancer biology & genetics help in cancer prevention, detection & treatment?. 56ème Congrès du GIRSO. Penafiel, Portugal. 2012.
José C. Machado. Update on gastric cancer pathogenesis and risk factors. United European Gastroenterology Week. Amsterdam, The
Netherlands. 2012.
José C. Machado. IBD pathogenesis and microbiota: state of the art introduction. United European Gastroenterology Week. Amsterdam, The Netherlands. 2012.
José C. Machado. Non-optical massive parallel DNA sequencing: towards the diagnostic setting. Next Generation Sequencing: Applications in Genetic and Infectious Diseases. Lisbon, Portugal. 2012.
Fernando Schmitt. P-Cadherin in breast cancer: from research to clinical practice. University Health Network. Toronto, Canadá. 2012.
Fernando Schmitt. From the cells to the molecules: an overview of molecular applications on cytology. Massachusetts General Hospital
– Harvard Medical School. Boston, EUA. 2012.
Fernando Schmitt. From the cells to the molecules: an overview of molecular applications on cytology. Institut de Pathologie – University of Lausanne. Lausanne, Suiça. 2012.
Fernando Schmitt. Molecular Methods in Non GYN Cytology. International Tutorial of the International Academy of Cytology. Hong
Kong, China. 2012.
Fernando Schmitt. Cancro da mama: uma ou múltiplas doenças? Reflexões sobre prevenção e tratamento. 6ªs Jornadas de Educação
para a Saúde. Barcelos, Portugal. 2012.
Fernando Schmitt. Thyroid Cytology: Is FNA still the best diagnostic approach?. XIII Congresso Técnico de Anatomia Patológica. Figueira
da Foz, Portugal. 2012.
Fernando Schmitt. Molecular Cytopathology in the Breast. 53th Annual Spring Meeting of the Japanese Society of Clinical Cytology.
Tokyo, Japão. 2012.
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Activity Report
2012
Fernando Schmitt. Desvendando os Aspectos Moleculares em Citopatologia. XXII Congresso Brasileiro de Citopatologia. Recife, Brasil.
2012.
Fernando Schmitt. Novos Horizontes na Aplicação prática da Imunocitoquímica no Diagnóstico Citopatológico. XXII Congresso Brasileiro
de Citopatologia. Recife, Brasil. 2012.
Fernando Schmitt. Metastatic Cancer: Mechanisms and Opportunities for Treatment. 37th European Congress of Cytology. DubrovnikCavtat, Croácia. 2012.
Fernando Schmitt. Novas dianas terapêuticas no cancro de mama. XI Xornada sobre Cancro de Mama. Cangas, Espanha. 2012.
Fernando Schmitt. Cytopathology beyond borders 2012: Interventional Cytopathologists as front-line players in the Clinical Setting: an
across the Atlantic Perspective. 60th Annual Scientific Meeting of the American Society of Cytopathology. Las Vegas, USA. 2012.
Fernando Schmitt. Molecular marker analysis in Cytologic Specimens. 22nd Congress of the Turkish Society of Pathology. Antalya,
Turquia. 2012.
Fernando Schmitt. Endoscopic Ultrasound guided FNA of GI Tract and Pancreas: Cytology, Pitfalls and Clinical implications. 22nd Congress of the Turkish Society of Pathology. Antalya, Turquia. 2012.
Fernando Schmitt. Breast, Fine needle aspiration cytology. Workshop Fine Needle Aspiration Cytology. Faculty of Medicine, Jabriya.
Jabriya, Kuwait. 2012.
Fernando Schmitt. Recent advances in Lung cytopathology. Workshop Fine Needle Aspiration Cytology. Faculty of Medicine, Jabriya.
Jabriya, Kuwait. 2012.
Fernando Schmitt. Molecular Cytopathology in the Breast. Workshop Fine Needle Aspiration Cytology. Faculty of Medicine, Jabriya.
Jabriya, Kuwait. 2012.
Fernando Schmitt. Aspiration cytology of soft tissue tumors. Workshop Fine Needle Aspiration Cytology. Faculty of Medicine, Jabriya.
Jabriya, Kuwait. 2012.
Oral Presentations
Joana Carvalho, Patrícia M. Pereira, Nicole C. van Grieken, Gerrit Meijer, Raquel Seruca, Beatriz Carvalho, Manuel A. S. Santos, Carla
Oliveira. Can microRNAs regulate the E-cadherin-Catenin Complex in Gastric Cancer?. Portuguese RNA meeting 2012. Lisbon, Portugal.
2012.
Joana Carvalho, Giovanni Corso, Nicole C. van Grieken, Sónia Sousa, Tineke E Buffart, Carla Vindigni, Begoña Diosdado, Gerrit Meijer,
Manuel A. S. Santos, Franco Roviello, Raquel Seruca, Beatriz Carvalho, Carla Oliveira. Modulating the expression and function of Ecadherin in Gastric cancer: classical and alternative mechanisms. Second FMUP PhD students meeting. FMUP. . Porto, Portugal. 2012.
Renata Bordeira-Carriço, Daniel Ferreira, Denisa D. Mateus, Hugo Pinheiro, Ana Paula Pêgo, Manuel A. S. Santos, Carla Oliveira. Suppressor-tRNA restores functional E-cadherin expression in CDH1 mutant cancer cells: a potential approach to treat Hereditary Diffuse Gastric
Cancer. Second FMUP PhD students meeting. FMUP. Porto, Portugal. 2012.
Daniel Ferreira, Joana Carvalho, Patrícia Oliveira, Heike Grabsch, Gerrit Meijer, Nicole CT van Grieken, Raquel Seruca, Beatriz Carvalho,
Carla Oliveira. Systematic analysis of novel and previously characterized E-cadherin expression repressors/modulators in gastric cancer.
Third I3S Scientific Retreat. Póvoa do Varzim, Portugal. 2012.
Sérgia Velho and Kevin Haigis. Restoring apoptotic sensitivity in N-Ras mutant colorectal cancer cells by targeting IAPs. Center for Cancer Research retreat. Fallmouth, USA. 2012.
Ferreira RM, Machado JC, Figueiredo C. Variation in Helicobacter pylori cagA promoter region is associated with CagA expression in
Portuguese strains. XXVth International Workshop of the Helicobacter Study Group. Ljubljana, Slovenia. 2012.
Magalhães A, Marcos-Pinto R, Nairn A, dela Rosa M, Santos MR, Ferreira R, Bugaytsova J, Figueiredo C, Dinis-Ribeiro M, Carneiro F, Moremen K, David L, Borén T, Reis CA. Helicobacter pylori switches the host cells glycosylation pathways to remodel the gastric mucosa glycophenotype. 10th International Workshop on Pathogenesis and Host Response in Helicobacter Infections. Helsingør, Denmark. 2012.
Vieira AF, Ribeiro AS, Dionísio MR, Sousa B, Nobre AR, Albergaria A, Santiago-Gómez A, Schmitt F, Clarke RB, Paredes J. P-cadherin intracellular signalling is dependent on a6b4 integrin activation to induce breast cancer stem cell and invasive properties. 7th International
Meeting of the Portuguese Society for Stem Cells & Cell Therapies. Porto, Portugal. 2012.
Ribeiro AS, Albergaria A, Sousa B, Seruca R, Schmitt F, Paredes J. P-cadherin aberrant expression promotes cell migration and invasion
through activation of Rac1 and alteration of the cadherin/catenin complex in a Src-dependent way. Third I3S Scientific Retreat. Póvoa
do Varzim, Portugal. 2012.
Costa JL, Ribeiro C, Justino A, Fernandes R, Sousa S, Canedo P, Pina MJ, Cirnes L, Machado JC. Validation of next generation DNA sequencing for the molecular diagnosis of idiopathic hypertrophic cardiomiopathy. 16ª Reunião Anual da Sociedade Portuguesa de Genética Humana. Porto, Portugal. 2012.
Justino A, Dias P, Pina MJ, Ribeiro C, Sousa S, Cirnes L, Costa JL, Sousa AB, Machado JC. Non-optical massive parallel DNA sequencing
for the genetic diagnosis of the RAS/MAPK related syndromes. 16ª Reunião Anual da Sociedade Portuguesa de Genética Humana. Porto,
Portugal. 2012.
22
Activity Report
2012
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
The Structural Plasticity of the Human Genome: Chromosomal Inversions Revisited
We reviewed the landscape of human genomic structural variants paying special attention to balanced rearrangements. A particular
subtype of balanced rearrangement – inversions – was found to be far more common than had been predicted from traditional cytogenetics. Chromosomal inversions alter the orientation of a specific genomic sequence and, unless involving breaks in coding or regulatory
regions (and, disregarding complex trans effects, in their close vicinity), appear to be phenotypically silent. Such a surprising finding,
which is difficult to reconcile with the classical interpretation of inversions as a mechanism causing subfertility (and ultimately reproductive isolation), motivated a new series of theoretical and empirical studies dedicated to understand their role in human genome evolution and to explore their possible association to complex genetic disorders. We analysed the latest methodological improvements and
explored the possible implications of inversion rearrangements on the evolution of the human genome.
Linkage between HPRTB STR alleles and Lesch–Nyhan syndrome: no implications in forensic casework
Although located inside the coding gene, HPRTB STR polymorphism can safely be used for forensic purposes without revealing any
health risks of the subjects; no association could be established between STR alleles or
genotypes and LNS phenotype at a population level.
An alignment-free approach for sequence comparison based on suffix tree and L-words frequency
Sequence comparison usually requires an alignment step, requiring a number of assumptions on evolutionary history, and sometimes is
very difficult or impossible to perform due to the abundance of gaps (insertions/deletions). In such cases, an alternative alignment-free
method would prove valuable. Our method starts by a computation of a generalized suffix tree of all sequences, which is completed in
linear time. Using this tree, the frequency of all possible words with a preset length L-L-words--in each sequence is rapidly calculated.
Based on the L-words frequency profile of each sequence, a pairwise standard Euclidean distance is then computed producing a symmetric genetic distance matrix, which can be used to generate a neighbor joining dendrogram or a multidimensional scaling graph. We
present an improvement to word counting alignment-free approaches for sequence comparison, by determining a single optimal word
length and combining suffix tree structures to the word counting tasks. Our approach is, thus, a fast and simple application that proved
to be efficient and powerful when applied to mitochondrial genomes.
Comparing the performance of alternative batteries of genetic markers for kinship investigations
Whenever the kinship under forensic investigation is remote or the alternative hypotheses are deficiently formulated due to the lack
of information (e.g. an unknown relationship between alleged and true father), beyond the routinely used marker set, other types of
markers are analysed. We compare d the results obtained after complementing an initial set of autosomal STRs with indels or with Xchromosome-specific STRs in simulated paternity cases where the alleged father is a close relative of the real one. Results show that in
paternity cases where a low number of incompatibilities are observed, the best strategy is to increase the number of autosomal STRs
under analysis. Nevertheless, if these are not available, we show that in father-daughter duos, a set of 12 X-STRs is more advantageous
than 38 highly diverse autosomal biallelic markers. Additionally, the usefulness of X-STRs was also evaluated in cases where only a close
relative of the alleged parent (father or mother) is available for testing. For those situations where these markers have the power to
exclude, strong LR values are obtained. In the remaining cases, LRs are usually weak and sometimes the results are more likely under
the wrong kinship hypothesis.
Development and applications of genetic tools for domesticates, their wild relatives and their parasites
A protocol for the multiplex PCR amplification and capillary electrophoresis of nine autosomal STRs and two fixed-size markers for sex
identification in dogs and wolves is described, complying with the recommendations of the International Society for Forensic Genetics.
Mitochondrial DNA diversity of the most important olive tree (Olea europaea) pest, the olive fly (Bactrocera oleae) was comparatively
analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the world. Strong population
substructure was found in the Central and Western Mediterranean area, a fact that can be instrumental in management and control
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Activity Report
2012
strategies for the pest.
An analytical tool for food control and authentication of dairy products manufactured from the milk of cow, sheep, goat, and buffalo
was developed, based on multiplex polymerase chain reaction (PCR) of species-specific mitochondrial DNA (mtDNA) targets followed by
fragment size analysis by capillary electrophoresis. The method includes (a) simultaneous detection of four species, (b) internal control
for DNA extraction and PCR, (c) mtDNA as a target for PCR, (d) amplicons of <200 bp, and (e) flexibility in the electrophoresis and fragment size detection method and proved able to detect an at least 1% (v/v) relative proportion of milk in binary mixtures.
Identification of Aspergillus fumigatus and discrimination from other pathogenic species within section Fumigati
Our microsatellite-based PCR multiplex previously designed for A. fumigatus was tested in a set of species belonging to section Fumigati, proving to be able of discriminating other pathogenic species within section Fumigati.
A multifunctional workbench for species identification using insertion/deletion variations
Molecular identification of species currently is based on pairwise sequence divergences between the query and reference sequences
(DNA barcoding). We have recently shown that a high level of species discrimination is attainable in all taxa of life simply by considering the length of hypervariable regions defined by insertion/deletions. We now describe a multifunctional computational workbench
(named SPInDel for SPecies Identification by Insertions/Deletions) to assist researchers providing a step-by-step environment for the
alignment of target sequences, selection of informative hypervariable regions, design of PCR primers and the statistical validation
of the species-identification. Our method can complement conventional DNA barcoding systems without the required step of DNA
sequencing.
Development of genetic markers suitable for degraded samples
Short amplicon genetic markers (SNPs and Indels) were used developed in order to genotype difficult samples, namely bones of 35 years
“post-mortem”. Results compared favourably with those obtained from standard commercial STR and mini-STR DNA typing kits making
this development especially useful in missing person and disaster victim identification.
A software to automatically assign human mtDNA sequences to haplogroups
We developed an automated process (mtDNAoffice software) to determine distances between mtDNA sequences allowing their subsequent clustering and haplogroup assignment increasing the speed of data analysis and avoiding human errors. The method uses the
protein coding region and a vectorial representation method and was made freely available on the web.
Identification of a highly frequent pyruvate kinase missense mutation and association with malaria
We have studied the occurrence of PK deficiency in several areas of Africa and a common PKLR mutation G829A (Glu277Lys) was identified. A global geographical co-distribution between malaria and high frequency of PK deficiency suggests that malaria may be a selective force raising the frequency of this variant.
The origins of a male lineage shared between Europeans and Africans
Human Y chromosomes belonging to the haplogroup R1b1-P25, although very common in Europe, are usually rare in Africa. High frequencies of this haplogroup in central-western Africa suggested a ‘back-to-Africa’ migration during prehistoric times. We characterised
the paternal genetic background of a population in Equatorial Guinea, a Central-West African country located near the region in which
the highest frequencies of the R1b1 haplogroup in Africa have been found to date. The frequency of the R1b1 haplogroup in our sample
(17.0%) was higher than that previously observed for the majority of the African continent, nine of these harbouring the V88 marker,
recently discovered in Africa. As high microsatellite variance was found inside this haplogroup in Central-West Africa and a decrease towards Northeast Africa, our findings do not support the previously hypothesised movement of Chadic-speaking people from the North
as the explanation for these lineages in Central-West Africa. Our findings are best explained by an origin of the V88-derived allele in the
Central-West Africa, and its presence in North Africa resulting from a migration from the south during the mid-Holocene.
Inference of ancestry and admixture proportions through insertion/deletion polymorphisms
A panel of 46 ancestry-informative insertion/deletion polymorphisms selected to measure population admixture proportions of four
different origins (African, European, East Asian and Native American) was developed and tested. The set showed to be highly efficient at
inferring the ancestry of individuals and providing good estimates of ancestry proportions at the population level.
Deterministic protein inference from peptides assigned to mass spectrometry data
We present here SIR (spectra based isoform resolver) that uses a novel transparent and systematic approach for organizing and presenting identified proteins based on peptide spectra assignments. The algorithm groups peptides and proteins into five evidence groups and
calculates sixteen parameters for each identified protein that are useful for cases where deterministic protein inference is the goal. The
novel approach has been incorporated into SIR which is a user-friendly tool only concerned with protein inference based on imports of
Mascot search results. SIR has in addition two visualization tools that facilitate further exploration of the protein inference problem.
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Activity Report
2012
Carbamoyl phosphate synthetase I: the structural role of the unknown function domains
CPSI is organized into six domains, three of which have no established role in the mammalian enzyme. Taking advantage of the high
degree of conservation between the human and the Escherichia coli homologue a comparative study was carried out in order to infer
about the biological role of these less characterized domains. We show that among the residues involved in the maintenance of quaternary structure of the E. coli enzyme, several are highly conserved between human and bacterial enzyme and match the homologous
positions of the “unknown function” domains in human enzyme, suggesting they are involved in the structural stability of the human
enzyme as they are in bacteria.
Unraveling the origin of Machado-Joseph disease in the Australian Aboriginal communities
The presence of Machado-Joseph disease (spinocerebellar ataxia type 3, SCA3 among Australian aborigines has been explained either
by a new mutational event or by the introduction from other populations. We have shown that an extended haplotype is shared by Australian aborigines and some Asian families suggesting a common ancestor for all, dating back more than 7000 years.
Reconstruction of the population history of European Romani
We expanded and deepened our previous studies on Gypsies, now using genome-wide data and X-chromosome markers. The Romani
diaspora was shown to stem from a single initial founder population from north/northwestern India ~1.5 thousand years ago (kya). Our
results further indicate that after a rapid migration with moderate gene flow from the Near or Middle East, the European spread of the
Romani people was via the Balkans starting ~0.9 kya. Our study also revealed high levels of population substructure and homozygosity
as well as differential gene flow in time and space with non-Romani Europeans. We further present evidence for asymmetries in female
and male effective population sizes during the admixture.
Mitochondrial DNA deletions are associated with non-B DNA conformations
We have shown that site-specific breakage hotspots exist in the mtDNA and that the most frequent deletion breakpoints occur within or
near predicted non-B DNA conformations. Moreover, there is a significant association between the folding energy of an mtDNA region
and the number of breakpoints it harbours.
Unraveling the mechanisms underlying the transcriptional regulation of the human mitochondrial peptide deformylase
In previous work we clarified how the gene overlap – a very rare event in eukaryotic genomes - between PDF and COG8 has occurred. We
have now investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF
has several transcription start sites, the most important of which only 18 bp from the initiation codon. A 97 bp minimal promoter region
for human PDF, capable of very strong transcriptional activity, contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription
factor, and possibly of other members of the Sp family. Since the entire minimal promoter region is located after the end of COG8’s coding region, human PDF most likely preserves an independent regulation from its overlapping partner.
Internationalization/Networking
Cintia Alves
Member of the Executive Committee of the Spanish and Portuguese Speaking Working Group of the International Society for Forensic
Genetics (GHEP-ISFG)
GEMINI - Genetics of Male Infertility Initiative
Alexandra Lopes is one of the founding members of this international network for research on the genetics of male infertility, launched
in April 2012 at the American Society of Andrology.
Antonio Amorim
National Contact Point of EUROFORGEN-NoE - European Forensic Genetics Network of Excellence
Section of Forensic Genetics, Univ. Copenhagen, Denmark (Morling N, Tomas C)
X chromosome in population genetics and forensics
Laboratory of Molecular Genetics, Immunology and Human Pathologies, Faculty of Sciences, University of Tunis El Manar, Tunisia (Khodjet-el-Khil H)
Population profiling of North Africa
Laboratório de Genética Humana e Médica, Universidade Federal do Pará, Belém, Brazil (Santos SE)
Ancestry markers and admixture
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Activity Report
2012
DNA Diagnostic Laboratory - Institute of Biology, State University of Rio de Janeiro, UERJ, Brazil (Carvalho EF)
Population profiling, ancestry markers and admixture
Max Planck Inst. Biology of Ageing, Cologne, Germany (Stewart JB)
Mitochondrial DNA
Center for Human Genetic Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA (Samuels
DC)
Mitochondrial DNA
Department of Electronics and Telecommunications, University of the Basque Country, UPV/EHU, Spain (Prieto
G)
Bioinformatics
Proteomics Core Facility-SGIKER, University of the Basque Country, UPV/EHU, Spain (Aloria K)
Proteomics
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, UPV/
EHU, Spain (Fullaondo A)
Proteomics
Department of Biochemistry and Molecular Biology, University of the Basque Country, UPV/EHU, Spain (Arizmendi JM)
Proteomics
Departamento de Toxicología y Legislación Sanitaria, Facultad de Medicina, Universidad Complutense de Madrid, Spain (Arroyo-Pardo E)
Y chromosome
Faculdade de Medicina, Universidade Eduardo Mondlane, Maputo, Mozambique (Fernandes N)
Malaria
Banco de Sangue do Hospital Central de Maputo, Maputo, Mozambique (Langa L)
Malaria
Hospital Pediátrico David Bernardino, Luanda, Angola (Miranda J)
Malaria
Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, Madrid, Spain (Cano J)
Malaria
Genetic Laboratory, Universidad Industrial de Santander, Bucaramanga, Colombia (Gil A)
Linkage in forensics
Norwegian University of Life Sciences, IKBM, Aas, Norway (Egeland T)
Biostatistics and forensics
Faculty of Medicine of Monastir, Monastir, Tunisia (Jaafar N)
Molecular genetics of inborn errors of metabolism
Department of Forensic Molecular Biology, Erasmus MC University Medical Center Rotterdam, The Netherlands (Kayser M)
Population genetics of Gypsies, forensics
Departament de Ciències de la Salut i de la Vida, Institut de Biologia Evolutiva, Universitat Pompeu Fabra, Barcelona, Spain (Comas D)
Population genetics of Gypsies
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Activity Report
2012
Forensic Genetics Unit, University of Santiago de Compostela, Spain (Carracedo A)
Ancestry markers and admixture; X chromosome in forensics
Department of Zoology, University of Oxford, Oxford, UK (Capelli C)
Y chromosome
Leonor Gusmão
Member of the Executive Committee of the International Society for Forensic Genetics (ISFG)
Future Research
Linguistic and religious isolates in northeastern Portugal: new insights from Genetics and Demography
We aim to understand the mating and reproductive strategies of two culturally isolated populations, one of linguistic basis (Leonese
dialects speakers) and the other due to its past religion affiliation (crypto-Jews descendants) by analyzing their demographic and genetic structure and contextualizing their genetic profiles within the present day population of the Bragança district (Trás-os-Montes
province, NE Portugal) where both minorities can be found. Specifically we intended to analyze a representative sample of all twelve
municipalities in the district, paying special attention to the Leonese dialects speakers (living in the Miranda do Douro municipality) and
the Crypto-Jews communities (scattered over the region: Bragança town, Argoselo, Mogadouro, Carção and Vilarinho dos Galegos).
Genetic determinants of male fertility
Although environmental factors may contribute to male subfertility, the complexity of pathways involved in spermatogenesis implies
that an appreciable number of genes should play a role. Most of the efforts to understand the genetic basis of impaired spermatogenesis have been focused on the Y chromosome. We have performed a genome-wide search for structural variants in individuals with
azoospermia in order to evaluate the contribution of presently unknown genetic defects to severe spermatogenic impairment and will
explore the data to unravel yet unidentified genetic determinants of male fertility.
Development of a PCR multiplex system to genotype markers of pharmacogenetic importance.
We aim to develop a multiplex PCR system to simultaneously genotype ten single nucleotide polymorphisms of established pharmacogenetic relevance present in the following genes: CYP2C9, CYP2D6, CYP3A4, VKORC1, NAT2 and TPMT.
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. In this study we intend to identify these mutations on MSUD Tunisian
patients.
The influence of different sampling strategies in lineage marker analysis in Native Colombian populations
With this work we aim to provide new insights on the distribution and diversities of the main Amerindian mtDNA and Y chromosome
haplogroups within Colombia. Given the particular history of South America in what concerns the maintenance of restricted Amerindian
reserves whilst still subjected to the introduction of post-European colonization populations, we also focus on understanding how the
different sampling strategies and the different characteristics of the sampled populations influence the distribution and diversities of
these uniparental transmission lineages in Colombia.
NUMT polymorphism and the history of human populations
Insertions of mitochondrial DNA (mtDNA) sequences into the nuclear genome (NUMTs) have been reported for many Eukaryotes. Since
NUMT insertion is a continuous process, some NUMTS are shared with other primates while others are human-specific, and in this last
group a few have been reported as polymorphic in human populations. Here we propose to give a new perspective of the history of human populations and of the genome evolution through the analysis of NUMT polymorphism at different levels: presence/absence polymorphism in human populations, internal polymorphism and flanking region polymorphism. We thus aim to demonstrate that NUMTs
are a valuable tool in the genetic analysis of human populations, by making available for the scientific community a wide knowledge of
the variation of these unique sequences and their flanking regions and by clarifying specific questions in the fields of population genetics and genome evolution.
Genetic history of South American populations
We intend to continue and deepen the genetic characterization of extant South American populations in order to reconstruct the history of male and female lineages through mtDNA and Y chromosome analyses.
Comparative behaviour of autosomal and X-chromosomal paternity exclusion power in standard and deficient
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Activity Report
2012
cases with inbreeding
We intend to study the behaviour of statistic exclusion power in situations where the randomness assumption is not met, because either
(a) the alleged - and false - father is related to the true one, or (b) there is background relatedness in the population.
Phylogeny and Worldwide Dispersion of Closely Related Nematode Species, Bursaphelenchus xylophilus (the
‹pinewood nematode›) and B. mucronatus
Using nuclear and mitochondrial DNA markers, we intend to study B. xylophilus and B. mucronatus phylogeography, sampling areas
comprising SW Europe, America and E Asia.
The evolutionary constraints in the ß-globin cluster: the paradox of the conservation of the delta-globin locus
We intend to show, using an evo-devo approach, that the surprising degree of conservation of the delta-globin locus is associated with
the regulation of the fetal/adult switch in gene expression.
Molecular Dynamics Simulations on Short Tandem Repeats
Microsatellites, or short tandem repeats (STRs) show a high mutation rate but the role that strand-slippage replication mechanisms
have on the process is unclear. We intend to perform molecular dynamics
simulations in tetranucleotide motif STRs from the Y chromosome (to avoid recombination interference).
Phylogeography and population structure of the powdery mildew fungus, Erysiphe necator, from Vitis cultivars
in Portugal
We aim to determine the population structure and phylogeny of the grapevine pathogenic fungi Erysiphe necator(powdery mildew) in
Portugal using molecular markers.
Hydrogen peroxide-induced cell death in Aspergillus fumigatus conidia
Aspergillus fumigatus conidia have been linked to a wide range of respiratory and allergic problems mainly in immunocompromised
patients. We are developing a methodology to detect cell death markers in fungi adapted to conidia cells, where the mechanism of
hydrogen peroxide-induced cell death is studied for the first time in A. fumigatus conidia
Aging in conidia of pathogenic fungi: an interspecies comparison
A. fumigatus, A. flavus, A. terreus, A. niger, and A. nidulans are amongst the species with more impact on human health. A methodology to study the mechanisms involved in cell death in fungi adapted to conidia cells is used to comparatively analyze these species.
From this comparison we expect to get insights into the details of cell death machinery providing a better knowledge of the molecular
mechanisms involved in conidia-mediated toxicity to the respiratory tract, and consequently provide research guidelines for therapeutic approaches.
Participation in PhD Programs
Alexandra Lopes, António Amorim - GABBA - Graduate Program in Areas of Basic and Applied Biology, UP - PhD student supervision (Ana
Cristina Lima, since September 2012)
Leonor Gusmão - Doutoramento em Ciências da Saúde no ramo de Ciências Biomédicas; Faculdade de Medicina da Universidade de
Coimbra; thesis supervision - Ana Mónica de Oliveira e Silva Rodrigues Garcia Ramos de Carvalho; (‹Identificação de linhagens humanas
através do estudo do ADN mitocondrial e do cromossoma Y e sua aplicação à genética forense›; 2012) .
Leonor Gusmão - Facultad de Ciencias Exactas y Naturales. Universidad de Antioquia. Medellin, Colombia; thesis supervision. Adriana
Alexandra Ibarra Rodríguez (since 2011)
Antonio Amorim - GABBA UP - Coordination and lecturing - Formal and Population Genetics module - Biology, FCUP, Director and supervisor.
António Amorim - Programa Pos-Graduação em Biociências – UERJ, Rio de Janeiro, Brazil; module «Biodiversidade, Genética e Forense».
nov/dez2012
Antonio Amorim - Matemática Aplicada FCUP; theses supervision - Nádia Maria Gonçalves de Almeida Pinto (‹General Algorithms for
computing genetic kinship likelihoods›, 30/07/2012) - Maria Inês Gomes Soares (‹Construction of algorithms to model phylogenetic trees›,
20/06/2012)
Leonor Gusmão - Biologia (FCUP) theses supervision: Maria Inês Nogueiro (with Antonio Amorim), Cristina Valente and Vania Pereira
(with Mª João Prata)
Luísa Azevedo - Biomedicina, Faculty of Medicine of the University of Porto; Thesis co-supervision. Isabel Pereira de Castro (“Unravelling
the function and regulation of human NLZ1 and COG8/PDF”, 22/11/2012)
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Activity Report
2012
Participation in Master Programs
António Amorim - Forensic Genetics UP - Coordination and lecturing: modules Forensic Genetics’, Non-Human Applications
Alexandra Lopes - Molecular and Celular Biology, FCUP - lecturer for the Molecular Diagnostics module
Alexandra Lopes - Molecular Biomedicine, U. Aveiro; thesis supervision (Catarina Seabra).
Sofia Quental - Molecular and Celular Biology, FCUP; lecturer for the Molecular Diagnostics module
Raquel Silva - Molecular Biomedicine, School of Health, University of Aveiro; thesis supervision (Mariana Isabel Gonçalves Correia) - and
co-supervision (Ariana Borges Moutinho)
Luis Fernandez - Forensic Genetics, FCUP; Invited lecture and theses co-supervision (Sofia Marques and Joana Pinto)
Ana Goios - Forensic Genetics, FCUP; Invited lecture and theses supervision (Catarina Xavier) and co-supervision (Ana Mafalda Rocha)
Sofia Quental - Forensic Genetics, FCUP; thesis co-supervision Marisa Sofia Sousa Oliveira)
Sandra Martins - Molecular Biomedicine, U. Aveiro; thesis supervision (Maria Inês Martins).
Sandra Martins - Molecular and Celular Biology, FCUP - lecturer in Molecular Diagnostics module
Cíntia Alves - Forensic Genetics, FCUP; Invited lecturer and theses supervision (Andreia Filipa Cabral de Melo and Ana Mafalda Rocha)
Ricardo Araújo - Forensic Genetics, FCUP; thesis co-supervision (Alexandre Almeida)
Leonor Gusmao / Antonio Amorim - Forensic Genetics FCUP; Supervision of Lidia Birolo thesis ‹Análise de 15 loci STRs autossómicos na
população do estado do Acre, norte do Brasil ‹ presented 12/12/2012
Maria João Prata - Forensic Genetics FCUP, Director and Lecturer - Cell and Molecular Biology, FCUP, Lecturer
Leonor Gusmão - Forensic Genetics FCUP, theses supervision (Catarina Xavier, with Ana Goios Ana Mafalda Rocha, with Cintia Alves)
Maria João Prata - Forensic Genetics; FCUP; thesis supervison Marisa Oliveira
Maria João Prata - Biodiversity, Genetics and Evolution, FCUP; thesis supervision Joana Pinto
Luísa Azevedo - Forensic Genetics FCUP, thesis supervision Ana Carolina Carlos Teixeira da Silva
Prizes
Antonio Amorim / Leonor Gusmão/ Cíntia Alves - Seed of Science Special 2012 - As a recognition for the ranking among the top ten publishing groups in Legal Medicine and Forensic Sciences in the ISI Web of Knowledge
Invited Talks
Alves JM - . Chromosomal inversions: Evolutionary brakes and/or accelerators?. IV Symposium on Bioengineering. Porto, Portugal.
23/11/2012.
Cíntia Alves - . GHEP-ISFG 2012 Collaborative Exercise Results – Non-human sample M7. XVII Jornadas GHEP-ISFG. San Andres, Colombia.
05/06/2012.
Cíntia Alves - . Quantiplex and ESSplex Plus for Human ID – IPATIMUP’s Experience. Encontro Forense (Forensic Meeting). Workshop
organized by Izasa and Qiagen. Histocompatibility Centre in Coimbra, Portugal. 30/10/2012.
Antonio Amorim - . Genética Forense. VI Jornadas de Ciências do ISCS-N “O Crime no séc. XXI”, Museu de Penafiel, 20-21/04/2012 - .
Penafiel. 20/04/2012.
Antonio Amorim - . Genética e Evolução, Lda - . Projeto / Ciclo de Conferências «Ler Ciência» . Biblioteca Municipal Vila Nova de Cerveira.
01/03/2012.
Antonio Amorim - . Biodiversidade - de que estamos falando?. ciclo de palestras «Ciência ao Meio-Dia». UERJ, Rio de Janeiro, Brazil .
06/12/2102.
Leonor Gusmão - . Linajes paternos en poblaciones de África subsariana: las migraciones Bantu y Nilota. . Universidad Nacional de Colombia. Bogotá, Colombia. 23/02/2012.
Leonor Gusmão - . Relevância dos Marcadores dos cromossomos sexuais em identificação genética. . IV Seminário Nacional de DNA e
Laboratório Forense. Associação Brasileira de Criminalística. . Cuiabá, Brazil. 10/05/2012.
Leonor Gusmão - . Origen y dispersión del hombre moderno. . Universidad Industrial de Santander. Bucaramanga, Colombia. 04/06/2012.
Leonor Gusmão - . Avaliação e intrepretação da prova pericial sobre DNA frente aos tribunais: Intrepretação do laudo.. Centro de Perícias Científicas Renato Chaves. Polícia Científica - Instituto Médico legal. . Belém, Pará, Brazil. 18/12/2012.
Luísa Azevedo - . Origin and loss of eukaryotic genes: a dynamic process undelying genome variability. Scientific seminar. CIIMAR, Porto.
26/01/2012.
Oral Presentations
Mairal, Q; Santos, C; Prata, M; Aluja, M; Amorim, A; Alvarez L.. Linguistic isolates in Portugal: insights from the mitochondrial DNA pat-
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Activity Report
2012
tern. DNA in Forensics 2012: Exploring Phylogenies. Innsbruck, Austria. 06/09/2012.
Silva M, Prata MJ, Amorim A. Alvarez L. Sequence diversity analysis of the human mtDNA control region: comparative analysis in two
populations from the Portuguese-Spanish border, en IV Jornadas Nacionais de Genética e Biotecnologia. IV Jornadas Nacionais de Genética e Biotecnologia. UTAD. 01/03/2012.
Seabra C. Validation of rare structural variants in Portuguese azoospermic patients. XV Portugaliae Genetica. Porto, Portugal. 22/03/2012.
Xavier C.. Insights into South-American colonization through mtDNA analysis in Colombian populations. 15th Portugaliæ Genetica. Porto, Portugal. 22/03/2012.
Moleirinho A.. Study of Hemoglobin A2: the paradox of d-globin gene conservation and its supposed physiological irrelevance.. EMPSEB
18. Virrat, Filand. 26/09/2012.
Moleirinho 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 . ENBE VIII. Oeiras, Portugal. 21/12/2012.
Nogueiro, I.; Alvarez, L.; Teixeira, J.; Gusmão, L.; Amorim, A.. Portuguese Jews after Inquisition: genetics and self-awareness. 32nd IAJGS
International Conference on Jewish Genealogy. Paris, France. 15/07/2012.
Marques, S.; Prata, M.J.; Gusmão, L.; Amorim, A.; Alvarez, L.. Leonese dialects in Portugal: linguistic-genetic relationships through Y
chromosome analysis. 15th Portugaliæ Genetica. Porto, Portugal. 22/03/2012.
Alves JM. Chromosomal inversions: Evolutionary brakes and/or accelerators?. 15th Portugaliæ Genetica. Porto, Portugal. 21/03/2012.
Melo F, Amorim A, Alves C. The New European Standard Set of Forensic Genetic Markers in the Portuguese Population. IJUP’12 – 5º Meeting of Young Researchers from the University of Porto. University of Porto, Porto, Portugal. 23/02/2012.
João Carneiro. Filipe Pereira, António Amorim, Irina de Sousa Moreira, Maria João Ramos
“The Single-Stranded Structural Conformations of STRs during Replication”. Cycle of Conferences 2012 - Theoretical Chemistry and
Computational Biochemistry Group. DQB/Faculty of Sciences, University of Porto Room A4. 16/05/2012.
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Activity Report
2012
Cancer Biology
Objectives
The main objective of the group is to progress in the understanding of the etiopathogenesis of some types of endocrine and neuroendocrine tumours with an emphasis on well differentiated thyroid cancer.
Within this frame, a special attention is paid to:
a) alterations in tyrosine kinase receptors and signal transducing molecules involved in the mitogen-activated protein kinases (MAPK)
pathway;
b) metabolic alterations secondary to mitochondrial DNA mutations/deletions or to mutations in nuclear genes encoding mitochondrial
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 and other neuroendocrine tumours
In the translational field, we have pursued the collaborative project (IPATIMUP-IPO-HSJ) initiated in the end of 2008. This study aims to
perform a thorough clinico-pathological re-evaluation of all cases of well differentiated thyroid carcinoma diagnosed and treated in the
three institutions that showed an aggressive behaviour.
In this setting we have evaluated the putative relationship between functioning TGF-beta/Smad-dependent pathway and BRAF mutation in PTC and we verified that nodal metastases are associated with poorly circumscribed, locally invasive PTCs that exhibit low levels
of nuclear Smad7 and a peripheral EMT phenotype. These invasive PTC display TGF-beta overexpression, regardless of the occurrence of
BRAF mutation. (Catarina Eloy PhD thesis and Eloy C et al, VA, 2012).
In the characterization of unique cases of thyroid tumours that we receive as consultation/collaboration we had the opportunity to
publish in 2012 three papers in collaboration with Cameselle-Teijero (Cameselle-Teijeiro et al, 2012, AJCP, AJCP and IJSP). The most relevant results refer to CDX2 expression in some variants of PTC, supporting the hypothesis, advanced many years ago by our group, of
an intestinal line of differentiation for PTCs, in contrast to an endocrine-like line for follicular carcinomas and a neuroendocrine-like line
for medullary carcinomas (Cameselle-Teijeiro et al,2012, AJCP).
Dissecting molecular pathways for therapeutic applications
Strategies for molecular-targeted therapy have to address not only the oncogenic pathways that are active in thyroid tumour cells, but
also the tumour-stroma interactions that support them. We decided to explore such tumour-stroma crosstalk, by studying the role and
regulation of STAT3, a pleiotropic transcription factor that mediates IL-6 cytokine family signaling, as well as growth factor signaling,
which are both important contributors to the tumor microenvironment.
Although tyrosine-phosphorylated or activated STAT3 (pY-STAT3) is a well described mediator of tumourigenesis, its role in thyroid
cancer remained to be investigated. Our studies demonstrate that, in the context of thyroid cancer, STAT3 is paradoxically a negative
regulator of tumour growth. These findings suggest that targeting STAT3 in these cancers could enhance tumour size and highlight the
complexities of STAT3’s role in tumourigenesis. (Joana Couto Silva, PhD thesis and Couto JP et al, PNAS, 2012).
Following our preliminary studies exploring signaling pathways activated by oncogenes, we tested the therapeutic effects of inhibiting
alternative pathways in thyroid cancer. Persistent RET activation is a frequent event in papillary thyroid carcinoma (PTC) and medullary
thyroid carcinoma (MTC) being able to activate the ERK/MAPK, the PI3K/AKT/mTOR and the JAK/STAT3 pathways. We tested the efficacy
of a JAK1/2- inhibitor, AZD1480, in the in vitro and in vivo growth of thyroid cancer cell lines expressing oncogenic RET. AZD1480 effectively blocks proliferation and tumour growth of activated RET- thyroid cancer cell lines, likely through direct RET inhibition in cancer
cells as well as by modulation of the microenvironment (e.g. via JAK/phospho-STAT3 inhibition in endothelial cells). We suggested that
AZD1480 could be considered as a therapeutic agent for the treatment of RET- activated thyroid cancers. (Joana Couto Silva, PhD thesis
and Couto JP et al, PlosOne, 2012).
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Activity Report
2012
Dissecting molecular pathways for therapeutic applications
To explore the potential use of MTOR inhobitors in thyroid cancer we characterized the expression and activation of the PI3K/AKT/
mTOR pathway in a series of human thyroid tumour specimens. We observed that the PI3K pathway was particularly activated in human
BRAFV600E-positive PTC cases, as well as in in vitro models, and demonstrated that BRAFV600E- mediated phosphorylation and inactivation of the tumour suppressor liver kinase B1 (LKB1) could mediate PI3K activation. Furthermore, we demonstrated that ERK/MAPK
over-activation was a strong indicator of sensitivity to mTOR pathway inhibition by rapamycin.( (Joana Couto Silva, PhD thesis, Faustino
A, et al, JCEM, 2012, Pópulo H, et al, Expert Opin Ther Targets, 2012 and Pópulo H, et al Int J Mol Sci 2012).
Dissecting molecular pathways – Fusion genes in thyroid tumours
In collaboration with the Department of Cancer Prevention of the Institute for Cancer Research of the Norwegian Radium Hospital of
Oslo University we used an oligo microarray-based approach for simultaneous analysis of all known and predicted fusion gene variants
in isolated RNA from 27 thyroid tumours. Within the thyroid tumours tested, only well known, previously reported fusion genes in thyroid oncology were identified. Our results reinforce the pathogenic role played by RET/PTC1, RET/PTC3, and PAX8-PPARG fusion genes in
thyroid tumourigenesis (Celestino R, PhD thesis and Celestino R, et al, Genes, Chromosomes & Cancer,2012).
The molecular alterations underlying follicular Hürthle cell carcinomas (FHCC) are largely unknown. In an attempt to clarify this issue,
we analyzed a series of Hürthle cell tumours for the presence of RET/PTC and PAX8/PPARG rearrangements and BRAF, HRAS and NRAS
mutations. Our study disclosed a significant association between RET/PTC rearrangement and FHCC with a solid growth pattern, raising
the possibility of using tyrosine kinase inhibitors for treating patients with FHCC which are often refractory to radioiodine treatment
(Celestino R, PhD thesis and de Vries MM & Celestino R, et al, Histopathology, 2012).
Mitochondrial alterations and cancer - Role of mtDNA mutations in tumourigenesis and in oncocytic transformation
The presence of somatic mitochondrial DNA (mtDNA) mutations in cancer cells has been interpreted in controversial ways, ranging from
random neutral accumulation of mutations to positive selection for high pathogenicity, or even conversely to negative selection of high
pathogenicity variants as occurs at the population level.
We evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compared these with the distribution
of variations observed in the global human population and all possible protein variations that could occur in human mtDNA. We focused
on oncocytic tumours which are clearly associated with mitochondrial dysfunction. The protein variant pathogenicity was predicted
using two computational methods, MutPred and SNPs&GO. The pathogenicity score of the somatic mtDNA variants were significantly
higher in oncocytic tumours compared to non-oncocytic tumours. Variations in subunits of Complex I of the electron transfer chain
were significantly more common in tumours displaying a oncocytic phenotype, while variations in Complex V subunits were significantly
more common in non-oncocytic tumours.
Our results show that the somatic mtDNA mutations reported over all types of tumours are indistinguishable from a random selection
from the set of all possible amino acid variations, and have therefore escaped the effects of purifying selection that act strongly at the
population level. We show that the pathogenicity of somatic mtDNA mutations is a determining factor for the oncocytic phenotype.
(Pereira L, et al, BMC Cancer. 2012)
Mitochondrial alterations and cancer- Role of GRIM-19 in human tumourigenesis
In order to clarify the role of GRIM-19 protein in tumourigenesis as well as in oncocytic phenotype development, and its interaction
with STAT3 protein, we assessed GRIM-19 expression and cellular location, by immunohistochemistry, in a series of thyroid and kidney
tumours, with oncocytic and non-oncocytic features, and correlated these data with STAT3 expression, cellular localization and activation. We also started an in vitro comprehensive study of the role of GRIM-19 downregulation, in order to understand the mechanisms by
which GRIM-19 exerts its tumour suppressor activity.
In thyroid tumours we saw that GRIM-19 is specifically downregulated in Hürthle cell tumours, corroborating the hypothesis that mitochondrial respiratory chain complex I is impaired in Oncocytic tumours. In renal cell tumours we observed that GRIM-19 is downregulated in all tumour histotypes, suggesting that GRIM-19 has a broader role in kidney tumourigenesis. Furthermore, in renal cell tumours,
we observed that phosphorylated forms of STAT3 were downregulated.
In in vitro studies, using cell lines from two clear cell renal cell carcinomas; we saw that GRIM-19 play an important role in cytoskeleton
and mitochondrial network organization. We also found that GRIM-19 downregulation alters cell metabolism, inducing a glycolytic phenotype.(Cameselle-Teijeiro J,et al, Am J Clin Pathol, 2012).
Mitochondrial alterations and cancer- Tumourigenic and metabolic effects of mitochondrial dysfunction
In this project we aimed to prove that OXPHOS dysfunction, caused by mutations in mtDNA or nuclear-encoded OXPHOS genes, underlies the Warburg effect in tumour cells and is essential for the survival of tumour cells, leading to the acquisition of phenotypic properties associated to tumour progression.
We have built a model of OXPHOS dysfunction, where we have 143B parental cells, the 143B¿0 (derived from 143B after mtDNA depletion), cybrids with WT mtDNA (normal OXPHOS, control cells), cybrids with a mutation in the tRNAleu (tRNA cybrid, defective OXPHOS)
and cybrids containing a mutation in ND gene1 (ND1 cybrid, defective OXPHOS).
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Activity Report
2012
After showing that tRNA cybrids display higher motility and higher migration capacity than WT cybrids, as seen by wound healing assays
and single-cell motility, we demonstrated also that tRNA cybrids showed increased levels of activated integrin-beta1 and fibronectin.
Experiments are under way to prove that the increased motility and migration are due to increased activated integrin beta1.
Using the same cell line models with OXPHOS dysfunction, we started a study aiming to unveil how pathogenic mutations in mtDNA
and/or mutations in nuclear genes encoding mitochondrial proteins may lead to epigenetic changes, namely alterations in the pattern
of nuclear DNA methylation and protein acetylation.
Mitochondrial alterations and cancer- Screening of mutations in SDH genes in human cancers
In collaboration with IPO (Portuguese Institute of Oncology), Porto we are studying Portuguese patients with paraganglioma from
northern Portugal for the presence of germline succinate dehydrogenase (SDH) mutations. At the moment, we have analysed 41 patients Our findings revealed that a very high proportion of apparently sporadic PGLs in the Portuguese population arises as a consequence of germline mutations and that most of the patients presented a large SDHB deletion encompassing exon 1 and the promoter;
the finding that the deleted allele shows the same haplotype in all patients suggests a founder effect for this particular deletion, which
would be the first founder mutation in SDHB in the Portuguese population. (Lima J, et al, In revision in Endoc Related Cancer).
Loss of function of succinate dehydrogenase (SDH) complex is an alternative molecular mechanism in GISTs. 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.
Our results confirm the occurrence of germline SDH genes mutations in isolated, apparently sporadic WT GISTs. GISTs without SDHB or
SDHA/SDHB expression may correspond to Carney–Stratakis dyad or Carney triad. (Celestino R, et al, Fam Cancer. 2012; Celestino R, et
al, Eur J Hum Genet. 2012)
Internationalization/Networking
Role of mitochondrial function and metabolism in human tumourigenesis.
Valdemar Máximo & Jorge Lima (IPATIMUP) & Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, USA – Principal
Investigator involved: Keshav Singh - Professor Keshav Singh is Associate Professor of Oncology/Full Member at the Department of
Cancer Genetics of RPCI, founding Editor-In-Chief of the Journal Mitochondrion and founder of The Mitochondria Research Society,
among other relevant facts. He was also co-supervisor of the post-doctoral studies of Jorge Lima and specialized in cybrid construction
and mitochondrial function. We have established a collaboration under the project “Targeting the Warburg effect for cancer therapy”.
Role of mitochondrial function and metabolism in human tumourigenesis
Valdemar Máximo & Jorge Lima (IPATIMUP) & Centre for Neuroscience and Cell Biology (CNC), Coimbra, Portugal – Principal Investigator
involved: Carlos Palmeira & Institute of Experimental and Technologic Biology (IBET), Oeiras, Portugal – Principal Investigator involved:
Ana Teixeira - In order to understand as fully as possible the role of mitochondrial function and metabolism in human tumourigenesis
we developed contacts with two of the most reputed Portuguese group [one from Coimbra (CNC) other Lisbon IBET)] specialists in
metabolic diseases and cellular metabolic pathways analysis. These contacts led to the establishment of a network between IPATIMUP
(experts in oncobiology), CNC (experts in metabolic disorders) and IBET (experts on cell metabolism pathways) and to the design of a
joint project entitled “Targeting the Warburg effect for cancer therapy” that was funded by IPATIMUP (See Approved Projects).
Role of GRIM-19 in Human tumorigenesis
Valdemar Máximo & Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, USA – Principal Investigator involved: Dhan Kalvakolanu. - Professor Dhan Kalvakolanu is an expert in the regulation of gene transcription and signal
transduction by cytokines; tumour cell growth control; and regulation of cell death-activating genes. Professor Dhan Kalvakolanu has
identified GRIM-19 and its role in cell death regulation. Professor Kalvakolanu on a visit to Europe he visited us and suggested a collaboration under the project: “Role of GRIM-19 in cancer development”.
Role of the mitochondrial biogenesis machinery in mitochondrion-rich tumours
Valdemar Máximo (IPATIMUP) & Department of Cell Physiology and Metabolism of the University of Geneva, Geneva, Switzerland –
Principal Investigator involved: Luca Scorrano. - Connected with the PhD projecto of the BSc André Emanuel Ferreira da Silva (entitled
“Role of the mitochondrial biogenesis machinery in mitochondrion-rich tumours”) a collaboration with Professor Luca Scorrano was
established. - Professor Luca Scorrano is an expert in the regulation of mitochondrial biogenesis, namely in the mitochondrial fission
processes as well as in the role of mitochondria in programmed cell death and autophagy.
GRIM-19, a novel protein involved in cell apoptosis: structure-function characterization
European Synchrotron Radiation Facility (ESRF), Grenoble, France – Principal Investigator involved: Daniele de Sanctis. - Valdemar Máximo & Instituto de Tecnologia Química e Biológica (ITQB), Oeiras, Portugal – Principal Investigator involved: Isabel Bento & European
Synchrotron Radiation Facility (ESRF), Grenoble, France – Principal Investigator involved: Daniele de Sanctis. - In the beginning of 2010
we started a collaboration with Doctor Isabel Bento from ITQB and Doctor Daniele de Sanctis from ESRF in order to progress in the understanding of the role of GRIM-19 in cell biology. These contacts led to the establishment of a network between ITQB, IPATIMUP and
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Activity Report
2012
ESRF and to the design of a joint project entitled “GRIM-19, a novel protein involved in cell apoptosis: structure-function characterization” . The proposal was funded by FCT – Ref.: PTDC/BIA-PRO/113064/2009
Fusion gene microarray-based approach and RNA-sequencing technology in thyroid tumours
M Sobrinho-Simões and Paula Soares (IPATIMUP) and Ragnhild Lothe and Rolf Stokheim from the Institute for Cancer Research, Oslo,
Norway. - Collaboration for the use of a fusion gene microarray-based approach that allow the simultaneous analysis of all known and
predicted fusion gene variants in thyroid tumours, and the use of RNA-sequencing technology in follicular carcinomas.Work developed
on behalf of Ricardo Celestino PhD project.
The Metabolic Needs of Epithelial to Mesenchymal Transition
Collaboration with Dr John Blenis from the Department of Cell Biology, Harvard Medical School, Boston, USA. - Dr Blenis is currently
co-supervising the PhD student Joana Nunes under the project
In depth genetic characterization of familial and sporadic thyroid tumours
Paula Soares, Valdemar Máximo and Hugo Prazeres (IPATIMUP) and Robert Hofstra from the University Medical Center Groningen,
Groningen, Holland. - Use of deep sequencing methods in order to identify molecular alterations in familial forms of thyroid cancer
Ethiopatogenesis of thyroid cancer
M Sobrinho-Simões, Catarina Eloy and Paula Soares (IPATIMUP) and José Cameselle-Teijeiro from the Universidade de Santiago de Compostela, Spain. - Several works in the molecular characterization of thyroid tumors with emphasis in rare cases
Genetic characterization of familial forms of paraganglioma and pheochromocytoma
Jorge Lima (IPATIMUP) & Department of Endocrinology, Portuguese Institute of Oncology (Porto, Portugal), Dr Isabel Torres. - A collaboration for the genetic screening of patients with sporadic and hereditary forms of paraganglioma and pheochromocytoma
ENETs Tumour registry
José Manuel Lopes and Paula Soares.As a member of GE-TNE of SPEDM we are evaluating the future enrolment of a Portuguese NETs
registry in the ENETs tumour registry. Work developed on behalf of João Vinagre PhD project.
STAT3 regulation and role in thyroid cancer
New therapies in thyroid cancer: the use of MEK inhbitiors and JAK inhibitors and combination therapies. Work developed in MSKCC,
New York, NY, in collaboration with Dr Jacqueline F. Bromberg, on behalf of Joana Silva’s PhD project.
Future Research
Clinico-pathological studies on thyroid and other neuroendocrine tumours
We will pursue the studies exploring the studies exploring signaling pathways activated by the presence of BRAF mutations, giving
attention to the expression of iodine metabolizing genes and response to therapy (Miguel Melo, MD, PhD project). - In collaboration
with Raghnild Lothe and Rolf Stockeinhem we will pursue the studies using high throughput technologies to evaluate the presence of
new genetic alterations (mutations/rearrangements) in thyroid tumours. - We will pursue the genetic characterization of sporadic and
hereditary (MEN1, MEN2) neuroendocrine tumours (João Vinagre PhD project)
Dissecting molecular pathways
NIS restoration in thyroid tumors is crucial to radioiodine therapy success, but the molecular mechanisms underlying its silencing in
cancer have not been disclosed. Even though it was reported that BRAFV600E interferes with NIS expression in human thyroid tumors,
whether mTOR pathway contributes or is involved with BRAV600E related NIS underexpression is yet to be investigated. We intend to
test and validate new drugs and combination of drugs that contribute to NIS reexpression and functionality (Catarina Tavares, PhD
project). - To go further in the study of BRAF V600E mutation in thyroid cancer and to address therapeutic options we intend to develop
a suitable animal model. We are establishing a thyroid targeted- BRAFV600E transgenic zebrafish model so we can study the role of
activated BRAFV600E in thyroid cancer development and progression. (Ana Almeida, PhD project)
Irradiation and disease
We are still following a cohort of individuals irradiated in childhood for tinea capitis treatment. The association between low dose (LD)
exposure and later cardiovascular disease (CVD) is still controversial. Scalp irradiation for tinea capitis treatment performed at childhood, may be a risk for the development of carotid stenosis in adult age. The aim of the study is to evaluate the atherosclerosis risk of
the tinea capitis irradiated individuals inviting to the study the 1367 individuals that we have already observed.
Mitochondrial alterations and cancer- Tumourigenic and metabolic effects of mitochondrial dysfunction
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Activity Report
2012
Using the cybrids cell line models with OXPHOS dysfunction, we started a study aiming to unveil how pathogenic mutations in mtDNA
and/or mutations in nuclear genes encoding mitochondrial proteins may lead to epigenetic changes, namely alterations in the pattern
of nuclear DNA methylation and protein acetylation. - Experiments are under way to prove that the increased motility and migration we
observed are in the same cell model due to increased activated integrin beta1.
Mitochondrial alterations and cancer- IDH1 and IDH2 Mutations in Human Gliomas and Correlation with Metabolic Players
We have collected a series of 48 low grade and high grade gliomas to screen for the presence of IDH1 and 2 mutations and study the expression of metabolic players such as glucose transporters (GLUT1 and 3), lactate transporters (MCT1 and 4) and glutamine transporters
(SNAT1). We could verify that 78% of low grade oligodendrogliomas, 38% of grade II oligodendrogliomas and 33% of grade II astrocytomas
harboured the IDH1 R132 mutation. No mutations were disclosed in glioblastomas or pylocytic astrocytomas.
Participation in PhD Programs
Jorge LimaGABBA Doctoral Programme
Joana Nunes Student (1st year) of the Doctoral Programme in Biomedicine, FMUP
Paula SoaresGraduate Program in Areas of Basic and Applied Biology (Porto); Gulbenkian PhD Program (Oeiras);
Professor of the Module of Oncobiology
Doctoral Program in Medicine and Molecular Oncology (FMUP); Doctoral Program in Genetics and Molecular Patholog (ICBAS/FMUP)y;
Doctoral Program in Biomedicine (FMUP); Doctoral Program in Biomedical Sciences (ICBAS)
Jorge LimaGraduate Program in Areas of Basic and Applied Biology (Porto); Gulbenkian PhD Program (Oeiras);
Professor of the Module of Oncobiology
Doctoral Program in Medicine and Molecular Oncology (FMUP); Doctoral Program in Genetics and Molecular Patholog (ICBAS/FMUP)y;
Doctoral Program in Biomedicine (FMUP); Doctoral Program in Biomedical Sciences (ICBAS)
Valdemar MáximoGraduate Program in Areas of Basic and Applied Biology (Porto); Gulbenkian PhD Program (Oeiras);
Professor of the Module of Oncobiology
Doctoral Program in Medicine and Molecular Oncology (FMUP); Doctoral Program in Genetics and Molecular Patholog (ICBAS/FMUP)y;
Doctoral Program in Biomedicine (FMUP); Doctoral Program in Biomedical Sciences (ICBAS)
M. Sobrinho-SimõesGraduate Program in Areas of Basic and Applied Biology (Porto); Gulbenkian PhD Program (Oeiras);
Professor of the Module of Oncobiology
Doctoral Program in Medicine and Molecular Oncology (FMUP); Doctoral Program in Genetics and Molecular Patholog (ICBAS/FMUP)y;
Doctoral Program in Biomedicine (FMUP); Doctoral Program in Biomedical Sciences (ICBAS)
Participation in Master Programs
Paula Soares, Valdemar Máximo, Jorge Lima, M Sobrinho-SimõesMaster of Science Program in Medicine and Molecular Oncology,
FMUP. Professor of the Modules of Oncobiology and Endocrinology
Paula Boaventura Begun the first year of the Master in Science of Communication, FLUP
Ana Isabel Mendes Dias “The role of GRIM-19 and STAT3 interaction in renal tumorigenesis: clear cell renal cell carcinoma”. Supervisor
Valdemar Máximo.Master in Human Biology and Environment, Faculdade de Ciências da Universidade de Lisboa. Completed on December 2012.
Prizes
Raquel G. Martins, Joana Couto, Ana Paula Santos, Paula Soares, Joana Nunes, Jorge Lima, Isabel Torres. Prize for the best series - poster
entitled “Pheocromocytomas And Paragangliomas: High Prevalence Of SDH Mutations In Patients Of Portuguese Institute Of Oncology
Of Oporto”.
Poster presented at the “XIII Congresso Português de Endocrinologia”, Coimbra, 2012.
Raquel G. Martins, Joana Couto, Ana Paula Santos, Paula Soares, Joana Nunes, Jorge Lima, Isabel Torres. Best poster in Translational
Research - “Pheocromocytomas And Paragangliomas: High Prevalence Of SDH Mutations In Patients Of Portuguese Institute Of Oncology Of Oporto”.
Poster presented at the XXI Porto Cancer Meeting, IPATIMUP, Porto, 2012.
Joana Nunes, Paula Soares, Adriana Rocha, Maria Oliveira, Joana Paredes, Anabela Pinto Rolo, Nuno Mendes, Keshav Singh, Manuel
Sobrinho-Simões, Valdemar Máximo & Jorge Lima. Best poster in Basic Research - “mtDNA-induced OXPHOS dysfunction causes enhanced tumour growth and metastatic potential: a role for OXPHOS in regulating migration?”.
Poster presented at the XXI Porto Cancer Meeting, IPATIMUP, Porto, 2012.
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Activity Report
2012
Invited Talks
Catarina Eloy . Diagnóstico diferencial entre tumores benignos e malignos da supra-renal. III Reunião de Cirurgia endócrina e cervical do
Centro Hospitalar de São João. Porto. 21/10/12.
Jorge Lima. SDH mutations in inherited neoplasias. XXI Porto Cancer Meeting. IPATIMUP. 20/04/2012.
Jorge Lima. Genetics of Paragangliomas and Pheochromocytomas. XIII Congresso Português de Endocrinologia. Coimbra. 29/01/2012.
M Sobrinho-Simões. Molecular genotyping of papillary thyroid carcinoma (A pathologist’s view). European Congress of Endocrinology.
Florence, Italy. 06/05/2012.
M Sobrinho-Simões. Introdução à biologia e patologia molecular do cancro. XXXVII Reunião do GRELL. Porto, Portugal. 16/05/2012.
M Sobrinho-Simões. Melhoria humana: um desafio genético ou, sobretudo, multifactorial?. Cerimónia de lançamento do livro «Pensar
Saúde Hoje». Lisboa, Portugal. 31/01/2012.
M Sobrinho-Simões. Trying to understand papillary carcinoma of the thyroid (1970-2012). Ciclo de conferências no Hospital Garcia de
Orta. Hospital Garcia de Orta, Almada, Portugal. 28/03/2012.
M Sobrinho-Simões. Investigação clínica em meio hospitalar: da teoria à prática em Ciências da Saúde. Ciclo de conferências no Hospital
Garcia de Orta. Hospital Garcia de Orta, Almada, Portugal. 28/03/2012.
M Sobrinho-Simões. Importância da genética molecular no diagnóstico, prognóstico e selecção terapêutica dos tumores da tireoide. 101ª
Reunión de la Asociación Territorial Valenciana de la Sociedad Española de Anatomia Patológica. Xativa, Valencia, Espanha. 27/04/2012.
M Sobrinho-Simões. Thyroid cancer: an update on cytology, histology and molecular pathology. Belgium Thyroid Club 40th Meeting.
Belgium. 28/04/2012.
M Sobrinho-Simões. Introduction to oncobiology. II Workshop on Cancer Research: biological and molecular basis. Porto, Portugal.
15/05/2012.
M Sobrinho-Simões. The potential of molecular biology in thyroid cytology and histology. Pathological Society Meeting, Summer Meeting 2012. Sheffield, England. 04/06/2012.
M Sobrinho-Simões. Molecular data in the diagnosis and prognosis of thyroid carcinoma. Poorly and undifferentiated carcinomas. 24th
European Congress of Pathology 2012 (ECP 2012). Prague, Czech Republic. 10/09/2012.
M Sobrinho-Simões. Recepção aos novos alunos da Faculdade de Medicina da Universidade do Porto . Recepção aos novos alunos da
Faculdade de Medicina da Universidade do Porto . Faculdade de Medicina da Universidade do Porto, Porto, Portugal. 17/09/2012.
M Sobrinho-Simões. A construção de seres multicelulares: do Homem à Cidade. 3º Conferência do Ciclo “Construtores do Mundo” promovido pela EPUL . Lisboa, Portugal. 24/09/2012.
M Sobrinho-Simões. Sociedade, Saúde e o S.N.S.. Primeira Reunião da Fundação para o Serviço Nacional de Saúde. Museu do Oriente,
Lisboa, Portugal. 27/09/2012.
M Sobrinho-Simões. «A Qualidade na Clínica Diária. Desafios Actuais, integrado na Mesa “A Diabetes na Prática da Medicina Actual». XII
jornadas de Diabetes da Madeira e 3º Simpósio Satélite de Tiróide. Madeira, Portugal. 18/10/2012.
M Sobrinho-Simões. Molecular pathology in cytology and pathology of thyroid cancer: The importance of understanding. Annual Educational Meeting for the Joint Endocrine Tumour Group. Manchester, England. 21/11/2012.
M Sobrinho-Simões. As doenças das próximas décadas: A medicina personalizada. Faculdade de Medicina Dentária da Universidade do
Porto. Faculdade de Medicina Dentária da Universidade do Porto, Porto, Portugal. 11/12/2012.
M Sobrinho-Simões. Envelhecimento Activo & Solidariedade entre Gerações. Comemoração do Ano Europeu do Envelhecimento Activo
e da Solidariedade entre Gerações. Porto, Portugal. 16/12/2012.
Paula Soares . Biomarkers of Thyroid Malignancy. XVIII Curso Pós-graduado de Endocrinologia, Diabetes e Metabolismo: Curso Avançado de Endocrinologia. Porto . 31/03/2012.
Hugo Prazeres. Clinical trials of reasearcher’s innitiative, a perspective in cancer. Investigação clínica da iniciativa do investigador - relevância de novas estruturas em Portugal. AIBILI, Coimbra, Portugal. 16/11/2012.
Oral Presentations
Catarina Eloy. “Morphology and immunohistochemistry in the study of thyroid tumours.”. 3rd Belgian week of Pathology. Ghent - Belgium. 19/4/2012.
Catarina Eloy. “TGFbeta expression in thyroid cancer.”. Cancer Biology Group Scientific Retreat. Vila Praia de Âncora . 25/1/2012.
Catarina Eloy. “Primary non endocrine small cell carcinoma of the thyroid with primary neuroectodermal tumour features”. 24th European Congress of Pathology. Prague. 10/9/2012.
Catarina Eloy. “Diagnóstico diferencial entre tumores benignos e malignos da supra-renal”. III Reunião de Cirurgia endócrina e cervical
do Centro Hospitalar de São João. Porto. 21/10/12.
Catarina Eloy. “Molecular diagnosis in thyroid fine needle aspirates”. 31st Meeting of CAEK & 42nd Annual Meeting of the Thyroid Section of DGE. Regensburg, Germany. 19/11/2012.
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Activity Report
2012
Carcinogenesis
Objectives
The main objective of the group is to identify alterations of mucins and mucin glycosylation, associated with gastric carcinoma and
precancerous lesions,that may be relevant for the development of diagnostic and therapeutic strategies. We are also engaged in understanding the molecular mechanisms involved in the development of gastric carcinogenesis with emphasis on the identification of
transcriptional pathways responsible for cancer/pre-cancer transdifferentiation, as well as to extend our expertise on other cancer
models (ex: mammary cancer from dogs).
Main Achievements
Glycosylation alterations in serum proteins of patients with gastric pathologies
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., 2013 Jan 31. [Epub ahead of print].
Evaluation of the toxicity of Pteridium aquilinum in gastric cells and its synergistic effect with Helicobacter
pylori infection
We have evaluated an environmental factor Pteridium aquilinum-bracken fern- and its toxin, ptaquiloside, for its capacity to induce
molecular damage effects in gastric cells and in mice gastric mucosa. We have demonstrated the direct DNA damaging and mutagenic effects of P. aquilinum in gastric cells (Gomes et al 2012,Toxicol Sci. 2012). We have also evaluated the glycophenotypic alterations induced
by Pteridium aquilinum and its synergistic effect with Helicobacter pylori infection (Gomes PLoS One. 2012;7(6):e38353).
Role of N-glycosylation of E-cadherin in gastric cancer
We have demonstrated for the first time 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
GnT-V-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 was further validated in human gastric carcinoma clinical samples (Pinho SS et al. Biochim Biophys Acta. 2012 Nov 5 [Epub ahead of print]).
The characterization of the role of Mgat3 gene (GnT-III glycosyltransferase) in Epithelial to Mesenchymal Transition
We have shown that Mgat3 gene (that encodes the GnT-III glycosyltransferase) is a gene involved in Epithelial to Mesenchymal Transition
(EMT) and the reverted process Mesenchymal to Epithelial Transition (MET). Interestingly, we found that E-cadherin is specifically regulated by GnT-III-mediated glycosylation during EMT/MET .This was the first report addressing the role of a glycosyltransferase modulating E-cadherin function during the EMT/MET process. (Pinho SS et al PLoS One. 2012;7(3):e33191 ).
Regulation of CDX2 and SOX2 expression by the BMP pathway and Helicobacter pylori
Using cellular as well as in vivo models we have shown that Helicobacter pylori, the main cause of intestinal metaplasia and gastric
cancer, upregulates the BMP pathway and CDX2 and downregulates SOX2 expression. Therefore, we show that the BMP pathway might
constitute a link between the infection with this microorganism and the development of intestinal metaplasia (Camilo et al, Carcinogenesis, 2012; Barros et al, Trends Mol Med, 2012)
Overexpression of the BMP pathway in the gastrointestinal tract in a transgenic mouse
We have successfully established a new mouse model with overexpression of a constitutively active BMPRIA in the gastrointestinal tract
using the Ah promoter. The aim is to assess in vivo whether the BMP pathway is able to induce intestinal metaplasia.
Development of a strategy to deliver siRNAs directed to CDX2 in vivo, using a chitosan nanoparticle approach
We have developed, in collaboration with INEB, a novel strategy to deliver siRNAs in vivo. This included so far the development of
chitosan nanoparticles and the optimisation of the formulation and of the delivery conditions in cell lines. We have successfully downregulated CDX2 expression in gastric carcinoma cell lines using two different formulations. We further assessed the diffusion of these
nanoparticles through the mucus layer, using mouse intestinal explants (manuscript in preparation). This was performed with the collaboration of the group of Prof. Gunnar Hanssen, in Gothemburg, Sweden.
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Activity Report
2012
CDX2 regulation by the RNA-binding protein MEX3A
We have characterized the regulation of CDX2 expression by the RNA binding protein MEX3A. We have shown that MEX3A binds to CDX2
RNA, repressing its translation. We further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects
cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show
that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties (Pereira et al, Nucleic Acids Res, in press).
Glycosylation alterations in cancer and new biomarker detection systems
Using Proximity Ligation Assays (PLA) we have identified new cancer-associated glycopeptide pairs and by screeing a series of mucinous
carcinomas from different locations we could demonstrate increased specificity compared to detection of peptides or glycans alone
(Pinto R et al, J cell Mol Med 2012). A series of engineered ovarian carcinoma cell lines, producing MUC16 with different glycoforms,
allowed characterization of glycosylation dependence of monoclonal antibodies used in the CA125 assay (M11 and CA125). A new monoclonal antibody for MUC16 was produced and is at an advanced stage of characterization.
Regulation of glycoproteome alteration in gastric carcinogenesis
We observed a significant association between CDX2 expression and the presence of sialil-Tn antigen and this led us to explore the possibility that CDX2 was directly transactivating the ST6GalNAcI responsible for sialil-Tn biosynthesis. The next steps are under way and
we have shown that CDX2 binds to the ST6GalNAcI promoter using ChIP assays and that CDX2 induces transactivation of a luciferase
reporter construct. A set of studies after induction and upon silencing of the CDX2 gene are being performed to confirm this important
and new mechanism of regulation of cancer associated glycoproteome alterations.
Internationalization/Networking
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. We have been collaborating with this group for production of MUC16 antibodies. Two PhD students of the group (Lara Silva
and Diana Campos) are doing joint PhD thesis and another student, Rita Pinto, started a regulatr collaboration for CDX2 knockout-knockin. We regularly collaborate in pos-graduate teaching activities at the University of Copenhagen – PhD Glycobiology Course.
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 an interesting possibility of cooperation of Helicobacter pylori in
glycoengeneering the host for certain Calicivirus strain infections.
University of Uppsala - Ola Soderberg
This collaboration has allowed the successful establishment of Proximity-Ligation assays for identification of glycopeptide structures in
situ and is allowing expansion of the approach for serum glycopeptide identification.
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.
Umeå University, Sweden – Thomas Borén.
This collaboration has contributed for the study of H.pylori adhesion to gastric mucosa and for developing binding assays.
Institut Pasteur, France - Eliette Touati
This collaboration is essential for the study of Helicobacter pylori using animal models. A PhD thesis (Joana Gomes) has been completed
between the two groups according to the International Convention for the joint supervision of theses.
Naoyuki Taniguchi - Osaka University and RIKEN, Japan. Jianguo Gu - Tohoku Pharmaceutical University, Japan
These collaborations have been important in addressing the role of N-glycosylation modifications in the regulation of pivotal molecules
involved in gastric development and progression.
INSERM, Strasbourg, France - Jean-Noel Freund
We collaborate with Jean-Noel Freund in the characterization of CDX2 regulation, namely by the MEX3A protein.
Institut Albert Bonniot, Grenoble, France - Marc Billaud
This collaboration was critical for the study of CDX2 regulation by MEX3A.
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Activity Report
2012
Future Research
The “Glycobiology in Cancer” group will aim to further characterize the role of glycans for Helicobacter pylori
adhesion/infection and to develop novel anti-adhesion therapeutic strategies based on carbohydrates/analogs.
We will further evaluate in human gastric biopsies of individuals with Helicobacter 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 previous in vitro findings. These glycophenotypic analyses will be combined with the detailed
characterization of the adhesins of the Helicobacter pylori strains infecting each individual. This comprehensive study of both the Helicobacter pylori adhesins expression, the glycosyltransferases expression and the glycophenotype will provide insights into mechanisms
contributing for the Helicobacter pylori chronic infection of the gastric mucosa.
The “Glycobiology in Cancer” group will continue the development of novel strategies for inhibition of Helicobacter pylori adhesion and infection.
We will further develop strategies for inhibition of Helicobacter pylori adhesion and infection. This approach is being developed within
an on-going collaboration with Dr. Cristina Martins from INEB. We are developing and testing chitosan microspheres modified with
synthetic carbohydrates receptors for inhibition of Helicobacter pylori adhesion, using cell line models expressing specific glycan receptors, gastric mucosa of genetically modified mice (previously characterized and published by our group), and human gastric mucosas.
The “Glycobiology in Cancer” group will continue to evaluate the role of glycans in cancer addressing the molecular mechanisms controlling glycosylation of key proteins.
We will continue to analyse the role of E-cadherin glycosylation in gastric cancer cell behaviour. We plan to map the structure-function 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. We will further validate our results in a set of human gastric carcinoma cases displaying E-cadherin dysfunction not
explained by genomic alterations.
The “Glycobiology in Cancer” group will also evaluate the role of terminal sialylation of glycoproteins in cancer
We will have particular focus on sialylation of glycan structures on important key receptors and study the effect on cellular signalling. We
intend to clarify the mechanisms underlying intermittent levels of sialic acid capping of glycoproteins in malignant tumours by assessing
the role of sialyltranferases in cancer cells.
Within this frame, and in order to translate to clinical applications, we will further continue to apply glycoproteomic strategies for the
identification of aberrantly glycosylated glycoproteins that can be used for cancer or pre-cancerous lesions detection.
The «Differentiation and Cancer» group will continue to characterize the animal model with overexpression of
the BMP pathway in the GI tract.
After having generated a new transgenic mouse model overexpressing the BMP pathway in the GI tract we will now characterize the the
phenotype obtained, particularly in the stomach, using different antibodies for gastric and intestinal markers, as well as proliferation
markers. We will also set up new experiments to collect mice at different time points.
The «Differentiation and Cancer» group will continue to characterize the regulation of the BMP pathway, CDX2
and SOX2 by Helicobacter pylori.
We will continue developing this model, exploring the mechanism by which Helicobacter pylori regulates the expression of these proteins. We will test whether interaction between bacteria and epithelial cells is required or whether this regulation is induced by soluble
factors. For this we will use conditioned media obtained from Hp liquid cultures as well as vesicles purified from Hp cultures. This last
part will be done in collaboration with the group of Anne Hanqvist, from Umea, sweden.
The «Differentiation and Cancer» group will characterize the expression of Sox2 in the stomach and esophagus.
We will characterize the expression of Sox2, which is emerging as a transcriptional factor necessary for esophageal and gastric differentiation, in the stomach and esophagus. Despite the function attributed to this proteins, its expression in adult esophagus and stomach
is not well characterized. In addition to the characterization of its expression in normal tissues, we will also study the expression in
preneoplastic lesions and in cancer.
The «Differentiation and Cancer» group will continue to develop a strategy to deliver in vivo siRNAs against
CDX2 using chitosan nanoparticles.
After having defined the optimal formulations of the nanoparticles we will now explore delivery strategies in vivo, first in the colon and
then using an animal model with CDX2 expression in the stomach.
The «Differentiation and Cancer» group will continue to characterize MEX3A functions in the GI tract.
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Activity Report
2012
We will continue to explore, using in vitro models and whenever possible in vivo models, the regulation of cell polarity and stemness
features by Mex3A. We will also characterize the expression of Mex3A in intestinal metaplasia of the stomach and in neoplasias from
intestine. In both cases CDX2 regulation is altered and we want to assess if it can be modulated by Mex3A.
The «Differentiation and Cancer» group will continue to explore CDX2 regulation of cancer glycoproteome.
We will pursue our studies on CDX2 regulation of ST6GAlNAcI and expand the system into a zinc-finger knock-in/knock-out CDX2 system
to search for a large scale effect on glycosyltransferase regulation and glycoproteome modification.
The «Differentiation and Cancer» group will continue studying differentiation-associated mucin-glycan pairs
relevant in gastric and ovarian carcinogenesis using PLA assays both in tissues and in serum.
We are using our PLA approach for glycopeptide identification, in collaboration with Ola Soderberg in Uppsala and the Olink company,
to map mucin-glycan combinations in benign and malignant gastric and ovarian lesions. To expand our preliminary observations in tissues we are building tissue microarrays and simultaneously producing all the needed PLA probes. After tissue characterization we will
apply the same approach to serum detection of the mucin-glycan pairs of interest.
Participation in PhD Programs
Celso Reis, Hugo Osorio, Salomé Pinho, Fatima Gartner, Raquel Almeida, Leonor David - Workshop on Cancer Research: biological and
molecular basis Local: IPATIMUP Instituição: IPATIMUP/ICBAS Âmbito: Curso de formação contínua
Raquel Almeida, Celso Reis, Leonor David - Programa Doutoral de Medicina e Oncologia Molecular of the Medical Faculty of the University of Porto.
Raquel Almeida, Celso Reis, Fatima Gartner, Leonor David - Programa Doutoral em Patologia e Genetica Molecular do Instituto de Ciencias Biomedicas Abel Salazar
Raquel Almeida, Celso Reis, Leonor David, Salome Pinho, Hugo Osório - PhD program GABBA (Graduate program in areas of Basic and
Applied Biology) of the University of Porto
Raquel Almeida, Celso Reis, Leonor David, Hugo Osório - Programa doutoral para medicos das Fundações Gulbenkian e Champalimaud.
Raquel Almeida, Celso Reis, Leonor David. - Programa Doutoral em Biomedicina
Leonor DavidPrograma Doutoral em Glicobiologia - Universidade de Copenhaga.
Participation in Master Programs
Fatima Gartner, Raquel Almeida, Celso Reis, Leonor David - Mestrado em Oncologia Molecular do Instituto de Ciencias Biomedicas Abel
Salazar
Prizes
Ana Magalhães EMBO short term Fellowship - EMBO short term Fellowship to Ana Magalhães to visit the laboratory of Thomas Borén at
the Medical Biochemistry and Biophysics department, Umeå University.
Salomé Pinho “Young Investigator Award” - “Young Investigator Award” sponsored by the European Association for Cancer Research
(EACR). October 2012.
Bruno Pereira - Best oral presentation –
II ED-FMUP Meeting, 2012, Porto, Portugal
Bruno Pereira - Best oral communication in Basic Research 16th Annual Meeting of the Portuguese Society of Human Genetics (SPGH), 2012, Porto, Portugal
Vânia Camilo - Travel grant - 10th International Workshop on Pathogenesis and Host Response in Helicobacter Infections
European Society of Clinical Microbiology and Infectious Diseases
Invited Talks
Celso Reis. “Role of glycosylation in cancer”. Seminar at the Copenhagen Center for Glycomics. Copenhagen, Denmark. 03/02/2012.
Celso Reis. . The Role of Glycans in H pylori infection and gastric carcinogenesis. The Jenner Glycobiology and Medicine Symposium. The
Royal Society of Medicine. Den Haag, The Netherlands. 01/04/2012.
Salomé Pinho. “O papel da glicosilação no cancro”; Glicosilação no cancro de estômago: implicações na função da E-caderina”. Programa
de Pós-graduação em Biociências, Universidade Estadual do Rio de Janeiro. Universidade Estadual do Rio de Janeiro. Brasil. 27/08/2012.
Celso Reis. «Introdução à glicobiologia e Glicobiomarcadores em cancro». Programa de Pós-graduação em Biociências, Universidade
Estadual do Rio de Janeiro. Brasil. 27/08/2012.
Raquel Almeida. Regulation of transdifferentiation in cancer development.. SINAL 2012 - 6th National Meeting on Cell Signaling. ICVS –
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Activity Report
2012
ECSaúde, Braga, Portugal,. 14/04/2012.
Raquel Almeida. Regulation of transdifferentiation in cancer development. Barrett›s esophagus advanced course. IPO, Lisboa. 06/2012.
Raquel Almeida. Regulatory mechanisms underlying metaplasia reprogramming. The model of CDX2 in intestinal metaplasia of the
stomach. IPO Porto. Portugal. 07/2012.
Leonor David.. Biomarcadores no cancro gástrico – da investigação até à prática clínica - . Curso sobre cancro gástrico - . Faculdade de
Medicina da Universidade do Porto. 10/12.
Leonor David. Regulação da diferenciação tecidular no desenvolvimento tumoral: da bancada a potenciais aplicações clínicas - . Conferencia convidada.. Instituto Nacional de Cancer - Rio de Janeiro, Brasil.. 7/12.
Leonor David. Biology of intestinal metaplasia. Phenotype with emphasis on mucin glycoproteome - . Barrett›s esophagus advanced
course.. IPO Lisboa. 6/12.
Leonor David. Regulation od Mucin Glycoproteome in Gastric Carcinogenesis - . Conferencia convidada. . Universidade de Umea, Suecia.
5/12.
Oral Presentations
Ana Magalhães. Helicobacter pylori switches the host cells glycosylation pathways to remodel the gastric mucosa glycophenotype. 10th
International Workshop on Pathogenesis and Host Response in Helicobacter Infections. Helsingør, Denmark. 04/07/2012.
Salomé Pinho. N-acetylglucosaminyltransferases III and V regulate E-cadherin stability at the cell membrane. Implications in the Epithelial to Mesenchymal Transition. Glyco-T 2012 meeting.. Hannover, Germany. 05/06/2012.
Salomé Pinho. Glycosylation regulating E-cadherin stability: a mechanism with implications for cancer and for Epithelial to Mesenchymal
Transition. I3S retreat. Póvoa do Varzim. 10/05/2012.
Rita Barros. The Bone Morphogenetic Protein (BMP) pathway in gastric intestinal metaplasia. . International Meeting of the Portuguese
Society for Stem Cells & Cell Therapies. Porto, Portugal. 04/2012.
Bruno Pereira. RNA-binding protein MEX3A: a new player in the establishment of the intestinal proliferation/differentiation axis. . II EDFMUP Meeting. Porto, Portugal. 12/2012.
Bruno Pereira. Modulation of CDX2 expression by the RNA-binding protein MEX3A: impact on intestinal-type differentiation and stemness. . 16th Annual Meeting of the Portuguese Society of Human Genetics (SPGH). Porto, Portugal.. 11/2012.
Bruno Pereira. Post-transcriptional regulation of CDX2 expression by MEX3A: impact on intestinal differentiation.. XXXème Club d›Etudes
des Cellules Epithéliales Digestives (CECED). Grenoble, France. 03/2012.
Vânia Camilo. Helicobacter pylori and the BMP pathway regulate CDX2 and SOX2 expression in gastric cells. 10th International Workshop
on Pathogenesis and Host Response in Helicobacter Infections. Helsingor, Denmark. 07/2012.
41
Activity Report
2012
Cancer Drug Resistance
Objectives
Resistance of cancer cells to traditional chemotherapy and targeted therapeutics is both a major clinical problem and a scientific challenge. Different approaches are currently being pursued with a view to overcoming this problem. Our group is mainly focused on translating basic science findings 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 is aimed at investigating the role of selected microRNAs (miRs) in drug resistance/drug response, in acute myeloid leukaemia,
in collaboration with Hospital São João. Project financed by Fundação Calouste Gulbenkian (co-PIs: Profs. M.H. Vasconcelos and J.E.
Guimarães).
We have found that miR-21 is involved in autophagy and drug resistance (work presented at the “10th International Congress on Targeted Anticancer Therapies - TAT 2012”, Amsterdam, Netherlands, 8-10 March 2012 and at the “22nd Biennial Congress of the European
Association for Cancer Research”, Barcelona, Spain, 7–10 July 2012; manuscript submitted for publication).
Additionally, we have conducted preliminary work on the role of miR-128-1 in the response of acute myeloid leukemia cells to chemotherapeutic drugs.
Role of P-glycoprotein (P-gp) in drug resistance and identification of anti-Pgp small molecules
This work has been carried out as a collaboration with CEQUIMED-UP, funded by FCT (FFUP as the Proponent Institution, PI Prof. M.
Pinto, PTDC/SAU-FCF/100930/2008). We have, together with our colleagues from CEQUIMED-UP, identified new molecules with a dual
activity: anti-Pgp activity and antitumor activity (Palmeira A. et al., Biochem Pharmacol 2012; Palmeira A. et al., J Pharm Pharmaceut Sci
2012; Palmeira A. et al., Curr Med Chem 2012; Palmeira A. et al., Curr Pharm Design 2012; Sousa E. et al., Med Chem Res 2012).
Furthermore, we are starting to study the significance of P-gp found in microvesicles and exosomes isolated from drug resistant cells.
Molecular targets involved in drug resistance in Cancer Stem Cells (CSCs)
The presence of targets related to drug resistance in CSCs is being investigated in an FCT funded project (PTDC/EBB-BIO/099672/2008), in
which the PI is Gabriela Almeida. This work is performed in collaboration with CEQUIMED-UP and with Dr. L.F. Santos-Silva at IPATIMUP.
CSCs enrichment with chemotherapeutic agents was attempted. These cells presented increased drug resistance, possibly due to overexpression of Bcl-2 (in pancreatic cancer cells), or ABCG2, XIAP and P-gp (in lung cancer cells). Additionally, the study of differential
expression of drug resistance proteins – in putative CSCs and non-CSCs - was also attempted by 2D gels (in collaboration with the Proteomics Unit at IPATIMUP). The proteins found to be overexpressed in the drug-resistant cell populations are therefore potential targets
to be used to circumvent drug resistance in the putative CSCs. Additionally, some preliminary studies (in collaboration with Dr. Carla
Oliveira, IPATIMUP) have been performed to investigate the induction of EMT upon cell drug-selection.
PLGA-PEG and PEI containing nanoparticles were developed in collaboration with CEQUIMED-UP. Technological parameters affecting
the entrapment of siRNAs were studied in order to optimize the formulations regarding loading capacity and stability. Six different formulations were developed and their physico-chemical properties evaluated. High siRNA incorporation efficiencies were achieved and
the in vitro release of siRNA from selected nanoparticle formulations was studied. Nanoparticles incorporating selected siRNAs are now
being tested for targeting drug resistance in the putative drug selected CSCs.
Development of small molecules with antitumor potential
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 has been carried out as a collaboration with CEQUIMED-UP, funded by FCT (FFUP as the
Proponent Institution, PI Prof. M. Pinto, PTDC/SAU-FCF/100930/2008), or as a collaboration with University of Minho (Prof. M.J. Queiroz),
Instituto Politécnico de Bragança (Prof. Isabel Ferreira) or Robert Gordon University, Aberdeen, Scotland (Prof. Paul Kong).
We have identified some compounds capable of decreasing human tumor cell growth and interfering with cell cycle or apoptosis. Some
results were published (Neves M.P. et al., Bioorganic and Medicinal Chemistry 2012; Calhelha et al., Molecules 2012; Belaz K.R.A. et al., J
Pharm Biomed Analysis 2012; Neves M.P. et al., Chemistry and Biodiversity 2012).
In addition, we have identified small molecules that were found to be in vitro and in silico inhibitors of hSIRT-2 (Lima et al., Anticancer
Agents Med Chem 2013), inhibitors of EBV (Lima et al., Chemical Biology & Drug Design, in press) or as activators of caspase-7 (manuscript submitted for publication).
In addition, we have been searching for small molecule modulators of p53 family proteins. This work was initiated as a collaboration
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Activity Report
2012
with Prof. Lucília Saraiva (project approved having ICETA-Porto/UP as the Proponent Instituion, PI Prof. Lucília Saraiva, PTDC/SAUFAR/110848/2009).
Searching for natural products with antitumor potential
Several studies have been carried out, in collaboration with CEQUIMED-UP and Instituto Politécnico de Bragança, to identify natural
products with antitumor potential. Part of this work is funded by FCT (Instituto Politécnico de Bragança as Proponent Institution, PI Prof
I.C.F.R. Ferreira, PTDC/AGR-ALI/098402/2008) and was also funded by the University of Porto.
Several publications resulted from this (Vaz J.A. et al., Food Chemistry 2012a; Vaz J.A. et al., Food Chemistry 2012b; Cazal et al., Anticancer Agents Med Chem, in press).
Internationalization/Networking
European COST Action CM1106 “Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells” (GM
Almeida).
GM Almeida was a member of the Management Committee of this COST Action.
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).
Robert Gordon University, Aberdeen, Scotland (Prof. Paul Kong).
Collaboration through a research paper.
Dublin City University and National Institute for Cellular Biotechnology, Ireland (Dr. Robert O’Connor).
Dr. O’ Connor is a collaborator of a PhD 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 training of students, a PhD student (V. Rodrigues) and projects financed by FCT (PTDC/SAU-FCF/100930/2008 and
PTDC/EBB-BIO/099672/2008).
Hospital São João (Prof. José Eduardo Guimarães e Doutor Manuel Sobrinho Simões).
Collaboration through a PhD student (H. Seca) and a Project financed by Fundação Calouste Gulbenkian.
Instituto Politécnico de Bragança (Prof. Isabel Ferreira and Prof. Anabela Martins).
Collaboration through a PhD student and two projects (one from UP and one from 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 a research paper.
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).
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Future Research
Validation of microRNAs with a role in cancer drug resistance
It is our expectation to further investigate the mechanisms by which some microRNAs are involved in drug resistance (PhD project of H.
Seca). In addition, we aim to confirm if some of the microRNAs that are responsible for drug resistance are transferred between cells,
via exosomes.
Identification and validation of small molecule p53 modulators
Several functions initially attributed to p53 are now believed to be shared by other members of the p53 family. Even though this opens
new expectations into the therapy of cancer, to date no pharmacological modulators of individual p53 isoforms have been described.
We will continue our collaboration with Prof. Lucília Saraiva to identify small molecules modulators of p53 family proteins (project approved having ICETA-Porto/UP as the Proponent Instituion, PI Prof. Lucília Saraiva, PTDC/SAU-FAR/110848/2009).
Investigation of the mechanism of action of small molecules which are inducers of cell death or/and inhibitors
of P-gp
The aim will be to further investigate the mechanism of action of one hit molecule from the CEQUIMED-UP library, which has potent human tumor cell growth inhibitory potential (post-doctoral project of R.T. Lima). We are currently considering the study of the activity of
this compound in lung cancer xenographs in nude mice. We expect to obtain more conclusive evidence about the cellular mechanisms
activated by this compound. This project is integrated in a research project funded by FCT (PTDC/SAU-FCF/100930/2008), as collaboration between CEQUIMED-UP and IPATIMUP.
Furthermore, we aim to better understand the molecular mechanisms, and identify novel inhibitors, of P-gp mediated cancer drug
resistance (PhD project of V. Rodrigues).
Identification of natural products with antitumor potential
We aim to continue identifying natural products with antitumor potential. This work is funded by FCT (Instituto Politécnico de Bragança
as Proponent Institution, PI Prof I.C.F.R. Ferreira, PTDC/AGR-ALI/098402/2008).
Participation in PhD Programs
M. Helena Vasconcelos. Programa Doutoral em Segurança e Saúde Ocupacionais, Faculdade de Engenharia da Universidade do Porto
(participação na Unidade Curricular “Seminários Multidisciplinares”).
M. Helena Vasconcelos. Programa Doutoral em Medicina e Oncologia Molecular, Faculdade de Medicina da Universidade do Porto (participação na Unidade Curricular “Técnicas de Biologia Molecular”).
M. Helena Vasconcelos. Programa Doutoral em Patologia e Genética Molecular, Instituto de Ciências Biomédicas Abel Salazar (participação na Unidade Curricular “Técnicas de Biologia Molecular”).
M. Helena Vasconcelos and Gabriela M. Almeida.Programa Doutoral GABBA, “Programa Graduado em Áreas da Biologia Básica e Aplicada” da Universidade do Porto (participação no módulo de Oncobiologia).
Participation in Master Programs
Raquel T. Lima. Mestrado em Terapias Moleculares, Instituto Superior de Ciências da Saúde – Norte.
Raquel T. Lima. Mestrado em Medicina Legal, Instituto de Ciências Biomédicas Abel Salazar.
M Helena Vasconcelos.Mestrado em Genética Molecular, Departamento de Biologia, Universidade do Minho.
Invited Talks
M.H. Vasconcelos. “Resistência a fármacos antineoplásicos”. Presented at the Congress “Oncologia” organized by the students of the
5th year of Pharmaceutical Sciences, Universidade da Beira Interior. Covilhã, Portugal. 06/01/2012.
M.H. Vasconcelos. “Aplicações práticas da cultura de células na investigação em oncobiologia”. Presented at a «Cell Culture Course”,
Escola Superior Agrária do Instituto Politécnico de Bragança. Bragança, Portugal. 20/04/2012.
Josiana Vaz . “Cultura de células tumorais humanas”. Presented at a «Cell Culture Course”, Escola Superior Agrária do Instituto Politécnico de Bragança. Bragança, Portugal. 21/04/2012.
Josiana Vaz . “Potencial antitumoral de cogumelos silvestres do Nordeste Transmontano”. Presented at the «IV SEMINÁRIO + Idade +
Saúde - Contributos para a Saúde na População Sénior». Bragança, Portugal. 05/05/2011.
Gabriela M. Almeida . «Mechanisms of drug resistance in cancer stem cells, from drug efflux pumps to apoptosis related proteins». Presented at the «1st Workshop COST Action CM1106». Milan, Italy . 03/07/2012.
Tiago dos Santos. “Uptake of polystyrene nanoparticles of different size into multiple cell lines: approaches to control and understand
bio-nano interactions”. Presented at the “Seminars of CEQUIMED-UP”, Faculdade de Farmácia da Universidade do Porto. Porto, Portu-
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2012
gal. 06/07/2012.
M.H. Vasconcelos. «Understanding and Overcoming Cancer Drug Resistance”. Presented at The National Institute for Cellular Biotechnology, Dublin City University (on the occasion of a laboratory visit). Dublin, Ireland. 13/08/2012.
Hugo Seca. “miR-21 e autofagia em células leucémicas”. Presented at the «I Jornadas de Saúde Pública e Farmacoterapia», Faculdade de
Farmácia da Universidade do Porto. Porto, Portugal. 22/11/2012.
Oral Presentations
Coutinho I., Lopes-Rodrigues V., Neves M.P., Lima R.T., Pereira C., Kijjoa A., Pinto M., Cidade H., Vasconcelos M.H., Saraiva L.
Presented by C. Pereira. “Discovery of potente small-molecule caspase-7 activators using yeast target-based screening assays”. XIX
Jornadas de Biologia de Leveduras “Professor Nicolau van Uden”, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa.
Lisboa, Portugal. 16/06/2012.
Lima R.T., Seca H., Palmeira A., Lopes-Rodrigues V., Guimarães J.E., Fernandes M.X., Pinto M.M., Sousa E., Vasconcelos M.H.
Presented by M.H. Vasconcelos. “Sensitizing drug resistant leukaemia cells to drugs”. I3S third Scientific Retreat - IBMC, INEB, IPATIMUP.
Póvoa de Varzim, Portugal. 11/05/2012.
Ferreira I.C.F.R., Vaz J.A., Barros L., Almeida G.M., Martins A., Vasconcelos M.H.
Presented by ICFR Ferreira. “Cogumelos silvestres portugueses: valorização como alimentos funcionais e fonte de nutracêuticos”.
Fórum CIMO (Centro de Investigação de Montanha), “Ciência e Desenvolvimento”.. Bragança, Portugal. 20/11/2012.
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Proteolysis in diseases
Objectives
A 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
Identification of signatures of natural selection among genes of serine protease inhibitors - “Loss and Gain of
Function in SERPINB11: an Example of a Gene under Selection on Standing Variation, with Implications for HostPathogen Interactions” Work published by Seixas et al in Plos ONE.
Serine protease inhibitors (SERPINs) are crucial in the regulation of diverse biological processes including inflammation and immune
response. SERPINB11, located in the 18q21 gene cluster, is a polymorphic gene/pseudogene coding for a non-inhibitory SERPIN. In a
genome-wide scan for recent selection, SERPINB11 was identified as a potential candidate gene for adaptive evolution in Yoruba. The
present study sought a better understanding of the evolutionary history of SERPINB11, with special focus on evaluating its selective
signature. Through the resequencing of coding and noncoding regions of SERPINB11 in 20 Yorubans and analyzing primate orthologous
sequences, we identified a full-length SERPINB11 variant encoding a non-inhibitory SERPIN as the putative candidate of selection – probably driven to higher frequencies by an adaptive response using preexisting variation. In addition, we detected contrasting evolutionary
features of SERPINB11 in primates: While primate phylogeny as a whole is under purifying selection, the human lineage shows evidence
of positive selection in a few codons, all associated with the active SERPINB11. Comparative modeling studies suggest that positively
selected codons reduce SERPINB11’s ability to undergo the conformational changes typical of inhibitory SERPINs – suggesting that it is
evolving towards a new non-inhibitory function in humans. Significant correlations between SERPINB11 variants and the environmental
variables, pastoralism and pathogen richness, have led us to propose a selective advantage through host-pathogen interactions, possibly linked to an adaptive response combating the emergence of infectious diseases in recent human evolution. This work represents the
first description of a resurrected gene in humans, and may well exemplify selection on standing variation triggered by drastic ecological
shifts.
Comparative genomics of the KLK family - “Birth-and-death of KLK3 and KLK2 in primates: adaptation driven by
reproductive biology” - Work published by Marques et al in Genome Biology and Evolution
The Kallikrein (KLK) gene family, located at chromosome 19q13.3-4 comprises the largest uninterrupted locus of serine proteases in the
human genome, and represents a notable case for studying the evolutionary fate of duplicated genes. In primates, a recent duplication
event gave rise to KLK2 and KLK3, both encoding essential proteins for the cascade of seminal plasma liquefaction. Briefly, upon ejaculation, the epididymal fluid is mixed with prostate and seminal vesicles secretions containing semenogelins (SEMG1 and SEMG2) to form
a coagulum that entraps sperm. Later, these are released with the hydrolysis of SEMGs by KLK3 and KLK2. We reconstructed the evolutionary history of KLK2 and KLK3 by comparative analysis of the orthologous sequences from 23 primate species, testing the hypothesis
of concerted evolution with SEMGs. Our findings support the placement of the KLK2-KLK3 duplication in the Catarrhini ancestor, and
unveil the frequent loss of KLK2 throughout primate evolution by different genomic mechanisms, including unequal crossing-over,
deletions, and pseudogenization. Furthermore, we found an association between mating system, the number of SEMG repeat units
determining the semen coagulum thickness and the number of functional KLK2 and KLK3. In most cases, higher repeat numbers and
polyandry are associated with active KLK2 and KLK3, while lower repeat numbers and monoandry are linked to the lack of one or both
of them. Finally, we propose an adaptive evolution of KLK2 and KLK3 in concert with SEMGs in a process driven by sperm competition.
Studying Genetic Variation and Positive selection at the WFDC locus in Hominids - “Reproduction and Immu46
Activity Report
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nity Driven Natural Selection in the Human WFDC Locus” - Work by Ferreira et al accepted for publication in
Molecular Biology and Evolution The whey acidic protein (WAP) 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). WFDC and SEMG genes have a strikingly high rate of amino
acid replacement (dN/dS), indicative of responses to adaptive pressures during vertebrate evolution. To better understand the selection
pressures acting on WFDC genes in human populations, we resequenced 18 genes and 54 non-coding segments in 71 European (CEU),
African (YRI), and Asian (CHB+JPT) individuals. Overall, we identified 484 single-nucleotide polymorphisms (SNPs), including 65 coding
variants (of which 49 are nonsynonymous differences). Using classic neutrality tests we confirmed the 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 of 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. This study provides further evidence that the WFDC
and SEMG loci have been under strong adaptive pressure within the short timescale of modern humans.
Glycophenotypic alterations induced by Pteridium aquilinum in mice gastric mucosa: synergistic effect with
Helicobacter pylori infection. - Gomes J, Magalhães A, Carvalho AS, Hernandez GE, Papp SL, Head SR, Michel V,
David L, Gärtner F, Touati E, Reis CA.
The bracken fern Pteridium aquilinum is a plant known to be carcinogenic to animals. Epidemiological studies have shown an association between bracken fern exposure and gastric cancer development in humans. The biological effects of exposure to this plant within
the gastric carcinogenesis process are not fully understood. In the present work, effects in the gastric mucosa of mice treated with
Pteridium aquilinum were evaluated, as well as molecular mechanisms underlying the synergistic role with Helicobacter pylori infection.
Our results showed that exposure to Pteridium aquilinum induces histomorphological modifications including increased expression of
acidic glycoconjugates in the gastric mucosa. The transcriptome analysis of gastric mucosa showed that upon exposure to Pteridium
aquilinum several glycosyltransferase genes were differently expressed, including Galntl4, C1galt1 and St3gal2, that are mainly involved
in the biosynthesis of simple mucin-type carbohydrate antigens. Concomitant treatment with Pteridium aquilinum and infection with
Helicobacter pylori also resulted in differently expressed glycosyltransferase genes underlying the biosynthesis of terminal sialylated
Lewis antigens, including Sialyl-Lewis(x). These results disclose the molecular basis for the altered pattern of glycan structures observed
in the mice gastric mucosa. The gene transcription alterations and the induced glycophenotypic changes observed in the gastric mucosa
contribute for the understanding of the molecular mechanisms underlying the role of Pteridium aquilinum in the gastric carcinogenesis
process.
Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation. Pinto R, Carvalho AS, Conze T, Magalhães A, Picco G, Burchell JM, Taylor-Papadimitriou J, Reis CA, Almeida R,
Mandel U, Clausen H, Söderberg O, David L.
Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins
expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus
far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We
therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity
of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1,
MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a)) and SialylLe(x) (SLe(x)) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung,
breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLe(a)/SLe(x)-MUC1 and STn/SLe(a)/SLe(x)-MUC2 glycoforms
in =50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLe(a)-MUC2, STn/T/SLe(a) SLe(x)-MUC5AC and
STn/T/SLe(a)/SLe(x)-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs.
In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing
specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.
Strategies to Identify Recognition Signals and Targets of SUMOylation. - Da Silva-Ferrada E, Lopitz-Otsoa F,
Lang V, Rodríguez MS, Matthiesen R.
SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques have been used to explore the functional consequences of protein SUMOylation. Most approaches consider the identification of sequences on substrates, adaptors, or
receptors regulating the SUMO conjugation, recognition, or deconjugation. The large majority of the studied SUMOylated proteins
contain the sequence [IVL]KxE. SUMOylated proteins are recognized by at least 3 types of hydrophobic SUMO-interacting motifs (SIMs)
that contribute to coordinate SUMO-dependent functions. Typically, SIMs are constituted by a hydrophobic core flanked by one or two
clusters of negatively charged amino acid residues. Multiple SIMs can integrate SUMO binding domains (SBDs), optimizing binding, and
control over SUMO-dependent processes. Here, we present a survey of the methodologies used to study SUMO-regulated functions
and provide guidelines for the identification of cis and trans sequences controlling SUMOylation. Furthermore, an integrative analysis of
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known and putative SUMO substrates illustrates an updated landscape of several SUMO-regulated events. The strategies and analysis
presented here should contribute to the understanding of SUMO-controlled functions and provide rational approach to identify biomarkers or choose possible targets for intervention in processes where SUMOylation plays a critical role.
PAnalyzer: A software tool for protein inference in shotgun proteomics. - Prieto G, Aloria K, Osinalde N, Fullaondo A, Arizmendi JM, Matthiesen R.
Protein inference from peptide identifications in shotgun proteomics must deal with ambiguities that arise due to the presence of
peptides shared between different proteins, which is common in higher eukaryotes. Recently data independent acquisition (DIA) approaches have emerged as an alternative to the traditional data dependent acquisition (DDA) in shotgun proteomics experiments.
MSE is the term used to name one of the DIA approaches used in QTOF instruments. MSE data require specialized software to process
acquired spectra and to perform peptide and protein identifications. However the software available at the moment does not group the
identified proteins in a transparent way by taking into account peptide evidence categories. Furthermore the inspection, comparison
and report of the obtained results require tedious manual intervention. Here we report a software tool to address these limitations for
MSE data.
RESULTS: In this paper we present PAnalyzer, a software tool focused on the protein inference process of shotgun proteomics. Our
approach considers all the identified proteins and groups them when necessary indicating their confidence using different evidence
categories. PAnalyzer can read protein identification files in the XML output format of the ProteinLynx Global Server (PLGS) software
provided by Waters Corporation for their MSE data, and also in the mzIdentML format recently standardized by HUPO-PSI. Multiple files
can also be read simultaneously and are considered as technical replicates. Results are saved to CSV, HTML and mzIdentML (in the case
of a single mzIdentML input file) files. An MSE analysis of a real sample is presented to compare the results of PAnalyzer and ProteinLynx
Global Server.
CONCLUSIONS: We present a software tool to deal with the ambiguities that arise in the protein inference process. Key contributions
are support for MSE data analysis by ProteinLynx Global Server and technical replicates integration. PAnalyzer
SIR: Deterministic protein inference from peptides assigned to MS data. - Matthiesen R, Prieto G, Amorim A,
Aloria K, Fullaondo A, Carvalho AS, Arizmendi JM.
Currently the bottom up approach is the most popular for characterizing protein samples by mass spectrometry. This is mainly attributed to the fact that the bottom up approach has been successfully optimized for high throughput studies. However, the bottom up
approach is associated with a number of challenges such as loss of linkage information between peptides. Previous publications have
addressed some of these problems which are commonly referred to as protein inference. Nevertheless, all previous publications on the
subject are oversimplified and do not represent the full complexity of the proteins identified. To this end we present here SIR (spectra
based isoform resolver) that uses a novel transparent and systematic approach for organizing and presenting identified proteins based
on peptide spectra assignments. The algorithm groups peptides and proteins into five evidence groups and calculates sixteen parameters for each identified protein that are useful for cases where deterministic protein inference is the goal. The novel approach has been
incorporated into SIR which is a user-friendly tool only concerned with protein inference based on imports of Mascot search results. SIR
has in addition two visualization tools that facilitate further exploration of the protein inference problem.
Integrative analysis of the ubiquitin proteome isolated using Tandem Ubiquitin Binding Entities (TUBEs). Lopitz-Otsoa F, Rodriguez-Suarez E, Aillet F, Casado-Vela J, Lang V, Matthiesen R, Elortza F, Rodriguez MS.
The successful use of proteasome inhibitors in clinical trials revealed the potential of the Ubiquitin Proteasome System for drug development. Protein remodeling through ubiquitylation is known to regulate the stability and activity of essential cellular factors through
largely uncharacterized mechanisms. Here, we used Tandem repeated Ubiquitin Binding Entities (TUBEs) under non-denaturing conditions followed by mass spectrometry analysis to study global ubiquitylation events that may lead to the identification of potential drug
targets. Using this approach we identified 643 proteins including known and unknown ubiquitin targets from human breast adenocarcinoma MCF7 cells treated with Adriamycin. Coherent with a global cellular response to this genotoxic insult, cellular factors identified are
involved in protein synthesis, cellular transport, RNA post-transcriptional modification and signaling pathways regulating early stress
responses. This includes components of large macromolecular complexes such as subunits and regulators of the proteasome, supporting the use of this method to characterize networks of molecular interactions coordinated by ubiquitylation. Further in vitro and in
silico analysis confirmed that 84% of the total proteins identified here, are ubiquitylated. More importantly the enrichment of known
biomarkers and targets for drug development, underlined the potential of this approach for the identification of this clinically relevant
information.
Book chapters and one edited book “2nd edition of Mass Spectrometry Data Analysis in Proteomics”
http://www.springer.com/new+%26+forthcoming+titles+%28default%29/book/978-1-62703-391-6
1. Introduction to mass spectrometry-based proteomics (Update from first edition), PhD Rune Matthiesen and Jakob Bunkenborg,
Methods Mol Biol., 2013
2. LC-MS spectra processing (Update from first edition), PhD Rune Matthiesen, Methods Mol Biol., 2013
3. Algorithms for database dependent search of MSMS data, PhD Rune Matthiesen, Methods Mol Biol., 2013
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4. Interpretation of Collision-Induced Fragmentation Tandem Mass Spectra of Posttranslationally Modified Peptides (Update from first
edition) PhD Jakob Bunkenborg and PhD Rune Matthiesen, Methods Mol Biol., 2013
5. Methods and algorithms for quantitative proteomics by mass spectrometry (Update chapter), PhD Ana Sofia Carvalho and PhD Rune
Matthiesen, , Methods Mol Biol., 2013
6. Tools for Protein Post Translational Modifications Analysis: FAK a case study? Paula Voabil, Catarina Fonseca, PhD Ana Sofia Carvalho
and PhD Rune Matthiesen, Methods Mol Biol., 2013
Mechanistic insights into anti-cancer drugs by MS-based drug profiling or pharmacoproteomics
We have established a complete pipeline for analysis of clinical samples or in vitro models using state of art MS instrument which supports all quantitative MS technologies. There are other laboratories that have pipelines for specific technologies. The unique feature
of the software is that it can include many variable modifications in the search against a protein database and have standardized the
analysis of all main MS-based quantitative methods. We can demonstrate that current state of art MS is so sensitive that a large number of protein modifications can be found directly without tedious peptide enrichment for specific modifications. This technology will
become even more significant as MS instruments get cheaper and more sensitive. We have used this software to analyze large MS data
sets obtained in a pharmacoproteomics project where we have fully characterized the molecular effect of specific cell proliferation
inhibitors of cancer cell lines on the molecular level (transcription arrays, MS-based proteomics, confocal microscopy, apoptosis, cell
cycle assays and pharmacological testing). This technology is also used to study 80 semen samples from control and infertile patients.
We have all MS methods established in my current group that is sample preparation for MS (in gel digestion, stable isotope labeling and
so on). We don’t have a MS instrument and are currently using a service in US. We prepare the samples in our laboratory and the service
only runs the MS (lowering the cost) and sends us back the raw data for us to analyze. We are able to identify and quantify 2000-3000
proteins (after collapsing the isoforms to the genes that encode them) from single two hour run on for example a nuclear extract. From
a fractionated raw extract more than 12000 proteins and 30000-50000 peptides with associated modifications can be identified and
quantified. This is a very powerful technology to obtain mechanistic insight into drug effects.
Internationalization/Networking
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.
Future Research
Evaluating the role of proteolysis in the male reproductive system through the study of KLK (19q13.4) and WFDC
(20q13) gene clusters. (PTDC/BEX-GMG/0242/2012)
Project submitted to FCT and recommended for funding
Many issues about the activities of KLKs and WFDCs still require clarification, including the contribution of genetic variation to male
reproduction and current patterns of health and disease. With this project, we propose: to uncover the genetic variation of KLK and
WFDC clusters that to some extent underlie phenotypic variation in male reproductive function in both healthy and infertile individuals;
to evaluate the biological significance of both common and rare variants with predicted functional repercussions, either beneficial or
deleterious, while simultaneously contributing to better characterization of these genes; and to address the long-term evolution of KLK
and WFDC to infer the importance of diverse reproductive factors and to detect major modifications with high impact on gene activity.
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Activity Report
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Participation in PhD Programs
Rune MatthiesenGABBA PhD program
Prizes
Luís Vaz Rodrigues, Filipa Costa, Patrícia Marques, Carlos Mendonça, Jorge Rocha, Susana SeixasPortuguese Society of Pneumology
(SPP) | Takeda Portugal 2012 - Prize for the best work published in a international scientific journal: - Rodrigues LV, Costa F, Marques P,
Mendonça C, Rocha J, Seixas S (2012). Severe alpha-1-antitrypsin deficiency caused by Q0ourém allele: clinical features, haplotype characterization and history. Clinical Genetics 81:462-469. - (Published online in 2011 Apr 25)
Ana Sofia CarvalhoDr. Ana Sofia Carvalho obtained three year post-doctoral fellowship from FCT starting summer 2013. - Mechanistic
insights into anti-cancer drugs by MS-based drug profiling or pharmacoproteomics
Rune MatthiesenDr. Matthiesen was recently selected in the 2012 FCT INVESTIGATOR PROGRAMME which was evaluated by panel of
European Research Council members for a position which corresponds to an associate professor position and was ranked among the
top 80 candidates whom received maximum score. - Mechanistic insights into anti-cancer drugs by MS-based drug profiling or pharmacoproteomics - Invited Talks
Susana Seixas. Patterns of diversity of alpha-I-antitrypsin and other genes of proteolysis.. 15th Portugaliæ Genetica. ‘Iberian Peninsula:
at the gate between the Mediterranean and the Atlantic worlds’. Porto, Porugal. 23/03/2012.
Rune Matthiesen. High-throughput clinical proteomics. ONCOBIOLOGIA: DO DIAGNOSTICO AO TRATAMENTO DO CANCRO. ICBAS, Porto. 05/12/2012.
Rune Matthiesen. New strategies for protein inference. Proteomics Workshop 2012. Aveiro, Portugal. 04/06/2012.
Rune Matthiesen. Overview of MS-based proteomics technologies. XIII Jornadas de Biologia Aplicada. Braga, University, Portugal.
08/03/2012.
Oral Presentations
Rune Matthiesen. Mass Spectrometry based peptide quantitation for studying epigenetic modulators used in cancer therapies. GABBA.
IPATIMUP, Porto, Portugal. 16/04/2012.
Rune Matthiesen. Overview of MS-based proteomics technologies. Invitation. INL, Braga, Portugal. 07/02/2012.
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Genetic Diversity
Objectives
The group aims to establish a bridge between population genetics and clinical genetics. This symbiosis is of major importance when
analysing non recombining genetic markers, such as mitochondrial DNA (mtDNA) and Y-chromosome. For these markers it is extremely
difficult to disentangle between neutral and pathologic diversities because of its transmission in block and its haplotypic distribution in
human populations (many rare haplotypes). We will pursuit a detailed characterisation of worldwide genetic diversity for the uniparental markers, and the design of studies to investigate complex phenotypes, namely cancer and Dengue. New developments in biostatistics and bioinformatics will be essential for an efficient evaluation of genetic diversity and mutation models in neutral and pathological
conditions.
In order to accompany the advances of the current genomic era, we are upgrading to the screening of genome-wide chips at a population level, taking advantage of the powerful links between anthropology, bioinformatics, population genetics and clinical genetics that
the group has already established.
Main Achievements
From Networks to Trees
Phylogenetic networks are a useful way of displaying relationships between nucleotide or protein sequences. They diverge from phylogenetic trees as networks present cycles, several possible evolutionary histories of the sequences analysed, while a tree presents a
single evolutionary relationship. Networks are especially useful in studying markers with a high level of homoplasy (same mutation happening more than once during evolution) like the control region of mitochondrial DNA (mtDNA), where the researcher does not need to
compromise with a single explanation for the evolution suggested by the data. However in many instances, trees are required. One case
where this happens is in the founder analysis methodology that aims at estimating migration times of human populations along history
and prehistory. Currently, the founder analysis methodology implicates the creation of networks, from where a probable tree will be
extracted by hand by the researcher, a time-consuming process, prone to errors and to the ambiguous decisions of the researcher. In
order to automate the founder analysis methodology an algorithm that extracts a single probable tree from a network in a fast, systematic way is presented here.
Publication: Alves et al. (2012) Adv Int Soft Comp
Somatic mitochondrial DNA mutations in cancer escape purifying selection and high pathogenicity mutations
lead to the oncocytic phenotype
We evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compare these to the distribution of
variations observed in the global human population and all possible protein variations that could occur in human mtDNA. Our results
showed that the somatic mtDNA mutations reported over all tumors are indistinguishable from a random selection from the set of all
possible amino acid variations, and have therefore escaped the effects of purifying selection that act strongly at the population level.
We showed that the pathogenicity of somatic mtDNA mutations is a determining factor for the oncocytic phenotype. The opposite
associations of the Complex I and Complex V variants with the oncocytic and non-oncocytic tumors implies that low mitochondrial
membrane potential may play an important role in determining the oncocytic phenotype.
Publication: Pereira et al. (2012) BMC Cancer
Mitochondrial DNA signals of Late Glacial recolonization of Europe from Near Eastern refugia
Human populations, along with those of many other species, are thought to have contracted into a number of refuge areas at the height
of the last Ice Age. European populations are believed to be, to a large extent, the descendants of the inhabitants of these refugia,
and some extant mtDNA lineages can be traced to refugia in Franco-Cantabria (haplogroups H1, H3, V, and U5b1), the Italian Peninsula
(U5b3), and the East European Plain (U4 and U5a). Parts of the Near East, such as the Levant, were also continuously inhabited throughout the Last Glacial Maximum, but unlike western and eastern Europe, no archaeological or genetic evidence for Late Glacial expansions
into Europe from the Near East has hitherto been discovered. We reported, on the basis of an enlarged whole-genome mitochondrial
database, that a substantial, perhaps predominant, signal from mitochondrial haplogroups J and T, previously thought to have spread
primarily from the Near East into Europe with the Neolithic population, may in fact reflect dispersals during the Late Glacial period,
~19–12 thousand years (ka) ago.
Publication: Pala et al. (2012) Am J Hum Genet.
Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication
High levels of genetic diversity in horse mtDNA have been detected when analyzing the control region; recurrent mutations, however,
tend to blur the structure of the phylogenetic tree. Here, we brought the horse mtDNA phylogeny to the highest level of molecular resolution by analyzing 83 mitochondrial genomes from modern horses across Asia, Europe, the Middle East, and the Americas. Our data
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reveal 18 major haplogroups (A–R) with radiation times that are mostly confined to the Neolithic and later periods and place the root
of the phylogeny corresponding to the Ancestral Mare Mitogenome at ~130–160 thousand years ago. All haplogroups were detected in
modern horses from Asia, but F was only found in E. przewalskii—the only remaining wild horse. Therefore, a wide range of matrilineal
lineages from the extinct E. ferus underwent domestication in the Eurasian steppes during the Eneolithic
period and were transmitted to modern E. caballus breeds. Importantly, now that the major horse haplogroups have been defined, each
with diagnostic mutational motifs (in both the coding and control regions), these haplotypes could be easily used to (i) classify wellpreserved ancient remains, (ii) (re)assess the haplogroup variation of modern breeds, including Thoroughbreds, and (iii) evaluate the
possible role of mtDNA backgrounds in racehorse performance.
Publication: Achilli et al. (2012) PNAS
Pleistocene-Holocene boundary in southern Arabia from the perspective of human mtDNA variation
It is now known that several population movements have taken place at different times throughout southern Arabian prehistory. One
of the principal questions under debate is if the Early Holocene peopling of southern Arabia was mainly due to input from the Levant
during the Pre-Pottery Neolithic B, to the expansion of an autochthonous population, or some combination of these demographic processes. Since previous genetic studies have not been able to include all parts of southern Arabia, we have helped fill this lacuna by collecting new population datasets from Oman (Dhofar) and Yemen (Al-Mahra and Bab el-Mandab). We identified several new haplotypes
belonging to haplogroup R2 and generated its whole genome mtDNA tree with age estimates undertaken by different methods. R2,
together with other considerably frequent southern Arabian mtDNA haplogroups (R0a, HV1, summing up more than 20% of the South
Arabian gene pool) were used to infer the past effective population size through Bayesian skyline plots. These data indicate that the
southern Arabian population underwent a large expansion already some 12 ka. A founder analysis of these haplogroups shows that this
expansion is largely attributed to demographic input from the Near East. These results support thus the spread of a population coming
from the north, but at a significantly earlier date than presently considered by archaeologists. Our data suggest that some of the mtDNA
lineages found in southern Arabia have persisted in the region since the end of the Last Ice Age.
Publication Al-Abri et al. (2012) Am J Phys Anthropol
Human neutral genetic variation and forensic STR data
The forensic genetics field is generating extensive population data on polymorphism of short tandem repeats (STR) markers in globally distributed samples. In this study we explored and quantified the informative power of these datasets to address issues related to
human evolution and diversity, by using two online resources: an allele frequency dataset representing 141 populations summing up to
almost 26 thousand individuals; a genotype dataset consisting of 42 populations and more than 11 thousand individuals. We show that
the genetic relationships between populations based on forensic STRs are best explained by geography, as observed when analysing
other worldwide datasets generated specifically to study human diversity. However, the global level of genetic differentiation between
populations (as measured by a fixation index) is about half the value estimated with those other datasets, which contain a much higher
number of markers but much less individuals. We suggest that the main factor explaining this difference is an ascertainment bias in forensics data resulting from the choice of markers for individual identification. We show that this choice results in average low variance of
heterozygosity across world regions, and hence in low differentiation among populations. Thus, the forensic genetic markers currently
produced for the purpose of individual assignment and identification allow the detection of the patterns
of neutral genetic structure that characterize the human population but they do underestimate the levels of this genetic structure compared to the datasets of STRs (or other kinds of markers) generated specifically to study the diversity of human populations.
Publication: Silva et al. (2012) Plos One
The expansion of mtDNA haplogroup L3 within and out of Africa
Although fossil remains show that anatomically modern humans dispersed out of Africa into the Near East 100 to 130 ka, genetic evidence from extant populations has suggested that non-Africans descend primarily from a single successful later migration. Within the
human mitochondrial DNA (mtDNA) tree, haplogroup L3 encompasses not only many sub-Saharan Africans but also all ancient nonAfrican lineages, and its age therefore provides an upper bound for the dispersal out of Africa. An analysis of 369 complete African L3
sequences places this maximum at 70 ka, virtually ruling out a successful exit before 74 ka, the date of the Toba volcanic supereruption
in Sumatra. The similarity of the age of L3 to its two non-African daughter haplogroups, M and N, suggests that the same process was
likely responsible for both the L3 expansion in Eastern Africa and the dispersal of a small group of modern humans out of Africa to settle
the rest of the world. The timing of the expansion of L3 suggests a link to improved climatic conditions after 70 ka in Eastern and Central
Africa rather than to symbolically mediated behavior, which evidently arose considerably earlier. The L3 mtDNA pool within Africa suggests a migration from Eastern Africa to Central Africa 60 to 35 ka and major migrations in the immediate postglacial again linked to
climate. The largest population size increase seen in the L3 data is 3–4 ka in Central Africa, corresponding to Bantu expansions, leading
diverse L3 lineages to spread into Eastern and Southern Africa in the last 3–2 ka.
Publication: Soares et al. (2012) Mol Biol Evol
Mitochondrial relicts of the first steps along the southern route out of Africa
A major unanswered question regarding the dispersal of modern humans around the world concerns the geographical site of the first
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human steps outside of Africa. The ‘‘southern coastal route’’ model predicts that the early stages of the dispersal took place when
people crossed the Red Sea to southern Arabia, but genetic evidence has hitherto been tenuous. We have addressed this question by
analyzing the three minor west-Eurasian haplogroups, N1, N2, and X. These lineages branch directly from the first non-African founder
node, the root of haplogroup N, and coalesce to the time of the first successful movement of modern humans out of Africa, ~60 thousand years (ka) ago.We sequenced complete mtDNA genomes from 85 Southwest Asian samples carrying these haplogroups and compared them with a database of 300 European examples. The results show that these minor haplogroups have a relict distribution that
suggests an ancient ancestry within the Arabian Peninsula, and they most likely spread from the Gulf Oasis region toward the Near East
and Europe during the pluvial period 55–24 ka ago. This pattern suggests that Arabia was indeed the first staging post in the spread of
modern humans around the world.
Publication: Fernandes et al. (2012) Am J Hum Genet
A ‘‘Copernican’’ Reassessment of the Human Mitochondrial DNA Tree from its Root
Mutational events along the human mtDNA phylogeny are traditionally identified relative to the revised Cambridge Reference Sequence,
a contemporary European sequence published in 1981. This historical choice is a continuous source of inconsistencies, misinterpretations, and errors in medical, forensic, and population genetic studies. Here, after having refined the human mtDNA phylogeny to an
unprecedented level by adding information from 8,216 modern mitogenomes, we propose switching the reference to a Reconstructed
Sapiens Reference Sequence, which was identified by considering all available mitogenomes from Homo neanderthalensis. This ‘‘Copernican’’ reassessment of the human mtDNA tree from its deepest root should resolve previous problems and will have a substantial
practical and educational influence on the scientific and public perception of human evolution by clarifying the core principles of common ancestry for extant descendants.
Publication: Behar et al. (2012) Am J Hum Genet
Internationalization/Networking
Characterisation of the main human population migrations in Southeast Asia
Martin Richards (School of Applied Sciences, University of Huddersfield, UK), Vincent Macaulay (Department of Statistics, University of
Glasgow, UK)
Analyses of pathogenicity in mitochondrial proteins coded by mitochondrial and nuclear genomes
David C. Samuels (Center for Human Genetics Research, Department of Molecular Physiology and Biophysics, Vanderbilt University
Medical Center, Nashville, TN, USA)
Analysis of pathogenicity in mitochondrial genes in oncocytic cancers
David C. Samuels (Center for Human Genetics Research, Department of Molecular Physiology and Biophysics, Vanderbilt University
Medical Center, Nashville, TN, USA)
Impact of human genetics on the outcome of Dengue infection
Anavaj Sakuntabhai and Richard Paul (Institut Pasteur, Paris, France) and colleagues of the EU funded project DENFREE
Network on the transatlantic slave trade
Thomas Gilbert (University of Copenhagen) and colleagues of the EU funded project EUROTAST
Characterisation of the settlement of the Arabian Peninsula
Farida Alshamali, (Dubai Police General Headquarters) and Viktor Cerny (Institute of Archaeology of the Academy of Sciences of the
Czech Republic, Prague)
Characterisation of the genetic diversity in the African Sahel through Genome-Wide screening
Viktor Cerny (Institute of Archaeology of the Academy of Sciences of the Czech Republic, Prague)
Future Research
An improved method for estimating migration times and a re-evaluation of the settlement of Europe
Together with Dr Vincent Macaulay we are testing an improved method for estimating migration times (founder analysis) that we will
apply in the context of the settlement of Europe, a long-term research area of the group.
Characterising mtDNA diversity in Southeast Asia and the Pacific
This is a long-ongoing aim, and it was funded in the last round of FCT projects (PI: Pedro Soares, project PTDC/IVC-ANT/4917/2012) and
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Activity Report
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has a PhD student (granted by FCT) dedicated to it. Southeast Asia is one of the regions worldwide that is less characterised if not really
the least characterized. This project aims to greatly increase our understanding of the population structure in the region. Our effort
will be initially focused in characterising complete mtDNA genomes. We will investigate both the role of Neolithic societies in the region
in the last 5 thousand years, as well as the time of the first settlement of Southeast Asia and the Pacific before 50 thousand years. We
will also characterize the genome-wide diversity of the region and obtain detailed picture of the likely complex population structure in
Southeast Asia.
Genome-wide screening in African Sahel
We are already analysing results for 2.5 million SNPs in around 170 individuals from several Sahel populations, and we aim to publish
these results during this year. Results testify that this African region was the main corridor for migrations between west and east, and
towards north and south, and is reveilling interesting genetic patterns in the nomadic Fulani. In this region certain phenotypes have
evolved, such as malaria susceptibility and food adaptations (e.g. lactose intolerance). The high coverage of our chip will allow to gain
insights on selection to these traits.
Genomics and complex traits - dengue infection
In this 5-year project funded by EU (beginning January 2012) we will contribute with the tasks of analysis of genome-wide screenings to
enlarged population sets in individuals with dengue infection from Thailand and Cuba. Cuba is a very interesting sample due to its high
level of admixture between African (not susceptible to dengue infection) and European (susceptible to dengue infection) gene pools.
We will also characterise the allelic frequencies accross Europe for candidate genes identified by us and other members of the project,
in order to evaluate the genetic susceptibility of European populations to Dengue infection. We will host the second annual meeting of
the project in May 2013.
To determine the range of somatic mutations in both mitochondrial and nuclear DNA responsible for the oncocytic phenotype in thyroid tumors
We will assemble mitochondrial DNA genomes for all thyroid tumor samples and paired normal samples available in the NIH/NCI resource The Cancer Genome Atlas (TCGA). We will identify somatic mtDNA changes in the tumor (both fixed and heteroplasmic) and
assess the pathogenicity of those somatic mutations. As well as to identify somatic changes in the nuclear DNA genes for oxidative phosphorylation and the TCA cycle and assess their pathogenicity. This will be coupled with a careful pathological revision of the oncocytic
phenotype of the thyroid tumors from the TCGA diagnostic slides. These tasks were included in a NIH grant application of our group,
the Cancer Biology group and our collaborator David C. Samuels - the project was not funded but we are still waiting for the detailed
evaluation. Meanwhile we got a Post-doc grant for two years who will initiate these tasks.
Development of an in vitro tool to evaluate the pathogenicity of nonsynonymous mtDNA mutations
This is a collaboration between our group, Cancer Biology (Valdemar Máximo) and the new group of Expression Regulation in Cancer
(Carla Oliveira). It gathers our expertise in the bioinformatic inference of pathogenecity, Valdemar’s expertise on metabolisms and Carla
expertise’s in gene expression. We aim to insert exogeneous mtDNA proteins (translated in the cytoplasm instead of in the mitochondrion) by adding a sign for migration of the protein to the mitochondria (based on the real pathway which carries proteins coded by
the nuclear genome but that migrate to the mitochondria). If we are sucessfull in this, we can then evaluate the effect of any inferred
pathogenic nonsynonymous mutation.
Participation in PhD Programs
Luisa Pereira - Programa Doutoral e Mestrado de Investigação Biomédica, Faculty of Medicine University of Coimbra
Invited Talks
Pedro Soares. A História dos Genes Humanos. V Jornadas Concelhias da Ciência. Riba de Ave. 09/03/2012.
Luisa Pereira. A population genetics perspective on human mitochondrial DNA diversity and clinical implications. . XXI Porto Cancer
Meeting. Metabolism and Cancer from Etiopathogenesis to Therapy.. IPATIMUP, Porto. 19/04/2012.
Luisa Pereira. O Património Genético Português. . Congresso Nação, Nacionalismos e Identidades Nacionais. Associação de Professores
de História. . Guimarães. 06/10/2012.
Luisa Pereira. Mitochondrial DNA genomics: human population history, selection and pathogenicity.. 16ª Reunião Anual da Sociedade
Portuguesa de Genética Humana. Porto. 23/11/2012.
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POST-GRADUATION
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2012
Post-Graduation Unit
Overview
In 2012 several activities were developed by the Post-Graduation unit of IPATIMUP. These activities included internal and external modules for Post-graduation programs.
Highlights
Módulo: ONCOBIOLOGY
Local: IPATIMUP
Instituição: Gulbenkian/Champalimaud
Âmbito: The Programme for Advanced Medical Education
Número de alunos: 10
Data: 9-13 january 2012
ONCOBIOLOGIA - GABBA
Módulo: ONCOBIOLOGIA
Local: GABBA
Instituição: UP
Âmbito: Programa Graduado em Biologia Básica e Aplicada - da Universidade do Porto
Número de alunos:12
Datas: 9 - 18 April 2012
Módulo: ONCOBIOLOGIA
Módulo: ONCOBIOLOGIA para a FMUP
Local: IPATIMUP
Instituição: Faculdade de Medicina do Porto
Âmbito: PD em Medicina Oncologia Molecular
Número de alunos: aprox. 12
Datas: March and April 2012.
Workshop on Cancer Research: biological and molecular basis
Módulo: Workshop on Cancer Research: biological and molecular basis
Local: IPATIMUP
Instituição: IPATIMUP/ICBAS
Âmbito: Curso de formação contínua
Número de alunos: 20
Data: 15-18 May 2012
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2012
OUTREACH
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Science Diffusion
Overview
The Science Diffusion Unit aims to promote scientific culture, interacting whether with schools or with the community. Thus, during 2012
the Science Diffusion Unit has developed/been involved in the following activities/projects:
1. “Laboratório Aberto”
2. “Porto de Crianças”
3. “Mostra UP”
4. “VI Feira da Ciência – Vila de Conde”
5. “Ciência Viva no Verão”
6. IPATIMUP’s Day
7. “Noite dos Investigadores”
8. “Escola das Ciências da Vida e Saúde”
9. School meetings
10. “Entrelaçar” project
11. “Traz um amigo também”
12. “Segunda há Ciência”
Highlights
Laboratório Aberto
In 2012 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 2012, 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 (32 total) and there was also an increase in funding.
Activity Statistics
Visits to Laboratório Aberto in 2012 per district
District
Students
Teachers
Total
Aveiro
616
52
668
Braga
511
36
547
Castelo Branco
29
3
32
Coimbra
93
10
103
Leiria
171
16
187
Porto
3651
371
4022
Setúbal
72
6
78
Viseu
43
4
47
5186
498
5684
TOTAL
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Activity Report
Visits to Laboratório Aberto in 2012 per month
Month
Students
Teachers
Total
Jan
862
75
937
Fev
725
64
789
Mar
703
78
781
Abr
379
38
417
Mai
661
59
720
Jun
240
28
268
Jul
143
21
164
0
0
0
Ago
Set
62
4
66
Out
656
58
714
Nov
634
61
695
Dez
121
12
133
5186
498
5684
TOTAL
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.
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Activity Report
2012
Public Awareness of Cancer
Overview
The unit of Public Awareness of Cancer during 2012 developed a set of innovative and comprehensive cancer education programs,
targeted for specific audiences (e.g. teens, teachers, families). There was a strong focus on the development, of initiatives that foster
cancer awareness in the community. The core projects were focused on students (CancerMobile), teachers (Cancer- Educate to Prevent)
and families (Cancer in the Family).
Highlights
Project CancerMobile
We continue to develop cancer prevention educational contents for the CancerMobile sessions of breast and colorectal cancers.
Project Cancer Educate to Prevent
We develop a b-learning course for teachers about cancer prevention.
1st Edition, January-April, 2012 – 60 teachers.
2nd Edition, September-December, 2012 – 10 teachers.
The data collected in this innitiatives (enquiries, direct observation and semi-structured interviews)
is currently being processed for optimization of format and contents. The impact of such trainning courses is being evaluated at schools
of these teachers using also control groups. The study encompass around 1200 students and 230 teachers.
Project Cancer in the Family
We develop a medical information system about Hereditary Breast and Colorectal cancers. We have designed a website
Targeted to Men and women concerned with the possibility of hereditary cancer in the family used to obtain information either through
the internet or through printed materials.
We conceive a different website with quality information for different degrees of knowledge, clarifying on familial and hereditary cancer, referring to medical services for risk assessment and connecting to the medical / research universe (clinical trials)
with a powerful image/ feeling. The contents encompass different types of formats: small applications, short videos,
quizzes and booklets/leaflets.
Other Activities
Workshop - Inclusive Science
This workshop was designed to train teachers with the best practices for blind students science teaching.
1st Edition, January 21st, 2012 – 16 teachers
2nd Edition, April 14th, 2012 – 40 teachers
MICE App
Development of a new version of the educational software for microscopy - MICE , for the Ipad platform.
Pain Management App
Collaboration on the development of a smartphone application during the meeting “Health and Wellness Innovation 2012” promoted
by the MIT Media Lab. This application allows patients with chronic pain to register the symptoms, the medication and to contact with
the physician.
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IPATIMUP Diagnostics
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2012
Activity Report
2012
IPATIMUP Diagnostics
Overview
In 2012, IPATIMUP Diagnostics maintained the high quality standards successfully achieved in the previous year through CAP accreditation. The CAP self-inspection in March 2012 was felt as a positive action, involving all collaborators, allowing for deep awareness of the
requirements and their relevance. Also, the ISO 9001:2008 certification was maintained through the follow-up audit held in May 2012.
Other goals for 2012, 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 partially or totally accomplished.
Highlights
- CAP accreditation and ISO 9001:2008 certification was successfully maintained by IPATIMUP Diagnostics;
- Participation in one international network began in 2012 – EUROFORGEN;
- Client satisfaction increased relatively to the previous year;
- Training increased considerably in relation to previous years;
- Participation in research projects with health institutions and pharmaceutical companies also increased in 2012.
Activity Statistics
EXAMS
LAP
Total
12025
Histology
1066
Cytology (includes 2862 cases of FNA cytology)
9456
Molecular Pathology
616
Consultations
235
LDG
Total
3798
Tumour mutation screening
1998
Genetic Diagnosis
1800
LPIG
Total
195
Genetic Characterizations (including lineage markers)
117
Parentage investigations and genetic profile comparisons
(sample/individual)
78*
* each exam involves analysis of two or more samples/individuals.
CONSULTATIONS
Brazil
10
Ireland
4
Turkey
United Kingdom
18
Norway
4
Germany
Portugal
48
Italy
3
Switzerland
1
USA
4
Mozambique
16
Israel
2
16
1
Spain
10
Croatia
1
The Netherlands
3
Algeria
41
France
12
U Arab Emirates
1
Belgium
8
England
5
CAP
27
Total
235
Quality Control
1. INTERNAL QUALITY CONTROL
A) LAP
The main findings, compared with the last 3 years, were:
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Activity Report
2010
2011
2012
Total nº of cases
11.746
12.478
12.025
Cases reviewed
470
479
456
Criteria
2010
2011
2012
Identification of specimen, archive and macroscopy
1.06%
0.0%
0.0%
Diagnosis
0.2%
0.0%
0.2%
2012
Discordant values:
(1/456)
Report evaluation
2.5%
0.0%
(12/470)
Coding
0.4%
(2/456)
0.0%
3.5%
2.6%
(17/479)
(12/456)
In 2012 the “turn-around time” (TAT) was the following:
- Histological Exams: 61,24% of reports are sent in 2 working days.
- Cytological Exams: 99,03% of reports are sent in 2 working days.
- Screening cytologies: 100% of reports are sent in 7 working days.
The main results for the Gynecological Cytology quality control analysis were:
2010
2011
2012
Total cases
6733
6977
6862
Reviewed cases
1253
1281
1349
2010
2011
2012
1150
1204
1245
(91.78%)
(93.99%)
(92.3%)
Concordance between pathologist and cytotechnician
Discordance between pathologist and cytotechnician
103
77
104
(8.22%)
(6.01%)
(7.7%)
The total amount of unsatisfactory cases for analysis was 1,43% (0.85% in 2011, 0.59% in 2010).
ASCUS/ACG were diagnosed in 184 cases with a ASCUS/Lesion reason of 1,84 (2,23 in 2011, 2,57 in 2010).
B) LDG
- EGFR
During 2012, 486 pulmonary carcinoma cases were analysed for the presence of somatic mutations in exons 18, 19, 20 and 21 of the EGFR
gene. In terms of global frequencies as well as in terms of frequencies of specific mutations (table below), our detection rates are similar
to those published in the literature (1, 2).
1- Penzel R et al. Virchows Arch 458:95-98, 2011
2- Sharma SV et al. Nature Rev Cancer 7:169-181, 2007
EGFR Mutation
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IPATIMUP Results
N
%
Literature Results
%
Exon 18
2
2%
2-10
Exon 19
47
52%
45-50
Exon 20
11
12%
5-14
Exon 21
30
33%
20-45
Total mutations
90
19%
10-20
Wild-type
396
81%
Total cases
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Activity Report
2012
- KRAS
During 2012, 1429 colorectal carcinoma cases were analysed for the presence of somatic mutations in codons 12 and 13 of the KRAS gene.
In terms of global frequencies as well as in terms of frequencies of specific mutations (table below), our detection rates are similar to
those published in the literature (1, 2).
1- Vaughn et al. Genes, Chromosomes & Cancer 2011 [Epub ahead of print].
2- Neumann J et al. Pathology - Research and Practice 205:858-862, 2009.
KRAS Mutation
IPATIMUP Results
Literature Results
N
%
Rank
%
Rank
12Asp
220
38%
1
31-47
1
12Val
143
24%
2
22-29
2
13Asp
108
18%
3
14-21
3
12Cys
42
7%
4
6-9
4
12Ser
27
5%
6
4-6
5
12Ala
37
6%
5
3-6
6
12Arg
5
1%
7
1-2
7
13Cys
1
0%
8
0,5-1
8
3
1%
0-0,5
9
12dup/13dup
1 mutation
586
>1 mutation
8
Total mutations
594
42%
Wild-type
835
58%
Total cases
1429
30-50%
C) LPIG
The main features comprising internal quality control are measured according to PR.QUA.10. The main finding concerns the overall turnaround time (Tat), where better results were obtained for 2012 relatively to 2011:
Type of exam
Tat
% exams where Tat was accomplished
2011
2012
1
Exams with simple reference
samples
10 working days
80,75%
82,26%
2
Exams with complex reference
samples
20 working days
62,75%
100,00%
3
Exams with limit samples
30 working days
87,50%
96,43%
All the other quality control measures are above 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 2012. Specific details of each program may be consulted in the 2013 edition of our Quality Manual.
1. CAP - Proficiency Tests
Program
% Concordance
Evaluation of Cervicovaginal Cytology (PAPM)
PAPM-A
100%
PAPM-B
100%
Evaluation of Non-gynecological Cytology (NGC)
NGC-A
60%
NGC-B
80%
NGC-C
100%
NGC-D
80%
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Activity Report
Prog. for performance improvement in Surgical Path (PIP)
PIP-A
100%
PIP-B
100%
PIP-C
100%
PIP-D
100%
Evaluation of Immunohistochemistry (MK)
MK-A
53%
MK-B
91%
Immunohistochemistry HER-2- (HER2)
HER2-A
100%
HER2-B
100%
Hybrid capture for HPV (CHPVJ)
CHPVJ A
100%
CHPVJ B
80%
CHPVJ C
100%
ER and PgR Immunohistochemistry (PM2)
PM2-A
100%
PM2-B
100%
ISH HER2 (ISH2)
ISH-A
100%
ISH-B
100%
Molecular Genetics (MGL1 e MGL3) – CAP/ACMG
MGL-A
MGL-B
100%
100%
(Genotype)
(Interpretation)
100%
100%
(Genotype)
(Interpretation)
Parentage/Relationship-Filter Paper (PARF)
PARF-A
100%
PARF-B
100%
PARF-C
100%
Molecular Genetics (SEQ) –CAP/ACMG
SEQ-A
92%
100%
(nucleotide alt.)
(protein alt.)
SEQ-B
NR
NR
Molecular Pathology (EGFR)
EGFR-A
100%
EGFR-B
100%
Molecular Pathology (KRAS)
KRAS-A
0%
KRAS-B
100%
Molecular Pathology (KIT)
KIT-A
100%
KIT-B
100%
Molecular Pathology (MSI)
MSI-A
100%
MSI-B
100%
2. Other Proficiency Tests
Breast
UK NEQAS for Immunohistochemistry
In House
16,50
13,00
UK-NEQAS
16,50
14,00
Quality Control Program of the European Society of Pathology
KRAS
100%
Quality Control Program of the GHEP-ISFG
Basic Parentage
100%
Adv. Parentage
100%
Basic Forensic
99%
Adv. Forensic
100%
NR - Results not received, as of 11 March 2013.
70
GP
2012
Activity Report
2012
Other Activities
TRAINING
In total, there were 23 people who underwent training at IPATIMUP Diagnostics during 2012, according to the following table:
Start Date
End Date
Alessia Di Lorito
Name
07/24/12
08/03/12
Italy
Country
Ana Silva
01/02/12
04/02/12
Portugal
Ana Lima
11/12/12
02/08/13
Portugal
Antonio Galzerano
09/17/12
12/14/12
Italy
Antônio Ladeia
12/01/12
01/31/13
Brazil
Carlos Santos
12/01/12
01/31/13
Brazil
Daniela Schwingel
05/01/12
06/30/12
Brazil
Diana Matos
11/01/11
07/31/12
Portugal
Emilly Franco
10/01/12
11/30/12
Brazil
Esther Rossi
07/03/12
07/21/12
Italy
Sofia Martins
09/17/12
11/19/12
Portugal
Inês Pimpão
07/16/12
08/24/12
Portugal
Joana Nogueira
03/01/12
05/01/12
Portugal
Katia Remane
11/01/12
11/30/12
Mozambique
Mariana Coelho
09/01/12
11/30/12
Portugal
Melissa Fontes
09/17/12
10/12/12
Portugal
Márcia Baixia
11/13/12
12/28/12
Portugal
Gené Hijós
01/02/12
02/28/12
Spain
Rosilene Leal
05/01/12
06/30/12
Brazil
Rui Barranha
12/17/12
06/30/14
Portugal
Diogo Rios
10/10/12
01/23/13
Portugal
Iara Rodrigues
01/11/11
04/30/12
Brazil
Lígia Lourenço
01/23/12
01/27/12
Portugal
PUBLICATIONS
1.
Abelha FJ, Fernandes V, Botelho M, Santos P, Santos A, Machado JC, Barros H. Apolipoprotein E e4 allele does not increase the risk
of early postoperative delirium after major surgery. Journal of Anesthesia (in press).
2.
Annaratone L, Marchio C, Renzulli T, Castellano I, Cantarella D, Isella C, Macri L, Mariscotti G, Balmativola D, Cantanna E, Deambrogio C, Pietribiasi F, Arisio R, Schmitt F, Medico E, Sapino A. High-Throughput Molecular Analysis from Leftover of Fine Needle
Aspiration Cytology of Mammographically Detected Breast Cancer. Translational Oncology doi: 10.1593, 10: 1-10, 2012.
3.
António Amorim e Cíntia Alves. Genética: Uma introdução à sua aplicação na investigação de parentescos. Em Testes de Paternidade: Ciência, Ética e Sociedade; Organização Helena Machado/Susana Silva. Edições Húmus, Lda., 2012.
4.
Catherwood MA, Schmitt F, Tellez-Salto M. Molecular diagnostics and the training of future tissue- and cell-based pathologists.
Cytopathology doi: 10.1111/cyt 12015.
5.
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. Human Mutation (in press).
6.
Di Lorito A, Rosini S, Falò E, Gustapane S, Gomes M. Costa JL, Schmitt F. Molecular alterations in endometrial archived liquid-based
cytology. Diagnostic Cytopathology doi: 10.1002/dc 2012.
7.
Gerhard R, Costa JL, Schmitt F. Bening and malignant apocrine lesions of the breast. Expert Rev Anticancer Ther 12: 215-221, 2012.
8.
Gomes C, Magalhães M, Alves C, Amorim A, Pinto N, Gusmão L (2012) Comparative evaluation of alternative batteries of genetic markers to complement autosomal STRs in kinship investigations: autosomal indels vs. X-chromosome STRs. Int J Legal Med
126(6):917-21.
9.
Gusmão L, Alves C, Gomes I, Sánchez-Diz P (2012) Capillary Electrophoresis of an X-Chromosome STR Decaplex for Kinship Deficiency Cases. Methods Mol Biol. 2012;830:57-71
10. Khodjet-el-Khil H, Fadhlaoui-Zid K, Gusmão L, Alves C, Benammar-Elgaaied A, Amorim A (2012) Allele frequencies for 15 autosomal
STR markers in the Libyan population. Ann Hum Biol. 2012 Jan;39(1):80-3.
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Activity Report
11.
2012
Lebreiro A, Martins E, Machado JC, Abreu-Lima C. Diagnostic challenges of Marfan syndrome in an XYY young man. Cardiol Young
4:1-3, 2012.
12. Marinsek Z. P, Nolde N, Skelin IK, 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 2012 doi:
10.1111/cyt.12024.
13. Pinto N, Magalhães M, Conde-Sousa E, Gomes C, Pereira R, Alves C, Gusmão L, Amorim A (2012) Assessing paternities with inconclusive STR results: The suitability of bi-allelic markers. Forensic Sci Int Genet. 7(1):16-21.
14. Santos LF, Pereira T, Rodrigues B, Correia E, Moreira D, Nunes L, Costa A, Elvas L, Machado JC, Castedo S, Henriques C, Matos A,
Oliveira Santos. Diagnostic criteria for the Brugada syndrome: Can they be improved? Rev Port Cardiol 31:355-362, 2012.
15. Santos LF, Rodrigues B, Moreira D, Correia E, Nunes L, Costa A,Elvas L, Pereira T, Machado JC, Castedo S, Henriques C, Matos
A,Oliveira Santos. Criteria to predict carriers of a novel SCN5A mutation in a large Portuguese family affected by the Brugada
syndrome. Europace 14:882-888, 2012.
16. Schmitt F, Vielh P, Zeppa P. Cytology for pathologists: two sides of the same coin or different views of the same side? Cytopathology
23: 340-346, 2012.
17. Schmitt FC, Vielh P. Molecular biology and cytopathology. Principles and applications. Annales de pathologie 32: e57-e63, 2012.
PROJECTS
•
Project with Life Technology – Participation in design and validation of Ion AmpliSeq BRCA1/2 Panel.
•
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:
72
•
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.
•
FCT Project – Portuguese study on Familial Dilated Cardiomyopathy.
Activity Report
2012
INNOVATION & TRANSLATION
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Activity Report
74
2012
Activity Report
2012
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 (IP) 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 products
derived from their core research activities. Having started in February 2012, Innovation Unit undertook four main lines of action: 1) implementation, 2) registering and reconciling industrial property (IP) with scientific publication, 3) prospection of projects with potential to
generate innovative products or services, 4) capacitance of these projects towards commercial exploitation.
Highlights
Summary of 2012 activities:
As part of implementation actions, presentations were made to the Institute’s Research Groups, contents for web-page were designed,
Regulations were proposed to the board and coordination with Innovation offices from University of Porto and INEB was operated.
In terms of IP, the Unit initially felt the need to overcome an identified handicap of IPATIMUP researchers concerning patents and
of how to reconcile patent submission with scientific publication. To overcome this shortcoming, the Innovation Unit conducted in
person sessions with IPATIMUP research groups to that addressed overall issues of patentability, searching through patent databases
and answered to frequent doubts about patents and reconciliation with scientific publication. In the spirit of making IP registration a
natural step of researcher’s activity, the Innovation Unit also made available an Invention Disclosure Form. In terms of patent submission, overall, in 2012 the Innovation Unit initiated a total of six IP processes in which IPATIMUP is applicant, with entitled ownership. This
represents a significant improvement relative to the previous track-record of IPATIMUP in this field.
The Innovation unit also took a pro-active role in prospecting knowledge and research which could constitute the basis for innovative
products or services. As a result of sessions with group-leaders and contacts with researchers undertaking both in-house and collaborative research, a total of eleven projects/technology/materials with some potential for innovation and commercialization came
forward to be supported by the Innovation Unit. These projects/technology/materials embody novel formulations of nanobiomaterials
for eradication of Helicobacter pylori, original sources of anticancer compounds (mushrooms and microalgae), new cell models for HCV
replication, simple in vivo assays, proprietary hybridomas to produce diagnostic and therapy selection antibodies, copy number alterations with prognostic value, serum biomarkers associated with gastric cancer and it’s precursor lesions, digital cytology for oral cancer,
a high throughput Mass-Spec analysis software and a Virtual Microscope.
By consulting and accompanying these projects to the end goal of taking them further in the innovation cycle and obtaining economic
revenue from promotion of their commercialization, the Innovation unit undertook several types of actions, established in a case by
case basis. These actions included definition of research steps that are further needed for the development of a product, identification
of an industrial partner to co-develop the product/technology, licensing of IP (proprietary hybridoma), fund raising through application
for innovation prizes/programs, coaching to pitch the technology to investors and venture capitals, presentation to venture capitals,
presentation to big pharma companies, writing of business plan, advising the steps for CE marking.
A) Implementation
As part of implementation, the following activities were conducted:
- Presentations Institute’s Research Groups: 10 sessions, comprising over 100 Researchers
- Contents for web-page were prepared and published
- Regulations were proposed to the board
- A Protocol for Coordination with Innovation offices from University of Porto (UPIN) was proposed.
- Frequent meetings with INEB’s Head of Business Development (João Cortez) took place.
- An invention disclosure form was operated and made available to researchers through the internal network.
B) Promotion of an IP Culture
Innovation Unit conducted a total of ten in person sessions with IPATIMUP research groups, reaching over IPATIMUP 100 researchers,
as a means to promote IP culture and overcome an identified handicap concerning the relatively low level of knowledge about patenting issues amongst IPATIMUP researchers. Sessions addressed overall issues of patentability, searching through patent databases and
answered to frequent doubts about patents and reconciliation with scientific publication.
C) Industrial Property: Patent submission
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Activity Report
2012
A total of six IP registration processes were initiated by the Innovation Unit in 2012.
It is worth mentioning that this represents a significant improvement over the previous track-record. IPATIMUP’s activity in IP submission had been residual (2 patents in 23 years) and researchers filed patents without attribution of any type of ownership to IPATIMUP. A
list of IP owned by IPATIMUP is presented below:
1. MICROSPHERES
TYPE: Provisional Application for Patent
OFFICE: US Patents and Trademarks Office
REFERENCE: 61642587
APPLICANTS: IPATIMUP/INEB/FCUP
INVENTORS: M. Cristina L. Martins, Inês Gonçalves, Paula Gomes, José Ricardo Oliveira, Celso Reis, Ana Magalhães
% OF IPATIMUP OWNERSHIP: 33,3%
2. USE OF SUILLUS COLLINITUS MUSHROOM EXTRACTS AS A SOURCE OF BIOACTIVE COMPOUNDS
TYPE: Provisional Application for Patent
OFFICE: INPI (Instituto Nacional de Propriedade Industrial)
REFERENCE: 106143
APPLICANTS: IPATIMUP/ IPB (Instituto politécnico de Bragança
INVENTORS: Isabel CFR Ferreira/Helena Vasconcelos
% OF IPATIMUP OWNERSHIP: 50%
3. ANIMAL AND CELL CULTURE MODELS FOR HCV REPLICATION STUDIES
TYPE: Provisional Application for Patent
OFFICE: INPI (Instituto Nacional de Propriedade Industrial)
REFERENCE: 106235 H
APPLICANTS: IPATIMUP/ICBAS
OFFICE: INPI (Instituto Nacional de Propriedade Industrial)
INVENTORS: Gertrude Averil Baker Thompson, Eliane Pimenta Da Silva, Sara Andreia Barros Costa Marques, Hugo Osório, Júlio Gil Vale
Carvalheira.
% OF IPATIMUP OWNERSHIP: 5%
4. USE OF THE LRP1B RECEPTOR, OR DERIVATES FROM IT, FOR MODULATION OF MULTIPLE FACTORS IN THE
EXTRACELLULAR ENVIRONMENT
TYPE: National Patent of Invention
OFFICE: INPI (Instituto Nacional de Propriedade Industrial)
REFERENCE: 105286
APPLICANTS: IPATIMUP
INVENTORS: Hugo João Marques Prazeres, Ana Paula Soares Dias Ferreira, Valdemar de Jesus Conde Máximo
% OF IPATIMUP OWNERSHIP: 100%
5. DNA COPY NUMBER ABERRATIONS ASSOCIATED TO GIST
TYPE: Provisional Application for Patent
OFFICE: INPI (Instituto Nacional de Propriedade Industrial)
REFERENCE: 106636 B
APPLICANTS: IPATIMUP
INVENTORS: António Manuel Ferreira Gouveia, Rune Matthiesen, José Manuel Pedrosa Baptista Lopes, Ana Paula Soares Dias Ferreira
% OF IPATIMUP OWNERSHIP: 100%
6. SERUM BIOMARKERS FOR THE DETECTION OF GASTRIC CANCER
TYPE: Provisional Patent
OFFICE: INPI (Instituto Nacional de Propriedade Industrial)
REFERENCE: 20131000002498
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Activity Report
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APPLICANTS: IPATIMUP
INVENTORS: Catarina de Sena Bastos Gomes, Celso Albuquerque Reis, Hugo Alexandre de Carvalho Pinheiro Osório
% OF IPATIMUP OWNERSHIP: 100%
D) Industrial Property: Proprietary Biological Materials
IPATIMUP stores unique and proprietary biological materials that have been synthetized by researchers and can also be transmitted for
the purpose of integrating commercial products. These refer mainly to hybridomas for the production of antibodies. The products of
this nature “handled” by the Innovation Unit in 2012 consisted of:
- Hybridomas for the production of MUC5A6 monoclonal antibodies:
Licensed to DAKO (20.000USD Upfront fee; 20.000USD Milestone fee, 1,5% of sales revenues).
- Hybridomas for the production of LRP1B monoclonal antibodies:
Useful to define a group of ovarian cancers resistant to treatment with liposomal doxorubicin – synthesized in 2012.
Aside from the above mentioned, IPATIMUP has some track-record in licensing hybridomas. It could be considered useful for Innovation
Unit to professionally manage these contracts, ensuring close follow-up on royalty payments, and providing skills to increase the commercial value that could be extracted from this pre-existing portfolio.
E) Innovation projects and respective valorization actions
The Innovation Unit has brought-out and put-forward a total of eleven projects that propose potential new products/compounds/biomaterials/services/technologies, which originated from in-house and collaborative IPATIMUP research.
The common desirable feature is that these products embody new paradigms (or major improvements) in addressing prevention, diagnosis, therapy or prediction of response to therapy of cancer or other aspects that are in line with IPATIMUP’s positioning. They should
have a highly differentiated, cutting-edge, science-based character and, most importantly, they must address a market that is economically interesting.
Valorization actions were conducted on a case-by-case basis and are briefly described for each project:
1. Microspheres for H. pylori
SHORT NON-CONFIDENTIAL DESCRIPTION: A microsphere cross-linked with specific glycans which binds to H. pylori.
USES: H. pylori eradication therapy, independent or in combination with antibiotics
VALORIZATION ACTIONS:
• Participation in patent filing
• Preparation and submission of an application to the BES Inovação Award 2012
• Coaching to pitch the project in the final phase of BES Innovation Award 2012
• Presentation to MERCK
• Presentation to the Venture Capital SROne, the venture arm of GSK
• Meeting with COTEC
• Starting of an application to Portugal Ventures
2. Wild mushroom extracts
SHORT NON-CONFIDENTIAL DESCRIPTION: Extracts from 3 species of wild mushrooms obtained by a team from Instituto Politénico de
Bragança in the Northeastern part of Portugal, with anti-proliferative effects.
USES: Anti-cancer compounds, nutraceutic additives in food, mushrooms with added value for agricultural production industry
VALORIZATION ACTIONS
• Participation in patent filing
• Preparation and submission of and application to the BES Inovação Award
• Presentation to MERCK
• Meeting with COTEC
3. LRP1B-based companion theragnostics and therapeutic uses.
SHORT NON-CONFIDENTIAL DESCRIPTION: An antibody-based assay to predict resistance to liposomal chemotherapy formulations and
a re-sensitization strategy.
USES: Theragnostics assay for liposomal formulations of chemotherapy.
VALORIZATION ACTIONS:
• Patent filing
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Activity Report
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• Presentation to MERCK
• Presentation to the Venture Capital SROne, the venture arm of GSK
4. HCV cell models
SHORT NON-CONFIDENTIAL DESCRIPTION: Cell lines that autonomously support replication and produce structural and non-structural
elements of HCV homolog genomic fragments in the European rabbit and hare.
USES: Cell-culture platform for screening of antiviral compounds and vaccine production.
VALORIZATION ACTIONS:
• Participation in patent filing
• Presentation to MERCK
• Presentation to the Venture Capital SROne, the venture arm of GSK
• Meeting with COTEC
• Definition of additional experiments to further substantiate possible patent claims
5. GIST prognostic CNAs
SHORT NON-CONFIDENTIAL DESCRIPTION: A method to stratify patients with GIST according to their risk of developing metastatic
disease.
USES: Prognostic IVD test
VALORIZATION ACTIONS:
• Patent filing
• Definition of validation studies to further substantiate possible patent claims
6. Mass-Spec Software
SHORT NON-CONFIDENTIAL DESCRIPTION: Software to analyze results from high throughput mass-spec.
USES: Web-based bioinformatics service.
VALORIZATION ACTIONS:
• Meeting with COTEC (definition of a business model)
7. MICE – Virtual Microscope
SHORT NON-CONFIDENTIAL DESCRIPTION: A software that teaches the components, working and simulates functioning of a microscope.
USES: Teaching software.
VALORIZATION ACTIONS:
• Presentation to Porto Editora
• Presentation to Critical Software
• Presentation to Leya
8. Expertus in vivo CAM assays
SHORT NON-CONFIDENTIAL DESCRIPTION: In vivo assays using the chick CAM assay.
USES: Outsourcing service for researchers, pharma and biotechs.
VALORIZATION ACTIONS:
• Review of value proposition, distinctive features, business model and prices. SWOT analysis and help in approaching the market
• Accompanying in meetings with potential clients
• Meeting with COTEC
• Starting an application process for InovPortugal, a spin-off financing contest.
9. Marine algae extracts and compounds
SHORT NON-CONFIDENTIAL DESCRIPTION: Extracts derived from marine algae and microalgae obtained by a team from University of
Algarve, which show potential anti-proliferative effects.
USES: Anti-cancer compounds, nutraceutic additives in food, algae with added value for aquaculture industry.
VALORIZATION ACTIONS:
• Establishment of a collaboration to validate extract’ bioactivity in terms of proliferation and apoptosis
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• Meeting with COTEC
10. Oral cytology staining
SHORT NON-CONFIDENTIAL DESCRIPTION: Oral cytology and respective staining method to provide means to detect oral cancer.
USES: A turn-key solution for oral cytology testing.
VALORIZATION ACTIONS:
• Review of distinctive features, scalability and logistics issues.
• Establishment of a partnership with systems and robotics provider for further product development
• Establishment of the classification and steps for CE marking
• Starting an application for QREN and Portugal Ventures
11. Glycobiomarkers
SHORT NON-CONFIDENTIAL DESCRIPTION: A serum-based biomarker (qualitative change in glycosylation profile of specific serum proteins) detected in patients with gastric cancer, as well as patients affected with the lesions that precede it.
USES: Non-invasive test for early detection of gastric cancer
VALORIZATION ACTIONS:
• Patent filing
• Starting of an application to Portugal Ventures
F) Other activities
• Innovation Unit has established multiple contacts for enrolment of partners to join IPATIMUP in organizing the “National Prize for
Innovation in the field of Cancer” initiative.
o Contacts were made with: ANJE, Porto Business Scholl, Associação Laço, Associação Acreditar, Associação Europacolon, Associação
Portuguesa de Bio-Indústrias, Associação Portuguesa de Investigação em Cancro (ASPIC), CEDOC, Millenium .
• IPATIMUP has established an agreement with Portugal Ventures so that the Innovation Unit integrates the network of “Ignition Partners”. As an elected Ignition Partner, IPATIMUP Innovation will prospect and help entrepreneurs to prepare applications for “Calls for
Entrepreneurship” spin-off financing initiatives. This partnership includes IPATIMUP Innovation amongst a network comprising the best
technology transfer offices in Portugal.
G) Main Achievements during 2012
• Six patent submissions (improvement in IPATIMUP’s track-record in IP filing).
• Installment of an IP submission routine among researchers, in reconciliation with scientific publication.
• Innovation Unit gathered a portfolio of twelve innovation projects with differentiated character and interesting market potential.
• Microspheres project ranked 2nd place in the Health and Biotechnology Sector of BES Inovação Award 2012.
• Expertus project selected for 2nd round of evaluation in the InovPortugal contest.
• Licensing of MUC5A6 hybridoma to DAKO.
• Recognition of IPATIMUP Innovation as a Portugal Ventures Ignition Partner Network.
Lessons and directions
Overall, the Innovation Unit made the best use of trust and proximity to IPATIMUP researchers to achieve proficiency in establishing an
innovation track for some of the knowledge generated by IPATIMUP’s core R&D activities. IP submission has become an integral part
of researcher’s activities. The Innovation Unit is well aware of need to focus in commercialization so as to keep costs with patent maintenance to a minimum. It is realistic to say that in the setting of IPATIMUP, most technologies/products are brought-out in premature
stages and significant revenue can only be expected in upcoming years, as some of the projects take the time to raise funds in the innovation setting or go all the way to the market. At this point, the most interesting asset brought by the Innovation Unit concerns that,
in view of upcoming paradigms for the R&D context, established by Horizon 2020 for instance, IPATIMUP may benefit from acknowledgment as an R&D plus Innovation Centre and as a source of Spin-offs. Scientific production and quality, however, should remain as the
fundamental cornerstone to keep feeding new applications to the innovation track.
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Activity Report
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IPATIMUP Translational Research
Overview
The Translational Research Unit (TRU) formally started its activity at February 2012 with the primary goal of being a partner of excellence for cutting-edge research, innovation and development projects implemented through strategic alliances with Pharma and Biotech companies.
The TRU is fully devoted to the design and management of tailor-made projects that fulfil the needs of the industry, from early stages
(target validation) to later stages (pre-clinical studies). The TRU team includes researchers with a deep knowledge of IPATIMUP’s scientific expertise and benefits from a privileged access to central University Hospitals and Oncology Institutes. The Unit has also a Scientific
Advisory Council that assembles IPATIMUP’s group leaders and/or Principal Investigators.
In 2012, TRU carry out four main actions: 1) internal implementation activities, 2) external diffusion activities, 3) prospection and management of IPATIMUP-Pharma Industry projects, 4) organization and creation of translational research promoting events and 4) promotion
of international networking at the translational research setting.
Description of the Activities in 2012
From February to April 2012 the TRU was dedicated to the process of its own implementation, characterized mainly by the creation of
specific statutes (i.e. Regulations) and a business/financial model. Another important part of this implementation process was the internal and external promotion of the TRU mission. Because a close interaction with IPATIMUP’s research groups is of crucial importance
to the Unit’s performance and to an efficient interface with the industry, several meetings were held with all the research groups of the
Institute in order to not only discuss the modus operandi of the Unit but also, essentially, to sensitize the group leaders for this readjustment of IPATIMUP’s scientific paradigm. Similar meetings also occurred at the São João Hospital, since the Institutional link between
IPATIMUP and this Hospital is essential for the implementation of a competitive Translational Research functional structure.
During 2012, external promotion was thus a major priority for the Unit. A “flyer” with a portfolio of scientific competences was created,
and a web page was designed and put on-line within IPATIMUP’s own web site providing the most important information about the TRU.
Perhaps most importantly, during 2012 the TRU was intensely dedicated to the establishment of formal contacts with companies in
the Pharma and Biotech communities and, in this context, more than 50 companies were contacted, mostly at an international level.
It is noteworthy to mention that the TRU held several meetings with Portuguese representatives of Big Pharma companies such as
GlaxoSmithKline, Novartis, Bayer, Sanofi, Merck, EISAI, Hoffman- La Roche and Astrazeneca, with the goal of involving them as key
intermediates between IPATIMUP and their Headquarters and Global R&D Departments. In almost all of these national-level meetings
the TRU proposed specific projects resulting from careful analysis of the companies’ priorities and matching with the installed expertise
of IPATIMUP.
As a result of this effort, high-level meetings with Global R&D Departments of large pharmaceutical companies were held, namely with
Merck-Serono and Bayer Healthcare. More importantly, the TRU prepared or helped organize twelve research projects, which were
considered for eventual submission to Pharma or Biotech companies. From these, the TRU submitted nine research projects to Pharma
companies, from which one has already given rise to a signed sponsored research contract, three are in advanced-stage negotiations
and two are under evaluation. Although in a slightly different setting, TRU is also setting up a collaboration project with a Portuguese
start-up, Coimbra Genomics, and the Beijing Genomics Institute (BGI), also at an advanced stage of negotiation.
Favouring the collaborative involvement with health care and biotech companies, and also looking forward to the improvement of the
international diffusion of IPATIMUP and TRU among these types of companies, we attended the BioSpain 2012 Partnering Event, where
more than 20 meetings where held and where several companies showed interest in joint Projects.
Nowadays, integration in International networks and consortia is not only a critical factor for attracting international grants but, most
importantly, it provides to the Institutions the possibility to share expertise and unique technologies. This is particularly important in
translation research.
In 2012, the TRU started managing the process that will lead to the inclusion of IPATIMUP (alone or together with other already associated institutions such as IPO and São João Hospital) into the European Infrastructure for Translational Medicine (EATRIS), a non-profit organization comprising European academic centers of excellence in translational research. In addition, TRU started the assembly of the
Porto Cancer Network, 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 2012 the TRU also began organizing the XXII Porto Cancer Meeting, to be held on April 11th and 12th, 2013 and 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 oncology-related 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 exciting discussions on Translation Research in Cancer.
Overall Appreciation and Directions
In summary, during its first year the TRU achieved most or all of its primary goals, effectively promoting IPATIMUP internationally among
the relevant industries, helping to organize events and networks that bring together good science and industrial players, and, most
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Activity Report
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crucially, having generated specific contracts for joint R&D projects between IPATIMUP researchers and major companies. Moreover,
the TRU has benefitted from the proximity, individual interaction and motivation of our researchers to the creation of a new translational research footpath which, making use of cancer science that is developed at IPATIMUP and pinpointing the potentiality of research
teams, will allow the increment of interactions with healthcare companies and the involvement with the Hospitals in a Translational
Research basis.
As a world critical issue, pharmaceutical companies experience a slew of patent expiries and revenues become much more exposed to
competition of generic drugs; thus, these companies are seeking strong ties with academia in a bid to speed up drug development and
introduce innovative research. In this scenario, from the input that TRU took from the several meetings at the international level, we
reinforce that maintenance of a robust and high quality scientific production rate of the IPATIMUP is of crucial importance, since this
constitutes the basis for strong ties with health companies in a translation research setting and to set up IPATIMUP a partner of reference for the development of projects. This is particular important in an era where pharmaceutical industry closes its research labs in response to falling profits and therefore start looking for academia in order to identify strategic partnerships supported by good science.
If by one hand, the critical but actual situation of worldwide pharmaceutical companies might constitute a threat for the partnership
process, we believe that this also constitutes a new window of opportunity for IPATIMUP, while an Institute focused in producing innovative cancer science.
One of the most stimulating challenges that TRU experienced during 2012 was, together with our researchers, to generate innovative
applications from their outstanding research.
Having researchers alongside with us and putting IPATIMUP alongside with Healthcare companies is what will make us greater.
Highlights
Internal Implementation Activities
1. Creation of specific statutes (i.e. Regulations) and a business/financial model.
2. Internal promotion of the TRU mission through several meetings with all the research groups and Hospitals.
3. Creation of a web page within IPATIMUP’s own web site.
4. Construction of a portfolio of IPATIMUP’s scientific competences.
5. Creation of a Pharma/Biotech companies database containing contacts and products pipeline characterization.
External Diffusion Activities
1. Establishment of contacts with over 50 Pharma and Biotech companies, mostly at the international level, but also at the national level.
2. Set up of meetings with Portuguese representatives of Big Pharma companies such as GlaxoSmithKline, Novartis, Bayer, Sanofi Aventis, Merck Serono, MSD, EISAI, Hoffman- La Roche, AstraZeneca and Abbvie.
3. Participation in BioSpain 2012 Partnering Event as IPATIMUP delegates.
IPATIMUP-Industry Projects Management
1. Management of 12 research projects, which were analyzed for an eventual submission to Pharma or Biotech companies.
2. Set up of meetings with Global R&D Departments of large pharmaceutical companies at the International level.
Contracted Research Performance
1. Submission of nine research projects to Pharma companies.
2. Three projects in advanced negotiations and two and under appreciation.
3. One project signed as sponsored research contract.
4. Set up a collaboration project with a Portuguese start-up, Coimbra Genomics, and the Beijing Genomics Institute (BGI), at an advanced stage of negotiation.
Translational Research Promoting Events
1. Organization of the XXII Porto Cancer Meeting, devoted to translational research in cancer, under the theme: Translational Research
in Cancer: Bringing Basic Science and Pathology to Clinical Oncology.
International Networking at the Translational Research Setting
1. Creation of the Porto Cancer Network Consortium, together with IPO-Porto and C.H.S. João, that aims to build a strong cluster in
Oncology centered in Porto.
2. Promotion of the integration in international networks, such as EATRIS (European Infrastructure for Translational Medicine).
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Activity Report
2012
Activity Statistics
IPATIMUP-Industry Projects Management
During 2012 the TRU was intensely dedicated to the establishment of formal contacts with companies in the Pharma and Biotech communities. More than 50 companies were contacted, mostly at an international level. As a result of this effort, the TRU prepared or helped
organize twelve research projects, which were considered for eventual submission to Pharma or Biotech companies. From these, the
TRU submitted nine research projects to Pharma companies, from which one has already given rise to a signed sponsored research
contract, three are in advanced-stage negotiations and two are under evaluation.
Project Stage
82
Number of Projects Handled
Management and preparation
12
Submission and appreciation
2
Negotiation
3
Contractualization
1
Activity Report
2012
INTERNAL SERVICES
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Activity Report
84
2012
Activity Report
2012
Sequencing Service
Overview
IPATIMUP’s sequencing service was initiated in 2006 to serve investigators and technicians who develop their work there. Nowadays,
IPATIMUP’s sequencing service is also open to the general scientific community. It performs the automated analysis of genotyping and
sequencing products by capillary electrophoresis. At an internal level, this service also performs sequencing reactions and its analysis
upon request.
The sequencing service is also in charge of the acquisition and distribution (only at an internal level) of some consumables
Highlights
Opening to the external general scientific community
In the final of 2012, the Sequencing Service was opened to the external general scientific community, offering services in automated
analysis of genotyping and sequencing products by capillary electrophoresis. For the external access to the sequencing service it was
created an on-line registration in the IPATIMUP webpage.
Activity Statistics
Number of samples processed per month in 2012
Samples
8000
7000
6000
5000
4000
3000
2000
1000
0
Number of samples processed per group in 2012
Samples
35000
30000
25000
20000
15000
10000
5000
0
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Activity Report
2012
Proteomics
IPATIMUP proteomics unit provided investigators access to the analysis of protein samples from solutions, bands or protein spots from
1D or 2D SDS PAGE gels.
The following proteomic analyses were performed:
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).
The provided services included: 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 search and identification using UniProt and/or NCBI protein sequence databases; result data analysis; and delivery of a report
with the identified protein(s), protein sequence, gene identified, molecular weight and isoelectric point.
In addition, the IPATIMUP proteomics unit has participated in 2 scientific projects, 3 scientific publications and has organized 2 Proteomics Workshops.
Highlights
IPATIMUP Proteomics Unit – Highlights 2012
The IPATIMUP Proteomics Unit developed its activity in 2012, similarly to the previous years, by performing research-based activities and
providing services to the inside and outside scientific community.
The groups requesting analysis were from IPATIMUP, IBMC, Faculty of Pharmacy, Faculty of Medicine and Faculty of Sciences from the
University of Porto. In addition, analyses were also requested by research groups from the Universidade Clássica de Lisboa, Universidade de Coimbra, and Instituto Superior de Agronomia.
The type of analysis performed included Molecular Mass determination, Protein identification by Peptide Mass Fingerprint, and Protein
identification and characterization by PMF followed by de novo peptide sequencing by MS/MS.
In 2012, IPATIMUP Proteomics Unit participated in scientific projects leading to the publication of 3 articles in international scientific
journals. Two new FCT projects started, involving the material and human resources of the IPATIMUP Proteomics Unit.
The IPATIMUP Proteomics Unit has organized, together with CIIMAR, two Proteomics Workshops open to the entire scientific community.
Activity Statistics
2000
1500
1000
IPATIMUP
IBMC
500
Other Institutes
Dec
Nov
Octb
Sep
Aug
Jul
Jun
May
Apr
Mar
Jan
Feb
0
Other Activities
Performed Analysis: Institutions
In 2012 were analyzed, by the IPATIMUP Proteomics Unit, a total of 405 samples, corresponding to 45 tasks from the different institutions / research groups:
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Activity Report
2012
IPATIMUP:
Cancer Biology (Valdemar Máximo)
Cancer Genetics (Carla Oliveira),
Carcinogenesis (Celso Reis).
Population Genetics (Raquel Silva)
IBMC:
Ageing and Stress
Biomolecular Structure
Cellular and Applied Microbiology
Molecular Biology of Nitrogen Assimilation
Molecular Genetics
Molecular Microbiology
Molecular Neurobiology
Nerve Regeneration
Parasite Disease
Unilipe
- Institutions inside Porto University: Faculty of Pharmacy, Faculty of Medicine and Faculty of Sciences
- Institutions outside Porto University: Instituto Superior de Agronomia, Technical University of Lisbon; Classical University of Lisbon;
and Coimbra University
Performed analysis: Numbers
The performed analysis of 405 samples, divided by 45 tasks, corresponded to:
- Molecular weight determination – 34 samples.
- Protein identification and characterization by Peptide Mass FingerPrint (PMF) – 185 samples.
- Protein identification and characterization by Peptide Mass FingerPrint (PMF) and MS/MS de novo peptide sequencing – 186 samples.
Participation in Scientific Projects
The IPATIMUP Proteomics unit has actively participated in the execution of various projctes funded by national and international funding agencies.
Participation in training / formation programs:
GABBA graduated program from University of Porto
II Workshop on Cancer Research
Organization of two Proteomics Workshops
Relevant publications
Pinheiro H, Carvalho J, Oliveira P, Ferreira D, Pinto MT, Osório H, Licastro D, Bordeira-Carriço R, Jordan P, Lazarevic D, Sanges R, Stupka
E, Huntsman D, Seruca R, Oliveira C (2012). Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that
increases gastric cancer cell invasion and angiogenesis. Hum Mol Genet. 21, 4253-4269.
Silva E, Marques S, Osório H, Carvalheira J, Thompson G (2012). Endogenous hepatitis C virus homolog fragments in European rabbit and
hare genomes replicate in cell culture. PloS one 7: e49820, 2012.
Freitas SC, Cereija TB, Figueiredo AC, Osório H, Pereira PJ, Barbosa MA, Martins MC (2012). Bioengineered surfaces to improve the blood
compatibility of biomaterials through direct thrombin inactivation. Acta biomaterialia 8: 4101-10, 2012.
Communications in scientific meetings:
Aveiro University Proteomics Workshop 2012, Oral Communication: Serum glycoprotein biomarkers in gastric cancer and precursor
lesions.
GlycoT Hannover 2012, Poster Communication: Role of SLea and SLex in gastric cancer cells.
I3S Scientific Retreat 2012, Póvoa de Varzim: IPATIMUP Proteomics Unit – Research activities and services to scientific community.
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Activity Report
2012
Animal Model Facility
Overview
During the year 2012, due to the necessity to allocate new strains of conventional mice and to perform more extensive xenograft research, IPATIMUP’s Animal House left the old Facilities located at “Hospital de São João” (facilities from the Medical Faculty of the University of Porto) and moved to the new CIM-FMUP Animal House Facilities. For this, a Collaboration Protocol was established between
the two Institutions. The “transference” process started in late June, with equipment, and finished in the beginning of October with the
complete abandon of the old Facilities. It was done, as expected, in stages and always keeping in mind animal well-being, animal microbiological status and, more importantly, that our immunodeficient mouse strain “survival” was ensured. In this all process, not always
easy, the support of our personnel, Dr.ª Luísa Guardão, responsible for CIM-FMUP Animal House and its personnel, and backstage help
of “Hospital de São João” Pathology Lab, namely Professor Fátima Carneiro and Maria José, was of extreme relevance. Despite this, the
Unit did not stop in terms of research and kept all the experiments that were already planned. Regarding Personnel working directly
in the Animal House/Animal Experimentation Unit these 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. We keep maintaining at the facility two types of animals distinguished
according to its microbiological status – immunodeficient strains and conventional strains - both of them having different physical conditions of the rooms and access permits. Animal maintenance areas are kept under controlled conditions of light and humidity. One of
these areas is maintained in Specific Pathogen-Free (SPF) conditions to allow the maintenance and breeding of our immunosuppressed
mouse strain (nude mouse model), not commercially available (strain: N: NIH(s) II-nu/nu). After the transference process to CIM-FMUP
Animal House, research with nude mouse model is made in a separated room in order to not interfere with the “SPF” conditions of the
breeding and maintenance areas and allow the routine surveillance and monitoring of the animals by all the members directly involved
in the experimental process. The other “main” area is for maintenance and expansion of conventional mice strains, transgenic and/or
knockout, received by IPATIMUP’s researchers. These mice have over-expression or no-expression of certain cancer-associated genes
under study. 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.
Highlights
Tumor biology research – tumorigenesis, invasion, metastization and response to stimuli
NIH(s) II-nu/nu mice are breeded in our Unit in SPF 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 2012 we started 8 in vivo assays – 6
were internal and 2 were performed in collaboration with an external Institution. The assays that started in late 2011 and kept in the
first months of 2012 are not taken into account. In terms of type of experiment, 4 were tumorigenesis assays, 3 were tumorigenesis and
metastization assays and 2 were tumorigenesis associated to response to therapy assays. This year, a new procedure was included and
already performed in our Unit that is the orthotopic injection of cells in internal organs. 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. During this year, we also
concluded 4 other assays that started in 2011. 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 February 2012 we received another transgenic mouse model (2 males and 2 females), from Glycobiology in Cancer group. In July 2012,
date that corresponded to the conventional mouse strains transference to the new facilities in CIM-FMUP, we had in clear expansion 7
mouse strains and sub-strains and 5 others in maintenance. All the manipulations done in the animals are performed by the personnel
from the Facility accompanied by the certified researchers. Number of cages maintained per day per month will be presented in the
point “Activity statistics”.
Activity Statistics
Animal House/Animal Experimentation Unit Activity - Year 2012
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Activity Report
2012
Jan-12
Fev-12
Mar-12
Abr-12
Mai-12
Jun-12
Jul-12
Ago-12
Set-12
Out-12
Nov-12
Dez-12
16
13
25
25
25
23
18
31
31
27
25
24
TABLE 1 : N: NIH(s) II-nu/nu reproduction maitained per month - year 2012
Jan-12
Fev-12
Mar-12
Abr-12
Mai-12
Jun-12
Jul-12
Ago-12
nude males
9
15
9
24
21
28
31
16
nude females
Set-12
Out-12
Nov-12
Dez-12
34
21
24
7
8
8
21
31
18
21
14
34
19
19
CBA/N females 1
1
12
23
30
25
25
7
20
8
26
Total*
24
29
68
82
71
77
37
88
48
69
17
0
TABLE 2 : Weaning NIH(s) II-nu/nu - males and females - and CBA/N - females - maintained per day per month - year 2012
Na
Nb
Nc
Nd
Internal 1 (CG)
15
Internal 2 (CG)
22
22
188
Internal 3 (CG)
12
12
199
Internal 4 (GiC)
10
5
28
Ne
144
22
Internal 5 (CDR)
10
10
25
180
Internal 6 (CDR)
30
30
22
450
External 1
20
20
46
External 2
32
32
57
Total
151
131
709
22
630
TABLE 3 : Number of NIH(s) II-nu/nu used in research, duration and procedures according to Internal /External Research Groups - year 2012
Strain
Jan-12
Fev-12
Mar-12
Abr-12
GA
9
10
8
6
7
CR
8
8
7
5
8
GW
7
6
8
9
9
FE
6
7
8
9
9
SU
12
11
12
10
8
7
6
BMPRIA
7
10
13
13
16
22
30
ATRX1
2
4
6
9
11
13
16
16
15
12
2
2
2
2
2
2
4
11
13
19
26
34
31
RIP-Cre
2
2
4
6
6
6
4
5
129J-AhCre
2
3
6
7
8
3
3
3
2
4
10
13
15
3
8
8
10
99
117
114
100
93
82
MGAT5
Mai-12
Jun-12
Jul-12
Ago-12
Set-12
9
7
8
6
6
5
8
7
5
5
6
5
4
4
5
9
7
8
7
5
5
4
7
7
6
4
4
34
24
7
6
7
BMPRIA*AhCre
ATRX1*RIP-Cre
Total
51
58
64
63
74
80
Out-12
Nov-12
Dez-12
4
4
4
6
4
4
TABLE 4: Conventional strains cages - WT and transgenic- maintained per day per month - year 2012
Other Activities
Other Activities
Animal Model Unit gives support to researchers in all phases of the licensing process of all the performed experimentations by the
National Regulator Entity (DGAV) in accordance with National Law.
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Activity Report
2012
Cell Lines Bank
Overview
IPATIMUP’s Cell Line Bank (CLB) 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. The vast majority of BLC cell lines were commercially obtained. However, others were
established at the Institute or provided by external institutions under scientific collaboration.
Highlights
IPATIMUP’s CLB Highlights
During the year 2012 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 2012, 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. Giving examples, during this year, a cell line provided by Cancer Biology Group, considered unable to recover, was provided to Cell Line Bank Technicians. After 2 months, this cell line
was completely characterized and available to use. Another 8 cell lines, 5 provided by Cancer Biology Group, 2 commercially obtained
by Cancer Genetics Group and 1 obtained from Carcinogenesis Group provided by an external collaborator were fully characterized.
BLC’s personnel also gave support in any case of cell culture problems, recommendations for the use of consumables (for example, FBS
control and acquisition) and any other question considered relevant for the improvement of in vitro quality performance. The continuous updating at IPATIMUP’s website of the considered relevant information, including pictures, about each particular cell line, available
internally to all researchers is a permanent task. For this purpose an “information management system” that had been developed in
collaboration with Informatics is also updated in accordance to the service needs.
Activity Statistics
IPATIMUP’s CLB Activity during 2012
Genotyped cell lines
25
20
15
10
5
0
Table 1: Genotyped cell lines - Year 2012
90
# of ext. orig.
# of int. orig.
Activity Report
2012
Stock cells and cultured cell lines per topography
300
250
200
150
100
# of frozen vials
50
# of maintained
0
Table 2: Numbers of stock cells and cultured cell lines per topography - year 2012
Services executed
80
70
60
50
40
30
20
10
0
Mycoplasma tests
Cell lines requested
Cell lines acquired
Table 3: Services executed by IPATIMUP’s Cell Line Bank - year 2012
Quality Control
Internal Quality Control
IPATIMUP’s BLC Technicians 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.
Other Activities
Other Activities
Continuous testing of new cell culture media and reagents in order to obtain the best results in terms of cost-effectiveness.
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Activity Report
2012
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.
After the successful establishment at IPATIMUP of assays to evaluate angiogenesis, tumorigenesis and metastization, Marta Teixeira
Pinto proposed the creation of a formal service unit at IPATIMUP. IPATIMUP’s “In vivo CAM assays Unit” is functioning as an internal
service since June 2012. The “Activity Statistics” refer only to the period from June to December 2012. The “Highlights” include some
CAM assays done previously to June 2012, that were included in published or submitted papers in 2012.
Personnel working directly in the “In vivo CAM assays unit” laboratory includes Marta Teixeira Pinto (responsible) and Ana Moreira
(research trainee),that worked in partial time from Jun2011-Dec 2012.
Highlights
Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric
cancer cell angiogenesis.
We used the CAM assay to evaluate the angiogenic response of CHO and MKN28 cells transfected with CDH1a isoform, an alternative
transcript of the CDH1 gene. Over-expression of CDH1a in cell lines expressing canonical E-cadherin (MKN28) promotes angiogenesis but
has no effect in cell without canonical E-cadherin (CHO). (Pinheiro eta al., Human Molecular Genetics, 2012).
Overexpression of ST3GAL4 leads an invasive phenotype in gastric carcinoma cells
We used the CAM assay do evaluate the angiogenic response and the invasion potential of MKN45 cells overexpressing ST3GAL4. Expression of ST3GAL4 leads to SLex expression and induces an invasive phenotype in MKN45 gastric carcinoma cells. No differences were
found in the Angiogenic response. (Gomes et al., submitted to PLOS ONE).
Conditioned medium from macrophages with different phenotypes (M0, M1 and M2) induces different angiogenic responses of gastric carcinoma cells
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 P et al., submitted to Oncogene).
Activity Statistics
Assays performed in “In vivo CAM assays unit”
The activity described in the attached table refers to the activity after formal implementation of the internal service of “in vivo CAM assays” at IPATIMUP (from June to December 2012). In the tables it is patented: the group and PI requesting the assay; the number of eggs
used in the assay (inoculated with cells) and the current status of the assay. The total number of eggs effectively used for the assays,
ie, inoculated with cells from June to December 2012 was 860 distributed through 12 distinct assays. To reach the necessary number of
embryos to be inoculated, a higher number of eggs had to be incubated and manipulated, this number was proximally 1300. Note that
around 35% of the eggs don’t reach inoculation day since incubation viability of the eggs is about 80% and around 15% don’t survive the
manipulation procedures.
Quality Control
Egg Quality Control
Eggs are acquired from a commercial source (Pintobar) and are free of Mycoplasma Galisepticum, Samonela, Enteritidis and Tiphymurium.
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Activity Report
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People Certification
The person responsible (Marta Teixeira Pinto) is certified by the DGAV under National and European Laws for animal handling. Although,
experiments using the chick embryo as a model do not require formal certification of the competences.
Other Activities
Development and optimization of other Chick embryo based assays
Chick embryo gut culture and CAM wound healing assay.
Service expansion
Explore the possible external market for the “In vivo CAM assay unit” services. As part of the developing strategy, we expect to divulge
our competencies in other institutes and biotech companies and we expect, in the future, to increase the number of requests for in vivo
CAM assays (in collaboration with IPATIMUP’s Innovation and Translational Research units)
Slides Preparation Service
Overview
The Slides Preparation Service (SPL) began its services in February 2012. In the period from February to December 2012, 21 requests were
submitted.
Highlights
In response to afore mentioned requests, the following activities took place:
•
Microtomy: 330 cuts were made in standard slides and 727 cuts in slides for immunohistochemistry.
•
Histochemistry: 376 slides were stained by Hematoxylin & Eosin.
Worth mentioning, the whole work proceeded without any complications of notice.
Activity Statistics
Jan
Feb
Requisition
-
4
Standard slides
-
Imuno slides
-
HE
-
Mar
Apr
May
1
1
1
100
5
57
120
90
0
100
5
57
0
Jun
Jul
Aug
Sept
Oct
4
Nov
2
1
1
2
3
0
0
48
0
44
76
40
60
0
9
38
60
91
0
0
44
76
3
Dec
TOTAL
1
21
0
0
330
300
10
727
0
376
93
Activity Report
94
2012
Activity Report
2012
CORE SERVICES
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Activity Report
96
2012
Activity Report
2012
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
2012 it was composed by Filipa Valente as the coordinator, by Ana Mafalda Rocha, Lara Henriques and Nuno Mendes as research technicians and by Maria José Ferreira and Mário Ramos as maintenance assistants.
Highlights
IPATIMUP INTERNAL SERVICES - Sequencing, Real Time PCR, QIAxcel and Cell Lines Bank
-Sequencing ServiceThe internal sequencing service processed in 2012 a total of 71970 samples (an average of 5998 samples sequenced/per month) in both
3130 and 3130xl sequencers units. Population Genetics group and IPATIMUP Diagnostics unit are the main users of this service. Both
sequencers were used at aproxim. 72% of its total capacity.
In the final of 2012, the Sequencing Service was opened to the external general scientific community, offering services in automated
analysis of genotyping and sequencing products by capillary electrophoresis. For the external access to the sequencing service was created a on-line resgistration in the IPATIMUP webpage.
Ana Mafalda Rocha is the technical responsible for this service.
- Real Time PCR Service In 2012 was used only one, of the two Real Time PCR equipments existent, to process all the samples and it is yet far from exhausting
its capacity. This results in a significant reduction of the costs (for half ) with preventive maintenance contracts for theses equipments.
The main users of this service were Cancer Genetics and Carcinogenesis research groups. Lara Henriques was the technical responsible
for this service.
- QIAxcel The QIAxcel Automated Electrophoresis System performed, in 2012, a total of 201 runs (an average of 17 runs/month), being the IPATIMUP
Diagnostics the main user of this service. This equipment is under sub-utilization and its maintenance is expensive.
QIAxcel service has been under the care of Ana Mafalda Rocha.
- Cell Lines Bank (BLC) BLC assured the maintenance of stocks cells of the institute composed by a collection of parental cell lines and commercial lines. This
maintenance includes the characterization of the genetic profile (genetic identity), microbiological status, of each bank line.
Nuno Mendes was the technical responsible for this service, with the technical support of Lara Henriques.
TRAINING
Training carried out, mainly in the area of health and safety at work:
- “QIACUBE: aplications and safety working procedures” (Feb14th,2012)
- “Chemical Spill Kits : How to use them & Group III and IV Residues: how to discard them safely? “ ( April 27th, 2012)
- “Internal security training: Reagents internal database/MSDS sheets” (Oct19th, 2012)
- “Risk management: risk perception and evaluation” (Nov 2nd, 2012)
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, Gel and cell counter room, Dark room, Radioactivity room, HPLC and Image acquisition room, Centrifuges and incubators
room, -80ºC and 4ºC Cold rooms).
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Activity Report
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The Technical Body permanently ensure that in these rooms, the equipments are operating good and in safety conditions, verifies the
correct use of the facilities and equipments, give specific formation, check the access to the rooms, and provide logbooks and Standart
Operating Procedures (SOP) for the equipments.
In fact, during 2012, Safety Rules (available in the IPATIMUP intranet) were created for these common rooms .They provide important
information related to the correct use of the different rooms (and associated equipments), safety working practices, safety residues
discard, good practices for equipment/room use and maintenance.
EQUIPMENT ACQUISITION
During 2012 some equipment was aquired, according to the needs observed (eg. for replacement of damaged equipment).
Equipment acquired:
- A CO2 incubator (for the Cell cuture room 2);
- Three mini-centrifuges (one per each cell culture room and biosafety level 2 room);
- A vortex (for the biosafety level 2 room);
- Ecopoints (as residues GIII & IV stations, for the research laboratories);
- Chemical Spill Kits (for the laboratories areas);
- Personal Protective equipments (eg. gloves for high tempertures, UV glasses, facial masks for gas and solid particles for all research
laboratories);
- Stirring plate (for 4ºC cold room II);
Reagents MSDS Database and Device Inventory
In 2012 was created an internal database for the reagents (and correspondent MSDS) existing in the institute.
The maintenance and growth of this database is ensured in a continuous mode by Filipa Valente and Ana Mafalda Rocha.
In what concerns the “Device Inventory” it was enriched with the equipment instruction manuals and it became accessible to IPATIMUP
researchers through the IPATIMUP helpdesk/devices. This work was coordinated by F. Valente and had the colaboration of Mafalda
Rocha.
Quality Control
PREVENTIVE MAINTENANCE - VERIFCATION CONTRACTS
It was realized Preventive Maintenance to the following equipments:
- Microtome, Thermo Electron Corporation Shandon Finesse 325, S/N: FI50850603
- 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
- Incubator Memmert UNE 200, S/N: C207.0147
- Incubator Memmert UNE 200, S/N: C207.0148
- High Speed BECKMAN Avanti centrifuge, S/N: JHY96J12
- 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
It was realized verification to 13 Laminar Flow Cabinets and 8 Carbon Dioxide Incubators from cell culture, biosafety level II and Pré-PCR
rooms (by an external supplier);
Other Activities
RISK MANAGEMENT PROJECT
Technical body was fully involved in the Risk Management Project, namely Filipa Valente as member of risk comission and responsible
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Activity Report
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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 produce several documents:
- Safety Rules of Cell Culture Rooms, Deep Freezers Room, Washing Room, Centrifuge & Incubators Room, Pre-PCR Room, Dark Room;
Radioactivity room;
- SOP’s for: Lentivirus and H. pilory work, microtome, gas burner, chemical spill kit, pH electrode, microwave, UV transiluminator, liquid
nitrogen and dry ice and centrifuge JA-25 handling;
- Good laboratory practices guide;
- Document with information related to the diferent categories of residues produced in IPATIMUP and how to discard them correctly
and safety;
- IPATIMUP risk evaluation table;
- Checklists development for verification of equipments/facilities;
- Document with information about incompatible chemicals & associated risk;
Reformulation of Internal Services
In December of 2012, the Internal services suffered changes:
- Nuno Mendes left the Technical body to be exclusively working in the Animal House of Hospital S. João;
- Lara Henriques became the technical responsible for the Cell lines bank service;
- Ana Mafalda Rochabecame the technical responsible for the Real time PCR service;
Other activities
- The Tickets system from Helpdesk is daily coordinated/maintained by Filipa Valente;
- It was created a Pricelist for the most used consumables in IPATIMUP (coordination by Filipa Valente with colaboration of Ana M. Rocha);
- The microtome device was transfered to the Slide Digitalization Unit and the service for its use was created, with Adriana Rocha as
responsible;
- Retention containers were supplied to the labs to be used for eg. with the electrophoresis systems in order to contain/prevent spills.
Some infrastructural changes were implemented based on the needs identified, they were:
- Metal plates were instaled in both cell culture rooms in order to redirect the forced air; This allowed to use the (from air conditioning
machines without compromise the sterility/work in the laminar flow cabinets;
- A bigger and stainless steel table was installed in the cold room II;
- Creation of two cabinets for flammable reagents storage;
- All the common rooms without sink were provided with hand hygienization units (eg. Deep Freezers Room, Centrifuge & Incubators
Room, HPLC’s & Image Acquisition Room, Advanced Microscopy Room; Biosafety Level II Room);
- Were instaled more bench modules in the Biosafety Level II Room;
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Activity Report
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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
Outsystems Platform [Development]
Migration of the platform of development in Outsystems of the version 5.1 to 6.0
Allowances Module [Development]
This Module allows users to submit or request all allowances related to a mission, like cash advance, expenses paid directly by IPATIMUP
or by the user which in turn can be reimbursed by IPATIMUP. The reimbursement could be done by expense or a daily value or by Kilometer.
This module also handles the sending of emails for approval of allowances, the generation of documents like receipts and mission report
and interacts with the existing requisitions module.
The mission can be closed when the Absence Workflow is finished and all allowances are conciliated.
Document Generator Manager [Development]
Reformulation of Document Generator, which includes new functionalities, such as, structured documents in end stage, possibility
to comment documents, send for issuu and show then in risk management area on ipatimup site, when documents are organized by
categories.
Development Webdocs which allows associate DocGen documents (directly upload) or others documents (upload in issuu) to risk management area in site.
External Requisitions [Development]
Implementation of external requisitions in the requisitions module, that allows customers to subscribe internal services. Automatization of sending results and customer management process.
Stock Manager [Development]
Form Generator Manager [Development]
The Stock Manager allows to registry and storage reagents data, Material Safety Data Sheet (MSDS) and its location in information
system. This module includes purchase, reception and query operations a reagents. All operations involving reagents force registry in
this module, including discart.
All regent’s MSDS used in IPATIMUP have shown on site.The Form Generator Manager allows to create dynamic forms, type Checklist,
Forms, Surveys, in which can use structures and pre-defined rules. These forms can be created with logic rules, submission dates and
sequencing forms. Then are sent immediately or scheduled way to one or more persons. This module introduces other features, such as
the query results, statistics, timeline and authorized submission.
Protocol Module [Development]
Development of an interface where is possible to insert all protocols IPATIMUP have with other institutions, as well as all information
and documents related with it.
Is also possible to associate users to the protocols which leads to the automatic creation of a new admission workflow for each one of
the users.
In IPATIMUP site, creation of an interface to list all Protocols and see the detail of each one and respective documents.
Absence Notification Module [Development]
Addition of a new workflow called Absence Change that allows users to change or cancel absences which are already validated.
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Activity Report
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It was also implemented automatic actions like sending email to the insurance company to inform when someone is going in mission to
other country.
WhoIsIn [Development]
Person Admission Module [Development]
The main purpose of this module is to control the access to the building. In the initial phase of the application was available outside of
normal working hours, weekends and holidays.
At the moment, the application is available all the time.
In emergency situations, offers up a listing in digital format and on paper with all the people present in the building, in order to confirm
the people that came out, and be able to identify people who are still inside the building.Inclusion of three more admission workflows,
Visiting Researcher Admission, Fellow Admission and Master Student Thesis Admission.
It was also done some improvements like automatic generation of the agreement which each person has to sign and deliver when it
enters at IPATIMUP.
Barcode Scanner Applications [Development]
Development of standalone applications where we can configure and listen several barcode scanner devices in order to trigger actions
like check-in/check-out in the building or meeting, or get information of reagents.
This application is also able to communicate with several Web Services, interact with the internet explorer, give feedback messages to
the screen with sound alerts and show a screensaver when is running outside of working hours. It was also done the integration with
the Module WhoIsIn.
Personal Calendar Module [Development]
Improvements of interface like the inclusion of a menu with all the available options for each day of absence.
It was also done all the changes to make possible the secretary see the personal calendar of any user.
Service Manager Module [Development]
Creation of new features like cloning technical files from on service type to another and define default values for each field.
Meeting Manager [Development]
Oracle Application Express
The Meeting Manager application is used to store meeting agenda information for a meeting organizer, assigned presenters, and other
attendees.
It is a collaborative environment for meeting participants to simultaneously review agenda items, and add relevant attachment files,
action items and/or decisions.
Action Planning [Development]
This module was developped in the scope of the Risk Management System.
The main purpose of this module is to manage the nonconformities of IPATIMUP.
These nonconformities has an origin, and during the process until it finishes its deadline, they pass through a several stages (Analysis,
Action plan and Conclusion).
Message Manager [Development]
This application offers the possibility of sending Messages for a particular user, an Organization Unit or an application. When a user is
browsing the application the message will be displayed. When the user view the message it has the option to agree the message or the
option to remind him later.
[Networking]
In January we changed our e-mail SPAM filter server, from Barracuda, with the Symantec Messaging Gateway.
With the end of Checkpoint support (it ended in January), we have switched to another firewall system, this happened in April.
Now is possible to release e-mails from quarantine and send authenticated e-mails from other clients, in the past this only could be
achieved by webmail, from outside our network. These operations were performed at May.
In August we and several outages with our file server, so we needed to install and configure a new file server using our new Iomega
Storage machine (full of disks) for this purpose. The migration from the old server to the new one started in September and ended in
October.
In October we had some issues with our wireless equipment, and it was needed to perform some firmware upgrades.
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Activity Report
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The remote access was reestablished in November, with a new system, and with the same access privileges policies used in the old Connectra.
We started to replace the computers from library in December, and this work is still in progress, with a new ones. Now we are trying to
replace all the old machines with the “leftovers” from the library.
It is expect that our department must perform some operations in the telephone network like create new telephones extensions, unlock telephones for external calling and resolution of other minor problems.
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.
In 2011 we started updating operative system from Windows XP to Windows 7. But there are still computers that need to be upgraded
and we are still working on it.
[Networking]
Devices that make image acquisition like Gel Doc or cameras that are attached to microscopes, need to be connected to computers or
directly to the network and we give technical assistance to the equipment’s responsible.
To deploy a PC we are using images stored in one server. In the past we used Tivoli Provisioning Manager and Clonezilla, but Clonezilla
was a little limited when you are trying to install a large set of PC’s at the same time and Tivoli has some issues when installing Windows
7, so we found a new system called FOG and now it is our main PC image server.
Platform evaluation [Development]
Study and evaluation of Web Platforms to develop future web modules.
The criteria were price, scalability, robustness, flexibility, performance, development environment and ease of development.
Site [Development]
Creation of an interface to list all Protocols and see the detail of each one and respective documents.
Activity Statistics
Networking - Helpdesk 2012 Statistics
Tickets
Informatics > Computer
270
Tickets resolved
265
Average delay
10 Day(s) 4 Hour(s) 3 Min(s)
Informatics > Computer > Hardware
38
37
19 Day(s) 12 Hour(s) 12 Min(s)
Informatics > Computer > Software
145
142
4 Day(s) 19 Hour(s) 31 Min(s)
44 Day(s) 22 Hour(s) 5 Min(s)
Informatics > Internet
23
23
Informatics > Printer
177
178
Informatics > Video Projector
1
1
5 Day(s) 3 Hour(s) 0 Min(s)
Helpdesk Team [Networking]
654
646
8 Day(s) 7 Hour(s) 2 Min(s)
3 Day(s) 8 Hour(s) 33 Min(s)
Networking - Helpdesk 2012 Statistics
Other Activities
Future Objectives [Development]
During 2013 we hope to stop software development on the Outsystems Plataform and develop new software in Oracle Application
Express (APEX).
Develop a module more agile and dynamic for our workflows.
Migrate papers module to APEX.
Include a list of prices from our suppliers in requisitions module so that you can choose the best price that suppliers offer us.
Extending stocks module for all kinds of articles purchased by IPATIMUP.
Future Objectives [Networking]
For 2013 it’s expected that all the workstations, except the machines that have some specific equipment attached, will have Windows
7. This is necessary because Microsoft will end the support for Windows XP at 2013. And with this change, we will upgrade our domain
from Windows 2003 to 2012.
We will upgrade our mail server to a new version, and at the same time the e-mail server will be substituted by a new one.
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Activity Report
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Secretary General
Overview
The General Secretary carries out the general administrative tasks of IPATIMUP and the management of treasury and research projects.
It comprises the accounting and all operations required for management of human resources and events. The accounting official technician and the legal advisor are outsourced.
It plays an important role on projects management, assuring all the tasks of financial reporting and related counselling to the principal
investigators.
Highlights
General highlights
During 2012, a controlling table for infrastructural expenditure has been monthly updated. The containment of costs became efective
at the end of the year, with a reduction of 165.000 euros on general costs (facilities and maintenance).
The management software developed inhouse was improved and it is crucial for the General secretary work.
IPATIMUP was able to pay all invoices in due time.
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 maintenace and renewal of the IPATIMUP’s library.
The helpdesk tool continues to be the key to solve technical problems that arise from the various equipments and infrastructures existing in the institution.
The administrative services receives and instructs all requests for human resources movements - entries, renewals or voluntary trainings - through worflows created for this purpose.
Over the years IPATIMUP has been organizing two annual mettings, Portugaliae Genetica and Porto Cancer Meeting. The General Secretary takes charge of the all organization and specific funding, as well as for other sporadic meetings.
Certification on occupational health and safety management systems.
IPATIMUP was certified in November 2012 by OSHAS 18001: Occupational Health and Safety Management. New administrative workflows were created in order to assure new standards of controlling accidents, monitoring of occupational health and keeping updated
a database of the applicable laws.
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Activity Report
2012
Programs’ Office
Overview
During the year 2012, 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 300 research project proposals (75% to national funding programs of which
70% to FCT (in its majority to the 2012 call for proposals to projects in all scientific domains) and 25% 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, such as licensing projects that involve animal experimentation, by the National Authority DGAV.
Like in previous years, the responsible for the office attended information sessions about the 7th Research Framework Program (European Commission’s Research & Development Funding Program) and other research funding programs. In July, she attended the “18th
Annual EARMA Conference – European Research and Innovation at the Horizon”, organized by EARMA, that took place in Dublin, Ireland. The programs’ office also had a relevant role in the coordination of the relations between the 3 institutes, concerning the I3S
Seminars and Workshops and other information and diffusion activities.
Risk Management System
Overview
In November 2012, IPATIMUP was certified by OSHAS 18001: Occupational Health and Safety. The work have started two years before,
with the project “Models of Risk Management in life sciences research institutes”, funded by ON.2 - Programa Operacional da Região
do Norte.
Highlights
The preparation for certification
The information system has developed important tools for risk analysis, management of non-conformities and archive of documents.
It was created a Risk Management Committee with representatives of each research group, with weekly meetings. A new competency
was implemented in each laboratory, the Local Safety Contact, that is, a researcher in charge of bridging the safety questions to the
management.
Risk assessement and safety rules were implemented, as well as conformity assessment and acident and incidents management.
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Activity Report
2012
ANNEXES
105
Activity Report
2012
RECENT PhDs
1.
13-02-2012
Patrícia Joana Morais Ferreira Oliveira
Identification of novel cis-regulatory elements in the cadherin superfamily and characterization of their role in cancer-related
processes
Supervisor: Prof.ª Carla Oliveira
2.
15-02-2012
Nair Susana da Costa Florim Ribeiro Lopes
The role of Vitamin D in the carcinogenesis and progression of breast cancer
Supervisor: Prof. Fernando Schmitt
3.
27-03-2012
Maria Natália Rios Vieira da Costa
Relevance of MUC1 mucin in gastric carcinogenesis
Supervisor: Prof. Luís Filipe Santos Silva
4.
03-04-2012
Ângela Maria Sousa Costa
T-Cell Factor 4 (TCF4) and the Groucho-Family Member AES: riders on the WNT
Supervisor: Prof. Luís Teixeira da Costa
5.
12-04-2012
Catarina Alexandra Pires Eloy
Carcinoma papilar da tireoide. Análise genotípica e fenotípica para identificação de biomarcadores de agressividade clínicopatológica
Supervisor: Prof. Manuel Sobrinho Simões
6.
04-05-2012
Carmen Ruiz de Valbuena Bueno
Fabry disease: pathogenesis and histopathology
Supervisor: Prof.ª Fátima Carneiro
7.
25-06-2012
Joana Cristina Tavares de Oliveira
Role of Galectin-3 in carcinogenesis and metastasis of canine malignant mammary tumours
Supervisor: Prof.ª Fátima Gärtner
8.
16-07-2012
Maria Inês Gomes Soares
Construction of Algorithms to Model Phylogenetic Trees
Supervisor: Prof. António Amorim
9.
17-07-2012
Joana Neto Cunha Gomes
Biological effects of Pteridium aquilinum and its toxin in gastric carcinogenesis: relationship with Helicobacter pylori infection
Supervisor: Prof. Celso Reis
10. 20-07-2012
Joana Pinto do Couto e Silva
STAT3 regulation and functional role in thyroid cancer. Therapeutic implications
Supervisor: Prof.ª Paula Soares
106
Activity Report
11.
2012
30-07-2012
Nádia Maria Gonçalves de Almeida Pinto
General algorithms for computing genetic kinship likelihoods
Supervisor: Prof. António Amorim
12. 24-09-2012
Maria Marta de Ascensão Teixeira Correia de Melo
The role of n3 polyunsaturated fatty acids in Helicobacter pylori mediated gastric inflammation and tumorigenesis
Supervisor: Prof. José Carlos Machado
13. 31-10-2012
Ângela Margarida Amorim Costa
Exploring the relationship between Helicobacter pylori and host cell invasion: from phenotypes to molecular mechanisms
Supervisor: Prof.ª Céu Figueiredo
14. 15-11-2012
Ricardo dos Santos Celestino
Structural and functional effects of chromosomal rearrangements in solid tumours: neoplastic thyroid lesions as model
Supervisor: Prof.ª Paula Soares
15. 20-11-2012
André Filipe de Barros Vieira
Stem cells in normal and malignant breast tissue: is P-cadherin a stem cell marker and a possible target for cancer stem cell
therapy?
Supervisor: Prof.ª Joana Paredes
16. 22-11-2012
Isabel Pereira de Castro
Unravelling the function and regulation of human NLZ1 and COG8/PDF
Supervisor: Prof. Luís Teixeira da Costa
17. 07-12-2012
Joana Teresa Lopes Carvalho
The involvement of MicroRNAs in Gastric Cancer
Supervisor: Prof.ª Carla Oliveira
18. 12-12-2012
Rui Manuel Mendes da Silva Ferreira
Clinical and functional relevance of Helicobacter pylori vacA intermediate region and CagA tyrosine phosphorylation motifs
Supervisor: Prof.ª Céu Figueiredo
19. 13-12-2012
Joana Pinto de Figueiredo
The role of E-cadherin trafficking regulation in epitelial cancer progression
Supervisor: Prof.ª Raquel Seruca
20. 19-12-2012
Josiana Adelaide Vaz
Study of antioxidant, antiproliferative and apoptosis-inducing properties of wild mushrooms from the Northeast of Portugal
Supervisor: Prof.ª Helena Vasconcelos
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Activity Report
2012
RESEARCH PROJECTS
108
•
Identificação de factores prognósticos e de selecção terapêutica em carcinomas diferenciados da tireoide [FCG]
•
Are genetic polymorphisms in inflammatory molecules risk factors for the development of autoimmune thyroiditis? (This project
implies the establishment of a tumour database) [IPATIMUP]
•
Identificação de vias de sinalização mediadas pelo oncogene MUC1 em linhas celulares de carcinoma gástrico e em células gástricas imortalizadas [FCT]
•
Study of mTOR pathway in human cancer [IPATIMUP]
•
miRNAs como alvos moleculares em leucemia humana [FCG]
•
Early detection of cancer using serum biomarkers based on aberrant post-translational modifications of O-glycoproteins, O-PTM-Biomarkers [FCT]
•
Cancer risk and irradiation: an epidemiological and genetic study of a cohort irradiated for Tinea Capitis [FCT]
•
Vitamin D, a candidate for new therapy of basal-like breast carcinomas [FCT]
•
Exploring the role of E-Cadherin-HER interaction in the search of molecular biomarkers for the clinical management of gastric
cancer patient [FCT]
•
O Nemátode-Da-Madeira-Do-Pinheiro (NMP), Bursaphelenchus Xyliophilus [IMAR]
•
Integrated Micro-Nano-OPTO Fluidic systems for high-content diagnosis and studies of rare cancer cells [UE]
•
Identification of Genetic markers for prediction of the clinical course and development of complications in portuguese Crohn’s
Disease patients [GEDII]
•
Unraveling the genetics of neuroendocrine tumors by high throughput methods [IPATIMUP]
•
Dissecção molecular do fenótipo de multinucleaçao das células de Hodgkin: validação de resultados [IPATIMUP]
•
mTOR expression in breast carcinomas and the ability of everolimus (RAD 001) to modulate its expression [Novartis Farma]
•
Interaction of HER receptors (EGFR and HER2) and E-Cadherin. Search of molecular biomarkers for clinical management of gastric
cancer patients [Roche]
•
Study of SDH alterations in GIST [IPATIMUP]
•
Analysis of the function and mechanisms of action of a novel Wnt pathway member in vertebrate tumourigenesis and development [IPATIMUP]
•
Males lineages in South Amerindian populations [IPATIMUP]
•
Bacterial protein azurin as a new candidate drug to trat poor-prognosis breast 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]
•
non-coding DNA structural information in phylogeny, evolution and disease [IPATIMUP]
•
Transcriptional and post-transcriptional mechanisms of LRP1B inactivation in sporadic and familial non-medullary thyroid cancer
[FCT]
•
Search for genomic structural variants in azoopermia:a study in the Portuguese population [FCT]
•
The causes and consequences of mitochondrial DNA deletions in animals cells [FCT]
•
Co-Evolutionary Study of Nad Biochemical Networks [FCT]
•
Development of siRNA-loaded nanoparticles to circumvent chemoresistance in cancer stem cells [FCT]
•
Modelos de Sistemas de Gestão do Risco na Investigação em Ciências da Vida e da Saúde [ON]
•
The Role of Protein Quality Control in the regulation of E-cadherin and its relevance in cancer [FCT]
•
Computational disease prediction systems based on molecular markers [FCT]
•
Dissection of the molecular role of O-GLcNAc in the multinucleation phenotype of the neoplasic cells in Hodgkin´s lymphoma
[FCT]
•
Is the mTOR pathway relevant in the initiation/progression and/or a putative therapeutic target in melanomas? [IPATIMUP]
•
Projecto de Desenvolvimento e Implementação de um modelo de ensino inclusivo da Ciência numa população de alunos cegos ou
com baixa visão [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]
•
Genetics of populations of Jewish origin [IPATIMUP]
•
Formação Avançada em Microscopia [IPATIMUP]
•
Unveiling GRIM-19 functional roles in cancer metabolism [IPATIMUP]
Activity Report
2012
•
Formação Pessoal [IPATIMUP]
•
Interacção entre Helicobacter pylori e as junções intercelulares de aclusão [IPATIMUP]
•
Genetic analysis of 15STR loci in the population of the Acre province, Northern Brazil [IPATIMUP]
•
Large inversion polymorphisms in the human genome [IPATIMUP]
•
Portuguese Wild Mushrooms: Chemical characterization and functional study of antiproliferative and proapoptotic properties in
cancer cell lines [FCT]
•
Identification of SNP backgrounds of the Androgen Receptor gene: an attempt to understand AR diversity and the mechanisms
of instability underlying CAG and GGC coding repeats [IPATIMUP]
•
The involvement of CDH1 in CG angiogenesis [IPATIMUP]
•
Programa de Acções Integradas Luso-Francesas PAULIF2011 [CRUP]
•
Development of tools for automatic comparison of biological sequences [IPATIMUP]
•
Fellowship para apoio ao Internato de Anatomia Patológica no Hospital de S. João [FCG]
•
Approaching basal-like breast carcinomas to target therapy. A project combining the reinforcement of logistic facilities with
translational research [IPATIMUP]
•
Galectins as modulators of tumor progression:Establishement of anti-cancer action of bioactive fragments from pectin by inhibiting galectin - 3 [IPATIMUP]
•
SOX2 and CDX2 negative cross-regulation in the establishment of intestinal metaplasia of the stomach [IPATIMUP]
•
Glycosylation alterations in cancer - characterization by proximity ligation (PLA) Assays and by production of glycopeptide specific monoclonal antibodies [IPATIMUP]
•
Banco de Tumores - Amostras Congeladas [IPATIMUP]
•
Polimorfismos em genes de moléculas pro-inflamatórias e risco de tireoidite de Hashimoto [Universidade do Porto]
•
A Study of the antitumour potential of three Portuguese Wild Mushrooms [Universidade do Porto]
•
Alterações metabólicas e cancro: GRIM-19 um gene do metabolismo celular e um gene supressor tumoral, que poderá ser um
target terapêutico interssante [Merck]
•
Cooperação Portugal/França Programa Pessoa 2011-2012 [FCT]
•
Study of mTOR pathway in GISTs [Novartis Farma]
•
Genetic and chronological characterisation of the European settlement by modern humans in the Upper Palaeolithic [FCT]
•
GRIM-19, a novel protein involved in cell apoptosis: structure-function characterization [FCT]
•
Helicobacter pylori diversity in pathogenesis, antibiotic resistance, and evasion from natural and vaccine-induced immune responses (HELDIVPAT) [FCT]
•
Effect of radio-and chemotherapy on cancer cell invasion: the role of macrophages [FCT]
•
Disorders on the Glycolytic and Pentose Phosphate Pathaways of the Red Blood Cell - how do they affect Plasmodium infection?
[IHMT]
•
E-cadherin Post-Translational Modifications by N-glycosylation: a potencial new mechanism of E-cadherin deregulation in cancer
[FCT]
•
Tumour spectrum in hereditary Diffuse Gastric Cancer [FCT]
•
Leonese dialects in Portugal: a genetic approach to a historical-linguistic issue [IPATIMUP]
•
Evaluation the role of proteolysis in the male reproductive system through the study of KLK (19q13,4) and WFDC (20q13) gene
clusters [IPATIMUP]
•
Protein glycosylation in epithelial cells: A glycomic approach to unravel cancer-related glycostructures [IPATIMUP]
•
Conhecer a doença: Os doentes em primeiro lugar [FCG]
•
Mucin MUC16-CA125 cancer biomarker - biological functions and development of biomarker assays [IPATIMUP]
•
Origin, evolution and functional divergence of ataxin-3 paralogs: ATXN3L1 and ATXN3L2 [IPATIMUP]
•
New E-cadherin RNAs: the dark side of a tumour suppressor gene [FCT]
•
Urease de helicobacter pylori: propriedades não enzimáticas que potencialmente contribuem para gastrite e cancro gástrico
[FCT]
•
Cancro-Educar para prevenir [ACS]
•
Identification of a susceptibility locus for gastric cancer in the IL1 gene cluster region [CRUP]
•
Acção dos ácidos gordos polinsaturados n-3 na modulação do processo da inflamação e tumorigénese [CRUP]
•
An approach to thyroid cancer targeted-therapy using transgenic zebrafish as a model [IPATIMUP]
•
Sorting out the genetics of neuroendocrine tumours [IPATIMUP]
•
Aging genes in model and non-model organisms a comparative approach [IPATIMUP]
•
Characterization of voriconazole susceptibility in relevant clinical molds [Pfizer]
109
Activity Report
110
2012
•
Telepathological Assessment of histopathological and cytological techniques [UE]
•
A European Initial Training Network on the History, Archaeology, and New Genetics of the Trans-Atlantic Slave Trade [UE]
•
Estudo do papel dos microRNAs como co-reguladores do complexo da desidrogenase dos alfacetoácidos ramificados [IPATIMUP]
•
Function studies of Germline Missense mutations in HDGC Families [IPATIMUP]
•
Laboratório Aberto [Câmara Municipal do Porto]
•
P-cadherin overexpression in breast carcinomas: an important player for cancer cell survival and invasion in response to tumour
hypoxic conditions [IPATIMUP]
•
Modelos experimentais de cancro (Drosphila e embrião de galinha) [IPATIMUP]
•
Helicobacter pylori infection and matrix metalloprotease [IPATIMUP]
•
EMT and MET in epithelial cancer [IPATIMUP]
•
HeliSysBio - Molecular Systems Biology Helicobacter pylori [FCT]
•
Functional, molecular and pharmacological studies of p53 family proteins: from yeast to human cells [FCT]
•
Glycomic Approach to unravel the role of Glycostructures in Gastric Carcinogenesis [Institut Mérieux]
•
Benign and malignant apocrine lesions of the Breast [IPATIMUP]
•
Wich is the role of both Cancer Stem Cells and Neural Stem Cells to the establishment of HER2+Breast Cancer Brain Metastases?
[Novartis Farma]
•
Consolidação e expansão do Banco de DNA e RNA acoplado ao Banco de Tecidos e Tumores do H.S. João [IPATIMUP]
•
Regulation of the glycomucinome in cancer and pre-neoplastic lesions [IPATIMUP]
•
Mouse models of intestinal metaplasia - exploring pathways from the TGF-beta superfamily [IPATIMUP]
•
Dengue research framework for resisting epidemics in Europe [UE]
•
L and U6 mtDNA lineages in Southern and Western Iberia: migratory links between Africa and Europe [CRUP]
•
HERICA - Histological and Endoscopic Evaluation of Remission induced by Infliximab in moderately to severely active ulcerative
Colitis Patients [GEDII]
•
Biomarcadores para avaliação de efectividade terapêutica no carcinoma colorectal [ADI - Programa IDEIA]
•
The role of sialylation in modulating galectin-3 functions in the metastatic process [FCT]
•
Helicobacter pylori genetic variation: An opportunity to identify individuals at increased risk for disease [FCT]
•
PYLORICIDAL - Engineered biomaterials with Helicobacter PYLORI bacteriCIDAL effect [FCT]
•
Unravelling a new Helicobacter pylori virulence factor involved in tight junction dysfunction [FCT]
•
Mapeamento de genes de susceptibilidade para tumores familiares da tireóide [FCT]
•
Adhesion, Differentiation and Invasion: different processes, common cadherin players [FCT]
•
Characterization of a population with increased risk of Gastric Cancer: First Degree Relatives of Early Onset Gastric Carcinoma
Patients [HSA]
•
Expression of Mesothelin in gastric cancer: its relevance in tumor differentiation and in prognosis (Mesothelin) [IPATIMUP]
•
TUMORTAG - Tumor-targeted nanoparticles for early diagnosis of gastric cancer [FCT]
•
Modulation of CDX2 expression in gastric intestinal metaplasia using na siRNA delivery system in vivo [SPG]
•
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]
•
Irradiation and atherosclerosic disease - epidemiologic and biologic evaluation of a cohort irradiated in childhood for tinea capitis
treatment [IPATIMUP]
•
Bioinformatics in HDGC cancer spectrum [IPATIMUP]
•
Identification of genetic markers for prediction of the clinical course and development of complications in Portuguese Crohn’s
Disease patients. [GEDII]
•
Phylogeography and population structure of the powdery mildew fungus, Erysiphe necator, from diverse Vitis vinifera cultivars
grown in Portugal [Reitoria Universidade do Porto]
•
Mucin MUC16 - CA125 cancer biomarker - biological functions and development of new biomarker assays [FCT]
•
Signalling pathoway in luminal B Breast Cancer [IPATIMUP]
Activity Report
2012
SCIENTIFIC PAPERS
1.
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;accepted. 2012 [] DOI: PMID:
IF= 18,372
2.
Clavel C, Grisanti L, Zemla R, Rezza A, Barros R, Sennett R, Mazloom AR, Chung CY, Cai X, Cai CL, Pevny L, Nicolis S, Ma’ayan A,
Rendl M. Sox2 in the Dermal Papilla Niche Controls Hair Growth by Fine-Tuning BMP Signaling in Differentiating Hair Shaft Progenitors.. Developmental cell;23: 981-94. 2012 [Article] DOI: 10.1016/j.devcel.2012.10.013 PMID: 23153495 IF= 13,946
3.
Van Der Werf CS, Wabbersen TD, Hsiao NH, Paredes J, Etchevers HC, Kroisel PM, Tibboel D, Babarit C, Schreiber RA, Hoffenberg
EJ, Vekemans M, Zeder SL, Ceccherini I, Lyonnet S, Ribeiro AS, Seruca R, Te Meerman GJ, van Ijzendoorn SC, Shepherd IT, Verheij
JB, Hofstra RM. CLMP is required for intestinal development, and loss-of-function mutations cause congenital short-bowel syndrome.. Gastroenterology;142: 453-462.e3. 2012 [Article] DOI: 10.1053/j.gastro.2011.11.038 PMID: 22155368 IF= 11,675
4.
Pala M, Olivieri A, Achilli A, Accetturo M, Metspalu E, Reidla M, Tamm E, Karmin M, Reisberg T, Hooshiar Kashani B, Perego UA,
Carossa V, Gandini F, Pereira JB, Soares P, Angerhofer N, Rychkov S, Al-Zahery N, Carelli V, Sanati MH, Houshmand M, Hatina J,
Macaulay V, Pereira L, Woodward SR, Davies W, Gamble C, Baird D, Semino O, Villems R, Torroni A, Richards MB. Mitochondrial
DNA signals of late glacial recolonization of europe from near eastern refugia.. American journal of human genetics;90: 915-24.
2012 [Article] DOI: 10.1016/j.ajhg.2012.04.003 PMID: 22560092 IF= 10,603
5.
Behar DM, van Oven M, Rosset S, Metspalu M, Loogväli EL, Silva NM, Kivisild T, Torroni A, Villems R. A “Copernican” reassessment of the human mitochondrial DNA tree from its root.. American journal of human genetics;90: 675-84. 2012 [Article] DOI:
10.1016/j.ajhg.2012.03.002 PMID: 22482806 IF= 10,603
6.
Fernandes V, Alshamali F, Alves M, Costa MD, Pereira JB, Silva NM, Cherni L, Harich N, Cerny V, Soares P, Richards MB, Pereira L.
The Arabian cradle: mitochondrial relicts of the first steps along the southern route out of Africa.. American journal of human
genetics;90: 347-55. 2012 [Article] DOI: 10.1016/j.ajhg.2011.12.010 PMID: 22284828 IF= 10,603
7.
Bordeira-Carriço R, Pêgo AP, Santos M, Oliveira C. Cancer syndromes and therapy by stop-codon readthrough.. Trends in molecular medicine;18: 667-78. 2012 [Review] DOI: 10.1016/j.molmed.2012.09.004 PMID: 23044248 IF= 10,355
8.
Barros R, Freund JN, David L, Almeida R. Gastric intestinal metaplasia revisited: function and regulation of CDX2.. Trends in molecular medicine;18: 555-63. 2012 [Review] DOI: 10.1016/j.molmed.2012.07.006 PMID: 22871898 IF= 10,355
9.
Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini GF, Gisbert JP, Graham DY, Rokkas T, El-Omar
EM, Kuipers EJ, European Helicobacter Study Group European Helicobacter Study Group. Management of Helicobacter pylori
infection--the Maastricht IV/ Florence Consensus Report.. Gut;61: 646-64. 2012 [] DOI: 10.1136/gutjnl-2012-302084 PMID: 22491499
IF= 10,111
10. Worthley DL, Phillips KD, Wayte N, Schrader KA, Healey S, Kaurah P, Shulkes A, Grimpen F, Clouston A, Moore D, Cullen D, Ormonde D, Mounkley D, Wen X, Lindor N, Carneiro F, Huntsman DG, Chenevix-Trench G, Suthers GK. Gastric adenocarcinoma and
proximal polyposis of the stomach (GAPPS): a new autosomal dominant syndrome.. Gut;61: 774-9. 2012 [Article] DOI: 10.1136/gutjnl-2011-300348 PMID: 21813476 IF= 10,111
11.
Caldeira J, Simões-Correia J, Paredes J, Pinto MT, Sousa S, Corso G, Marrelli D, Roviello F, Pereira PS, Weil D, Oliveira C, Casares
F, Seruca R. CPEB1, a novel gene silenced in gastric cancer: a Drosophila approach.. Gut;61: 1115-23. 2012 [Article] DOI: 10.1136/gutjnl-2011-300427 PMID: 22052064 IF= 10,111
12. Achilli A, Olivieri A, Soares P, Lancioni H, Kashani BH, Perego UA, Nergadze SG, Carossa V, Santagostino M, Capomaccio S, Felicetti M, Al-Achkar W, Penedo MC, Verini-Supplizi A, Houshmand M, Woodward SR, Semino O, Silvestrelli M, Giulotto E, Pereira L,
Bandelt HJ, Torroni A. Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication.. Proceedings of the National Academy of Sciences of the United States of America;109: 2449-54. 2012 [Article] DOI: 10.1073/
pnas.1111637109 PMID: 22308342 IF= 9,681
13. Couto JP, Daly L, Almeida A, Knauf JA, Fagin JA, Sobrinho-Simões M, Lima J, Máximo V, Soares P, Lyden D, Bromberg JF. STAT3 negatively regulates thyroid tumorigenesis.. Proceedings of the National Academy of Sciences of the United States of America;109:
E2361-70. 2012 [Article] DOI: 10.1073/pnas.1201232109 PMID: 22891351 IF= 9,681
14. Mendizabal I, Lao O, Marigorta UM, Wollstein A, Gusmão L, Ferak V, Ioana M, Jordanova A, Kaneva R, Kouvatsi A, Kucinskas V,
Makukh H, Metspalu A, Netea MG, de Pablo R, Pamjav H, Radojkovic D, Rolleston SJ, Sertic J, Macek M, Comas D, Kayser M. Reconstructing the population history of European Romani from genome-wide data.. Current biology : CB;22: 2342-9. 2012 [Article]
DOI: 10.1016/j.cub.2012.10.039 PMID: 23219723 IF= 9,647
15. Paredes J, Figueiredo J, Albergaria A, Oliveira P, Carvalho J, Ribeiro AS, Caldeira J, Costa AM, Simões-Correia J, Oliveira MJ, Pinheiro H, Pinho SS, Mateus R, Reis CA, Leite M, Fernandes MS, Schmitt F, Carneiro F, Figueiredo C, Oliveira C, Seruca R. Epithelial
E- and P-cadherins: Role and clinical significance in cancer.. Biochimica et Biophysica acta-Reviews on Cancer;1826: 297-311. 2012
[Review] DOI: 10.1016/j.bbcan.2012.05.002 PMID: 22613680 IF= 9,38
16. Amaro Helena M., Barros R, Guedes A. Catarina, Sousa-Pinto I., Malcata F. Xavier. Microalgal compounds modulate carcinogenesis in the gastrointestinal tract. Trends in Biotechnology;1. 2012 [] DOI: PMID: IF= 9,148
17. Damas J, Carneiro J, Gonçalves J, Stewart JB, Samuels DC, Amorim A, Pereira F. Mitochondrial DNA deletions are associated with
non-B DNA conformations.. Nucleic acids research;40: 7606-21. 2012 [Article] DOI: 10.1093/nar/gks500 PMID: 22661583 IF= 8,026
18. Pinheiro C, Longatto-Filho A, Nogueira R, Schmitt F, Baltazar F. Lactate-induced IL-8 pathway in endothelial cells--letter.. Cancer
research;72: 1901-2; author reply 1903-4. 2012 [Letter] DOI: 10.1158/0008-5472.CAN-11-1540 PMID: 22473315 IF= 7,856
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19. Vieira AF, Ricardo S, Ablett MP, Dionísio MR, Mendes N, Albergaria A, Farnie G, Gerhard R, Cameselle-Teijeiro JF, Seruca R, Schmitt
F, Clarke RB, Paredes J. P-cadherin is coexpressed with CD44 and CD49f and mediates stem cell properties in basal-like breast
cancer.. Stem cells (Dayton, Ohio);30: 854-64. 2012 [Article] DOI: 10.1002/stem.1075 PMID: 22389315 IF= 7,781
20. Ferreira AC, Suriano G, Mendes N, Gomes B, Wen X, Carneiro F, Seruca R, Machado JC. E-cadherin impairment increases cell
survival through Notch-dependent upregulation of Bcl-2.. Human molecular genetics;21: 334-43. 2012 [Article] DOI: 10.1093/hmg/
ddr469 PMID: 21989054 IF= 7,636
21. Pinheiro H, Carvalho J, Oliveira P, Ferreira D, Pinto MT, Osório H, Licastro D, Bordeira-Carriço R, Jordan P, Lazarevic D, Sanges R,
Stupka E, Huntsman D, Seruca R, Oliveira C. Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform
that increases gastric cancer cell invasion and angiogenesis.. Human molecular genetics;21: 4253-69. 2012 [Article] DOI: 10.1093/
hmg/dds248 PMID: 22752307 IF= 7,636
22. Martins S, Soong BW, Wong VC, Giunti P, Stevanin G, Ranum LP, Sasaki H, Riess O, Tsuji S, Coutinho P, Amorim A, Sequeiros J, Nicholson GA. Mutational origin of Machado-Joseph disease in the Australian Aboriginal communities of Groote Eylandt and Yirrkala.. Archives of neurology;69: 746-51. 2012 [Article] DOI: 10.1001/archneurol.2011.2504 PMID: 22351852 IF= 7,584
23. Ferreira RM, Figueiredo C, Bonet C, Pardo ML, Liso JM, Alonso P, Sala N, Capella G, Sanz-Anquela JM, González CA. Helicobacter
pylori vacA intermediate region genotyping and progression of gastric preneoplastic lesions.. The American journal of gastroenterology;107: 145-6. 2012 [Letter] DOI: 10.1038/ajg.2011.389 PMID: 22218041 IF= 7,282
24. González CA, Megraud F, Buissonniere A, Lujan Barroso L, Agudo A, Duell EJ, Boutron-Ruault MC, Clavel-Chapelon F, Palli D,
Krogh V, Mattiello A, Tumino R, Sacerdote C, Quirós JR, Sanchez-Cantalejo E, Navarro C, Barricarte A, Dorronsoro M, Khaw KT,
Wareham N, Allen NE, Tsilidis KK, Bas Bueno-de-Mesquita H, Jeurnink SM, Numans ME, Peeters PH, Lagiou P, Valanou E, Trichopoulou A, Kaaks R, Lukanova-McGregor A, Bergman MM, Boeing H, Manjer J, Lindkvist B, Stenling R, Hallmans G, Mortensen LM,
Overvad K, Olsen A, Tjonneland A, Bakken K, Dumeaux V, Lund E, Jenab M, Romieu I, Michaud D, Mouw T, Carneiro F, Fenge C,
Riboli E. Helicobacter pylori infection assessed by ELISA and by immunoblot and noncardia gastric cancer risk in a prospective
study: the Eurgast-EPIC project.. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO;23:
1320-4. 2012 [Article] DOI: 10.1093/annonc/mdr384 PMID: 21917738 IF= 6,425
25. Pareja F, Ferraro DA, Rubin C, Cohen-Dvashi H, Zhang F, Aulmann S, Ben-Chetrit N, Pines G, Navon R, Crosetto N, Köstler W, Carvalho S, Lavi S, Schmitt F, Dikic I, Yakhini Z, Sinn P, Mills GB, Yarden Y. Deubiquitination of EGFR by Cezanne-1 contributes to cancer
progression.. Oncogene;31: 4599-608. 2012 [Article] DOI: 10.1038/onc.2011.587 PMID: 22179831 IF= 6,373
26. Davalos V, Moutinho C, Villanueva A, Boque R, Silva P, Carneiro F, Esteller M. Dynamic epigenetic regulation of the microRNA-200
family mediates epithelial and mesenchymal transitions in human tumorigenesis.. Oncogene;31: 2062-74. 2012 [Article] DOI:
10.1038/onc.2011.383 PMID: 21874049 IF= 6,373
27. Ribeiro AS, Sousa B, Carreto L, Mendes N, Nobre AR, Ricardo S, Albergaria A, Cameselle-Teijeiro JF, Gerhard R, Soderberg 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;. 2012 [] DOI: 10.1002/path.4143 PMID: 23180380 IF= 6,318
28. Carvalho J, van Grieken NC, Pereira PM, Sousa S, Tijssen M, Buffart TE, Diosdado B, Grabsch H, Santos MA, Meijer G, Seruca R,
Carvalho B, Oliveira C. Lack of microRNA-101 causes E-cadherin functional deregulation through EZH2 up-regulation in intestinal
gastric cancer.. The Journal of pathology;228: 31-44. 2012 [Article] DOI: 10.1002/path.4032 PMID: 22450781 IF= 6,318
29. Faustino A, Couto JP, Pópulo H, Rocha AS, Pardal F, Cameselle-Teijeiro JM, Lopes JM, Sobrinho-Simões M, Soares P. mTOR pathway overactivation in BRAF mutated papillary thyroid carcinoma.. The Journal of clinical endocrinology and metabolism;97:
E1139-49. 2012 [Article] DOI: 10.1210/jc.2011-2748 PMID: 22549934 IF= 5,967
30. Lopes N, Paredes J, Costa JL, Ylstra B, Schmitt F. Vitamin D and the mammary gland: a review on its role in normal development
and breast cancer.. Breast cancer research : BCR;14: 211. 2012 [Review] DOI: 10.1186/bcr3178 PMID: 22676419 IF= 5,785
31. Camilo V, Barros R, Sousa S, Magalhães AM, Lopes T, Mário Santos A, Pereira T, Figueiredo C, David L, Almeida R. Helicobacter
pylori and the BMP pathway regulate CDX2 and SOX2 expression in gastric cells.. Carcinogenesis;33: 1985-92. 2012 [Article] DOI:
10.1093/carcin/bgs233 PMID: 22791809 IF= 5,702
32. Soares P, Alshamali F, Pereira JB, Fernandes V, Silva NM, Afonso C, Costa MD, Musilová E, Macaulay V, Richards MB, Cerny V, Pereira L. The Expansion of mtDNA Haplogroup L3 within and out of Africa.. Molecular biology and evolution;29: 915-27. 2012 [Article]
DOI: 10.1093/molbev/msr245 PMID: 22096215 IF= 5,55
33. Lutz MP, Zalcberg JR, Ducreux M, Ajani JA, Allum W, Aust D, Bang YJ, Cascinu S, Hölscher A, Jankowski J, Jansen EP, Kisslich R,
Lordick F, Mariette C, Moehler M, Oyama T, Roth A, Rueschoff J, Ruhstaller T, Seruca R, Stahl M, Sterzing F, van Cutsem E, van der
Gaast A, van Lanschot J, Ychou M, Otto F. Highlights of the EORTC St. Gallen International Expert Consensus on the primary therapy of gastric, gastroesophageal and oesophageal cancer - Differential treatment strategies for subtypes of early gastroesophageal cancer.. European journal of cancer (Oxford, England : 1990);48: 2941-53. 2012 [Article] DOI: 10.1016/j.ejca.2012.07.029 PMID:
22921186 IF= 5,536
34. Serafini M, Jakszyn P, Luján-Barroso L, Agudo A, Bas Bueno-de-Mesquita H, van Duijnhoven FJ, Jenab M, Navarro C, Palli D,
Boeing H, Wallström P, Regnér S, Numans ME, Carneiro F, Boutron-Ruault MC, Clavel-Chapelon F, Morois S, Grioni S, Panico S,
Tumino R, Sacerdote C, Ramon Quirós J, Molina-Montes E, Huerta Castaño JM, Barricarte A, Amiano P, Khaw KT, Wareham N,
Allen NE, Key TJ, Jeurnink SM, Peeters PH, Bamia C, Valanou E, Trichopoulou A, Kaaks R, Lukanova A, Bergmann MM, Lindkvist B,
Stenling R, Johansson I, Dahm CC, Overvad K, Jensen M, Olsen A, Tjonneland A, Lund E, Rinaldi S, Michaud D, Mouw T, Riboli E,
González CA. Dietary total antioxidant capacity and gastric cancer risk in the European prospective investigation into cancer and
nutrition study.. International journal of cancer. Journal international du cancer;131: E544-54. 2012 [Article] DOI: 10.1002/ijc.27347
PMID: 22072493 IF= 5,444
35. Alhopuro P, Sammalkorpi H, Niittymäki I, Biström M, Raitila A, Saharinen J, Nousiainen K, Lehtonen HJ, Heliövaara E, Puhakka J,
Tuupanen S, Sousa S, Seruca R, Ferreira AM, Hofstra RM, Mecklin JP, Järvinen H, Ristimäki A, Orntoft TF, Hautaniemi S, Arango D,
Karhu A, Aaltonen LA. Candidate driver genes in microsatellite-unstable colorectal cancer.. International journal of cancer. Journal international du cancer;130: 1558-66. 2012 [Article] DOI: 10.1002/ijc.26167 PMID: 21544814 IF= 5,444
36. Busby GB, Brisighelli F, Sánchez-Diz P, Ramos-Luis E, Martinez-Cadenas C, Thomas MG, Bradley DG, Gusmão L, Winney B, Bodmer W, Vennemann M, Coia V, Scarnicci F, Tofanelli S, Vona G, Ploski R, Vecchiotti C, Zemunik T, Rudan I, Karachanak S, Toncheva
D, Anagnostou P, Ferri G, Rapone C, Hervig T, Moen T, Wilson JF, Capelli C. The peopling of Europe and the cautionary tale of
112
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2012
Y chromosome lineage R-M269.. Proceedings. Biological sciences / The Royal Society;279: 884-92. 2012 [Article] DOI: 10.1098/
rspb.2011.1044 PMID: 21865258 IF= 5,415
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42. Freitas SC, Cereija TB, Figueiredo AC, Osório H, Pereira PJ, Barbosa MA, Martins MC. Bioengineered surfaces to improve the blood
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46. Gomes J, Magalhães A, Valérie M, Amado I, Aranha P, Ovesen RG, Hansen HCB, Gartner F, Reis C A, Touati E. Pteridium aquillinum
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53. Soares NCP, Teodoro AJ, Oliveira FL, Santos CAN, Takiya CM, Junior OS, Bianco M, Palumbo A, Ferreira LB, Gimba EP EP, Borojevic
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54. Silva E, Marques S, Osório H, Carvalheira J, Thompson G. Endogenous hepatitis C virus homolog fragments in European rabbit
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55. Machado P, Manco L, Gomes C, Mendes C, Fernandes N, Salomé G, Sitoe L, Chibute S, Langa J, Ribeiro L, Miranda J, Cano J, Pinto J, Amorim A, do Rosário VE, Arez AP. Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent
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56. Couto JP, Almeida A, Daly L, Sobrinho-Simões M, Bromberg JF, Soares P. AZD1480 Blocks Growth and Tumorigenesis of RET- Activated Thyroid Cancer Cell Lines.. PloS one;7: e46869. 2012 [Article] DOI: 10.1371/journal.pone.0046869 PMID: 23056499 IF= 4,092
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59. Pereira R, Phillips C, Pinto N, Santos C, Dos Santos SE, Amorim A, Carracedo A, Gusmão L. Straightforward Inference of Ancestry
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60. Seixas S, Ivanova N, Ferreira Z, Rocha J, Victor L. B. Loss and Gain of Function in SERPINB11: An Example of a Gene under Selection on Standing Variation, with Implications for Host-Pathogen Interactions. PloS one;1. 2012 [Article] DOI: 10.1371/journal.
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61. Simões-Correia J, Figueiredo J, Lopes R, Stricher F, Oliveira C, Serrano L, Seruca R. E-cadherin destabilization accounts for the
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65. Marcos-Pinto R, Carneiro F, Dinis-Ribeiro M, Wen X, Lopes C, Figueiredo C, Machado JC, Ferreira RM, Reis CA, Ferreira J, Pedroto
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69. Soares I, Amorim A, Goios A. mtDNAoffice: A software to assign human mtDNA macro haplogroups through automated analysis
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70. Costa A, Leite M, Figueiredo C. Adherens junctions as targets of microorganisms: a focus on Helicobacter pylori.. FEBS letters;1.
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71. Carneiro P, Fernandes MS, Figueiredo J, Caldeira J, Carvalho J, Pinheiro H, Leite M, Melo S, Oliveira P, Simões-Correia J, Oliveira
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72. Annaratone L, Marchiò C, Renzulli T, Castellano I, Cantarella D, Isella C, Macri L, Mariscotti G, Balmativola D, Cantanna E, Deambrogio C, Pietribiasi F, Arisio R, Schmitt F, Medico E, Sapino A. High-Throughput Molecular Analysis from Leftover of Fine Needle
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75. Celestino R, Sigstad E, Løvf M, Thomassen GO, Grøholt KK, Jørgensen LH, Berner A, Castro P, Lothe RA, Bjøro T, Sobrinho-Simões
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76. Coutinho MF, Prata MJ, Alves S. A Shortcut to the Lysosome: The Mannose-6-Phosphate-Independent Pathway. Molecular genetics and metabolism;1. 2012 [Review] DOI: dx.doi.org/10.1016/j.ymgme.2012.07.012 PMID: IF= 3,193
77. Vaz Rodrigues L, Costa F, Marques P, Mendonça C, Rocha J, Seixas S. Severe a-1 antitrypsin deficiency caused by Q0(Ourém)
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78. Manta F, Caiafa A, Pereira R, Silva D, Amorim A, Carvalho EF, Gusmão L. Indel markers: genetic diversity of 38 polymorphisms
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80. Pinto N, Gusmão L, Egeland T, Amorim A. Paternity exclusion power: comparative behaviour of autosomal and X-chromosomal
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81. Gelabert-Besada M, Ferreira S, García-Magariños M, Gusmão L, Sánchez-Diz P. Genetic characterization of Western Iberia using
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82. Pinto N, Silva PV, Amorim A. A general method to assess the utility of the X-chromosomal markers in kinship testing.. Forensic
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83. Ferreira RM, Machado JC, Leite M, Carneiro F, Figueiredo C. The number of Helicobacter pylori CagA EPIYA C tyrosine phosphorylation motifs influences the pattern of gastritis and the development of gastric carcinoma.. Histopathology;60: 992-8. 2012
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84. de Vries MM, Celestino R, Castro P, Eloy C, Máximo V, van der Wal JE, Plukker JT, Links TP, Hofstra RM, Sobrinho-Simões M, Soares P. RET/PTC rearrangement is prevalent in follicular Hürthle cell carcinomas.. Histopathology;61: 833-843. 2012 [Article] DOI:
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87. Araujo R, Amorim A, Gusmão L. Diversity and specificity of microsatellites within Aspergillus section Fumigati.. BMC microbiology;12: 154. 2012 [Article] DOI: 10.1186/1471-2180-12-154 PMID: 22838495 IF= 3,044
88. Pereira L, Soares P, Máximo V, Samuels DC. Somatic mitochondrial DNA mutations in cancer escape purifying selection and high
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90. Belaz KR, Denadai M, Almeida AP, Lima RT, Vasconcelos MH, Pinto MM, Cass QB, Oliveira RV. Enantiomeric resolution of albendazole sulfoxide by semipreparative HPLC and in vitro study of growth inhibitory effects on human cancer cell lines.. Journal of
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92. Neves MP, Cravo S, Lima RT, Vasconcelos MH, Nascimento MS, Silva AM, Pinto M, Cidade H, Corrêa AG. Solid-phase synthesis of
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93. Gonçalves J, Pereira F, Amorim A, van Asch B. New method for the simultaneous identification of cow, sheep, goat, and water
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96. Al-Abri A, Podgorná E, Rose JI, Pereira L, Mulligan CJ, Silva NM, Bayoumi R, Soares P, Cerný V. Pleistocene-Holocene boundary in
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98. Gerhard R, Costa JL, Schmitt F. Benign and malignant apocrine lesions of the breast.. Expert review of anticancer therapy;12: 21521. 2012 [Review] DOI: 10.1586/era.11.213 PMID: 22316369 IF= 2,652
99. Cameselle-Teijeiro J, Ferreira R, Caramés N, Abdulkader I, Máximo V, Soares P, Sobrinho-Simões M. Absence of the BRAF and the
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105. Boaventura P, Oliveira R, Pereira D, Soares P, Teixeira-Gomes J. Head and neck basal cell carcinoma prevalence in individuals submitted to childhood X-ray 1 epilation for tinea capitis treatment. European Journal of Dermatology;1. 2012 [Article] DOI: PMID:
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106. Nolte S, de Castro Damasio D, Baréa AC, Gomes J, Mello Zischler LF, Stuelp-Campelo PM, Elífio-Esposito SL, Roque-Barreira MC,
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110. Vinagre J, Falcão M, Eloy C, Soares P, Lopes JM. A clear cell renal carcinoma causing resistance to VEGF inhibitors in an age-related
macular degeneration (AMD)?. Virchows Archiv;S49. 2012 [Meeting Abstract] DOI: PMID: IF= 2,491
111. Valbuena C, Leitão D, Carneiro F, Oliveira JP. Immunohistochemical diagnosis of Fabry nephropathy and localisation of globotriaosylceramide deposits in paraffin-embedded kidney tissue sections.. Virchows Archiv : an international journal of pathology;460:
211-21. 2012 [Article] DOI: 10.1007/s00428-011-1182-y PMID: 22205110 IF= 2,491
112. Gerhard R, Ricardo S, Albergaria A, Gomes M, Silva AR, Logullo AF, Cameselle-Teijeiro JF, Paredes J, Schmitt F. Immunohistochemical features of claudin-low intrinsic subtype in metaplastic breast carcinomas.. Breast (Edinburgh, Scotland);21: 354-60. 2012
[Article] DOI: 10.1016/j.breast.2012.03.001 PMID: 22464177 IF= 2,491
113. Schmitt F, Ricardo S, Vieira AF, Dionísio MR, Paredes J. Cancer stem cell markers in breast neoplasias: their relevance and distribution in distinct molecular subtypes.. Virchows Archiv : an international journal of pathology;460: 545-53. 2012 [Review] DOI:
10.1007/s00428-012-1237-8 PMID: 22562130 IF= 2,491
114. Eloy C, Santos J, Cameselle-Teijeiro J, Soares P, Sobrinho-Simões M. TGF-beta/Smad pathway and BRAF mutation play different
roles in circumscribed and infiltrative papillary thyroid carcinoma.. Virchows Archiv : an international journal of pathology;460:
587-600. 2012 [Article] DOI: 10.1007/s00428-012-1234-y PMID: 22527019 IF= 2,491
115. Pereira-Castro I, Costa LT, Amorim A, Azevedo L. Transcriptional regulation of the human mitochondrial peptide deformylase
(PDF).. Biochemical and biophysical research communications;421: 825-31. 2012 [Article] DOI: 10.1016/j.bbrc.2012.04.097 PMID:
22554513 IF= 2,484
116. Ricardo S, Gerhard R, Cameselle-Teijeiro JF, Schmitt F, Paredes J. Claudin expression in breast cancer: High or low, what to expect?. Histology and histopathology;27: 1283-95. 2012 [Article] DOI: PMID: 22936447 IF= 2,48
117. Alves J M., Lopes A M, Chikhi L, Amorim A. On the Structural Plasticity of the Human Genome: Chromosomal Inversions Revisited.
Current genomics;13: 623-632. 2012 [Article] DOI: PMID: IF= 2,408
118. Calhelha Ricardo C., Ferreira Isabel C. F. R., Peixoto Daniela, Abreu Rui M. V., Vale-Silva Luís A., Pinto Eugénia, Lima Raquel T.,
Alvelos M. Inês, Vasconcelos M. Helena, Queiroz Maria-João R. P.. Aminodi(hetero)arylamines in the Thieno[3,2-b]pyridine Series:
Synthesis, Effects in Human Tumor Cells Growth, Cell Cycle Analysis, Apoptosis and Evaluation of Toxicity Using Non-Tumor Cells.
Molecules;3834-3843. 2012 [Article] DOI: 10.3390/molecules17043834 PMID: IF= 2,386
119. Caplin M, Sundin A, Nillson O, Baum RP, Klose KJ, Kelestimur F, Plöckinger U, Papotti M, Salazar R, Pascher A, Lopes JM. ENETS
Consensus Guidelines for the management of patients with digestive neuroendocrine neoplasms: colorectal neuroendocrine
neoplasms.. Neuroendocrinology;95: 88-97. 2012 [] DOI: 10.1159/000335594 PMID: 22261972 IF= 2,376
120. Alvarenga CA, Paravidino PI, Alvarenga M, Gomes M, Dufloth R, Zeferino LC, Vassallo J, Schmitt FC. Reappraisal of immunohistochemical profiling of special histological types of breast carcinomas: a study of 121 cases of eight different subtypes.. Journal of
clinical pathology;65: 1066-71. 2012 [Article] DOI: 10.1136/jclinpath-2012-200885 PMID: 22944625 IF= 2,306
121. Santos S, Bastos E, Baptista CS, Sá D, Caloustian C, Guedes-Pinto H, Gartner F, Gut IG, Chaves R. Sequence Variants and Haplotype
Analysis of Cat ERBB2 Gene: A Survey on Spontaneous Cat Mammary Neoplastic and Non-Neoplastic Lesions.. International journal of molecular sciences;1. 2012 [Article] DOI: PMID: IF= 2,279
122. Pópulo H, Lopes JM, Soares P. The mTOR Signalling Pathway in Human Cancer.. International journal of molecular sciences;13:
1886-918. 2012 [Review] DOI: 10.3390/ijms13021886 PMID: 22408430 IF= 2,279
123. Baptista CS, Santos S, Laso A, Bastos E, Avila S, Guedes-Pinto H, Gärtner F, Gut IG, Castrillo JL, Chaves R. Sequence variation and
mRNA expression of the TWIST1 gene in cats with mammary hyperplasia and neoplasia.. Veterinary journal (London, England :
1997);191: 203-7. 2012 [Article] DOI: 10.1016/j.tvjl.2011.01.011 PMID: 21330172 IF= 2,239
124. van Asch B, Pereira-Castro I, Rei F, da Costa LT. Mitochondrial haplotypes reveal olive fly (Bactrocera oleae) population substructure in the Mediterranean.. Genetica;140: 181-7. 2012 [Article] DOI: 10.1007/s10709-012-9669-2 PMID: 22825843 IF= 2,148
116
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125. Peleteiro B, Barros R, Carrilho C, Artiaga J, Cunha L, Modcoicar P, Ferreira R, Figueiredo C, Almeida R, La Vecchia C, David
L, Barros H, Lunet N. Determinants of gastric CDX2 expression: a study in Mozambique.. European journal of cancer prevention : the official journal of the European Cancer Prevention Organisation (ECP);21: 532-540. 2012 [Article] DOI: 10.1097/
CEJ.0b013e3283523480 PMID: 22407102 IF= 2,13
126. Lopes-Marques M, Pereira-Castro I, Amorim A, Azevedo L. Characterization of the human ornithine transcarbamylase 3’ untranslated regulatory region.. DNA and cell biology;31: 427-33. 2012 [Article] DOI: 10.1089/dna.2011.1391 PMID: 22054066 IF= 2,072
127. 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;. 2012 [Epub ahead of print] DOI: 10.1007/s10238-012-0184-7 PMID: 22543498 IF= 2
128. Santos LF, Rodrigues B, Moreira D, Correia E, Nunes L, Costa A, Elvas L, Pereira T, Machado JC, Castedo S, Henriques C, Matos A,
Santos JO. Criteria to predict carriers of a novel SCN5A mutation in a large Portuguese family affected by the Brugada syndrome.. Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing,
arrhythmias, and cardiac cellular electrophysiology of the European Society of Cardiology;14: 882-8. 2012 [Article] DOI: 10.1093/
europace/eur421 PMID: 22277643 IF= 1,98
129. Khodjet-el-Khil H, Fadhlaoui-Zid K, Gusmão L, Alves C, Benammar-Elgaaied A, Amorim A. Allele frequencies for 15 autosomal STR
markers in the Libyan population.. Annals of human biology;39: 80-3. 2012 [Article] DOI: 10.3109/03014460.2011.630678 PMID:
22039975 IF= 1,975
130. Botelho MC, Ribeiro R, Vale N, Oliveira P, Medeiros R, Lopes C, Machado JC, Correia da Costa JM. Inactivation of estrogen receptor by Schistosoma haematobium total antigen in bladder urothelial cells.. Oncology reports;27: 356-62. 2012 [] DOI: 10.3892/
or.2011.1552 PMID: 22089035 IF= 1,835
131. Neves MP, Lima RT, Choosang K, Vasconcelos MH, Pinto M, Silva AM, Cidade H. Synthesis of a natural chalcone and its prenyl
analogs--evaluation of tumor cell growth-inhibitory activities, and effects on cell cycle and apoptosis.. Chemistry & Biodiversity;9: 1133-43. 2012 [Article] DOI: 10.1002/cbdv.201100190 PMID: 22700231 IF= 1,804
132. Lopes N, Carvalho J, Durães C, Sousa B, Gomes M, Costa JL, Oliveira C, Paredes J, Schmitt F. 1alpha,25-dihydroxyvitamin D3 induces de novo E-cadherin expression in triple-negative breast cancer cells by CDH1 promoter demethylation. Anticancer research;1.
2012 [] DOI: PMID: 22213313 IF= 1,725
133. Vala H, Pópulo H, Mesquita JR, Esteves F, Santos C, Soares P, Lopes JM. Melanocytic tumour in a black sheep never exposed to
ultraviolet radiation.. Journal of comparative pathology;146: 160-4. 2012 [Article] DOI: 10.1016/j.jcpa.2011.04.001 PMID: 21612790
IF= 1,647
134. Tavares C, de Oliveira JT, Lopes C, Carvalheira J, Matos AJ, Rutteman G, Reis CA, Gärtner F. Mucin 6 and tn antigen expression in
canine mammary tumours: correlation with pathological features.. Journal of comparative pathology;147: 410-8. 2012 [Article]
DOI: 10.1016/j.jcpa.2012.03.007 PMID: 22595635 IF= 1,647
135. Catherwood MA, Schmitt F, Salto-Tellez M. Molecular diagnostics and the training of future tissue- and cell-based pathologists..
Cytopathology : official journal of the British Society for Clinical Cytology;23: 283-5. 2012 [Editorial Material] DOI: 10.1111/cyt.12015
PMID: 22985226 IF= 1,588
136. Schmitt F, Vielh P, Zeppa P. Cytology for pathologists: two sides of the same coin or different views of the same side?. Cytopathology : official journal of the British Society for Clinical Cytology;23: 345-6. 2012 [Letter] DOI: 10.1111/cyt.12018 PMID: 22985228 IF=
1,588
137. Marcos-Pinto R, Dinis-Ribeiro M, Carneiro F, Machado JC, Figueiredo C, Reis CA, Ferreira J, Areias J. First degree relatives and
familial aggregation of gastric cancer: who to choose for control in case-control studies?. Familial cancer;11: 137-43. 2012 [Article]
DOI: 10.1007/s10689-011-9488-0 PMID: 22057474 IF= 1,302
138. 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;1. 2012 [] DOI: 10.1007/s00044-012-0203-y
PMID: IF= 1,271
139. Di Lorito A, Schmitt FC. (Cyto)pathology and sequencing: Next (or last) generation?. Diagnostic cytopathology;40: 459-61. 2012
[Article] DOI: 10.1002/dc.21691 PMID: 21538954 IF= 1,16
140. Pinheiro C, Longatto-Filho A, Soares TR, Pereira H, Bedrossian C, Michael C, Schmitt FC, Baltazar F. CD147 immunohistochemistry
discriminates between reactive mesothelial cells and malignant mesothelioma.. Diagnostic cytopathology;40: 478-83. 2012 [Article] DOI: 10.1002/dc.22821 PMID: 22619123 IF= 1,16
141. Di Lorito A, Rosini S, Falò E, Gustapane S, Gomes M, Costa JL, Schmitt FC. Molecular alterations in endometrial archived liquid-based cytology.. Diagnostic cytopathology;. 2012 [] DOI: 10.1002/dc.22869 PMID: 22807394 IF= 1,16
142. Amorim A. Opening the DNA black box: demythologizing forensic genetics. New Genetics and Society;1. 2012 [Article] DOI:
10.1080/14636778.2012.687083 PMID: IF= 0,902
143. . Apolipoprotein E e4 allele does not increase the risk of early postoperative delirium after major surgery.. Journal of anesthesia;.
2012 [Epub ahead of print] DOI: 10.1007/s00540-012-1326-5 PMID: 22302107 IF= 0,831
144. Lebreiro A, Martins E, Machado JC, Abreu-Lima C. Diagnostic challenges of Marfan syndrome in an XYY young man.. Cardiology in
the young;22: 466-8. 2012 [Article] DOI: 10.1017/S1047951111001776 PMID: 22050831 IF= 0,759
145. Alvarenga CA, Lopes JM, Vinagre J, Paravidino PI, Alvarenga M, Prando A, Castilho LN, Soares P, Billis A. Paraganglioma of seminal vesicle and chromophobe renal cell carcinoma: a case report and literature review.. São Paulo medical journal = Revista paulista de medicina;130: 57-60. 2012 [Article] DOI: PMID: 22344361 IF= 0,711
146. Corso G, Seruca R, Roviello F. Gastric cancer carcinogenesis and tumor progression.. Annali italiani di chirurgia;83: 172-6. 2012 []
DOI: PMID: 22595727 IF= 0,231
147. Tiburcio M, Costa S M A, Duarte MF, Schmitt F, Filho AL. Characterization of PAR1 and FGFR1 expression in invasive breast carcinomas: Prognostic significance. Oncology Letters;1. 2012 [Article] DOI: 10.3892/ol.2012.806 PMID: IF= 0,108
117
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2012
148. Faria G, Cardoso MJ, Martins D, Bettencourt H, Amendoeira I, Schmitt F. P-cadherin as prognostic factor for loco-regional relapse
in breast cancer.. Acta médica portuguesa;25: 97-105. 2012 [Article] DOI: PMID: 22985920 IF= 0,091
149. Gusmão L, Alves C, Gones I, Sanchez-Diz P. Capillary Electrophoresis of an X-Chromosome STR Decaplex for Kinship Deficiency
Cases. DNA Electrophoresis protocols for forensic genetics;830: 57-71. 2012 [] DOI: 10.1007/978-1-61779-461-2_5 PMID: IF=
150. Oliveira M, OK L, Araúko R. Airborne fungi and bacteria in public indoor environments. Advances in Environmental Research;1.
2012 [] DOI: PMID: IF=
151. Amorim A. Biology/DNA Basic Principles. Encyclopedia of Forensic Sciences 2nd Edition;1. 2012 [] DOI: PMID: IF=
152. Figueira AC, Teodósio AS, Carvalheira J, Lacerda M, de Matos A, Gärtner F. P-cadherin expression in feline mammary tissues..
Veterinary medicine international;2012: 10.1155/2012/687424. 2012 [] DOI: 10.1155/2012/687424 PMID: 23091776 IF=
153. Santos LF, Pereira T, Rodrigues B, Correia E, Moreira D, Nunes L, Costa A, Elvas L, Machado JC, Castedo S, Henriques C, Matos
A, Santos O. [Diagnostic criteria for the Brugada syndrome: can they be improved?].. Revista portuguesa de cardiologia : orgão
oficial da Sociedade Portuguesa de Cardiologia = Portuguese journal of cardiology : an official journal of the Portuguese Society
of Cardiology;31: 355-62. 2012 [Article] DOI: 10.1016/j.repc.2011.09.023 PMID: 22475738 IF=
154. Falcão Manuel S., Vinagre J, Soares P, Lopes JM, Brandão E, Carneiro Ângela M.. A Clear Cell Renal Cell Carcinoma Inhibiting the
Response to Intravitreal Antivascular Endothelial Growth Factor Therapy in Wet Age-Related Macular Disease. Case Rep Ophthalmol;1. 2012 [] DOI: 10.1159/000346045 PMID: IF=
155. Drabek K, Gutiérrez L, Vermeij M, Clapes T, Patel SR, Boisset JC, van Haren J, Pereira AL, Liu Z, Akinci U, Nikolic T, van Ijcken W, van
den Hout M, Meinders M, Melo C, Sambade C, Drabek D, Hendriks RW, Philipsen S, Mommaas M, Grosveld F, Maiato H, Italiano JE,
Robin C, Galjart N. The microtubule plus-end tracking protein CLASP2 is required for hematopoiesis and hematopoietic stem cell
maintenance.. Cell reports;2: S2211-1247(12)00281-1. 2012 [] DOI: 10.1016/j.celrep.2012.08.040 PMID: 23084744 IF=
156. Da Silva-Ferrada E, Lopitz-Otsoa F, Lang V, Rodríguez MS, Matthiesen R. Strategies to Identify Recognition Signals and Targets of
SUMOylation.. Biochemistry research international;2012: 875148. 2012 [] DOI: 10.1155/2012/875148 PMID: 22811915 IF=
157. Soares I, Goios A, Amorim A. Sequence Comparison Alignment-Free Approach Based on Suffix Tree and L-Words Frequency. The
Scientific World Journal;4. 2012 [Article] DOI: 10.1100/2012/450124 PMID: 22997494 IF=
158. Leite M, Figueiredo C. A method for short-term culture of human gastric epithelial cells to study the effects of Helicobacter pylori.. Methods in molecular biology (Clifton, N.J.);921: 61-8. 2012 [] DOI: 10.1007/978-1-62703-005-2_9 PMID: 23015492 IF=
159. Lopes-Marques M, Igrejas G, Amorim A, Azevedo L. Human carbamoyl phosphate synthetase I (CPSI): insights on the structural
role of the unknown function domains. Elsevier Science;1. 2012 [] DOI: 10.1016/j.bbrc.2012.04.033 PMID: 22521883 IF=
160. Pereira R, Gusmão L. Capillary electrophoresis of 38 non-coding biallelic mini-Indels for degraded samples and as complementary
tool in paternity testing. Methods in molecular biology (Clifton, N.J.);1. 2012 [] DOI: 10.1007/978-1-61779-461-2 PMID: IF=
161. Gama A, Schmitt F. Cadherin cell adhesion system in canine mammary cancer: a review.. Veterinary medicine international;2012:
357187. 2012 [] DOI: 10.1155/2012/357187 PMID: 22973534 IF=
162. van Asch B, Amorim A. Capillary Electrophoresis Analysis of a 9-plex STR Assay for Canine Genotyping.. Methods in molecular biology (Clifton, N.J.);830: 231-40. 2012 [Article; Book Chapter] DOI: 10.1007/978-1-61779-461-2_16 PMID: 22139664 IF=
163. Sequeiros J, Martins S, Silveira I. Epidemiology and population genetics of degenerative ataxias.. Handbook of clinical neurology /
edited by P.J. Vinken and G.W. Bruyn;103: 227-51. 2012 [] DOI: 10.1016/B978-0-444-51892-7.00014-0 PMID: 21827892 IF=
118
Activity Report
2012
Members of IPATIMUP at Editorial Boards
1. American Journal of Cancer Therapy and Pharmacology
2. PloS one
3. Dataset Papers in Biology
4. ISRN Genomics
5. Dataset Papers in Oncology
6. Research in Cancer and Tumor
7. World Journal of Clinical Infectious Diseases
8. The Scientific World Journal
9. Frontiers in Genomic Assay Technology
10. World journal of gastroenterology : WJG
11. Journal of Integrated OMICS
12. World Journal of Biological Chemistry
13. Ultrastructural pathology
14. Seminars in diagnostic pathology
15. Patologia
16. Pathology, research and practice
17. Open Pathology Journal
18. Journal of Pathology
19. Histopathology
20.European Journal of Cancer Prevention
21. Current Diagnostic Pathology
22. The Open Forensic Science Jounal
119