relatório de actividades 2007

RELATÓRIO DE ACTIVIDADES
2007
ÍNDICE
PÁG.
1.
INTRODUÇÃO
2.
INVESTIGAÇÃO CIENTÍFICA
3
2a. Cancer Biology
7
2b. Cancer Genetics
17
2c. Carcinogenesis
34
2d. Genetics, Evolution and Pathology
41
2e. Population Genetics
55
2f. Tumour Evolution and Development
63
2g. Genetics Diversity and Bioinformatics
65
2h. Public Health and Cancer
70
3.
EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA
74
4.
SERVIÇO À COMUNIDADE
4a. UPS
79
4b. UPSi
84
4c. UPSs
87
5. RECÉM-DOUTORADOS
91
6. RESUMO DOS PROJECTOS
94
7. TRABALHOS PUBLICADOS
98
8. REVISTAS CIENTÍFICAS INTERNACIONAIS COM MEMBROS
DO IPATIMUP NOS SEUS EDITORIAL BOARDS
9. NÚCLEO DE AMIGOS DO IPATIMUP
107
108
2
INTRODUÇÃO
Por sugestão de algumas personalidades nacionais e estrangeiras que auscultámos a este propósito, inauguramos
este ano a elaboração de um Relatório de Gestão que incluirá uma parte dos elementos que habitualmente
incluíamos no Relatório de Actividades.
As actividades do IPATIMUP desenvolveram-se em 2007 de forma semelhante à de 2006, tanto no que diz respeito
à investigação científica, como à pós-graduação, divulgação e prestação de serviços, apesar das restrições
financeiras que nos foram inesperadamente impostas pela Reitoria da Universidade do Porto e pela Fundação para
a Ciência e a Tecnologia (ver Relatório de Gestão).
O ano de 2007 foi marcado pela preparação das assinaturas dos Consórcios com o IBMC e o INEB (Consórcio I3S)
e com o IPO-Porto, que se vieram a concretizar já no princípio de 2008. Foi também marcado pelo nosso
envolvimento na criação, em articulação com o IBMC e o INEB por um lado, e com a CCDRN, a BIAL e outras
instituições académcas e empresas, do Pólo de Competitividade em Saúde (“Health Cluster Portugal”)
Os investigadores do IPATIMUP publicaram, em 2007, 115 artigos científicos, 110 dos quais em revistas
internacionais e 5 em revistas nacionais. Foram ainda autores ou co-autores de 9 capítulos de livros/livros. Noventa
e dois (92) dos artigos foram publicados em revistas com Factor de Impacto. Cerca 52% desses artigos (n=47)
foram publicados em revistas com Factor de Impacto entre 3 e 29,1.
Nos quadros seguintes resume-se a evolução da produção científica nos útlimos anos.
3
Seguindo as recomendações internacionais temos vindo a prestar cada vez maior atenção à repercussão na
comunidade cientifica dos artigos dos nossos investigadores através da avaliação do numero de citações que eles
suscitamm na literatura indexada a nível mundial.
4
A WEB of KNOWLEDGE (ISI) tem uma lista dos artigos de todas as áreas científicas mais citados no mundo.
Portugal tinha, em Março de 2008, 330 artigos nesta lista. Destes 330 artigos, 12 têm como autores ou co-autores
investigadores do IPATIMUP.
Em 2007 a ISI registou vários milhares de citações a artigos publicados por investigadores do IPATIMUP desde
1989. A avaliação internacional desta realidade faz-se, por investigador, através de dois índices – o “índice h” que
mede o numero de artigos com elevado número de citações e o “índice m” que introduz na avaliação o peso do
número de anos de vida cientifica do investigador, de forma a permitir comparar, com equidade, os resultados dos
investigadores que começaram a publicar há mais tempo com os mais novos. Vinte e cinco (25) investigadores do
IPATIMUP têm “índices h” entre 10 e 40 e vinte e três (23) têm “índices m” entre 1 e 1,9. Os índices de citações dos
investigadores do IPATIMUP comparam-se (muito) favoravelmente com os dos investigadores das instituições
congéneres nacionais e estrangeiras.
Continuaram-se, em 2007, os Programas de Mestrado e Doutoramento em curso desde meados dos anos noventa
e realizaram-se, na Fundação Serralves, as Sessões do quarto Ciclo de Colóquios sobre Medicina Preventiva de
Cancro para muitas centenas de professores de Biologia e Físico-Química.
Continuaram-se também, em 2007, as actividades da divulgação científica junto de numerosas escolas básicas e
secundárias (Projecto de Auto-Laboratório) assim como os Programas Ciência Viva em Férias e Universidade
Júnior.
Instalou-se, preparou-se o pessoal docente e inaugurou-se no último trimestre de 2007, com a colaboração da
Agencia Ciencia Viva e da Câmara Municipal do Porto, o Laboratório Aberto. Este Laboratório está sediado na
antiga Escola Primária de Paranhos e recebe semanalmente, para trabalho prático, muitas dezenas de alunos de
escolas do Grande Porto e de todo o País.
Expandimos as actividades de apoio à comunidade, tanto de diagnóstico anatomo-patológico, como de patologia
molecular e de genética populaciona/genética forense. Continuámos o apoio no domínio da consultadoria
diagnóstica tendo realizado 198 exames de doenças cancerosas e precancerosas para cerca de uma centena de
instituições de vinte países europeus, americanos, asiáticos e africanos.
Os investigadores do IPATIMUP integram os Corpos Editoriais de 24 revistas científicas internacionais.
O IPATIMUP realizou, em 2007, como nos anos anteriores, três reuniões internacionais – uma sobre Genética
Populacional e Genética Forense (Portugaliae Genetica – 10ª edição), outra sobre Cancro (Porto Cancer Meeting
– 16ª edição) e a terceira sobre Ciência e Cultura (Conferência do Equinócio – 11ª edição).
5
Nota: O IPATIMUP voltou a contar, em 2007, com o apoio excepcional dos seus Associados Efectivos
(Universidade do Porto, Câmara Municipal do Porto, Fundação Luso-Americana Para o Desenvolvimento, Liga
Portuguesa Contra o Cancro, Comissão de Coordenação e Desenvolvimento da Região Norte, Instituto de
Genética Medica Dr. Jacinto de Magalhães e Cruz Vermelha Portuguesa) e Aderentes (Faculdade de Medicina,
Faculdade de Ciências, Instituto de Ciências Biomédicas Abel Salazar, Faculdade das Ciências da Alimentação e
Nutrição, Faculdade de Medicina Dentária, Faculdade de Farmácia, Hospital de S. João e Instituto Português de
Oncologia – Centro Regional do Norte), assim como de várias instituições públicas e privadas: Fundação Calouste
Gulbenkian, Fundação Millennium-bcp, Fundação Oriente, Fundação para a Ciência e Tecnologia, Fundação
Ciência Viva e Agência Portuguesa de Inovação. Conforme se pode ler no Relatório de Gestao e também,
discriminadamente, neste Relatório (ver listas dos Projectos em curso dos vários Grupos com a colaboração da
industria e a lista dos Amigos do IPATIMUP), o IPATIMUP continuou a contar, em 2007, com o apoio e a
colaboração de diversas empresas nacionais e multinacionais. A todos o nosso obrigado.
6
INVESTIGAÇÃO CIENTIFICA
CANCER BIOLOGY
Introduction
Following the suggestions of the External Scientific Board after the 2006 site visit the CANCER BIOLOGY Group
has started its reorganization.
The most important points of this reorganization concern: a) the concentration of the research activities on two
topics: Oncobiology of familial and sporadic thyroid tumours with an emphasis on papillary carcinoma (Group
coordinator and PI – Paula Soares) and Role of mitochondrial alterations in the etiopathogenesis of sporadic,
irradiation-induced and familial tumours (PI – Paula Soares and Valdemar Máximo );
b) the clarification of the internal structure of the Group in order to individualize the researchers and the fellows
involved in the aforementioned research topics as well as those involved in the three additional topics (Oncobiology
of neuroendocrine tumours – PI José Manuel Lopes ; Validation of molecular targets in cancer treatment or
chemoresistance – PI Helena Vasconcelos ).
Coordinator: Paula Soares (PS), BSc, MSc, PhD.
Scientific Consultant: Manuel Sobrinho-Simões (MSS), MD, PhD
Oncobiology of familial
and sporadic thyroid
tumours
PI’s
Others
Post-docs
PhD Stud.
MSc Stud.
BI
Undergrad.
PS
MSS,MCO+, AP, ASR,
PC
VT*, HugoP
JV, JS
IA, NB, MC.
Role
of
mitochondrial
alterations…..
tumours
VM** and PS
MSS, TG+
JL*, PB
NL, JSenra
IB, CG
IG, MdV
Oncobiology of
neuro endocrine
tumours
Validation
of
molecular targets
JML and PS
DP+
HV
HP, JP
RL, AP
RC
HS
LS, MIC
Principal Investigators: José Manuel Lopes (JML) MD, PhD, Valdemar Máximo (VM) BSc, PhD; Clara Sambade
(CS) MD, PhD; Helena Vasconcelos(HV), BSc, PhD.
Investigators/collaborators: Ana Preto(AP), BSc, PhD; Ana Sofia Rocha(ASR), BSc, PhD,
Post-Docs: Paula Boaventura (PB) BSc, PhD; Patrícia Castro(PC), BSc, PhD; Jorge Lima(JL), BSc, PhD.
PhD Students: Raquel Lima(RL), Andreia Palmeira (AP), Helena Popúlo (HP), Hugo Prazeres (Hugo P), Jonh Preto
(JP), Vitor Trovisco(VT)*.
MSc Students: Noémia Leal (NL)*, Raquel Portugal (RP), Joana Senra (JSenra)*.
BI’s:, Inês Bento (IB)#, Ricardo Celestino (RC), Catarina Gonçalves(JG), André Silva (AS), Joana Silva (JS), João
Vinagre (JV).
Undergraduate students: Inês Alvelos (IA), Nuno Blanc (NB), Miguel Cabral (MC), Sara Carvalho (SC), Maria Inês
Castro (MIC), Inês Gonçalves (IG), Liliana Santos (LS).
Pathologists: Isabel Amendoeira, MD, Catarina Eloy MD, Elsa Fonseca MD, PhD, João Magalhães, MD.
+ Clinicians: Manuel Cardoso de Oliveira (MCO), MD, PhD, Endocrine Surgeon; Duarte Pignatelli (DP), MD, PhD,
Endocrinologist; José Teixeira Gomes (JTG) , MD, PhD, Endocrine Surgeon.
Visiting researchers: Margriet De Vries (MdV), Medical student.
* - Concluded their theses in 2007 (see below).
** - Investigator who is in charge of starting a joint project with the IBMC, ICBAS and INSRA-Porto.
# - Move to IGC in September.
ASR Moved to Jacques Dumont Lab during 2007 but maintains a project with CB group.
AP will stay in part-time since she has got a job as Professor in UM.
PC is doing her Post –Doc under the supervision of Eugénio Santos (University of Salamanca) and PS.
7
Objectives/Goals of the research activity
1. The main objective is to progress in the understanding of the etiopathogenesis of some types of human cancer,
with an emphasis on thyroid and neuroendocrine tumours. Within this frame, a particular attention is paid to: a)
genetic alterations in tyrosine kinase receptors and signal transducing molecules involved in the mitogen-activated
protein kinases (MAPK) pathway; and b) mitochondrial alterations secondary to mitochondrial DNA
mutations/deletions or to mutations in nuclear genes encoding mitochondrial enzymes.
2. Some members of the group are also involved in clinical-pathological studies in other types of human tumours,
and in projects aiming to validate and/or identify molecular targets for cancer treatment, namely via the utilization of
cell signalling inhibitors and the down regulation of gene expression with siRNAs.
Main research topics
1 - Oncobiology of familial and sporadic thyroid tumours with an emphasis on papillary carcinoma.
2 - Role of mitochondrial alterations in the etiopathogenesis of sporadic, irradiation-induced and familial tumours
(The study of post-Chernobyl tumours is made under this frame).
Additional topics subheadings:
• Oncobiology of neuro endocrine tumours with an emphasis on digestive system
• Validation of molecular targets in cancer treatment and/or chemo-resistance
Background and major achievements in 2007 and plans for 2008
1. Oncobiology of familial and sporadic thyroid tumours with an emphasis on papillary carcinoma
We have continued collecting and studying a large series of papillary and follicular carcinomas with detailed clinicalpathological data, appropriately sampled material (paraffin-embedded and, whenever possible, also frozen
material), long follow-up, and reliable information on regional and distant metastases, with the following main
objectives:
1a) To clarify the influence on the metastatic pattern of the tumours and on the patients’ prognosis of BRAF
mutations, RAS mutations and PAX8/PPARγ translocation. In relation to this objective, we have analysed, in
collaboration with clinicians from H.S. João, Santiago de Compostela (Spain) and USP (Brazil) a cohort of PTC
patients in an attempt to find if there is an increase in the frequency or in any particular type of BRAF mutation
associated with nodal metastization (as reported by others and reviewed by us in Paper 5). In 2007 we finished
the study of 35 primary-metastasis paired PTC cases. Our data indicate that the acquisition of BRAF mutations
does not seem to be a requirement, nor even a frequent event, in the progression from localized to metastatic
PTC. Moreover, there seems to exist no association of a particular type of BRAF mutation (VK600-1E) to PTC
metastases. (Trovisco et al, Clin. Endoc, in press).
1b) To disclose the molecular characteristics of the follicular variant of PTC which is known to behave differently
from conventional PTC, namely regarding its tendency to give rise to lung and bone metastases. In previous
studies we have shown that this variant displayed a different BRAF mutation (BRAFK601E in about 7-9% of the
cases and no BRAFV600E), and that it shares some of the molecular features of follicular carcinomas: frequent
occurrence of PAX8/PPARγ translocation and of N-RAS mutations. In 2007 we pursued the study of these
characteristics in an independent tumour series from José Cameselle-Teijeiro (Santiago de Compostela,
Spain).
1c) To identify molecular alterations underlying the development of thyroid tumours. The first thing to do is to
investigate if particular molecular alterations that are associated with different histological patterns in thyroid
differentiated tumours are restricted to a specific component in thyroid mixed tumours. Mixed tumours are
classified as classical papillary thyroid tumours but have around 50% of follicles and in the present study we
analysed separately, using microdissection, both components of mixed tumours. Although our series is too
small to draw meaningful conclusions, we found an association between the PAX8-PPARgamma
8
rearrangement and the RAS mutation with the follicular compartment of the tumours, and an association
between RET/PTC rearrangement and BRAF mutation with the papillary compartment of the tumour. In this
topic, and in a pathology oriented approach, we have also analysed these genes in several consultancy cases,
corresponding to rare subtypes of thyroid tumours, in order to disclose the molecular identity of these variants
(Cameselle-Teijeiro et al., Human Pathol in press).
Our paper on the presence of mutations in THRB gene in a panel of thyroid carcinomas was published in 2007
(Paper 4).
We have also reinforced several in vitro studies with the following aims:
1.d) As a a sort of cell biology counterpart of the clinicopathological studies listed in Points 1a), b) and c) and to
progress in the understanding of the role played by BRAF mutations, we have established in vitro systems in
which we can evaluate the biological role (proliferation, apoptosis, motility and invasiveness) and the signalling
pathways activated by the different BRAF mutations in thyroid cancer derived cell lines.
Using transfection cell models, we have been comparing the transcription activation patterns of BRAF mutants
with those of RET/PTC; the results obtained to date show that STAT1/STAT3 appears to be differentially
activated by RET/PTC and by BRAF (PhD Project of VT and BI of JS). According to these results, STAT3
appears to be permanently phosphorylated in thyroid cancer cell lines harbouring BRAF V600E mutation or
RET/PTC rearrangements, shown by Western-blot analysis of cell lysates from 8505C and TPC-1 cell lines. In
cell lines without these genetic activations (XTC-1 and C643) very low levels of endogenous STAT3 Y705
phosphorylation were detected. In order to progress in this study we are performing luciferase-reporter assays
using the IRE-LUC (for STAT3) and GAS-LUC (for STAT1, current work) constructs, to detect possible
modifications of STATs transcriptional activity in presence of the different BRAF and RET/PTC mutants. We are
also performing sub cellular detection of STAT3 and STAT1 by immunofluorescence and subcellular protein
fractionation techniques and real-time quantitative PCR for known STAT3 and STAT1 dependent genes
(current work by JS).
Another part of the project consists in the “construction” of stable clones of thyroid cell lines harbouring both
mutated B-RAF and RET/PTC-1. We have transfect successfully TPC-1 cell line (RET/PTC-1 positive) with
wild-type and mutated B-RAF. Although we were able to isolate viable stable clones from both B-RAF forms we
could not detect changes in B-RAF protein expression by western-blot. To pursue this study we are now
engineering TPC-1 and 8505C cell lines with a tetracycline-regulated expression system (T-REx System). The
vectors will express mutant BRAF and RET/PTC under an inducible promoter(current study by JV).
1.e)To identify mechanisms underlying the occurrence of aneuploidy in thyroid tumours.
The two major histotypes of differentiated thyroid carcinoma display different patterns of DNA content: follicular
carcinomas are usually aneuploid whereas almost every papillary carcinoma is diploid or quasi–diploid. Our
group showed in a previous study an association between the presence of a polymorphism in H-RAS 81T→C
and the occurrence of aneuploidy in thyroid follicular tumors (Castro P, Oncogene, 2006), suggesting a
possible role of H-RAS in their aneuploidization. The link between the H-RAS 81T→C and aneuploidy may
reside on the fact that the allele 81 C was associated with significantly higher amounts of total H-RAS mRNA
and/or significantly higher relative amounts of p21ras /p19ras (Castro P, Oncogene, 2006). In 2007 we
continued to study the association between the presence of the H-RAS 81C polymorphism and the ploidy of
thyroid tumors having confirmed the association between the H-RAS 81T→C and ploidy in an independent
thyroid tumour series from Spain. The work main goal was to validate our previous findings (Castro P, 2006). In
order to access the relationship between the polymorphism and the two different H-RAS isoforms we cloned the
cDNA of 3 cases and a thyroid cell line heterozygous for the polymorphism. The results showed a significant
association (p=0.0023) between the allele C and the isoform p21ras and/or the allele T and the isoform p19 ras;
this is in accordance to our previous results by semi-quantitative RT-PCR (Castro P, Oncogene, 2006).
2. Role of mitochondrial alterations in the etiopathogenesis of sporadic, irradiation-induced and familial
tumours
It is not well understood the role of the mtDNA mutations/variants, nor the role of genetic alterations in nuclear
genes that codify for mitochondrial proteins, on the etiopathogenesis of several types of tumours exhibiting
mitochondrion rich neoplastic cells.
Mitochondrion-rich or oxyphilic tumours constitute an unusual form of neoplasm composed of cells with a
voluminous, granular, eosinophilic cytoplasm due to the huge amount of structurally abnormal mitochondria. In a
previous study (Máximo V et al, Am J Pathol, 2002), we analysed the mtDNA in benign and malignant thyroid
9
tumours and we concluded that mtDNA variants and mtDNA somatic mutations of Complex I and Complex IV genes
appear to be involved in thyroid tumourigenesis. The data we obtained in 2007 in a series of 19 Warthin tumours of
the salivary gland and 14 renal oncocytomas is similar to those found in Hürthle cell tumours and was used as basis
for the Master thesis of Noémia Leal (see below).
The existing literature in mtDNA alterations in oncocytomas was reviewed in a book chapter (Lima J et al,
Mitochondria and oncocytomas, in press).
We started a new project on this topic centred on cybrid cell lines, in order to verify the functional effects induced by
mtDNA alterations (JL post-doc project). These cybrids cell lines are obtained after fusion of a mitochondrial DNA
(mtDNA)-free cell line (designated as ρ0) and an enucleated cell line.
The mtDNA-free cell line used as mtDNA acceptor was the 143Bρ0. The cell lines used as mtDNA donors were the
XTC.UC1 (thyroid Hürthle cell tumour), MDA-MB435 (melanoma) and MDA-MB231 (breast cancer); of these three,
our attention will be mainly focused on XTC.UC1, which presents two mtDNA mutations – 3571insC in ND1 gene
and G15557A in CytB gene. To establish cybrids derived from the 143Bρ0 cell line and the mtDNA-donor cell lines
XTC.UC1 we eliminate the nucleus from the mtDNA-donor cell lines by treating the cells with the enucleating agent
actinomycin D. After this treatment, the resulting cytoplasts (cells without nucleus) were fused, using PEG, with the
143Bρ0 cell line, after which the resulting fused cells were cultured in selective medium (in which only cells with
mtDNA grow), in order to eliminate the non-fused 143Bρ0 cells. From this process we have obtained three clones
from the fusion 143Bρ0/XTC.UC1. The above mentioned clones were tested, by western-blot, for the mtDNAencoded protein COXII to demonstrate that all surviving clones from the fusions were positive for COXII, showing
that cybrids derived from the 143Bρ0 cell line and XTC.UC1 were successfully established.
Next we had to demonstrate that the cybrids displayed the nuclear DNA derived from the 143Bρ0 cell line and the
mtDNA derived from the XTC.UC1 cell lines. Using 15 nuclear STR (Powerplex kit – Promega) we have compared
the profiles of 143Bρ0, XTC.UC1 and the cybrids 143Bρ0/XTC.UC1. The results have shown that the STR profile of
the 143Bρ0/XTC.UC1 is identical to the 143Bρ0 STR profile and different from the XTC.UC1, proving that the
nuclear DNA of the 143Bρ0/XTC.UC1 cybrid was derived from the 143Bρ0 cell line. On the other hand, we have
sequenced repetitive regions of mtDNA (HVTI), normally used as markers for population genetics studies, as well as
the ND1 gene, where one of the mutations of interest is located, in the 143B, XTC.UC1 and 143Bρ0/XTC.UC1 cell
lines. The sequenciation results show that the mtDNA of the 143Bρ0/XTC.UC1 cybrids is similar to the XTC.UC1
mtDNA and different from the 143B mtDNA. At the moment, we are trying to enrich the 143Bρ0/XTC.UC1 cells with
ND1-mutated molecules, since during the cybridization process, the amount of mutated molecules has dropped from
~50% to ~10%.
GRIM-19 is a cell death regulatory gene that promotes apoptosis, is a negative regulator of cell growth, and is also
involved in mitochondrial metabolism. The dual role of GRIM-19 in apoptosis and mitochondrial biogenesis turned it
a good candidate for being a gene involved in the etiopathogenesis of oncocytic cell tumours. Its localization to the
same region as the previous region containing a gene [thyroid cell oxyphilia (TCO gene)] predisposing to Hürthle
cell familial tumours (chromosome 19p13.2) also suggested that it could be involved in the etiopathogenesis of
oncocytic cell tumours. Using small interfering RNAs we have studied the effect of GRIM-19 downregulation in
human thyroid cell lines, namely the effect in cell apoptosis and proliferation and in the expression of STAT3 and
STAT3 downstream genes, namely CyclinD1 and ICAM-1. We used two different siRNAs in two different cell lines
(8505C and XTC1). We observed a decrease in proliferation and no alteration in the apoptosis index in the cell lines
treated with siRNA specific for GRIM-19. We are now confirming the results and we started the study of the effect of
GRIM-19 downregulation by the analysis of the expression of STAT3 downstream genes. We detected an
association between the GRIM-19 downregulation and STAT3 phosphorylation and an increased expression of
CyclinD1 and ICAM-1 in the cell lines treated with siRNA against GRIM-19. Our hypothesis that downregulation of
GRIM-19 is necessary for complex I assembly and could induce a mitochondrial defect led us to perform an
immunocytochemical study of mitochondrial staining; we observed that cells in which GRIM-19 was down regulated
displayed an increase in mitochondrial number and a dispersed distribution of the mitochondria throughout the cell.
It is known that mitochondria appear to move along microtubules; so we did a co-staining for mitochondria and
tubulin, and to look for further alterations in the cytoskeleton we co-stained mitochondria and actin; the results from
both experiments reinforced the idea that GRIM-19 silencing induced changes in the cytoskeleton conformation,
mainly in cell size, polarity, and number/distribution of mitochondria Maximo V et al, unpublished results).
Concerning the study of the role of GRIM-19 in the etiopathogenesis of oncocytic tumours we checked for the
expression of GRIM-19, by immunohistochemistry, in a series of 41 renal cell tumours, including 11 Clear- Cell
10
carcinomas(CCR), 15 Papillary carcinomas, 5 Chromophobe carcinomas and 10 Oncocytomas. We found a
decrease in the expression of GRIM-19 in 11/11 CCR, in 12/15 Papillary carcinomas, in 3/5 Chromophobe
carcinomas and in 5/10 Oncocytomas (Master thesis of RP). We have also studied the expression of STAT3 in the
same series and we observed an association between low levels of GRIM-19 expression and STAT3 phosporylation
and nuclear localization, suggesting a role of GRIM-19 in renal cell tumours through STAT3 pathway. Now we are
checking for the expression of STAT3 downstream genes in order to verify our hypothesis.
We have searched for mutations in GRIM-19 gene in the aforementioned series and no mutations were found in any
of the tumours (Master thesis of JSenra).
2.a) Familial tumours
The succinate dehydrogenase (SDH) mitochondrial proteins (Complex II of the Mitochondrial Respiratory Chain and
Krebs cycle) were implicated in familial forms of neuroendocrine tumours (paraganglioma, pheocromocytoma). In
2007 we continued the genetic screening in sporadic and familial pheochromocytoma and paragangliomas from a
Portuguese multi-centre study involving 7 major institutions in Portugal. Presently we have pooled a large series
comprising a total of 68 sporadic and 4 syndromic/familial cases of pheochromocytoma and paraganglioma, in
which genetic screening for hereditary syndromes was performed. Mutation screening of the RET, VHL, SDHB and
SDHD genes was performed on peripheral blood leucocytes by PCR-SSCP and DNA sequencing. Causative
mutations were detected in all familial/syndromic cases. In sporadic cases, the overall frequency of mutations was
4%.(Prazeres H et al, manuscript in preparation).
Our paper on the frequency of germline succinate dehydrogenase mutations in sporadic cervical paragangliomas in
northern Spain was published in 2007 (Paper 2).
In a previous study (Prazeres H et al, 2006) we have characterized RET mutational spectrum in a series of
Portuguese hereditary and sporadic Medullary Thyroid Carcinoma. We detected 3 novel RET variants (Arg886Trp,
Ser649Leu and Glu511Lys), all of which were absent in normal controls, and will be the focus of further studies.
Functional studies of novel RET mutations (Arg886Trp, Ser649Leu and Glu511Lys, dinucleotide Cys634Arg: TGCTTT) were performed in 2007 by cloning the novel mutants of RET, obtained by PCR-based site-directed
mutagenesis, into the pRcCMV expression vector and transfection of HEK293 and NIH3T3 cells. We performed
focus formation assays and soft agar assays to assess the transforming capacity of the novel variants in relation
with other known RET mutations. Stable transfectants of the novel mutants were also produced with the objective of
assaying RET tyrosine-kinase activity, signalling through intracellular partners, and sensitivity to RET kinase
inhibitors. Our preliminary data suggest that the novel Arg886Trp variant has oncogenic potential in vitro, although it
is milder than that of Cys634Arg. Further studies of RET kinase activity (Tyrosine Kinase assays) and activation of
RET signaling targets are under way in order to confirm this observation (Prazeres H, PhD project).
The so-called non-medullary thyroid cancer (fNMTC) in general, and familial PTC in particular, are associated with
some of the highest risks among all cancer sites. The model of inheritance of fNMTC appears to be autosomal
dominant although the majority of families are small (sibs/trios) and therefore a multigenic inheritance is plausible.
Four loci have been identified through genetic linkages: MNG on 14q32, TCO1 on 19p13.2, fPTC on 1p21 and
NMTC1 on 2q21. Through clinical criteria (the existence of at least two first degree relatives with a follicular cellderived thyroid tumour and/or age below 35 y) we identified 14 fNMTC suspected families. Six families, 4 with
multinodular goiter and 2 showing Hürthle cell tumors were analysed for linkage at the aformentioned loci. We did
not find any association with the previous identified loci, suggesting that in those families a new locus may be
associated with the disease. In the future, we intend to perform Whole Genome Assay in some (up to 3) of the
families with most numerous menbers in an attempt to identify putative new loci predisposing to familial nonmedullary thyroid tumors in these families. In the study of the tumours from familial clusters of NMTC we found LOH
at the 19p13.2 and 2q21 loci, providing evidence that inactivation of putative genes in these regions, acting as
tumour-suppressors, may be involved in the development of tumours in the context of fNMTC. (Paper 3)
2.b) Radiation induced thyroid tumourigenesis
In the field of radiation induced carcinogenesis we initiated in 2006 a project (with financial support from Fundação
Calouste Gulbenkian) aiming to evaluate Head and Neck Tumours in a cohort of about 5000 individuals irradiated
for the treatment of Tinea Capitis in the 1950’s. Until now, 466 individuals (8.7%) from the original cohort have been
clinically observed and part of them showed lesions that can be potentially related with head and neck irradiation
(meningiomas, thyroid and skin tumours). From all the individuals peripheral blood and samples from oral mucosa
have been collected. We intend to continue, in 2008, the identification and clinical observation of the members of the
cohort through the National Health Service databases and also mailing a second and a third time the persons who
did not respond to the first mail contact. We made an agreement with ARS Norte that allows us to request ourselves
11
the complementary exams needed for evaluation of the individuals (thyroid scan and the calcium dosage). This
agreement allows also us to observe the individuals living in Póvoa de Varzim and Vila do Conde in the respective
Health Care Centre. This fact contributed, and will hopefully continue contributing to a greater availability from these
individuals to come to the clinical observation. Thyroid nodules, basal cell carcinoma (BCC) and meningioma are
the lesions more frequently associated with a therapeutic irradiation history; therefore we are dedicating our efforts
to screen them in our cohort. From the 466 observed individuals 7 had thyroid cancer (papillary carcinoma)
representing a prevalence of 1.5%, 28 had BCCs (6%; 35.7% of which were multiple BCCs) and 4 had meningioma
(0.86%). These figures are higher than those referred for non-irradiated populations and in accordance with the
ones referred for irradiated cohorts in Israel and USA.
Additional Topics
Oncobiology of neuro endocrine tumours (PI – José Manuel Lopes:JML).
Our Group has installed in the IPATIMUP the National DataBase of GastroIntestinal Stromal Tumours (GIST) and
NeuroEndocrine Tumours (NET), which have been designed as ReGIST and ReGENE, respectively under the
supervision of JML. The establishment and maintenance of the DataBase has been and will be supported by
NOVARTIS ONCOLOGY.
a) The results obtained in the clinical revision of a series of 100 NET tumours, as well as the histological reclassification of the tumours have been presented in ENETS conference. The biopathologic classification of the
series in terms of proliferation (and cell cycle proteins) and production of neuroendocrine markers started in 2007.
JML was involved in the Consensus Conference for establishment of new guidelines for clinical management of
these tumours (Paper B12).
Given the genetic and biologic similarities between papillary thyroid carcinomas and melanomas (BRAF and N-RAS
mutations and c-MET overexpression), we decided in 2007 to start a study in melanomas (Helena Pópulo PhD
project). For that, in 2007, 97 human melanoma samples were collected and classified according to their histotypes.
A database collecting several parameters related to the melanoma samples was organized and tissue microarrays
(TMA) blocks constructed from the melanoma collected samples. We have started the study of mTOR pathway (by
immunohistochemistry) and the genetic characterization of the melanoma samples.
Clearly distinct morphological and biologically tumours derive from the two distinct areas of the adrenal gland, the
cortex and the medulla. In particular, a great deal of attention has been given to those from the cortex mainly
because of their clinical characteristics which are frequently rather notorious. However, not many studies at a
cellular level have been done mostly due to the rarity of such tumours. In 2006, organization of the existing
histological material was recognized as an essential step to allow a rapid access to the samples of the different
types of tumours, in their different developmental stages. Simultaneously, with this systematic identification, a
correlation of the pathological with both the biochemical and the clinical data is in the process of completion. In 2008
we intend to do tissue arrays of the different types of tumours and then start performing immunohistochemical
studies using antibodies for proliferation (Ki67; p53; ciclin D; ERKs 1 e 2; p38; STAT’s) and apoptosis (Caspases 3
e 8; Bcl 2; HSPs 60 e 90) markers.The eventual results will be presented at two important meetings: The American
annual meeting of the Endocrine Society and the Annual meeting of the European group for the study of adrenal
tumors of which Duarte Pignatelli is a member.
Validation of molecular targets in cancer treatment or chemoresistance (PI – Helena Vasconcelos)
During the current year we have continued working on the validation of molecular therapeutic targets by gene
silencing approaches, namely RNAi (with siRNAs or shRNAs) (Paper B6). We have been mainly studying targets
involved in drug resistance. Furthermore, we have initiated collaboration with CEQOFFUP in order to try to identify
and validate small molecules that may be of therapeutic interest.
MSc Theses:
Joana Senra. “Role of GRIM-19 in kidney carcinogenesis: Evaluation of GRIM-19 expression and genetic alteration
analysis in normal and tumoural kidney samples.” Supervisor: Valdemar Máximo. MSc Course - Molecular Medicine.
IPATIMUP/ Cranfield University (United Kingdom), September 2007.
Noémia Leal. “Mutações e delecções do DNA mitocondrial (mtDNA) em oncocitomas renais e tumores de Warthin
das glândulas salivares: Comparação com tumores de células de Hürthle da tireóide.” Supervisor: Valdemar
Máximo.MSc Course - Molecular Genetics – University of Minho, December 2007.
12
Hugo Seca Teixeira. “Estudo da relevância da diminuição da expressão do XIAP por RNA de interferência na
resposta de células de LMC ao Imatinib.” Supervisor M. Helena Vasconcelos Meehan; Co-supervisor: José Eduardo
Guimarães. MSc Course - Molecular Medicine and Molecular Oncology, Medical Faculty of the University of Porto,
January 2007.
PhD Theses:
Victor Trovisco. “Role of BRAF gene alterations in the natural history of papillary thyroid carcinoma”. Supervisor
Manuel Sobrinho-Simões (Porto, Portugal).Thesis defended at the Medical Faculty of University of Porto, February
2008.
Papers: Soares P, Trovisco V, Lima J, Rocha A, Castro P, Preto A, Máximo V, Botelho T, Seruca R and SobrinhoSimões M. “BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC”.
Oncogene, 2003; 22:4578-4580.
Trovisco V, Soares P, Preto A, Vieira de Castro I, Lima I, Castro P, Máximo V, Botelho T, Moreira S, Meireles AM,
Magalhães J, Abrosimov A, Cameselle-Teijeiro J and Sobrinho-Simões M. “Type and prevalence of BRAF
mutations are closely associated to papillary thyroid carcinoma histotype and patients’ age but not with tumour
aggressiveness”. Virchows Archiv, 2005; 446:589-595.
Trovisco V, Vieira de Castro I, Soares P, Máximo V, Silva P, Magalhães J, Abrosimov A, Guiu XM and SobrinhoSimões M. “BRAF mutations are associated with some histological types of papillary thyroid carcinoma”. The
Journal of Pathology, 2004; 202:247-251.
Trovisco V, Soares P, Soares R, Magalhães J, Sá-Couto P and Sobrinho-Simões M. “A new BRAF gene mutation
detected in a case of a solid variant of papillary thyroid carcinoma”. Human Pathology, 2005; 36:694-7.
Trovisco V, Couto JP, Cameselle-Teijeiro J, Vieira de Castro I, Fonseca E, Soares P and Sobrinho-Simões M.
“Acquisition of BRAF gene mutation is not a requirement for nodal metastization of PTC”. Submitted for publication
to ‘Clinical Endocrinology’.
Soares P, Trovisco V, Rocha AS, Feijao T, Rebocho AP, Fonseca E, Vieira de Castro I, Cameselle-Teijeiro J,
Cardoso-Oliveira M, Sobrinho-Simões M. “BRAF mutations typical of papillary thyroid carcinoma are more
frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas”. Virchows
Archiv, 2004; 444:572-576.
Prizes:
Hugo Prazeres - Prize Prof. E. Limbert SPEDM/Genzyme in Thyroid Pathology awarded by the Portuguese Society
of Endocrinology, Diabetes and Metabolism and Genzyme for the project: “Tiró-alvo: Identificação e validação de
novos marcadores moleculares/alvos terapêuticos para o cancro da tiróide (LRP1B e TACSTD2)”.
Hugo Prazeres - Prize for the best basic research work presented at the Portuguese Congress of Endocrinology,
Porto, Portugal, January 2007.
Patricia Castro - Award Sérgio Vidal for the best work in Biomedical Research published by a
researcher with less than 35 years old (Castro P et al, JCME, 2006). Award from the
University of Santiago de Compostela (13/04/2007).
Raquel T. Lima - Prize for the best communication presented at the 2º Congresso Nacional de Virologia /VI
Encontro da Sociedade Portuguesa de Virologia, Porto, Portugal, September, 2007.
Publications
A. Within the main research topics
1. Meireles AM, Preto A, Rocha AS, Rebocho AP, Máximo V, Pereira-Castro I, Moreira S, Feijão T, Botelho
T, Marques R, Trovisco V, Cirnes L, Alves C, Velho S, Soares P, Sobrinho-Simões M. Molecular and
genotypic characterization of human thyroid follicular cell carcinoma-derived cell lines. Thyroid, 17:707-15,
2007
2. Lima J, Feijão T, Ferreira da Silva A, Pereira-Castro I, Fernandez-Ballester G, Máximo V, Herrero A,
Serrano L, Sobrinho-Simões M, Garcia-Rostan G.High frequency of germline succinate dehydrogenase
mutations in sporadic cervical paragangliomas in northern Spain: mitochondrial succinate dehydrogenase
structure-function relationships and clinical-pathological correlations. J Clin Endocrinol Metab, 92:4853-64,
2007
13
3. Prazeres HJ, Rodrigues F, Soares P, Naidenov P, Figueiredo P, Campos B, Lacerda M, Martins TC.Loss
of heterozygosity at 19p13.2 and 2q21 in tumours from familial clusters of non-medullary thyroid
carcinoma. Fam Cancer, 2007
4. Rocha AS, Marques R, Bento I, Soares R, Magalhães J, de Castro IV, Soares P. Thyroid hormone
receptor beta mutations in the 'hot-spot region' are rare events in thyroid carcinomas. J Endocrinol,
192:83-6, 2007
5. Trovisco V, Soares P, Preto A, Castro P, Máximo V, Sobrinho-Simões M. Molecular genetics of papillary
thyroid carcinoma: great expectations. Arq Bras Endocrinol Metabol 51:643-53, 2007.
6. Volante M, Collini P, Nikiforov YE, Sakamoto A, Kakudo K, Katoh R, Lloyd RV, LiVolsi VA, Papotti M,
Sobrinho-Simoes M, Bussolati G, Rosai J.Poorly differentiated thyroid carcinoma: the Turin proposal for the
use of uniform diagnostic criteria and an algorithmic diagnostic approach. Am J Surg Pathol 31:1256-64,
2007
7. Preto A, Soares P e Sobrinho-Simões M. “Novas terapêuticas no carcinoma da tireóide: novos alvos
moleculares?” Mundo Médico, n.º 54:8-12, 2007.
Accepted for publication in 2007:
8. Valdemar Máximo, Jorge Lima, Paula Soares, André Silva, Inês Bento, Manuel Sobrinho-Simões. GRIM19 in health and disease. Adv Anat Pathol,15:46-53, 2008
9. Ferreira AC, Gomes L, Máximo V, Amil J, Carneiro F, Machado JC, Tavarela-Veloso F. GRIM-19
mutations are not associated with Crohn's disease. Inflamm Bowel Dis, 14:434-5, 2008.
10. Preto A, Figueiredo J, Velho S, Ribeiro AS, Soares P, Oliveira C, Seruca R. BRAF provides proliferation
and survival signals in MSI colorectal carcinoma cells displaying BRAF(V600E) but not KRAS mutations. J
Pathol, 214:320-7, 2008.
11. Trovisco V, Pinto do Couto J, Cameselle-Teijeiro J, Vieira de Castro I, Fonseca E, Soares P, SobrinhoSimões M. Acquisition of BRAF gene mutation is not a requirement for nodal metastization of PTC. Clinical
Endocrinology (Accepted for publication)
12. Cameselle-Teijeiro J, Pardal F, Eloy C, Ruiz-Ponte C, Celestino R, Castro P, Soares P, Sobrinho-Simões
M. Follicular thyroid carcinoma with an unusual glomeruloid pattern of growth. Human Pathology (Accepted
for publication).
B. Related with the main or acessory research topics
1. Ferreira JG, Cruz CD, Neves D, Pignatelli D. Increased extracellular signal regulated kinases
phosphorylation in the adrenal gland in response to chronic ACTH treatment. J Endocrinol. 192:647-58,
2007
2. Gomes AL, Reis-Filho JS, Lopes JM, Martinho O, Lambros MB, Martins A, Schmitt F, Pardal F, Reis RM.
Molecular alterations of KIT oncogene in gliomas. Cell Oncol. 29:399-408, 2007
3. Gouveia A, Pimenta A, Lopes JM: Quando e como operar os tumours estromais gastrointestinais
primários. Revista Portuguesa de Cirurgia 1: 49-52, 2007
4. Lopes JM, Gouveia A, Pimenta A: O papel da anatomia patológica no diagnóstico e prognóstico dos
GISTs. Rev Port Cir 1: 35-39, 2007
5. Macedo JE, Machado M, Araújo A, Angélico V, Lopes JM. Orbital metastasis as a rare form of clinical
presentation of non-small cell lung cancer.J Thorac Oncol. 2:166-7, 2007
6. Lima R, Guimarães JE, Vasconcelos ME. Overcoming K562Dox resistance to STI571 (Gleevec) by
downregulation of P-gp expression using siRNAs. Cancer Therapy, 5:67-76, 2007
7. Pitteloud N, Zhang C, Pignatelli D, Li JD, Raivio T, Cole LW, Plummer L, Jacobson-Dickman EE, Mellon
PL, Zhou QY, Crowley WF Jr.Loss-of-function mutation in the prokineticin 2 gene causes Kallmann
syndrome and normosmic idiopathic hypogonadotropic hypogonadism. Proc Natl Acad Sci U S
A.30;104:17447-52, 2007
8. Preto A, Moutinho C, Velho S, Oliveira C, Rebocho AP, Figueiredo J, Soares P, Lopes JM, Seruca R: A
subset of colorectal carcinomas express c-KIT protein independently of BRAF and/or KRAS activation.
Virch Archiv 450:619-626, 2007
9. Rindi G, Kloppel G, Couvelard A, Komminoth P, Korner M, Lopes JM, McNicol AM, Nilsson O, Perren A,
Scarpa A, Scoazec JY, Wiedenmann B: TNM staging of midgut and hindgut (neuro) endocrine tumors: a
consensus proposal including a grading system. Virch Archiv 451:757-762, 2007
Accepted for publication in 2007:
14
10. Gomes AL, Gouveia A, Capelinha AF, de la Cruz D, Silva P, Reis RM, Pimenta A, Lopes JM. Molecular
alterations of KIT and PDGFRA in GISTs: evaluation of a Portuguese series.J Clin Pathol, 61:203-8, 2008
11. Steinmüller T, Kianmanesh R, Falconi M, Scarpa A, Taal B, Kwekkeboom DJ, Lopes JM, Perren A, Nikou
G, Yao J, Delle Fave GF, O'Toole D; Frascati Consensus Conference participants. Consensus guidelines
for the management of patients with liver metastases from digestive (neuro)endocrine tumors: foregut,
midgut, hindgut, and unknown primary. Neuroendocrinology, 87:47-62, 2008
C. Other topics
1. Barroca H, Farinha NJ, Lobo A, Monteiro J, Lopes JM. Deep-seated congenital juvenile xanthogranuloma:
report of a case with emphasis on cytologic features. Acta Cytol. 51:473-6, 2007
2. Cardoso H, Machado AS, Figueira P, Teixeira AV, Veloso FT, Lopes JM. Multiple myeloma presenting with
malabsorption.Dig Dis Sci. 52:1851-4, 2007
3. Costa BM, Ferreira P, Costa S, Canedo P, Oliveira P, Silva A, Pardal F, Suriano G, Machado JC, Lopes
JM, Reis RM.Association between functional EGF+61 polymorphism and glioma risk. Clin Cancer Res.
13:2621-6, 2007
Accepted for publication in 2007:
4. Marques M, Magro F, Cardoso H, Carneiro F, Portugal R, Lopes J, Costa Santos C: Infliximab-induced
lupus-like syndrome associated with autoimmune hepatitis. Inflammatory Bowel Diseases. 2007 Oct 10;
[Epub ahead of print].
5. Timonera ER, Paiva ME, Lopes JM, Eloy C, van der Kwast T, Asa SL. Composite adenomatoid tumor and
myelolipoma of adrenal gland: report of 2 cases. Arch Pathol Lab Med, 132:265-7, 2008.
6. de Carvalho M, Moreira ME, Lopes JM, Martins BM.Authors' reply. J Pediatr (Rio J),83:577,2007.
7. Lima-Ramos V, Pacheco-Figueiredo L, Costa S, Pardal F, Silva A, Amorim J, Lopes JM, Reis RM. TP53
codon 72 polymorphism in susceptibility, overall survival, and adjuvant therapy response of gliomas.
Cancer Genet Cytogenet, 180:14-9, 2008.
Ongoing Projects
“Teste para selecção in vitro de compostos com potencial actividade anti-oxidante, obtidos através de plantas
medicinais utilizadas tradicionalmente em Portugal.”
Principal Investigator: Valdemar Máximo
Duration:1/2/2005 to 1/2/2008;. Budget :48468 Euros
Funding: UNICER (beverage and food company)
“Role of raft domains in the modulation/alteration of the transducing pathways in normal thyroid and in papillary
thyroid carcinoma.”
Principal Investigator: Paula Soares
Duration:1/1/2004 to 1/7/2007. Budget: 72645 Euros
Funding: FCT (concluded in 2007)
“Biological role of BRAF oncogene activaction in thyroid carcinogenesis.”
Principal Investigators: Paula Soares
Duration: 1/9/2005 to 1/9/2008. Budget: 95250 Euros
Funding: FCT
“Neuroendocrine tumors: Clinico-pathological and immunohistochemistry characterization and identification of
biological factors of aggressiveness”
Principal Investigator: José Manuel Lopes
Duration: 23/9/2004; to 23/9/2008. Budget: 53150 Euros.
Funding: NOVARTIS
“Is the mTOR pathway relevant in the initiation/progression and/or a putative therapeutic target in melanomas?”
Principal Investigators: José Manuel Lopes and Paula Soares
Duration:1/5/2006 to 1/5/2008;. Budget :54500 Euros
Funding: NOVARTIS
15
“Relevance of mTOR pathway in thyroid carcinogenesis”
Principal Investigators: Ana Sofia Rocha
Duration: 1/9/2007 to 1/9/2010. Budget: 95250 Euros
Funding: FCT
“Mapping of Genes Predisposing to Familial Thyroid Tumours.”
Principal Investigators: Valdemar Máximo
Duration: 1/09/2006 to 1/09/2009. Budget: 86700€
Funding: The late David and Esther Bernstein Halpern fund
“Risco de cancro em indivíduos irradiados para tratamento da tinha do couro cabeludo (Tinea Capitis) durante a
infância. Estudo de follow-up de uma coorte do norte de Portugal.”
Principal Investigator: Teixeira Gomes
Duration: 1/3/2006 to 1/3/2008. Budget: 91000 Euros.
Funding: Fundação Calouste Gulbenkian
“Validação do papel do XIAP na resistência da leucemia mielógena aguda à quimioterapia: abrir caminho para uma
possível nova estratégia de tratamento tendo o XIAP como alvo terapêutico”
Principal Investigators: M. Helena Vasconcelos Meehan
Duration: 1/6/2005 to 1/9/2007. Budget: 15000 Euros. (2005: 7500 E; 2006:7500 E)
Funding: "Associação Portuguesa Contra a Leucemia"
“Molecular dissection of the multinucleation phenotype of the neoplastic cells in Hodgkin´s lymphoma.”
Principal investigator: Clara Sambade
Duration: 1/6/2005 to 1/6/2007. Budget: 15000 Euros. (2005: 7500 E; 2006:7500 E)
Funding: "Associação Portuguesa Contra a Leucemia"
16
CANCER GENETICS
Coordinator: Raquel Seruca
Principal Investigators: Carla Oliveira, Céu Figueiredo, Fátima Carneiro, Fernando Schmitt, Gianpaolo Suriano,
Joana Paredes, José Carlos Machado
Post-Docs: Cecília Durães, Maria José Oliveira, Marina Leite, José Luís Costa
PhD students: Ana Costa, Ana Machado, Ana Ferreira, Ana Rita Mateus, Ana Sofia Ribeiro, André Albergaria,
António Ferreira, Catarina Alves, Fernanda Milanezi, Gonçalo Regalo, Joana Correia, Marta Correia de Melo, Nuno
Guimarães, Patrícia Oliveira, Paulo Canedo, Rachid Karam, Sérgia Velho, Sílvia Carvalho
The research of our group focuses on the molecular genetics of three common types of epithelial cancer (gastric,
breast, and colorectal carcinoma). We aim at 1) identifying individuals at risk for the various forms of these tumours;
2) identifying pathological features and molecular markers occurring in the setting of familial and sporadic
carcinoma; and 3) identifying signalling pathways mediated by genetic and environmental factors in tumour
development, in order to find new molecular targets for therapeutic intervention.
1. Identifying individuals at risk for gastric carcinoma
1. a) Bacterial factors for sporadic gastric carcinoma
H. pylori virulence factors constitute an important source of variation in the outcome of the infection. In 2007, we
have assessed the clinical relevance of the CagA tyrosine phosphorylation motifs for gastric carcinoma
development. After infection, H. pylori CagA is injected into the host cells, where it undergoes tyrosine
phosphorylation on residues within EPIYA motifs, present as repeats in the C-terminus of the protein. EPIYA motifs
are classified as A, B, C, and D, and it has been shown that the number and type of EPIYA motifs influence the level
of tyrosine phosphorylation, the degree of SHP-2 binding, and the magnitude of induction of the hummingbird
phenotype. We compared the number and type of EPIYA motifs in strains infecting Portuguese patients with chronic
superficial gastritis and gastric carcinoma. Overall, in the 80 cagA-positive cases, the number of CagA EPIYA motifs
varied from one to five, and the majority of the strains contained three EPIYA motifs and ABC-type CagA. There was
no relationship between the number of EPIYA motifs and clinical outcome. However, chronic superficial gastritis
patients were more frequently infected with strains without an EPIYA C motif, whereas gastric carcinoma patients
were more frequently infected with strains with two EPIYA C motifs (p<0.05). These data suggest that the CagA
EPIYA C tyrosine phosphorylation motif may influence the risk for gastric carcinoma development, and that
characterization of the EPIYA-containing region may be important in more clearly defining H. pylori pathogenesis
and associated disease risk.
In keeping with previous lines of work of our group showing an association between H. pylori vacA s1, vacA m1 and
cagA-positive strains and more severe parameters of gastritis, gastric atrophy, intestinal metaplasia, and gastric
carcinoma in the Portuguese population, we have collaborated with the Carcinogenesis group in a project that aims
at understanding the differences in incidence of gastric carcinoma in Portugal and Mozambique. For that we have
genotyped H. pylori vacA s and m regions and assessed the cagA status in a series of 140 paraffin-embedded
gastric biopsy specimens from Mozambique. Result analysis is still ongoing.
1. b) Host factors for sporadic gastric carcinoma
In 2007, we have been actively involved in the identification of additional inflammation-related genetic
polymorphisms associated with risk of development of gastric cancer (GC). Besides our previous findings on the
association between polymorphisms in the IL1B, IL1RN and TNFA genes and risk of GC, during 2007 we added
new data on genes such as IL8 and IFNGR1. Our results do not support the existence of an association between
the IL8-251*T/*A polymorphism and risk of GC in Caucasian populations. In contrast, studies conducted in Asian
populations show that the association between the IL8-251*T/*A polymorphism and increased risk of GC is likely to
be ethnic-specific.
Recently, polymorphisms in the gene encoding the interferon gamma receptor 1 (IFNGR1) were found to be
associated with increased susceptibility to H. pylori infection. Hence, we aimed to determine the association
between polymorphisms in the IFNGR1 gene and development of chronic gastritis and GC. In a case-control study
including 733 controls, 213 individuals with chronic gastritis and 393 GC patients, the IFNGR1 -611*G/*A, -56*C/*T,
+1004*A/*C and +1400*T/*C polymorphisms were genotyped. The effect of the -56*C/*T promoter polymorphism in
the level of expression of the IFNGR1 gene was evaluated by an IFNGR1-56*C/*T allele specific luciferase reporter
assay. In individuals with early onset GC (defined as having less than 40 years of age at the time of diagnosis) we
17
found a significant over-representation of the IFNGR1-56*T/*T homozygous genotype with an odds ratio (OR) of 4.1
(95% confidence interval [CI] 1.6-10.6). In the luciferase reporter assay we observed a 10-fold increase (P < 0.001)
in luciferase expression associated with the IFNGR1-56*T allele.
Our results indicate that the IFNGR1-56C/T polymorphism is a relevant host susceptibility factor for GC
development. Our data also indicate that this genetic polymorphism is functionally relevant and may be related with
early development of GC.
1. c) Genetic factors for familial forms of gastric cancer
E-cadherin is the only gene identified, to date, with a causative role in Hereditary Diffuse Gastric Cancer. Germline
mutations in the E-cadherin gene CDH1, cause 30% of hereditary diffuse gastric cancer (HDGC), an autosomal
dominant gastric cancer susceptibility syndrome which also predispose to familial breast and colon cancer. In 6070% of the HDGC families, and in 90% of families with at least two gastric cancers but with diffuse histotpathology
confirmed in a single case (FDGC families), CDH1 germline inactivation failed to be identified. Although current
techniques of genotyping did not reveal CDH1 mutations, diffuse gastric tumours occurring in these families display
similar morphological features and E-cadherin immunostaining to those harbouring CDH1 germline mutations
(unpublished results). We have previously analysed 128 HDGC and FDGC patients, lacking molecular diagnosis, for
CDH1 large deletions using MLPA and found that none displayed this mechanism of inactivation of the CDH1 gene.
In 2007, we extended the series of patients to 150 and found five large deletions in 5 HDGC families. Two of these
families show the exact same deletion which is suggestive of a founder effect. This is the first alternative
mechanism, to CDH1 mutations, described to date in these families.
A remarkably high percentage (~80%) of CDH1 mutations in HDGC patients and carriers generate premature
termination codons (PTCs). In 2007, we examined whether CDH1 transcripts harboring PTCs are downregulated by
nonsense-mediated decay (NMD), an RNA surveillance pathway that degrades PTC-bearing transcripts. Using an
allele-specific expression (ASE) assay to differentiate between mutated and wild-type CDH1 alleles, we found that
PTC-bearing CDH1 mRNAs are strongly downregulated in normal gastric tissue from several CDH1 mutation
carriers. We showed that NMD is responsible for this robust downregulation, as CDH1 transcripts harbouring PTCs
in the KATO-III gastric tumor cell line were upregulated in response to protein synthesis inhibitors or depletion of the
NMD factors UPF1 and eIF4AIII. Analysis of HDGC patients harboring CDH1 alleles with PTCs at a wide variety of
different positions indicates an association of their predicted ability to induce NMD and an earlier age of onset of
gastric cancer. This suggests that NMD may be detrimental for HDGC patients and that therefore NMD is a
potentially useful therapeutic target in CDH1 mutation carriers (Paper in press).
Our team focuses on the role of E-cadherin germline mutations of the missense type found in the setting of both
hereditary diffuse gastric cancer and early onset diffuse gastric cancer. As reference laboratory of the International
Gastric Cancer Linkage Consortium, we have been involved in the functional characterization of new HDGCassociated E-cadherin germline missense mutations, aiming at unrevealing their pathogeneicity, using in vitro
assays. In 2007, we have completed the functional characterization of 2 new HDGC-associated E-cadherin
germiline missense mutations (in collaboration with Prof. Franco Roviello, University of Siena, Italy). One proved to
be pathogenic in vitro, whereas the second one displayed only mild or no effect in vitro and in silico. Interestingly the
same alteration was later found in healthy controls, suggesting is polymorphic nature. Results were submitted to the
respective laboratories, and shared with the clinicians responsible for the genetic counselling and clinical follow-up
of the carrier families. On the other hand, results were also integral part of research publications. This work has
been also pivotal for the establishment or strengthening of international collaborations.
In 2007, we had identified a subset of three HDGC-associated missense E-cadherin germline mutations in the
cytoplasmic region of E-cadherin which appeared to interfere with the correct localization of the protein at the cell
surface. Despite the advance in the understanding of the mechanisms that regulate E-cadherin expression, the
cause for its loss is yet unidentified in a significant percentage of the cases. Based on the observation that these
three missense mutations lead to protein loss in the membrane, we built the hypothesis that membrane trafficking
plays an important role in modulating E-cadherin levels and activity, therefore its subversion might be an alternative
mechanism for E-cadherin deregulation during cancer progression. To explore this hypothesis, we transduced CHO
cells with the E-cadherin cytoplasmic germline missense mutations. We verify that, despite normal RNA levels,
these cells exhibit reduced expression of E-cadherin at plasma membrane and accumulation in the ER. We
investigated the mechanism responsible for E-cadherin regulation and found that the variants are subjected to
Endoplasmic Reticulum Quality Control (ERQC) and their loss is due to ER-associated degradation (ERAD).
Additionally, we show that mutant E-cadherin surface expression can be rescued by specific Chemical Chaperones
and functionality restored. These findings suggest that overcoming ERAD might represent a possible therapeutic
approach for prevention of tumour development for carriers of specific E-cadherin variants (unpublished results).
18
In Familial Gastric Cancer (FGC) not linked to E-cadherin, no alternative genes have yet been identified. In 2007,
we aimed at increasing our knowledge on the genetic basis of FGC associated to mismatch repair (MMR) defects.
Only families negative for germline defects in CDH1 were selected to be screened for MSI in its tumours (family
number=70). We decided to screen these families for MMR repair defects using MSI as a pre-screening method to
detected putative MMR repair families. In order to do that microsatellite instability phenotype was determined in
tumour material. Tumour material was only available in 19 of these 70 CDH1 negative families. From these 19
probands analysed three cases had MSI-H phenotype in the tumour. The frequency observed in these familial
gastric cancer cases (15.8%) is similar to the frequency of MSI-H in sporadic gastric cases reported in the literature
(15-20%). We proceed with the analysis of promoter methylation of the MLH1 and MSH2 genes in tumour material
from MSI familial gastric cancer with using direct sequencing of the complete promoter region after PCR of bisulfite
treated DNA using flanking primers to the CpG islands for each gene. Two of the three MSI-H FGC tumours
displayed hypermethylation in MLH1 promoter region, whereas none (3 out of 3) was methylated in MSH2 promoter
region. We implemented the analysis of MMR repair (MSH2, MLH1 and MSH6) protein expression. MLH1 protein
expression was decreased on tumour cells from 2 out of the 3 FGC probands patients with MSI-H. These
immunoexpression results are in accordance with the presence of MLH1 promoter methylation found in those
patients. We further screened mutations in MSH2, MLH1 and MSH6 in tumour material. In one of the families a
missense mutation in MLH1 was detected in exon 16 (ATG→GTG) leading to Met587Val substitution in tumour
material. No germline mutations of the different MMR genes (MSH2, MLH1, and MSH6) were found in these families
(unpublished results).
2. Identifying pathological features and molecular markers occurring in the setting of familial and sporadic
cancer
2. a) Gastric cancer
Current knowledge on the morphological steps underlying the development of HDGC stems from detailed studies
performed in 22 stomachs that were totally mapped, encompassing prophylactic gastrectomy specimens (group A)
and total gastrectomies performed in patients referred from chromoendoscopic surveillance programmes (group B).
Till now there are only two publications reporting prophylactic gastrectomies performed in CDH1 germline mutation
carriers in which cancer was not identified. However, these two studies do not provide detailed data on the protocol
used for the study of the surgical specimens. In our experience, unless total sampling of the whole stomachs is
performed, the diagnosis of early invasive cancers can not be excluded. In 2007, we had the opportunity to see in
consultation several prophylactic gastrectomies, originally reported as negative for cancer, in which the detailed
microscopic study of the whole length of gastric mucosa (after complete embedding of the whole stomach) revealed
the presence of foci of early invasive signet ring-cell carcinomas in all cases. The neoplastic cells in the early
invasive cancers generally display the features of signet ring cells. In some cases a layered structure of the
carcinomas is observed, the cells being small and undifferentiated at deep levels and displaying the signet ring cell
phenotype at the surface. E-cadherin immunoexpression was shown to be reduced or absent in early invasive
gastric carcinomas, contrasting with the normal membranous E-cadherin expression in adjacent nonneoplastic
mucosa, in keeping with a clonal origin of the cancer foci.
As precursors of the invasive cancers, two distinct types of lesions were identified in prophylactic gastrectomies: (i)
in situ signet ring cell carcinoma, corresponding to the presence of signet ring cells within basal membrane,
generally with hyperchromatic and depolarised nuclei; and (ii) pagetoid spread of signet ring cells below the
preserved epithelium of glands/foveolae. In situ carcinomas are identified at the proximity of invasive carcinomas as
well as in areas distant from invasive cancer. The low number of in situ carcinomas in comparison to the number of
invasive cancers suggests that invasion of the lamina propria by signet ring cells may occur without a
morphologically detectable in situ carcinoma.
It is accepted that the gastric mucosa in CDH1 germline mutation carriers is normal until the second CDH1 allele is
inactivated. It is postulated that this down-regulation occurs in multiple cells in the gastric mucosa, accounting for
the multifocal tumour lesions. E-cadherin expression is mislocated or absent in neoplastic cells from primary gastric
tumours from HDGC patients carrying CDH1 mutations, which is consistent with biallelic inactivation of the CDH1
gene by a 2nd hit mechanism. To date a low number of these tumours was analyzed for second-hit inactivation
mechanisms. So far, CDH1 hypermethylation, a second somatic mutation and an intragenic deletion have been
found in 11 tumours from HDGC patients. In 2007, we decided to undertake the characterization of the 2nd hit in 18
neoplastic lesions (13 primary and 5 metastases) from 16 CDH1 germline mutation carriers belonging to 14 different
families in order to determine whether the reversion of the second hit can be used as a therapeutical tool in HDGC
patients. We found that CDH1 hypermethylation and LOH are the most frequent second hit mechanism in HDGC
19
tumours and that they may occur simultaneously in the same lesion. We also found that primary tumours and
metastasis from the same patient may display different second hit mechanisms (unpublished results).
In 2007, we analysed a panel of 179 sporadic gastric tumours for the microsatellite instability (MSI) status. Thirtythree (18.4%) of these sporadic gastric cancers showed MSI phenotype. From the MSI sporadic gastric cancer
cases, twenty-five were further investigated for MLH1 promoter methylation. Twenty of these tumours showed MLH1
promoter methylation. Five MSI tumours were negative for MLH1 promoter methylation and were further screened
for MMR gene mutations. In sporadic MSI gastric cancer we also searched for somatic mutations in MLH1, MSH2,
MSH6 and MLH3 genes. We found four truncating mutations, three in MSH6 and one in MLH3. Interestingly two of
the four truncating mutations were identified in MSI-H cases, which lack hypermethylation of the MLH1 promoter
region. Since all truncating mutations were found in polyA tracts they are likely to represent mutations in target
repeat sequence due to an increase of mutation rate in MSI tumours rather than the cause of MSI phenotype (Paper
in press).
2. b) Colorectal cancer
Activating mutations of BRAF and KRAS are frequently found in sporadic colorectal (CRC) cancer. BRAF mutations
occur in 30 to 45% of MSI CRC and are rare in MSS CRC and whenever present are associated to advanced
carcinomas. KRAS mutations occur in both MSI (in about 20%) and MSS (in about 35%) subsets of sporadic CRC.
Within the MSI subset of CRC KRAS mutations do not associate with the presence of MLH1 promoter
hypermethylation neither with the presence of CIMP-High. PIK3CA mutations are present in 14% to 25% of the
cases and no differences in frequency and type of PIK3CA mutations were found between MSI and MSS subsets. In
2007, we analyzed a series of 17 mixed hyperplastic and adenomatous colorectal polyps and study the association
of these mutational oncogenic events with CIMP, MLH1 methylation and MSI phenotype. Furthermore, we
compared the frequency of KRAS, BRAF and PIK3CA mutations found in polyps with the frequency found in a
series of 103 colorectal tumours, 50 MSI CRC and 53 MSS CRC, in order to determine the timing of occurrence of
KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis. We found that KRAS, PIK3CA or
BRAF occur in 70% of these pre-malignant lesions in a mutually exclusive manner. KRAS mutations occur in 35% of
the polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations were found in 29% of the cases. CIMPH phenotype occurred in 25% of the polyps and all were mutated for BRAF. None of the polyps harboured
methylation at MLH1 and all were microsatellite stable. The comparison between the frequency of oncogenic
mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede
both types of CRC. In contrast, BRAF mutations are likely to be pivotal to precede MSI carcinomas since the
frequency found in polyps is similar to what is found in MSI CRC (P=0.9112), but statistically different from what is
found in MSS tumours (P=0.0191). Our results show that mutations of BRAF, KRAS and PIK3CA occur prior to
malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal
carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal
polyps while KRAS mutations are found alone. Moreover we verified that colorectal polyps and MSI CRC show a
similar frequency of BRAF mutations. These results support that BRAF mutations harbour a mild oncogenic effect in
comparison to KRAS and suggest that BRAF mutant colorectal cells need to accumulate extra epigenetic alterations
in order to acquire full transformation and evolve to MSI CRC (unpublished results).
Further in 2007, we analysed, in MSI and MSS colorectal carcinomas, the frequency of mutations in a MAP3K that
activates MAPK pathways and it has been reported to be a component of a multiprotein BRAF/RAf1 complex. This
MAP3K activates MAPK pathways and it has been reported that its integrity is required for ERK activation,
suggesting that plays a pivotal role in mitogen regulation of BRAF, ERK and cell proliferation. Therefore, it is likely
that it may have oncogenic properties but gene alterations were, so far, never found in cancer. In 2007, we
performed a mutation screening of the entire gene in HNPCC (n= 41) and sporadic MSI (n= 35) and MSS (n= 55)
colorectal tumors. We found mutations in 29% (12/41) of HNPCC tumours and in 23% (8/35) and 3.6% (2/55) of
sporadic MSI and MSS colorectal tumors, respectively. None of the mutations observed in colorectal tumors were
present in the normal counterpart and none were found in DNA from a population control of 50 individuals, indicating
that these mutations are tumour specific events. We found two different types of mutations – missense and
frameshift. In general, missense mutations were found in 68.2% (15/22) and frameshift mutations were identified in
31.8% (7/22) of the mutant tumours. We found that mutations in this MAP3K cluster in the kinase and the
proline/serine/threonine-rich domains (unpublished results).
2. c) Breast cancer
Inflammatory breast cancer (IBC) is an example of a highly aggressive form of mammary cancer; IBC often
overexpresses HER2 and manifests a large degree of lymphovascular invasion. In 2007, we analysed 18 HERB-2
positive IBC patients for cten expression. cten was positive in 56% of tumour biopsies. In addition, we found a
20
strong correlation between EGFR activation (phospho-EGFR) and cten expression (P < 0.0001). Furthermore,
because a structurally related dual specificity TKI, lapatinib (Tykerb), is currently being tested in clinical trials, we
examined the effect on cten in sequential IBC biopsies obtained prior to and during lapatinib monotherapy.
Representative immunohistochemical analyses of tumour biopsies were taken from the patients, before and after a
21-day treatment with the drug. We observed a statistically significant reduction in cten, pEGFR, pHER2 and pErk
following lapatinib treatmen. We conclude that cten expression in breast cancer is highly dependent on EGFR
activation, and may predict the response to EGFR/HER2-targeted therapy.
The association in mammary tumours between cten expression and active EGFR as well as the observed reduction
in cten expression in lapatinib- treated patients, suggests that cten is a marker for EGFR-driven tumours and is
capable of predicting patient response to EGFR-targeted therapy.
In breast cancer epidemiological studies, in vitro and animal models demonstrated that 1,25(OH)2D3 has anti-cancer
benefits, affecting both progression and breast cancer metastasis. Alike the role in calcium Vitamin D3 regulation, its
anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is
lacking. In 2007, we aimed at unveiling the molecular mechanism behind the anti-proliferative action of 1,25(OH)2D3
using genomic tools. For that purpose four independently developed 1,25(OH)2D3 sensitive/resistant MCF7 cell line
pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to
omit the use of 1,25(OH)2D3. We deem this omission crucial since in the presence of 1,25(OH)2D3 downstream
genes involved in proliferation and cell cycle are identified rather than causal resistance genes which we are
interested in here. The variety of genomic techniques included expression, NMD and oligo CGH arrays. In total a set
of 22 genome-wide microarray hybridizations were performed and analyzed. The identification, both at the DNA and
RNA level, of the genetic alterations responsible for the different phenotype concerning 1,25(OH)2D3 response,
allowed us to identify in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered
expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation
were identified in this analyses (unpublished results).
3. Identifying signaling pathways mediated by genetic and environmental factors in the distinct cancer
models
3. a) Signaling pathways mediated by H. pylori
The interaction of H. pylori with the host gastric epithelium is the basis for the development of gastric disease. In
2007, we have explored the relationship between H. pylori and cell invasion. We had previosly shown that H. pylori
strains containing a functional type IV secretion system induce cell invasion in a c-Met receptor dependent manner.
To identify the signalling pathways activated downstream c-Met involved in H. pylori-mediated cell invasion, we used
an RNAi-based approach targeting c-Met adaptors, c-Met effectors, and GTPases. This work is to be continued in
2008 in an ongoing collaboration with the group of Thomas Meyer at the Max Planck Institute for Infection Biology,
Berlin, Germany.
Because E-cadherin is a cell-cell adhesion molecule with an invasion-suppressor function, we also investigated
whether E-cadherin functions as inhibitor of H. pylori-mediated invasion. For that, we stably transduced AGS cells
using a lentivirus transduction system with E-cadherin (AGSEcad) and observed that after infection with H. pylori,
AGSEcad cells displayed significantly lower levels of invasion than AGS cells, suggesting that E-cadherin is
sufficient to suppress H. pylori-mediated cell invasion. We also investigated the effect of H. pylori on the Ecadherin/catenin complex, and showed that H. pylori interferes with the subcellular localization of molecules of the
complex. Following this line of investigation, we demonstrated that after H. pylori infection, in both AGSEcad and in
the NCI-N87 gastric cell line endogenously expressing E-cadherin, H. pylori CagA binds to E-cadherin and to p120catenin, affecting the interaction between these and other elements of the E-cadherin/catenin complex.
Furthermore, our results suggest that within the host cell, CagA participates with c-Met, E-cadherin, and p120catenin in the formation of a tetrameric complex. This work is to be continued in 2008 in collaboration with Prof.
Marc Mareel, from the Ghent University Hospital, Ghent, Belgium.
3. b) Signaling pathways mediated by Slug
The transcriptions factor Slug (SNAI2) plays an important role in epithelial-mesenchymal transition (EMT) during
embryonic development by down-regulating the expression of epithelial genes such as E-cadherin, conferring
increased migration capabilities to cells. We have previously shown that Slug is overexpressed in primary gastric
carcinomas (GC) and that its expression together with E-cadherin down-regulation was associated with the
presence of distant metastasis and advanced TNM stages. In 2007, we further evaluate the putative role of Slug in
GC progression in an in vitro GC model. For that, gastric carcinoma-derived NCI-N87 cells were stably transfected
with a cDNA coding for human Slug. Despite the well described relationship between Slug expression and E-
21
cadherin down-regulation, we found that this adhesion molecule was not the main target of Slug in our model.
Instead, Slug expression resulted in the disassembly of tight junctions, through the down-regulation of Occludin and
Claudins-4 and -5, as well as de novo expression of N-cadherin. Furthermore, Slug expression resulted in increased
migration capabilities, invasion on Matrigel and in the decrease in cell-cell adhesion abilities of these cells. Our
findings suggest that Slug overexpression plays a critical role in the context of GC progression and invasion, leading
to the loss of expression of important tight junction proteins and promoting invasive and motile behavior of cells.
3. c) Signaling pathways mediated by P-cadherin
P-cadherin molecule is overexpressed in basal-like breast carcinomas, predicting a worse prognosis for patients.
We showed that the pro-invasive activity of P-cadherin is awarded to its juxtamembrane domain, and probably also
to its interaction partners, like p120-catenin (p120ctn). Analogously to P-cadherin, p120ctn has been implicated in
the carcinogenesis process, although there are few studies about its role in breast cancer.
In 2007, we aimed to clarify if there is an effective role of P-cadherin together with p120ctn in breast cancer. We
were particularly interested in studying the influence of P-cadherin expression on p120ctn subcellular localization in
human invasive breast carcinomas. We investigated the expression and subcellular localization of p120- and βcatenin in a series of human invasive breast carcinomas, and correlated it with biological markers and
clinicopathological parameters. We demonstrated that both catenins frequently exhibit a reduced membranous or
cytoplasmic staining pattern. These alterations were significantly correlated with lack of both E-cadherin and
estrogen receptor-α expression. Considering β-catenin, it was also possible to associate its expression with
histological grade, tumour size and nodal status, suggesting a relevant role for this catenin as a prognostic factor.
Interestingly, we found that the majority of E- and P-cadherin co-expressing tumours were related with cytoplasmic
expression of p120-catenin, being this group of breast carcinomas the one with poor patient survival. These results
indicate that p120-catenin cytoplasmic accumulation may play an important role in mediating the oncogenic effects
derived from P-cadherin aberrant expression, including enhanced motility and invasion, particularly in tumours which
maintain E-cadherin expression.
We still investigated the expression of p120-catenin isoforms in a collection of breast cancer cell lines with distinct
molecular profiles and expressing different cadherins. We assessed the expression by RT-PCR and Westernblotting analysis and we observed that the expression of p120-catenin isoforms was associated with the genomic
and transcriptional phenotype of breast cancer cells. Besides, the recruitment of p120-catenin isoforms was not
apparently related with the particular expression of E-, P- or N-cadherin. We demonstrate that mammary tumour
cells exhibit a characteristic p120-catenin isoform expression profile, depending from their specific genomic and
transcriptional properties. These particular expression patterns, combined with other regulatory proteins and in a
specific cellular context, may explain how p120-catenin can either contribute to strength intercellular adhesions or
instead to promote cell motility.
In order to elucidate the role of P-cadherin in cell invasion, we also investigated the effect of this molecule on cell
migration, as well as the expression and activity profile of matrix metalloproteases (MMPs) using a breast cancer
cell line (MCF-7/AZ) retrovirally transduced with human P-cadherin. Using wound-healing migration assay and timelapse microscopy, we demonstrated that P-cadherin overexpression is also able to promote breast cancer cell
migration and cell motility. Considering the pattern of MMPs expression and activity, we showed that P-cadherin
overexpressing cells cultured in a collagen type I matrix, have an induction in activity of MMP-1 and MMP-2.
Concerning MMPs secretion to the medium, an increase in expression of MMP-1, MMP-2 and TIMP-1 was observed
in P-cadherin transduced cells, as well as a decrease in expression of MMP-3 and MMP-9. Concomitantly with the
increase in activity and expression of some MMPs in the conditioned medium of MCF-7/AZ.P-cad cells, it was also
detected a soluble P-cadherin fragment, with 80kDa. Since we found that the conditioned medium from MCF7/AZ.P-cad cells is enough to induce invasion of the non-invasive parental cell line, we decided to look for the proinvasive activity of this soluble fragment. To achieve this, we performed a matrigel invasion assay where we treated
the cells with the medium with and without the sP-cad, and we found that, depletion of the sP-cad from the medium,
abrograted completely cell invasion, showing that this fragment has pro-invasive activity and may explain in part the
invasive capacity of MCF-7/AZ.Pcad cells. We are now trying to address how this soluble fragment is able to induce
invasion.
Additionally, we decided to clone the P-cadherin promoter in a luciferase reporter vector (pGL3.CDH3 promoter).
Using a cell line ER negative (U2OS), transfected with an ER-α Tet-On transfection system, we could observe that
the induction of ERα is crucial for the down-regulation of the P-cadherin promoter activity. This observation is more
obvious if the cells are treated with the ERα natural ligand (E2), and are confirmed at the protein level. These
results suggest once again the existence of a molecular cross-talk between ERα and CDH3 expression, in which the
ERα signalling pathway modulate the CDH3 activity at the promoter level. Moreover, we are planning to perform
22
ChIP (chromatin immunoprecipiation) to see if the ERα directly binds to the CDH3 promoter, as well as transfections
with transcription factors ERα-downstream targets, like FOXA1 (a co-activator of ER), to see if we find potential
CDH3 promoter regulators.
In contrast, in ER positive cell lines, the treatment of these cells with ER inhibitors did not change the activity of Pcadherin promoter, although there are obvious increases at the mRNA and protein level. These results make us
think that in ER-dependent breast cancer cell lines, the regulatory mechanism of ER on CDH3 protein and mRNA
expression is possibly due to the function of ER-target miRNAs. We are now studying this hypothesis.
We still aimed to analyse the genes that are altered, relatively to the control, when the MCF-7/AZ breast cancer cell
line is treated with estradiol, tamoxifen, ICI and siRNA for ER. This was thought to be performed by means of cDNA
microarrays, which is a technique that offers a molecular approach to single gene expression analysis, being a
useful tool for the classification and categorization of human solid tumours and cancer cell lines, based upon their
molecular gene expression profiles. However, based on the problem explained in the previous reports, just during
this third year we were able to send the samples to the Microarray Facility from the VU Medical Centre (Prof. Bauke
Ylstra), in Amsterdam. Until now, we still did not obtain these results.
3. d) Signaling pathways mediated by E-cadherin
E-cadherin missense mutations represent a unique and subtle research opportunity to understand the role of Ecadherin deregulation in cancer, but also to address questions of a more general interest in cell biology:
E-cadherin and cell adhesion
Following the publication of breakthrough findings which demonstrated that a quaternary complex between Ecadherin, beta-catenin, alfa-catenin and actin cytoskeleton is not formed, whether alfa-catenin plays a direct role in
cell-cell adhesion, especially at early times of contact became unclear. Taking advantage of our E-cadherin
mutations in vitro system and in collaboration with Denis Wirtz at the Johns Hopkins Baltimore, we applied live-cell
single-molecule force spectroscopy to probe the strength of interactions between individual cadherin pairs on the
adjoining surface of two E-cadherin-expressing CHO cells. These measurements indicated that an individual WT Ecadherin molecular bond between two cells, for which a-catenin can bind the E-cadherin/b-catenin complex, is
significantly stronger than an individual mutant E-cadherin bond, for which a-catenin cannot bind the complex
(V832M HDGC-associated E-cadherin germline missense mutatnt). While single WT E-cadherin bonds between two
cells increase their strength for increasing time of contact, single mutant cadherin bonds weaken rapidly. Moreover,
the probability of forming multiple intercellular bonds increases quickly for WT cadherin cells, while is remains low
and constant for mutant cadherins. These results reveal that, through binding to the E-cadherin/b-catenin complex,
alfa-catenin plays two critical roles in adhesion: it enhances the initial intercellular binding of E-cadherins and it
catalyzes the transformation of a single-bond contact between WT cells into a nascent junction by mediating the
formation of multiple bonds between cells. These results are now being confirmed on CHO cells in which alfacatenin expression is being abrogated by siRNA (unpublished results).
E-cadherin and cell motility
Using cell lines stably expressing mutant forms of E-cadherin we have shown that mutations on the extracellular
domain of the protein exhibit enhanced cell motility mediated through RhoA activation. In 2007, we have challenged
the hypothesis of the existence of a bidirectional cross-talk between E-cadherin and EGFR activity, and
characterized in vitro the effect of its subversion on cell behaviour. We built this hypothesis on the knowledge that
EGFR and E-cadherin colocalize at the basolateral areas of polarized epithelial cells. EGFR activation has been
shown to inhibit E-cadherin-dependent adhesion; but also EGF-dependent activation of EGFR has been reported to
be inhibited in an E-cadherin adhesion-dependent manner. Furthermore, EGFR has been found in many human
tumours and it has been associated to enhanced cell motility and invasion. Using a subset of E-cadherin germline
missense mutations we demonstrated the existence of a physical interaction between E-cadherin and EGFR, likely
taking place at the extracellular side. We further explored this model and showed that E-cadherin might function as
a natural inhibitor of EGFR, through the formation of inactive heterodimers. Loss of such interactions (i.e. caused by
mutations on the extracellular domain of E-cadherin) leads to the activation of EGFR and consequently to enhanced
cell motility. In keeping with our previous observation, the effect of EGFR activation on cell motility was shown to be
transmitted through the activation of the small GTPase RhoA.
In gastric tumours data concerning structural alterations of EGFR remains controversial. Given its possible
therapeutic relevance, we aimed to determine the frequency and type of structural alterations of the EGFR gene in a
series of primary gastric carcinomas. Within the 77 primary gastric carcinomas we found two EGFR somatic
mutations and several EGFR polymorphisms in exon 20. Six different intronic sequence variants of EGFR were also
found. Four gastric carcinomas showed balanced polysomy or EGFR gene amplification. We verified that gastric
carcinoma with alterations of EGFR (somatic mutations or copy number variation) showed a significant increase of
23
tumour size (p=0.0094) in comparison to wild-type EGFR carcinomas. We demonstrated that EGFR structural
alterations are rare in gastric carcinoma, but whenever present, they lead to tumour growth. We considered that
searching for EGFR alterations in gastric cancer is likely to be clinically important in order to identify patients
susceptible to respond to tyrosine kinase inhibitors.
3. e) Signaling pathways mediated by EGFR in breast cancer
EGFR is involved in various cellular processes, including proliferation and motility, and constitutive receptor
activation contributes to the transition from a localized primary tumour mass into an invasive state leading to
secondary metastases. Several mechanisms have been identified, through which EGFR may regulate cell migration,
but the identity of key genes underlying EGF-induced cell motility remained largely unknown. In 2007 we aimed at
identify EGF-induced genes that are potentially involved in cell migration in breast cancer. To that goal, we used a
previously established transcriptional profiling data set derived from EGF-treated human HeLa cells. Analysis of this
data set revealed that prolonged EGF treatment induced the expression of various genes that are involved in the
regulation of cell adhesion or organization of the actin cytoskeleton. In addition, among other alterations, we
observed a reduction in transcripts that encode mediators of cell-to-cell interactions. Notably, we observed
reciprocal alterations in the expression of two members of the tensin family. This family comprises four proteins
(tensin-1, -2 and -3, and cten (C-terminal tensin-like protein), which are involved in cell migration, and are localized
to focal adhesion sites. All four tensin family members contain SH2 and PTB domains in their carboxyl termini.
Through the PTB domain, tensins interact with the cytoplasmic tail of β integrins, which are transmembrane celladhesion molecules. Although tensin-1, -2 and -3 contain an actin-binding domain in their amino termini, cten is a
shorter protein that lacks this domain. Following EGF treatment, TNS3 (tensin-3) mRNA undergoes downregulation,
whereas TNS4 (cten) expression exhibits remarkable upregulation. Notably, expression of TNS1 (tensin-1) is barely
detectable, whereas the moderate expression of tensin-2 is unchanged following EGF treatment. Protein
immunoblotting confirmed the induction of cten. In addition, cell-to-substrate adhesion — a signal that is known to
allow cellular migration — induced cten expression through a mechanism that does not involve autocrine EGFR
activation. In conclusion, these results raise the possibility that reciprocal regulation of cten and tensin-3 plays a
general role in cell adhesion and migrations. We propose that this tensin-3–cten switch is key mediator of EGFdriven mammary cell migration.
3. f) Signaling pathways mediated by C/EBP transcription factors and gastric carcinogenesis
Transcription factors from the CCAAT/enhancer binding protein (C/EBP) family are known to have a crucial role in
the control of differentiation and proliferation of adult tissues. We have recently shown that in the normal gastric
mucosa C/EBPbeta expression is restricted to the proliferative neck zone, and is subsequently overexpressed in
pre-neoplastic lesions and gastric carcinoma (GC). C/EBPalpha plays a crucial role in terminal differentiation and is
an established tumour suppressor gene in several models. In 2007, we analyzed the expression of C/EBPalpha in
normal and neoplastic gastric tissues, and assessed the role of C/EBPalpha on the proliferation of gastric carcinoma
cells. In normal gastric mucosa, C/EBPalpha is expressed in differentiated foveolar epithelium and co-localizes with
the gastric differentiation marker TFF1. The expression of C/EBPalpha was found to be lost in a significant
percentage of GC samples. To evaluate the role of C/EBPalpha in cell proliferation we transfected AGS cells with an
expression vector for the full length C/EBPalpha protein. We observed, by BRDU incorporation, a significant
decrease in proliferation in the transfected cells. Accordingly, the decrease on proliferative activity was accompanied
by a decrease in the expression of the cell cycle regulator cyclin D1, and an increase in the expression of the cell
cycle inhibitor p27. We also demonstrated that C/EBPalpha expression is able to induce expression of the gastric
differentiation marker TFF1 in transfected cells. We also sought to establish if CEBPA mutations could be present in
GC.
For this purpose, we sequenced the entire coding region of CEBPA in 142 cases of GC. We found alterations
1164C>T and 1281G>T in three and 38 cases, respectively, that were previously reported as polymorphisms. The
1302del7 mutation was detected in one case and has not been previously reported in the literature. This mutation
originates a truncated protein that lacks the terminal part of the bZIP-domain, which is fundamental for the DNA
binding of the CEBPA protein. The 1302del7 alteration was tumour-specific since it was not present in constitutional
DNA from the same patient. Alteration 1175-1180dup was observed in 7% (10/142) of the cases. We sequenced
constitutional DNA of the patients with this alteration and found that it was present in the germline of all 10 cases.
This result suggests that the 1175-1180dup is not a mutation but is instead a polymorphism. To verify this
hypothesis we screened a series of 25 healthy blood donors for the presence of this alteration.
The 1175-1180dup was observed in 6 out of the 25 controls (24%), confirming that we are indeed dealing with a
common population polymorphism. Our results suggest that C/EBP transcription factors may play an active role in
maintaining a balance between proliferation and differentiation in the normal gastric mucosa. In a theoretical model
24
of gastric carcinogenesis, C/EBPbeta would have a pro-proliferative activity in gastric epithelial stem-like cells and,
along neoplastic transformation, it would be expressed in cells retaining a proliferative phenotype such as those
seen in dysplastic and cancer lesions. C/EBPalpha, on the other hand, would be associated with induction of
differentiation in normal foveolar gastric mucosa cell lineages and, along neoplastic transformation, it would be
associated with a tumour suppressor-like activity.
Collaborative Projects between Cancer Genetics and other groups at IPATIMUP
In the ambit of a project which was funded by FCT and carried out by Gianpaolo Suriano (PI) in collaboration with
Luísa Azevedo (Population Genetics group) and the University of Torino, Italy, we demonstrated in vitro the
existence of a genetic mechanisms of intra-locus compensation using the ornithine transcarbamylase protein as
model. Ornithine transcarbamylase deficiency (OTCD) is an X-linked inborn defect of metabolism of the urea cycle,
which causes hyperamonemia. Mutations of the OTC gene have been recognized as the genetic cause underlying
the OTC deficiency. The severity of the disease is associated with the type of mutation, leading either to neonatal
onset of hyperammonemia or to a later appearance of the disease. The mutation Thr125Met is associated with
neonatal hyperammonemia. Recently, the disease-causing Thr125Met mutation in humans was reported as wildtype neutral allele in chimpanzees. Further analysis confirmed the presence of Met125 fixed in chimpanzees
together with Thr135, representing the only two divergent positions between human and chimpanzee OTCs. Thr125
and Thr135 were identified as ancestral mammalian combination, so the Thr135Ala substitution occurred as humanspecific event, whereas the substitution of Thr125Met was characteristic of the chimpanzee linage. Only when
Met125 emerges in a background with the human-specific Ala135, a highly deleterious effect is observed,
suggesting among other hypotheses the existence of a compensatory effect in chimpanzee. To explore this
hypothesis, we built an in vitro cell model system to study the effect of the three distinct genetic backgrounds
(Ala135-Thr125; Ala135-Met125 and Thr135-Met125) on the OTC protein function. We observed that the human
Thr125Met mutant is inactive, whereas the chimp OTC shows an enzymatic activity comparable with the wild-type
human OTC. We concluded that the presence of a threonine at position 135 in chimps rescues the deleterious effect
of the methionine at position 125, in a mechanism of intra-locus compensation.
Funded Projects
PI: Carla Oliveira
− Identificação de mecanismos celulares subjacentes ao desenvolvimento de cancro gástrico em familiares
portadores e não por-mutações germinativas da caderina-E, Fundação para a Ciência e a Tecnologia
(POCI/SAU-OBS/58111/2004)
− Papel de novos mechanismos regulatórios na perda de expressão da caderina-E, Fundação para a Ciência e a
Tecnologia (PTDC/SAU-GMG/72168/2006)
PI: Céu Figueiredo
− Effects of Helicobacter pylori on gastric epithelial cells, Fundação para a Ciência e a Tecnologia (POCI/SAUIMI/56681/2004)
− A global RNAi approach to unravel eukaryotic host functions that modulate bacterial infections (acronym RNAiNET), Fundação para a Ciência e a Tecnologia/Era-Net PathoGenoMics (ERA-PTG/0002/2006)
− Potencial oncogénico de Helicobacter pylori: da Biologia à Clínica, Fundação Calouste Gulbenkian (Proj.
356327-S)
PI: Fátima Carneiro
− Análise funcional dos repressores da caderina-E (Slug, ZEB1 e E12/E47) em carcinomas do estômago,
Fundação para a Ciência e a Tecnologia (POCI/SAU-OBS/57275/2004)
− Environmental factors, Helicobacter pylori infection, genetic susceptibility and the gastric cancer risk in the
European population (acronym EUR-GAST), European Commission (FP5.QLTR-2000-01049). Coordinator of
the Panel of Pathologists.
− Archives’s tissues; improving molecular medicine research and clinical practice (acronym: IMPACTS –
Integration of Molecular Pathology and Cell and Tissues Structure) (Contract: 037211)
− European Pathology Assessment & Learning System (acronym: EUROPALS), (Ref. 133852-LLP-1-2007-1-NLERASMUS-ENW)
25
PI: Fernando Schmitt
− P-cadherin in Breast Cancer: what regulates its aberrant expression and how it can induce invasion of
neoplastic cells?, Fundação para a Ciência e a Tecnologia (POCTI/BIA-BCM/59252/2004)
− P-cadherin Expression: its effect in breast cancer metastasis and angiogenesis using in vivo animal models
(Sanofi - Aventis - Produtos Farmacêuticos, SA)
− Approaching basal-like breast carcinomas to target theraphy. A project combining the reinforcement of logistics
facilities with translational research (GlaxoSmithKline)
− P53 e Resistência a Quimoterapia no cancro da Mama (Novartis Farma, SA)
PI: Gianpaolo Suriano
− E-cadherin germline missense mutations and hereditary diffuse gastric cancer: a model for the identification of
the E-cadherin-dependent signaling pathways pivotal for cell invasion, Fundação para a Ciência e a Tecnologia
(POCI/SAU-OBS/57670/2004)
− Estudo da heterogeneidade fenotípica patológica usando a proteína ornitina transcarbamilase (OTC) como
modelo, Fundação para a Ciência e a Tecnologia (POCI/CVT/58082/2004)
PI: José Carlos Machado
− The role of chronic infections in the development of cancer (acronym INCA), European Commission, FP6
Priority 1, Integrated Project (Contract no.: 018704)
− Teste de susceptibilidade genética para determinação de risco de desenvolvimento de Doença de Crohn na
população Portuguesa, Programa IDEIA, Agência de Inovação (TSG-CROHN)
− Evaluation and Control of Neglected Mucosal Enteric Infections in Childhood (acronym CONTENT), European
Commission, FP6, INCO Specific Measures (Contract no.: 018704)
− Parasite and host genetic diversity in Helicobacter pylori infections (acronym HELDIVNET), Fundação para a
Ciência e a Tecnologia/Era-Net PathoGenoMics (ERA-PTG/0004/2006)
PI: Raquel Seruca
− No cancro colorectal com instabilidade de microssatélites são os genes BRAF e KRAS novos marcadores de
prognóstico e novas terapêuticas?, Fundação para a Ciência e a Tecnologia (POCI/SAU-OBS/56921/2004).
− Prevention, diagnosis and molecular characterisation of mismatch repair defect-related hereditary cancers of
the digestive system, European Commission (FP6-2004-LIFESCIHEAL TH-5).
− Mechanisms of cell invasion: possible application to oncobiology”. Programa IDEIA, Agência de Inovação (INVONC-DPN).
Participation of elements of the Cancer Genetics group in other Projects
− Identificação de vias de sinalização envolvidas na regulação do Cdx2 em dois modelos humanos de
diferenciação intestinal: metaplasia intestinal e polipose juvenil. Fundação para a Ciência e a Tecnologia
(POCI/SAU/-OBS/55840/2004); PI: Raquel Almeida; Team Member: Carla Oliveira.
− Characterization of nine members of the major outer membrane proteins family in Helicobacter pylori strains
isolated from Portuguese patients, Fundação para a Ciência e a Tecnologia (POCI/SAU-IMI/57297/2004); PI:
Lurdes Monteiro, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa; Team Member: Céu Figueiredo.
− PYLORI L&EPS: Structural Characterisation of Lipo- and Exopolysaccharides from Helicobacter pylori Establishment of the chemical and biochemical basis for the development of vaccines and for the
understanding of the adhesion processes, Fundação para a Ciência e a Tecnologia (POCI/QUI/56393/2004);
PI: Manuel A. Coimbra, Dep. Química da Universidade de Aveiro, Aveiro; Team Member: Céu Figueiredo.
− Identification of glycosylation-associated genes induced by Helicobacter pylori in gastric cells: a glycomic
approach, Fundação para a Ciência e a Tecnologia (POCI/SAU-OBS/56686/2004); PI: Celso Reis, IPATIMUP;
Team Member: Céu Figueiredo.
− Clarification of the relevance of Muc1 polymorphism in the Helicobacter pylori infection, Fundação para a
Ciência e a Tecnologia (POCI/SAU-IMI/56895/2004); PI: Luís Filipe Santos Silva, IPATIMUP; Team Member:
Céu Figueiredo.
− Lesions of the gastric mucosa in Mozambique and Portugal: the “African enigma”. Study of biologic factors and
lifestyle that contribute to understand the differences in incidence of gastric carcinoma in two countries with a
high prevalence of Helicobacter pylori infection: Mozambique and Portugal. Fundação Calouste Gulbenkian
(Project FC-68697); PI: Leonor David, IPATIMUP; Team Members: Céu Figueiredo and José Carlos Machado.
26
−
Targeting nanoprobes for early detection of invasive cancer cells using Hereditary Diffuse Gastric Cancer as a
model, Fundação para a Ciência e a Tecnologia (PTDC/BEB/69901/2006); PI: Pedro Granja, Instituto de
Engenharia Biomédica (INEB), Porto; Team Member: Raquel Seruca.
Master Thesis
Student: Hugo Celso Coelho Pinheiro
Supervisors: Cândida Lucas (Universidade do Minho, Braga, Portugal) and Carla Oliveira
Title: E-cadherin silencing: More than inactivating mutations. Molecular mechanisms modulating CDH1 expression:
HDGC as a model
Mestrado em Genética Molecular, Escola de Ciências, Universidade do Minho
Date of conclusion: 21st December 2007
PhD Thesis
Student: Cristina Rocha Felgueiras Sousa Nogueira
Supervisors: Lynda Chin (Danna Farber Institute, USA) and Fátima Carneiro
Title: Molecular genetics of melanoma initiation and progression
Date of conclusion: 7th May 2007
Publications in 2007
1. Alazzouzi H, Suriano G, Guerra A, Plaja A, Espin E, Armengol M, Alhopuro P, Velho S, Shinomura
Y, Gonzalez-Aguilera JJ, Yamamoto H, Aaltonen LA, Moreno V, Capella G, Peinado MA, Seruca
R, Arango D, Schwartz S Jr. Tumour selection advantage of non-dominant negative P53 mutations
in homozygotic MDM2-SNP309 colorectal cancer cells. J Med Genet 44: 75-80, 2007.
2. Azevedo NF, Guimarães N, Figueiredo C, Keevil CW, Vieira MJ. A New Model for the
Transmission of Helicobacter pylori: Role of Environmental Reservoirs as Gene Pools to Increase
Strain Diversity. Crit Rev Microbiol 33: 1-13, 2007.
3. Baltazar F, Filho AL, Pinheiro C, Moreira MA, Queiroz GS, Oton GJ, Junior AF, Ribeiro LF, Schmitt
FC. Cyclooxygenase-2 and Epidermal Growth Factor Receptor Expressions in Different
Histological Subtypes of Cervical Carcinomas. Int J Gynecol Pathol 26: 235-41, 2007.
4. Buffart TE, Carvalho B, Mons T, Reis RM, Moutinho C, Silva P, van Grieken NC, Vieth M, Stolte M,
van de Velde CJ, Schrock E, Matthaei A, Ylstra B, Carneiro F, Meijer GA. DNA copy number
profiles of gastric cancer precursor lesions. BMC Genomics 8: 345, 2007.
5. Canedo P, Figueiredo C, Machado JC. After Helicobacter pylori, Genetic Susceptibility to Gastric
Carcinoma Revisited. Helicobacter 12 (Suppl 2): 45-9, 2007.
6. Canedo P, Machado JC. Genetic susceptibility to gastric carcinoma. Acta Endoscopica 37: 239-47,
2007.
7. Carneiro F, Moutinho C, Pera G, Caldas C, Fenger C, Offerhaus J, Save V, Stenling R, Nesi G,
Mahlke U, Blaker H, Torrado J, Roukos DH, Sabourin JC, Boeing H, Palli D, Bueno-de-Mesquita
HB, Overvad K, Bingham S, Clavel-Chapelon F, Lund E, Trichopoulou A, Manjer J, Riboli E,
Gonzalez CA. Pathology findings and validation of gastric and esophageal cancer cases in a
European cohort (EPIC/EUR-GAST). Scand J Gastroenterol 42: 618-27, 2007.
8. Cassali GD, Gobbi H, Malm C, Schmitt FC. Evaluation of accuracy of fine needle aspiration
cytology for diagnosis of canine mammary tumours: comparative features with human tumours.
Cytopathology 18: 191-6, 2007.
9. Castro Alves C, Rosivatz E, Schott C, Hollweck R, Becker I, Sarbia M, Carneiro F, Becker KF. Slug
is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the
down-regulation of E-cadherin. J Pathol 211: 507-15, 2007.
10. Corso G, Roviello F, Paredes J, Pedrazzani C, Novais M, Correia J, Marrelli D, Cirnes L, Seruca R,
Oliveira C, Suriano G. Characterization of the P373L E-cadherin germline missense mutation and
implication for clinical management. Eur J Surg Oncol 33: 1061-7, 2007.
11. Costa BM, Ferreira P, Costa S, Canedo P, Oliveira P, Silva A, Pardal F, Suriano G, Machado JC,
Lopes JM, Reis RM. Association between functional EGF+61 polymorphism and glioma risk. Clin
Cancer Res 13: 2621-6, 2007.
5.087
3.829
2.184
4.029
2.477
1.869
0.989
5.759
6.177
27
12. Costa S, Pinto D, Pereira D, Rodrigues H, Cameselle-Teijeiro J, Medeiros R, Schmitt F. DNA repair
polymorphisms might contribute differentially on familial and sporadic breast cancer susceptibility: a
study on a Portuguese population. Breast Cancer Res Treat 103: 209-17, 2007.
13. Davalos V, Dopeso H, Velho S, Ferreira AM, Cirnes L, Diaz-Chico N, Bilbao C, Ramirez R,
Rodriguez G, Falcon O, Leon L, Niessen RC, Keller G, Dallenbach-Hellweg G, Espin E, Armengol
M, Plaja A, Perucho M, Imai K, Yamamoto H, Gebert JF, Diaz-Chico JC, Hofstra RM, Woerner SM,
Seruca R, Schwartz S Jr, Arango D. High EPHB2 mutation rate in gastric but not endometrial
tumors with microsatellite instability. Oncogene 26: 308-11, 2007.
14. Dufloth RM, Matos I, Schmitt F, Zeferino LC. Tissue microarrays for testing basal biomarkers in
familial breast cancer cases. Sao Paulo Med J 125: 226-30, 2007.
15. Filho AL, Baltazar F, Bedrossian C, Michael C, Schmitt FC. Immunohistochemical expression and
distribution of VEGFR-3 in malignant mesothelioma. Diagn Cytopathol 35: 786-91, 2007.
16. Gomes AL, Bardales RH, Milanezi F, Reis RM, Schmitt F. Molecular analysis of c-Kit and PDGFRA
in GISTs diagnosed by EUS. Am J Clin Pathol 127:89-96, 2007.
17. Gomes AL, Reis-Filho JS, Lopes JM, Martinho O, Lambros MB, Martins A, Schmitt F, Pardal F,
Reis RM. Molecular alterations of KIT oncogene in gliomas. Cell Oncol 29: 399-408, 2007.
18. Gray W, Bayer-Pietsch E, Chieco P, Cochand-Priollet B, Desai M, Drijkoningen M, Griffin M,
Hagmar B, Kapila K, Kloboves-Prevodnik V, Kobayashi T, Krogerus L, Majak B, Mihailovici M,
Olsewski W, Schenk U, Schmitt F, Shabalova I, Shapiro N, Smith J, Tani E, Totsch M, Vass L,
Wiener H, Herbert A. The future of cytopathology in Europe. Will the wider use of HPV testing have
an impact on the provision of cervical screening? Cytopathology 18: 278-82, 2007.
19. Guimaraes N, Azevedo NF, Figueiredo C, Keevil CW, Vieira MJ. Development and application of a
novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsies.
J Clin Microbiol 45: 3089-94, 2007.
20. Katz M, Amit I, Citri A, Shay T, Carvalho S, Lavi S, Milanezi F, Lyass L, Amariglio N, Jacob-Hirsch
J, Ben-Chetrit N, Tarcic G, Lindzen M, Avraham R, Liao YC, Trusk P, Lyass A, Rechavi G, Spector
NL, Lao SH, Schmitt F, Bacus SS, Yarden Y. A reciprocal tensin3-cten switch mediates EGF-driven
mammary cell migration. Nat Cell Biol 9: 961-9, 2007.
21. Kaurah P, MacMillan A, Boyd N, Senz J, De Luca A, Chun N, Suriano G, Zaor S, Van Manen L,
Gilpin C, Nikkel S, Connolly-Wilson M, Weissman S, Rubinstein WS, Sebold C, Greenstein R,
Stroop J, Yim D, Panzini B, McKinnon W, Greenblatt M, Wirtzfeld D, Fontaine D, Coit D, Yoon S,
Chung D, Lauwers G, Pizzuti A, Vaccaro C, Redal MA, Oliveira C, Tischkowitz M, Olschwang S,
Gallinger S, Lynch H, Green J, Ford J, Pharoah P, Fernandez B, Huntsman D. Founder and
recurrent CDH1 mutations in families with hereditary diffuse gastric cancer. JAMA 297: 2360-72,
2007.
22. Lee AH, Paish EC, Marchio C, Sapino A, Schmitt FC, Ellis IO, Reis-Filho JS. The expression of
Wilms' tumour-1 and Ca125 in invasive micropapillary carcinoma of the breast. Histopathology 51:
824-8, 2007.
23. Longatto-Filho A, Costa SM, Milanezi F, Montruccoli D, Montruccoli GC, Baltazar F, Schmitt FC.
Immunohistochemical expression of VEGF-A and its ligands in non-neoplastic lesions of the breast
sampling assisted by dynamic angiothermography. Oncol Rep 18:1201-6, 2007.
24. Longatto-Filho A, Oliveira TG, Pinheiro C, de Carvalho MB, Curioni OA, Mercante AM, Schmitt FC,
Gattás GJ. How useful is the assessment of lymphatic vascular density in oral carcinoma
prognosis? World J Surg Oncol 5: 140, 2007.
25. Longatto-Filho A, Pinheiro C, Pereira SM, Etlinger D, Moreira MA, Jube LF, Queiroz GS, Baltazar
F, Schmitt FC. Lymphatic vessel density and epithelial D2-40 immunoreactivity in pre-invasive and
invasive lesions of the uterine cervix. Gynecol Oncol 107: 45-51, 2007.
26. Longatto-Filho A, Schmitt FC. Gynecological Cytology: too old to be a pop star but too young to die.
Diagn Cytopathol 35: 672-3, 2007.
27. Lunet N, Valbuena C, Vieira AL, Lopes C, Lopes C, David L, Carneiro F, Barros H. Fruit and
vegetable consumption and gastric cancer by location and histological type: case-control and metaanalysis. Eur J Cancer Prev 16: 312-27, 2007.
28. Masciari S, Larsson N, Senz J, Boyd N, Kaurah P, Kandel MJ, Harris LN, Oliveira C, Troussard A,
Miron P,Tung N, Pinheiro HC, Collins L, Schnitt S, Garber JE, Huntsman D. Germline E-Cadherin
mutations in familial lobular breast cancer. J Med Genet 44: 726-31, 2007.
4.671
6.582
0.786
2.939
2.979
0.989
3.445
18.485
23.175
3.216
1.567
2.319
0.786
1.993
5.087
28
29. Mateus AR, Seruca R, Machado JC, Keller G, Oliveira MJ, Suriano G, Luber B. EGFR regulates
RhoA-GTP dependent cell motility in E-cadherin mutant cells. Hum Mol Genet 16:1639-47, 2007.
30. Mendez MA, Pera G, Agudo A, Bueno-de-Mesquita HB, Palli D, Boeing H, Carneiro F, Berrino F,
Sacerdote C, Tumino R, Panico S, Berglund G, Manjer J, Johansson I, Stenling R, Martinez C,
Dorronsoro M, Barricarte A, Tormo MJ, Quiros JR, Allen N, Key TJ, Bingham S, Linseisen J, Kaaks
R, Overvad K, Jensen M, Olsen A, Tjønneland A, Peeters PH, Numans ME, Ocké MC, ClavelChapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Slimani N, Jenab M, Ferrari P, Riboli E,
González CA. Cereal fiber intake may reduce risk of gastric adenocarcinomas: the EPICEURGAST study. Int J Cancer 121: 1618-23, 2007.
31. Milne AN, Sitarz R, Carvalho R, Carneiro F, Offerhaus GJ. Early onset gastric cancer: on the road
to unraveling gastric carcinogenesis. Curr Mol Med 7:15-28, 2007.
32. More H, Humar B, Weber W, Ward R, Christian A, Lintott C, Graziano F, Ruzzo AM, Acosta E,
Boman B, Harlan M, Ferreira P, Seruca R, Suriano G, Guilford P. Identification of seven novel
germline mutations in the human E-cadherin (CDH1) gene. Hum Mutat 28:203, 2007.
33. Nagel G, Linseisen J, Boshuizen HC, Pera G, Del Giudice G, Westert GP, Bueno-de-Mesquita HB,
Allen NE, Key TJ, Numans ME, Peeters PH, Sieri S, Siman H, Berglund G, Hallmans G, Stenling
R, Martinez C, Arriola L, Barricarte A, Chirlaque MD, Quiros JR, Vineis P, Masala G, Palli D,
Panico S, Tumino R, Bingham S, Boeing H, Bergmann MM, Overvad K, Boutron-Ruault MC,
Clavel-Chapelon F, Olsen A, Tjonneland A, Trichopoulou A, Bamia C, Soukara S, Sabourin JC,
Carneiro F, Slimani N, Jenab M, Norat T, Riboli E, Gonzalez CA. Socioeconomic position and the
risk of gastric and oesophageal cancer in the European Prospective Investigation into Cancer and
Nutrition (EPIC EURGAST). Int J Epidemiol 36: 66-76, 2007.
34. Oliveira MJ, Costa AM, Costa AC, Figueiredo C. Helicobacter pylori induit l'invasion dês cellules
gastriques via un mécanisme qui nécessite le c-Met cellulaire et un système de sécrétion bactérien
de type IV fonctionnel. La Lettre de L’ Infectiologue Tome XXII – Hors-Série: 3-6, 2007.
35. Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval A, Hamelin
R, Machado JC, Schwartz S Jr, Carneiro F, Seruca R. KRAS and BRAF oncogenic mutations in
MSS colorectal carcinoma progression. Oncogene 26:158-63, 2007.
36. Palli D, Masala G, Del Giudice G, Plebani M, Basso D, Berti D, E Numans M, Ceroti M, Peeters
PH, de Mesquita HB, Buchner FL, Clavel-Chapelon F, Boutron-Ruault MC, Krogh V, Saieva C,
Vineis P, Panico S, Tumino R, Nyren O, Siman H, Berglund G, Hallmans G, Sanchez MJ,
Larranaga N, Barricarte A, Navarro C, Quiros JR, Key T, Allen N, Bingham S, Khaw KT, Boeing H,
Weikert C, Linseisen J, Nagel G, Overvad K, Thomsen RW, Tjonneland A, Olsen A, Trichoupoulou
A, Trichopoulos D, Arvaniti A, Pera G, Kaaks R, Jenab M, Ferrari P, Nesi G, Carneiro F, Riboli E,
Gonzalez CA. CagA+ Helicobacter pylori infection and gastric cancer risk in the EPIC-EURGAST
study. Int J Cancer 120: 859-67, 2007.
37. Paredes J, Correia AL, Ribeiro AS, Albergaria A, Milanezi F, Schmitt FC. P-cadherin expression in
breast cancer: a review. Breast Cancer Res 9: 214, 2007.
38. Paredes J, Correia AL, Ribeiro AS, Schmitt F. Expression of p120-catenin isoforms correlates with
genomic and transcriptional phenotype of breast cancer cell lines. Cell Oncol 29: 467-76, 2007.
39. Peleteiro B, Lunet N, Figueiredo C, Carneiro F, David L, Barros H. Smoking, Helicobacter pylori
virulence, and type of intestinal metaplasia in Portuguese males. Cancer Epidemiol Biomarkers
Prev 16: 322-6, 2007.
40. Peleteiro B, Lunet N, Santos-Silva F, David L, Figueiredo C, Barros H: Short mucin 1 alleles are
associated with low virulent H pylori strains infection. World J Gastroenterol 13: 1885-6, 2007.
41. Paredes J, Lopes N, Milanezi F, Schmitt FC. P-cadherin and cytokeratin 5: useful adjunct markers
to distinguish basal-like ductal carcinomas in situ. Virchows Arch 450: 73-80, 2007.
42. Preto A, Moutinho C, Rebocho AP, Velho S, Oliveira C, Figueiredo J, Lopes JM, Soares P and
Seruca R. A subset of colorectal carcinomas express c-KIT protein independently of KRAS and/or
BRAF activation. Virchows Archiv 450: 619-26, 2007.
43. Resende C, Regalo G, Duraes C, Carneiro F, Machado JC. Genetic changes of CEBPA in cancer:
mutations or polymorphisms? J Clin Oncol 25: 2493-4, 2007.
44. Ricardo S, Milanezi F, Carvalho S, Leitão D, Schmitt F. HER2 evaluation through the novel rabbit
monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinoma. J Clin
Pathol 60: 1001-5, 2007.
8.099
4.693
4.850
6.473
4.517
6.582
4.693
4.157
2.979
4.289
2.251
2.251
13.598
2.245
29
45. Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark-Mansson H. The role of osteopontin
in tumor progression and metastasis in breast cancer. Cancer Epidemiol Biomarkers Prev 16: 1-11,
2007.
46. Roviello F, Corso G, Pedrazzani C, Marrelli D, De Falco G, Berardi A, Garosi L, Suriano G, Vindigni
C, De Stefano A, Leoncini L, Seruca R, Pinto E. Hereditary diffuse gastric cancer and E-cadherin:
description of the first germline mutation in an Italian family. Eur J Surg Oncol 33: 448-51, 2007.
47. Roviello F, Corso G, Pedrazzani C, Marrelli D, De Falco G, Suriano G, Vindigni C, Berardi A,
Garosi L, De Stefano A, Leoncini L, Seruca R, Pinto E. High incidence of familial gastric cancer in
Tuscany, a region in Italy. Oncology 72, 243-7, 2007
48. Sarmento-Castro R, Horta A, Vasconcelos O, Coelho H, Mendez J, Tavares AP, Seabra J, Duarte
M, Chaves L, Fortes O, Recalde C, Ventura A, Pires N, Pinho L, Dias N, Carneiro F. Impact of
peginterferon alpha-2b and ribavirin treatment on liver tissue in patients with HCV or HCV-HIV coinfection. J Infect 54: 609-16, 2007.
49. Savage K, Lambros MB, Robertson D, Jones RL, Jones C, Mackay A, James M, Hornick JL,
Pereira EM, Milanezi F, Fletcher CD, Schmitt FC, Ashworth A, Reis-Filho JS. Caveolin 1 is
overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a
morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis. Clin Cancer
Res 13: 90-101, 2007.
50. Schmitt FC, Gomes AL, Milanezi F, Reis R, Bardales R. Mutations in gastrointestinal stromal
tumors diagnosed by endoscopic ultrasound-guided fine needle aspiration. Minerva Med 98: 385-8,
2007.
51. Schmitt FC. Cells carry the clue for targeted treatment: a new horizont for cytopathology.
Cytopathology 18: 275-7, 2007.
52. Schmitt FC. Is BRCA1 a possible predictor of response to neo-adjuvant chemotherapy ? Hereditary
Cancer in Clinical Practice 5: 136-7, 2007.
53. Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A. Sequence diversity at the
proximal 14q32.1 SERPIN subcluster: evidence for natural selection favoring the pseudogenization
of SERPINA2. Mol Biol Evol 24: 587-98, 2007.
54. Suriano G, L Azevedo, M Novais, B Boscolo, R Seruca, A Amorim, E M Ghibaudi. In vitro
demonstration of intra-locus compensation using the Ornithine transcarbamylase protein as model.
Hum Mol Genet 16: 2209-14, 2007
55. Svrcek M, El-Bchiri J, Chalastanis A, Capel E, Dumont S, Buhard O, Oliveira C, Seruca R, Bossard
C, Mosnier J-F, Berger F, Leteurtre E, Lavergne-Slove A, Chenard M-P, Hamelin R, Cosnes J,
Beaugerie L, Tiret E, Duval A, Fléjou J-F. Specific clinical and biological features characterize
inflammatory bowel disease.associated colorectal cancers showing microsatellite instability. J Clin
Oncol 25: 4231-8, 2007.
56. Turner NC, Reis-Filho JS, Russell AM, Springall RJ, Ryder K, Steele D, Savage K, Gillett CE,
Schmitt FC, Ashworth A, Tutt AN. BRCA1 dysfunction in sporadic basal-like breast cancer.
Oncogene 26: 2126-32, 2007.
57. Vollset SE, Igland J, Jenab M, Fredriksen A, Meyer K, Eussen S, Gjessing HK, Ueland PM, Pera
G, Sala N, Agudo A, Capella G, Del Giudice G, Palli D, Boeing H, Weikert C, Bueno-de-Mesquita
HB, Carneiro F, Pala V, Vineis P, Tumino R, Panico S, Berglund G, Manjer J, Stenling R, Hallmans
G, Martínez C, Dorronsoro M, Barricarte A, Navarro C, Quirós JR, Allen N, Key TJ, Bingham S,
Linseisen J, Kaaks R, Overvad K, Tjønneland A, Büchner FL, Peeters PH, Numans ME, ClavelChapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Slimani N, Ferrari P, Riboli E, González
CA. The association of gastric cancer risk with plasma folate, cobalamin, and
methylenetetrahydrofolate reductase polymorphisms in the European Prospective Investigation into
Cancer and Nutrition. Cancer Epidemiol Biomarkers Prev 16: 2416-24, 2007.
4.289
2.037
6.177
0.989
6.726
8.099
13.598
6.582
4.289
Publications in press
58. Canedo P, Castanheira-Vale AJ, Lunet N, Pereira F, Figueiredo C, Gioia-Patricola L, Canzian F, 1.993
Moreira H, Suriano G, Barros H, Carneiro F, Seruca R, Machado JC. The interleukin-8-251*T/*A
polymorphism is not associated with risk for gastric carcinoma development in a Portuguese
population. Eur J Cancer Prev in press.
59. Carneiro F, Oliveira C, Suriano G, Seruca R. Molecular pathology of familial gastric cancer, with an 2.245
30
emphasis on hereditary diffuse gastric cancer. J Clin Pathol in press.
60. Costa S, Pinto D, Pereira D, Rodrigues H, Cameselle-Teijeiro J, Medeiros R, Schmitt F. Importance
of TP53 codon 72 and intron 3 duplication 16bp polymorphisms in prediction of susceptibility on
breast cancer. BMC Cancer in press.
61. Di Palma S, Collins N, Bilous M, Sapino A, Mottolese M, Kapranos N, Schmitt FC, Isola J. A quality
assurance exercise to evaluate the accuracy and reproducibility of CISH for HER2 analysis in
breast cancer. J Clin Pathol in press.
62. Ferreira AC, Gomes L, Máximo V, Amil J, Carneiro F, Machado JC, Tavarela-Veloso F. GRIM-19
mutations are not associated with Crohn's disease. Inflamm Bowel Dis in press.
63. Gama A, Paredes J, Gärtner F, Alves A, Schmitt F. Expression pattern of adhesion molecules (Ecadherin, P-cadherin and b-catenin) and their relationship with clinicopathological parameters,
proliferation and survival in canine mammary malignant tumours. Vet J in press.
64. Jenab M, Ferrari P, Riboli E, Slimani N, Norat T, Tjønneland A, Olsen A, Overvad K, Boutron,
Ruault M-C, Clavel-Chapelon F, Linseisen J, Nagel G, Boeing H, Schulz M, Trichopoulou A,
Mattielo A, Sieri S, Tumino R, Palli D, Sacerdote C, Bueno-de-Mesquita HB, Buchner FL, Peeters
PH, Numans ME, Skeie G, Pera G, Sánchez M-J, Quirós JR, Tormo Díaz MJ, Dorronsoro M,
Barricarte A, Berglund G, Manjer J, Hallmans G, Stenling R, Key TJ, Allen NE, Bingham S, Khaw
K-T, Kaaks R, Carneiro F, Save V, Gonzalez C: Plasma and Dietary Carotenoid, Retinol,
Tocopherol and Vitamin C Levels and Risk of Esophageal Adenocarcinoma: Preliminary Results
from the European Prospective Investigation into Cancer and Nutrition (EPIC-EurGast). Int J
Cancer in press.
65. Karam R, Carvalho J, Bruno I, Graziadio C, Senz J, Huntsman D, Carneiro F, Seruca R, Wilkinson
MF, Oliveira C. The NMD mRNA Surveillance Pathway Downregulates Truncated E-Cadherin
Transcripts in Gastric Cancer Cells and in CDH1 Mutation Carriers. Oncogene in press.
66. Longatto-Filho A, Pinheiro C, Ferreira L, Scapulatempo C, Alves VA, Baltazar F, Schmitt F.
Peritumoural, but not intratumoural, lymphatic vessel density and invasion correlate with colorectal
carcinoma poor-outcome markers. Virchows Arch in press.
67. Macedo FY, Baltazar F, Almeida PR, Távora F, Ferreira FV, Schmitt FC, Brito GA, Ribeiro RA.
Cyclooxygenase-2 expression on ifosfamide-induced hemorrhagic cystitis in rats. J Cancer Res
Clin Oncol in press.
68. Macedo FY, Baltazar F, Mourão LC, Almeida PR, Mota JM, Schmitt FC, Ribeiro RA. Induction of
COX-2 expression by acrolein in the rat model of hemorrhagic cystitis. Exp Toxicol Pathol in press.
69. Marques M, Magro F, Cardoso H, Carneiro F, Portugal R, Lopes J, Costa Santos C: Infliximabinduced lupus-like syndrome associated with autoimmune hepatitis. Inf Bowel Dis in press.
70. Moutinho C, Mateus AR, Milanezi F, Carneiro F, Seruca R, Suriano G. Epidermal growth factor
receptor structural alterations in gastric cancer. BMC Cancer in press.
71. Paredes J, Correia AL, Ribeiro AS, Milanezi F, Cameselle-Teijeiro J, Schmitt FC. Breast
carcinomas that co-express E- and P-cadherin are associated with p120-catenin cytoplasmic
localization and poor patient survival. J Clin Pathol in press.
72. Pinheiro C, Longatto-Filho A, Scapulatempo C, Ferreira L, Martins S, Pellerin L, Rodrigues M,
Alves VA, Schmitt F, Baltazar F. Increased expression of monocarboxylate transporters 1, 2, and 4
in colorectal carcinomas. Virchows Arch in press.
73. Pinto M, Wu Y, Mensink RG, Cirnes L, Seruca R, Hofstra RM. Somatic mutations in mismatch
repair genes in sporadic gastric carcinomas are not a cause but a consequence of the mutator
phenotype. Cancer Genet Cytogenet in press.
74. Preto A, Figueiredo J, Velho S, Ribeiro AS, Soares P, Oliveira C, Seruca R. BRAF provides
proliferation and survival signals in MSI colorectal carcinoma cells displaying BRAF(V600E) but not
KRAS mutations. J Pathol in press.
75. Schmitt FC, Longatto-Filho A, Valent A, Vielh P. Molecular techniques in cytopathology practice. J
Clin Pathol in press.
76. Woo MMM, Salamanca CM, Miller M, Symowicz J, Leung PCK, Oliveira C, Ehlen TG, Gilks CB,
Huntsman D, Auersperg N. Serous borderline ovarian tumours in long term culture:phenotypic and
genotypic distinction from invasive ovarian carcinomas. Int J Gynecol Cancer in press.
2.359
2.245
3.912
1.589
4.693
6.582
2.251
2.469
0.755
3.912
2.359
2.245
2.251
1.544
5.759
2.245
1.469
31
Patents
“PNA Probe, Kit and Procedure for Specific Detection of Helicobacter pylori and applications”, PN103767 deposited
21.06.2007. Inventors: Nuno Azevedo, Nuno Guimarães, Céu Figueiredo, Charles Keevil, and Maria J. Vieira.
Prizes
− 1st Prize LabMed Laboratories for the paper: Oliveira MJ, Costa AC, Costa AM, Henriques L, Suriano G,
Atherton JC, Machado JC, Carneiro F, Seruca R, Mareel M, Leroy A, Figueiredo C. Helicobacter pylori induces
gastric epithelial cell invasion in a c-Met and type IV secretion system-dependent manner.J Biol Chem 281:
34888-96, 2006.
− Karam R , Carneiro F, Oliveira C, Wilkinson MF: The NMD RNA Surveillance Pathway Degrades Aberrant ECadherin Transcripts in Hereditary Diffuse Gastric Cancer. University of Texas M.D. Anderson Cancer Center,
Houston, TX. Trainee recognition day (Second place winner), May 2007.
− Prize of the Portuguese Society of Oncology and Schering-Plough for the project "Vitamin D has a therapy for
basal-like breast carcinomas: fiction or reality?", PI Fernando Schmitt.
International Collaborations
Belgium
− VIB Department for Molecular Biomedical Research, University of Ghent (Frans Van Roy),
Ghent, Belgium, ”E-cadherin signalling”, Gianpaolo Suriano
− Ghent University Hospital (Marc Mareel), Ghent, Belgium, “H. pylori-mediated cell invasion”,
Céu Figueiredo; “P-cadherin-mediated cell invasion”, Joana Paredes; “E-cadherin-mediated
cell invasion”, Gianpaolo Suriano and Raquel Seruca
− Jules Bordet Institute (Martine Piccart), Brussels, Belgium, "European MINDACT project",
Fernando Schmitt
Brazil
− Faculdade de Medicina (Dulciene Queiroz), Belo Horizonte, Brazil, “H. pylori and other
Helicobacter species”, Céu Figueiredo
− Department of Genetics, FFFCMPA (Carla Graziadio), Porto Alegre, Brazil, “Hereditary
gastric cancer”, Carla Oliveira and Raquel Seruca
− São Paulo University (Venâncio Alves), São Paulo, Brazil, "Lymphangiogenesis", Fernando
Schmitt
Canada
− Genetic Pathology Evaluation Centre, University of British Columbia (David G Huntsman),
Vancouver, Canada, “Hereditary Diffuse Gastric Cancer”, Carla Oliveira, Gianpaolo Suriano,
Raquel Seruca, and Fátima Carneiro
− Department of Obstetrics and Gynecology, University of British Columbia (Auersperg Nelly),
Vancouver, Canada, “Borderline ovarian cancer”, Carla Oliveira
China
− Chinese University of Hong-Kong (Joseph Sung), Hong-Kong, China, “Gastric cancer”, José
Carlos Machado
Denmark
− Roskilde University (Lene J Rasmussen), Roskilde, Denmark, “MMR related cancer”, Céu
Figueiredo and Raquel Seruca
Finland
− University of Helsinki (Lauri A Aaltonen, Paivi Peltomaki), Helsinki, Finland, “MMR related
cancer”, Raquel Seruca
France
− INSERM U762 CEPH, INSERM (Richard Hamelin, Alex Duval), Paris, France, “MMR related
cancer genes”, Raquel Seruca
− Institut Pasteur (Eliette Touati), Paris, France, “Mouse model of H. pylori infection”, Céu
Figueiredo, José Carlos Machado
Germany
− Technische Universität München (Birgit Luber, Gisela Keller, Karl Becker), Germany, “Ecadherin related cancer”, Gianpaolo Suriano and Raquel Seruca
− Max Planck Institute for Infection Biology (Thomas Meyer), Berlin, Germany, “Host responses
to H. pylori”, Céu Figueiredo
− University of Heidelberg (Federico Canzian), Heidelberg, Germany, “Genetic susceptibility”,
José Carlos Machado
− University of Tuebingen (Nikolaus Blin), Heidelberg, Germany, “Gastric cancer”, José Carlos
Machado
32
− Division of Surgical Oncology, University of Siena (Franco Roviello), Siena, Italy, “Hereditary
Diffuse Gastric Cancer”, Carla Oliveira, Gianpaolo Suriano, and Raquel Seruca
− CBM S.c.r.l. AREA SCIENCE PARK (Elia Stupka), Trieste, Italy, “Non-coding sequences”,
Carla Oliveira
Israel
− Weizmann Institute of Science (Yosef Yarden), Rehovot, Israel, "Growth factor receptors and
cell signalling", Fernando Schmitt
Japan
− National Cancer Center Research Institute (Jun Yokota), Tokyo, Japan, “Hereditary Diffuse
Gastric Cancer”, Raquel Seruca
New
− University of Otago (Parry Guilford), Dunedin, New Zealand, “Hereditary Diffuse Gastric
Zealand
Cancer”, Gianpaolo Suriano
Scotland
− Aberdeen University (Emad El-Omar), Aberdeen, Scotland, “Genetic susceptibility”, José
Carlos Machado
Spain
− Cancer Epigenetics Laboratory CNIO (Manel Esteller), Madrid, Spain, “Epigenetics of
Cancer”, Raquel Seruca and Carla Oliveira
− Molecular Biology and Biochemistry Research Center (CIBBIM), Valle Hebron Hospital
Research Institute (Simo Schwartz Jr), Barcelona, Spain, “MMR related cancer genes”,
Raquel Seruca
− CABD-Centro Andaluz de Biologia del Desarrollo, Univ. Pablo de Olavide (Fernando
Casares), Sevilla, Spain, “E-cadherin in Drosophila”, Raquel Seruca
Sweden
− Dept of Cell and Molecular Biology (CMB) Karolinska Institutet (Liam Good), Stockholm,
Sweden, “RNA processing”, Carla Oliveira
The
− University of Groningen (Robert MW Hofstra), Groningen, The Netherlands, “MMR related
Netherlands
cancer”, Raquel Seruca
− Leiden University Medical Center (Niels de Wind), Leiden, The Netherlands, “MMR related
cancer”, Raquel Seruca
− UMC Nijmegen (Marjolijn Ligtenberg), Nijmegen, The Netherlands, “Hereditary cancer
genes”, Carla Oliveira and Raquel Seruca
− Delft Diagnostic Laboratory (Leen-Jan van Doorn), Delft, The Netherlands, “Molecular
methods for H. pylori detection and typing”, Céu Figueiredo
UK
− Oncology Department Hutchison/MRC Research Centre, University of Cambridge (Carlos
Caldas), Cambridge, UK, “Second hit mechanisms in HDGC”, Fátima Carneiro
− St James's University Hospital, (Jean Crabtree), Leeds, UK, “Genetic susceptibility”, José
Carlos Machado
− Institute of Infection, Immunity and Inflammation, University of Nottingham (John Atherton),
Nottingham, United Kingdom, “H. pylori virulence”, Céu Figueiredo
− Breakthrough Breast Cancer Center (Alan Ashworth and Jorge Sérgio Reis- Filho), London,
UK, "Basal-like breast carcinomas", Fernando Schmitt
− Imperial College School of Medicine (Eric Lam), London, UK, "Forkhead Transcription
Factors", Fernando Schmitt
USA
− Department Chemical Engineering, Johns Hopkins University (Denis Wirtz), Baltimore, MD,
USA, “cell-cell adhesion”, Gianpaolo Suriano
− University of Texas M.D. Anderson Cancer Center (Miles Wilkinson), Houston, TX, USA,
“NMD and cancer”, Carla Oliveira and Fátima Carneiro
− Dana-Farber/Harvard Cancer Center (DF/HCC) (Gregory Lauwers), Boston, USA,
“Gastrointestinal pathology”; Fátima Carneiro
Italy
33
CARCINOGENESIS
Group Leader: Leonor David, MD PhD, Full Professor of Pathology from the Medical Faculty of the University of
Porto and senior Researcher of IPATIMUP
Staff members: Fátima Gartner, PhD, Full Professor at ICBAS; Celso Reis, PhD, senior Investigator; Raquel
Almeida, PhD, senior Investigator; Ana Carvalho, post-Doc; Hugo Osório, post-Doc; Nuno Marcos, PhD student;
Rita Barros, PhD student; Salomé Pinho, PhD student; Liliana Silva, PhD student (jointly with the Dental Faculty);
Ana Magalhães, PhD student; Joana Gomes, PhD student; Maria Manuel Azevedo, BI; Joana Oliveira, BI; Bruno
Pereira, BI; Vania Camilo, BI; Lara Marcos, BI; Nuno Mendes, technician.
Objectives/Goals of the research activity - 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 such alterations, including the identification of transcription factors
responsible for cancer/pre-cancer transdifferentiation, as well as to extend our expertise on other cancer models
(ex: mammary cancer from dogs).
Major achievements during 2007 – The group continued previous studies on the clarification of the mechanisms
involved in the development of intestinal metaplasia (IM). The group progressed in this line of research and
succeeded in showing that matrix derived BMP2/4, through the Smad4 pathway, are responsible for regulation of
the CDX2 homeobox transcription factor in gastric cells (paper submitted). We are therefore gathering strong and
innovative evidence in favor of a matrix-derived initiation signaling pathway for IM development. We further showed
that GATA factors regulate CDX2 transcription in gastric cell lines and GATA 6 potentiates the effect of Smad4 in
CDX2 transcription. This is being done in collaboration with Jean-Noel Freund from Strasbourg. Furthermore, we
initiated the characterization of the CDX2 autoregulatory mechanism and obtained a CDX2 expression construct
with loxP sites, allowing the expression and removal of CDX2, that will be used in functional assays to study this
autoregulatory mechanism in living cells.
We continued last year work on the genotype/phenotype associations in the Secretor and Lewis systems and their
relevance for gastrointestinal infectious diseases – Helicobacter pylori and Calicivirus. We
e demonstrated that
Secretor status, together with ABO histo-blood group phenotype for some viral strains, is determinant for adhesion
of Helicobacter pylori and Calicivirus (the work on Helicobacter is submitted for publication and the work on
calicivirus is being prepared by Jacques Le Pendu from Nantes). We continued the collaboration with the group of
Jorge Rocha from IPATIMUP, to elucidate the phenotype/genotype discrepancies in the Secretor and Lewis
systems. So far we demonstrated that FUT2 G428A is the major responsible for the non-Secretor phenotype in the
Portuguese population, similarly to the observation in most western countries. We have further confirmed that
mutations previously described by our group – G739A and T839C – greatly reduce enzyme activity towards Type 1
acceptor substrates. Haplotype/phenotype associations will be analysed and further enzyme activity studies will be
undertaken to elucidate the in vivo role of the new mutant variants.
We have previously demonstrated that ST6GalNAc-I is the enzyme responsible for the biosynthesis of the cancerassociated carbohydrate antigen Sialyl-Tn. Using a novel monoclonal antibody, produced in collaboration with Dr.
Ulla Mandel from the University of Copenhagen, we demonstrated that, both in intestinal metaplasia and in gastric
carcinomas, the immunodetection of enzyme ST6GalNAc-I co-localizes with expression of Sialyl-Tn. We have also
demonstrated in vitro that the Sialyl-Tn antigen modulates the malignant phenotype in gastric carcinoma cells. A
paper describing these results has been published.
The group demonstrated that the most virulent H. pylori strains, cagPAI+, lead to increased expression of
glycosyltransferases, namely β3GnT5, participating in the biosynthesis of glycans such as Sialyl-Lex antigen, the
ligand of H. pylori SabA adhesin. We further demonstrated that overexpression of β3GnT5 leads to de-
34
novo/increased Sialyl-Lex expression in gastric cells and that this expression increases adhesion of H. pylori. A
manuscript reporting these findings has been submitted for publication.
Our results also showed that virulent H. pylori strains induced a remarkable expression of Syndecan-4, suggesting
that this proteoglycan may be involved in the modulation of H. pylori-associated gastritis. We observed that H. pylori
infected individuals show increased expression of syndecan-4 in gastric mucosa and this expression was associated
with the cagPAI status of the infecting strain. We further demonstrated that induction of syndecan-4 in gastric cells is
associated with the cagPAI status of the H. pylori strain.
The group initiated the characterization of the role that Sialyl-transferases (ST3Gal-III, ST3Gal-IV, ST3gal-VI) play in
building the terminal sialylation of the carbohydrate chain, leading to the formation of Sialyl-Lewisa and Sialyl- Lewisx
antigens. We demonstrated that ST3Gal-III is involved in the sialylation of both type-I and type-II chains,
participating in the biosynthesis of Sialyl-Lewisa and Sialyl-Lewisx, whereas ST3Gal-IV preferentially sialylates typeII chains leading to the biosynthesis of Sialyl-Lewisx. Finally, we demonstrated that the final products of the complex
carbohydrate structures depend on the set of fucosyltrasferases and their activities in the cell lines. The cellular
models developed are presently being tested and will be used in functional assays.
Transgenic mice were obtained from the Consortium for Functional Glycomics (Celso Reis is a member of this
consortium) with knock-out of one of the following glycosyltransferases - Core2-GlcNAc (controlling mucin type Oglycosylation braching), FUT2 (controlling terminal H-type 1 terminal chain), ST3Gal-IV (controlling terminal
sialylation forming the Sialyl-Lewis structures), and the control wild type mice with the C57BL6 background. We
characterized the glycan antigenic profile of the gastric mucosa of the knock-out mice and demonstrated major
alterations in the glycan structures, such as lack of H-type 1 and Lewisb in the FUT2 K.O. mice. These mice models
will be used for evaluating the in vivo relevance of the different glycans for H. pylori infection.
During 2007 we extended our study of gastric lesions in a Mozambican population to evaluate: the rate of
Helicobacter pylori infection; the characteristics of Hp strains (in collaboration with Céu Figueiredo, from the Cancer
Genetics group); IL1B and IL1RN polymorphisms (in collaboration with José Carlos Machado, from the Cancer
Genetics group); dietary and smoking habits (in collaboration with Nuno Lunet, from the Department of
Epidemiology of the Medical Faculty of Porto). Our objective is to clarify the reasons why Mozambique, despite
having a high rate of Helicobacter pylori infection, has a low rate of intestinal metaplasia and gastric carcinoma – the
so-called “African enigma”. During 2007 samples/questionnaires from 180 Mozambicans were completed. The initial
analysis showed that Helicobacter pylori infection affects 93% Mozambicans and that intestinal metaplasia is
observed in 10% (contrasting to 85% infection, 30% intestinal metaplasia in a population from northern Portugal) suggesting that progression of gastric lesions upon infection is limited by factors yet unknown.
Fátima Gartner leads the veterinary division of the group in close line to our main objectives. We analysed the
Sialyl-Lewisx expression in canine malignant mammary tumours and evaluated if the presence of Sialyl-Lewisx
correlates with the expression of E-Cadherin and with clinicopathological features. We showed that Sialyl-Lewisx
expression was present in all cases of canine mammary carcinomas and the levels of expression are significantly
associated to lymph node metastases. Moreover, an inverse relationship was observed for E-Cadherin and SialylLewisx expression, which among other reasons may stem from impediment of E-Cadherin recognition in the
presence of extended glycosylation.
This result led us to evaluate the importance of E-cadherin glycosylation in the process of tumour development and
tumour progression, namely to characterize the overall glycosylation pattern of E-cadherin in canine mammary
gland tumour progression using two different tumour models, adenoma and carcinoma. Our results provide
evidence that E-cadherin with sialylated glycans, β1,6 branched complex type and high mannose N-glycan
structures, are implicated in the process of progression of canine mammary tumours.
We explored the possibility that mucin 1 (MUC1) might be overexpressed in canine mammary tumours as previously
shown for human breast carcinomas. We demonstrated for the first time that in canine mammary tumours high
MUC1 expression was significantly associated with a higher rate of distant metastasis and worse prognosis. The
relevance of this observation goes beyond the observation per se and opens the possibility of using the canine
35
model for validation of relevant clinical applications in humans that derive from this mucin alteration – from serum
detection assays to potential immunotherapy applications.
A poorly differentiated mammary carcinoma cell line - CMT-U27 - generously supplied by Eva Hellmén, Uppsala
University, was characterized and showed positivity for Sialyl-Lewis X, Lewis X and Lewis A as well as a strong
immunoexpression for E-cadherin. Female athymic nude mice were evaluated for its tumorigenic and metastatic
capacity and metastization was observed in the lung, heart, liver, spleen and ovary. Cell lines from two complex
carcinomas and two complex adenomas, cultured from mammary canine primary tumours, are being established
and characterized.
The group, under the leadership of Celso Reis and with a strong input from Hugo Osório, established a Proteomics
Unit at IPATIMUP. The unit uses MALDI-TOF mass spectrometry technology. The unit is currently serving
researchers from IPATIMUP and from other research institutes in Porto – IBMC, CIMAR, Medical Faculty of Porto,
and the Faculty of Engineering of Porto. The Proteomics Unit had a central role in our successful application to FP7
framework program (see projects below).
Apart from the mainstream of the group objectives we collaborated in several publications, mainly clinico-pathologic
descriptions (see “Other” publications).
Financing/Projects
The group won Royalties from monoclonal antibodies sells: 2007 – 751€
•
•
•
•
•
MUC5AC (MAB2011, Clone CLH2). CHEMICON INTERNATIONAL INC. 28835 Single oak drive,
Temecula, CA 92590; California, USA.
MUC5AC (NCL-MUC5). NOVOCASTRA LABORATORIES Ltd. Balliol Business Park West,
Benton Lane, Newcastle upon Tyne; NE12 8EW, United Kingdom.
MUC5AC (CLH2 sc-33667).SANTA CRUZ BIOTECHNOLOGY Inc., USA.
MUC6 (NCL-MUC6). NOVOCASTRA LABORATORIES Ltd. Balliol Business Park West, Benton
Lane, Newcastle upon Tyne; NE12 8EW, United Kingdom.
MUC6 (CLH5 sc-33668).SANTA CRUZ BIOTECHNOLOGY Inc., USA.
The group gathered 251.764€ for funding during 2007 and has approved funding for 2008 (337.401€). The funding
for 2007, after deduction of 20% overheads (50.352€), and salaries: 69.401€: one technician – 15.436€/year; one
pos-doc – 17.940€/year; a student who performed dietary questionnaires in Mozambique - 2.500€/year and 33.525€
for the 6BIs/variable periods, leaves 132.011€ for Lab running costs.
1. “Molecular mechanisms of biosynthesis of cancer associated mucin carbohydrate antigens in intestinal
metaplasia”. Fundação para a Ciência e a Tecnologia (POCTI/CBO/44598/2002). PI: Celso Reis. Total
budget for 2003-2006 - 66.600€, extended until April 2007. (2007 – 22.200€).
2. “Molecular mechanisms of biosynthesis of cancer associated mucin carbohydrate antigens in gastric
carcinoma”. Association for International Cancer Research (AICR grant Ref: 05-088). PI: Celso Reis. Total
budget for 2005 – 2008 - 112.320€ (2007 – 37.440€; 2008 -9.360€).
3. “Identification of Glycosylation-associated genes induced by H. pylori in gastric cells: a glycomic approach”.
Fundação para a Ciência e a Tecnologia. POCI/SAU-OBS/56686/2004 PI: Celso Reis. Total budget for
2005-2007: 55.000€ (2007 -13.470€).
4. “A strategy for preventing H. pylori-associated gastric cancer based on materials with specific receptors to
the bacteria – from SAM´s to Gly-R chitosan microspheres” (Project PTDC/CTM/65330/2006). P.I. Cristina
Martins (INEB). Total budget for the group 2008-2010: 71.948€ (2008 – 20.000€).
36
5. “Genes associated to intestinal metaplasia of the gastric mucosa (MUC2 mucin and fucosyltransferase
FUT3): transcriptional regulation and relevance for Helicobacter pylori adhesion” (Project POCI/SAUOBS/55549/2004). PI: Leonor David. Total budget for 2005-2008: 96.680€ (2007 – 29.420€; 2008 –
12.000€).
6. “Lesions of the gastric mucosa in Mozambique and Portugal: the “African enigma”. Study of biologic factors
and lifestyle that contribute to understand the differences in incidence of gastric carcinoma in two countries
with a high prevalence of Helicobacter pylori infection: Mozambique and Portugal”. Fundação Calouste
Gulbenkian (Project FC-68697). PI: Leonor David. Total budget for 2005-2008: 100.000€ (2007 - 33.000€;
2008 – 15.000€).
7. “Identification of signalling pathways involved in Cdx2 regulation in two human models of altered intestinal
differentiation: intestinal metaplasia and juvenile polyposis”. Fundação para a Ciência e a Tecnologia
(Project POCTI/SAU-OBS/55840/2004). PI: Raquel Almeida. Total budget for 2005-2008: 98.500€ (2007 –
28.635€; 2008 – 10.000€).
8. “CDX2 autoregulation in the reversibility/irreversibility of gastric intestinal metaplasia”. Fundação para a
Ciência e a Tecnologia (Project PTDC/SAU-OBD/64490/2006). PI: Raquel Almeida. Total budget for 20082011: 148.500€ (2008 – 48.560€).
9.
“Biological characterization of canine mammary mixed tumours: histogenesis, tumour progression and
genetic alterations”. Fundação para a Ciência e a Tecnologia (POCI/CVT/57795/2004). PI: Fátima Gartner.
Total budget for 2005-2008: 92.225€ (2007 – 22.914€; 2008 – 12.000€).
10. “Cat mammary tumours-Pathologic, Molecular and Cytogenetic approaches”. Fundação para a Ciência e a
Tecnologia (POCI/CVT/62940/2004). PI: Fátima Gartner. Total budget for 2005-2008: 82.927€ (2007 7.500€; 2008 – 17.508€).
11. “In vitro and in vivo manipulation of the expression of Sialyl lexisx and E-cadherin in canine malignant
mammary tumours”. Fundação para a Ciência e a Tecnologia (PTDC/CVT/65537/2006). PI: Fátima
Gartner. Total budget for 2007-2009: 156.178€ (2007 – 50.205€; 2008 – 57.533€).
12. “Secretor and Lewis phenotypes/genotypes in Helicobacter pylori and calicivirus infection”. Fundação para
a Ciência e a Tecnologia (PTDC/SAU-MII/64153/2006). PI: Leonor David. Total budget for 2007-20072010: 130.000€ (2007 – 6.980€; 2008 – 47.440€).
13. “Discovery of novel cancer serum biomarkers based on aberrant post-translational modifications of Oglycoproteins, O-PTM-Biomarkers, and their application to early detection of cancer”. Seventh Framework
Program – Health (Grant agreement nº: 201381). PI: Joyce-Taylor Papadimitriou, King’s College London.
Total budget for the group 2008 - 2010: 264.000€ (2008 – 88.000€).
International collaborations
•
Faculty of Health Sciences of the University of Copenhagen – Ulla Mandel and Henrik Clausen. This
collaboration has been fundamental for characterization of carbohydrate antigens and glycosyltransferases
using unique monoclonal antibodies. We will collaborate with this group for building glycopeptides arrays
with the context of the European project.
•
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 such
complex field.
•
INSERM, Strasbourg – Jean-Noel Freund. This collaboration started in 2006 with the acceptance of JeanNoel Freund to be co-supervisor of the PhD thesis of Rita Barros. Raquel Almeida and Jean-Noel Freund
37
successfully applied for travel money for one year at the CRUP. This collaboration will be essential for the
study of CDX2 regulation in intestinal metaplasia.
•
Universite des Sciences et Technologies de Lille – Anne Harduin-Lepers. This collaboration has been
useful in the cloning and recombinant expression of sialyltransferases.
•
Umea University, Sweden – Thomas Borén. This collaboration has contributed for the establishing of
H.pylori adhesion assays. Thomas Borén acceptance to be co-supervisor of the PhD thesis of Ana
Magalhães.
•
Consortium for Functional Glycomics – The Scripps Research Institute, CA, USA - James Paulson. This
collaboration has provided the use of resources of the Consortium, including Microarray analysis and
knock-out mice.
•
University of Uppsala - Ola Soderberg. This collaboration has allowed the successful establishment of
Proximity-Ligation assays for identification of glycopeptides structures in situ.
•
University of Cologne - Tilo Schwientek. This collaboration is essential for the identification of the cancer
serum glycoproteome in the context of the European project.
Master Thesis
In 2007 one Master student of the group, Joana Gomes, defended her thesis at the Medical Faculty of Porto
“Establishment and characterization of in vitro and in vivo canine mammary tumours cell lines”.
The aim of the work was to establish and characterize canine mammary carcinoma cell lines and to establish their
glycosylation profiles, in an attempt to evaluate their potential as model systems for human carcinoma. Joana
established three canine mammary carcinoma cell lines using fragments of tumors surgically excised. After in vitro
characterization these cell lines were heterotlansplanted in nude mice and they were not tumorigenic. In the second
part of the work an in vitro and in vivo characterization of a previously established cell line, derived from a canine
mammary ductular carcinoma, was performed. Cell motility assays showed that the cells conserved an epithelial-like
architecture and had the ability to move between the edges of an artificial wound in a compact front of migration,
with the cells moving undirectionally. These cells had immunoreactivity for E-cadherin and showed a high
proliferative index. In vivo assays showed that these cells grew when inoculated subcutaneously in the mammary fat
pad of female nude mice and metastasized to lymph nodes, lungs, heart, spleen, kidney and liver. Tumour masses
were histologically identical to the primary mammary tumour lesion and the carbohydrate expression profile, tested
in primary tumours and respective metastases, was not altered.
On the whole, the results indicate that canine
mammary tumours are good models for the study of human breast cancer, both regarding tumour behaviour and
glycosylation profiles.
Plan of activity for 2008
In 2008 the group aims to clarify the mechanisms of intestinal transdifferentiation of the gastric mucosa, including
the clarification of reversibility/irreversibility of the process, to elucidate the FUT2 haplotype – Secretor phenotype
associations and to evaluate the role of glycosylation, in mice knocked out for several glycosyltransferases, in
Helicobacter pylori adhesion/infection. A new strategy will be implemented to visualize in situ peptide-glycan
associations. The group will also study the serum glycoproteome of cancer patients and initiate the synthesis of
glycopeptides for building glycopeptide arrays. Finally, we will use the canine mammary cancer model to evaluate
the role of E-cadherin glycosylation in cancer cell beahaviour.
To clarify the mechanisms of intestinal transdifferentiation (intestinal metaplasia – IM) of the gastric
mucosa, including reversibility/irreversibility of the process. A)The group will explore further mechanisms of
transactivation of the homeobox gene CDX2, by testing the role played by GATA transcription factors as co-factors
38
for the BMP/SMAD pathway, using luciferase assays. B) We will study the auto-regulatory CDX2/CDX2 loop in
gastric and intestinal cell lines using a model where a construct with CDX2 gene flanked by loxP sites will be
transfected with and without removal by cre-recombinase to test for activation of endogenous CDX2 and
reversibility/irreversibility of the activation. We plan to further explore this line by building an in vivo mouse model
that expresses B-galactosidase regulated by CDX2 promoter, where siRNAs for CDX2 will be delivered to the small
intestine, endogenous CDX2 will be knocked-down, and expression of B-galactosidase, if decreased, will confirm
the presence of an active auto-regulatory loop.
To clarify the FUT2 haplotype - Secretor phenotype associations. A) The group will extend haplotyping of the
2nd exon of FUT2 and use the haplotypes to clarify Secretor/non-Secretor phenotypes in saliva and in the gastric
mucosa. B) We will use both systems to explore for implications in two human infections: Helicobacter pylori and
Calicivirus.
To implement a new strategy to visualize in situ peptide-glycan associations. A) A modification of proximity
ligation assay will be implemented to see, in situ, if a given protein is the actual carrier of a glycan – specifically we
will use the technique to visualize which mucins are carrying the Sialyl-Tn structure in intestinal metaplasia and
gastric carcinomas.
To use mice knocked-out for several glycosyltransferases as models for Helicobacter pylori
adhesion/infection . A) Mice knocked-out for several glycosyltransferases (Core2-GlcNAc, FUT2 and ST3Gal-IV)
will be fully characterized for the profile of glycans expression using immunohistochemistry and mass spectrometry.
B) Helicobacter pylori adhesion to sections from the gastric mucosa as well as in vivo infection assays will clarify the
relevance of the glycosylation profiles for adhesion/infection and will also allow to see if upon infection Helicobacter
induces by itself changes in the glycosyltransferase repertoire.
To define the serum glycoproteome of cancer patients and initiate the synthesis of glycopeptides for
building glycopeptide arrays. A) We will use serum samples from gastric, breast, pancreas, lung and ovary
cancer patients, obtained in the context of the European project, to generate 2D-maps from serum glycoproteins,
after depletion of abundant proteins and purification with lectins, where cancer-associated glycopeptides (Tn, STn,
T, ST) will be identified with moAbs and the protein component will be identified by Mass spectrometry. B) The
cancer-serum glycoproteome identification will allow us to use recombinant glycosyltransferases to synthesize, in
vitro, the desired glycoforms, using synthetic peptides or recombinant proteins expressed in E. coli Rosetta 2
strains. The glycopeptides will be used by our Danish partners to build glycopeptide arrays to screen patient’s serum
for antibodies at a pre-clinical stage.
To evaluate the role of E-cadherin glycosylation in canine tumour cells. A) We will use tumour tissues and cell
lines to immunoprecipitate E-cadherin and characterize the glycan structures by mass spectrometry. B) We will
transfect cells with E-cadherin mutant forms for potential N-glycosylation sites and indirectly evaluate the role of Nglycosylation in the behaviour (in vitro and using mouse models) of the manipulated cells.
The group will continue the collaboration with the Epidemiology team and the Cancer Genetics group to
understand the reasons underlying the low incidence of gastric carcinoma and intestinal metaplasia in
Mozambique, despite a high rate of Helicobacter pylori infection - the so called "African enigma".
39
Selected publications:
1. Pinho S, Marcos NT, Ferreira B, Carvalho AS, Oliveira MJ, Santos-Silva F, Harduin-Lepers A, Reis CA:
Biological significance of cancer-associated silyl-Tn antigen: modulation of malignant phenotype in gastric
carcinoma cells. Cancer Letters 249: 157-170, 2007.
2. Peleteiro B, Lunet N, Figueiredo C, Carneiro F, David L, Barros H: Smoking, Helicobacter pylori virulence, and
type of intestinal metaplasia in Portuguese males. Cancer Epidemiology, Biomarkers and Prevention 16: 322326, 2007.
3. Pinho SS, Matos AJ, Lopes C, Marcos NT, Carvalheira J, Reis CA, Gartner F: Sialyl Lewis x expression in
canine malignant mammary tumours: correlation with clinicopathological features and E-cadherin
expression. BMC Cancer 7:124, 2007.
“Other” publications:
Brito C, Escrevente C, Reis CA, Lee VM, Trojanowski JQ, Costa J: Increased levels of fucosyltransferase IX and
carbohydrate Lewis(x) adhesion determinant in human NT2N neurons. J Neurosci Res 85: 1260-1270, 2007.
David L: Lewis antigens. Encyclopedia of Cancer, Springer (in press).
Reis CA: Gastric Cancer. Encyclopedia of Cancer, Springer (in press).
Reis CA: Glycosylation. Encyclopedia of Cancer, Springer (in press).
Saraiva AL, Gartner F, Pires MA: Expression of p63 in normal canine skin and primary cutaneous glandular
carcinomas. Vet J (in press).
Matos AJ, Duarte S, Lopes C, Lopes JM, Gartner F: Splenic hamartomas in a dog. Vet Rec 161: 308-310, 2007.
Gil da Costa RM, Matos E, Rema A, Lopes C, Pires MA, Gartner F: CD117 immunoexpression in canine mast
cell tumours: correlations with pathological variables and proliferation markers. BMC Vet Res 3:19, 2007.
Gil da Costa RM, Rema A, Payo-Puente P, Gartner F: Immunohistochemical characterization of a sebaceous
gland carcinoma in a gerbil (Meriones unguiculatus). J Comp Pathol 137: 130-132, 2007.
Gama A, Paredes J, Gartner F, Alves A, Schmitt F: Expression of E-cadherin, P-cadherin and beta-catenin in
canine malignant mammary tumours in relation to clinicopathological parameters, proliferation and
survival. Vet J (in press).
Do Vale A, Costa-Ramos C, Silva A, Silva DS, Gartner F, dos Santos NM, Silva MT: Systemic macrophage and
neutrophil destruction by secondary necrosis induced by a bacterial exotoxin in a Gram-negative
septicaemia. Cell Microbiol 9: 988-1003, 2007.
Lunet N, Valbuena C, Vieira AL, Lopes C, David L, Carneiro F, Barros H: Fruit and vegetable consumption and
gastric cancer by location and histological type: case-control meta-analysis. Eur J Cancer Prev 16: 312-327,
2007.
Peleteiro B, Lunet N, Santos-Silva F, David L, Figueiredo C, Barros H: Short mucin 1 alleles are associated with
low virulent H pylori strains infection. World J Gastroenterol 13: 1885-1886, 2007.
40
GENETICS, EVOLUTION AND PATHOLOGY
1. Group Composition
Group Leader: Jorge Rocha, BSc, PhD, University of Porto
Post-doc fellows: Susana Seixas (BSc, PhD, University of Porto) Sandra Beleza, (BSc, PhD,
University of Porto) and Nevyana Ivanova (MSc, PhD, University of Sofia; joined the group in
October 2007).
PhD students: Margarida Coelho (BSc, University of Porto)
Project grant students: João Pedro Oliveira (BSc, University of Coimbra), Zélia Ferreira (BQ;
University of Porto joined the group in November 2007), Joana Campos (BSc; University of
Coimbra, joined the group in October 2007)
Undergraduate students: Isabel Alves, Cláudia Martinho (Biological Sciences students, University
of Porto; from March to July 2007)
2. Research interests and goals
The major research interest of this group is to study the evolutionary forces that shaped the
current patterns of human genetic diversity and their implications in health and disease. Our
research focus both on the study of specific populations and on the analysis of the evolutionary
history of particular genes. Presently, most of our scientific activities are structured around the
following three major, interconnected, areas: 1) the genetic structure and history of African
populations (including Cape Verde, Mozambique, São Tomé and Angola); 2) the evolutionary
history of genes involved in genetic disease and human adaptation to environmental changes
(including serine protease inhibitor (SERPIN), malaria resistance (HBB, HBA and FY), lactose
tolerance (LCT) and skin colour genes (SLC24A5, SLC45A2 TYRP, KITLG); 3) the study of
admixed populations (like Cape Verde) to understand the genetic basis of complex traits with
anthropological relevance (like skin colour) or biomedical interest (like obesity and
hypertension).
3. General overview of 2007
3.1 Funding
The year of 2007 was a turning point in the consolidation and growth of our group. All the
projects that we had submitted in 2006 were finally approved. The projects are nested in our
three major areas of research and are coordinated by different Principal Investigators (see
Annex 1):
a) On the edge of the Bantu expansions: inference of recent population history with
independently evolving haplotypic systems. (Funded by FCT; PTDC / BIABDE / 68999/
2006; 122 000 Euros)
b) Looking for evidences of human adaptation in the proteolysis universe: the case study of
serine protease inhibitors. (Funded by FCT; PTDC / SAU-GMG / 64043 / 2006; 178 700
Euros)
41
c) Understanding the genetic architecture and evolution of human pigmentary traits:
admixture mapping studies in Cape Verde. (Funded by FCT; PTDC / BIA-BDE / 64044 /
2006; 121 000 Euros)
d) Using Admixed Populations to Study the Genetic Basis of Obesity and Hypertension: the
Case-Study of Cape Verde and of the Portuguese Population from the Regions of Tejo,
Sado and Guadiana River Basins. (Funded by the pharmaceutical company SanofiAventis; 111 000 Euros).
The first project, coordinated by Jorge Rocha, is linked to our interest in the genetic structure
and history of African populations. The second project, coordinated by Susana Seixas, is related
with our interest in the evolutionary history of genes that may have a medical interest or may be
potential targets for selection. The third and the fourth projects, coordinated by Sandra Beleza,
are both centered on the use of the admixed population of Cape Verde to study the genetic basis
of complex traits. The third project, financed by FCT, is focused on the analysis of pigmentation
traits; the fourth project, financed by Sanofi-Aventis, is centred in the study of obesity and
hypertension.
3.2 Participation in projects from other groups
In addition to the projects coordinated by our own researchers, we became involved in research
teams of projects led by colleagues from other groups in IPATIMUP. Jorge Rocha is now a
team member of the project entitled Secretor and Lewis phenotypes/genotypes in Helicobacter
pylori and calicivirus infection led by Leonor David, which was approved by FCT during 2007.
This collaboration, aimed to study of the relationship between the diversity of FUT genes and
susceptibility to gastro-intestinal infection, is rooted on a previous co-supervision of the graduation
work of Luís Pedro Resende, who studied the contribution of polymorphisms in human FUT2 gene
to the secretor phenotype. Still within the realm of the relationship between host’s genetic variation
and susceptibility to infectious and carcinogenic agents, we are collaborating with José Carlos
Machado in the Work Package on Human genetic markers and risk of infection with Helicobacter
pylori, from the European consortium on Parasite and host genetic diversity in Helicobacter
infections (HELDIVNET) financed by ERA-NET PathoGenoMics.
3.3 Changes in group composition
Thanks to the new financial support we were able to recruit a new pos-Doctoral fellow
(Nevyana Ivanova) and two young graduate students (Zélia Ferreira and Joana Campos) that
started to work in the SERPIN and Cape Verde projects. A third graduate student (Isabel Alves)
has been recruited just before the end of 2007 to work in the project on the Bantu expansions.
During 2007, two researchers changed the nature of their link to the group. Susana Seixas was
selected for a job position on the terms of the Program Ciência 2007 aimed at the recruitment of
post doctoral researchers and promoted by FCT. Susana will remain in the group as a researcher
of IPATIMUP, starting May 2008. On the other hand, João Pedro Oliveira, has ceased his
activity as a graduate student financed by the project entitled “Biocultural Adaptation: human
adaptive responses to changes in subsistence economy”, and successfully submitted an application
for a PhD grant to FCT with a project entitled “The evolutionary history and geographic spread
of advantageous variants that confer resistance to malaria in Sub-Saharan African
populations”. The project will start in January 2008 with co-supervision of Jorge Rocha and
42
Prof. Anna Di Rienzo, from the Department of Human Genetics of the University of Chicago (see
below).
3.4 Undergraduate training
As part of our support to the training of undergraduate students, we hosted Isabel Alves and
Cláudia Martinho, from the Faculty of Sciences of Porto University, who stayed in the lab from
March to July 2007, and made graduation works on the evolutionary history of genes involved
in skin pigmentation (see below).
In pace with our commitment to add a strong educational/formative component to the Cape Verde
project, we hosted two Biology students from the local Instituto Superior de Educação (ISE),
Jailson Lopes and Venceslau Sanches, who stayed in our lab during February and March 2007. Both
students were trained in basic laboratory methods currently used in molecular population genetics,
and gathered small datasets that could be included in their graduation works. Using samples
collected in the island of Santiago, Jailson made a pilot screen of the β-globin S mutation and G6PD
deficiency in two locations of the island. Venceslau has studied the distribution of two functionally
relevant ancestral informative markers (AIMs)- the Duffy blood group (FY) and lactose tolerance
(LCT)- to evaluate admixture levels in Santiago. In July 2007 Jorge Rocha visited Cape Verde to
supervise the final steps of the graduation work of Jailson Lopes entitled “Hereditary diseases
with Mendelian inheritance: concepts, methods of study, screening and prevention”. The visit to
Cape Verde also served to further promote our research proposal in the local University. After his
graduation Jailson Lopes made an application to the Masters Course in Biodiversity, Genetics
and Evolution, from the Faculty of Sciences (Porto University). However, despite being
accepted in the course, he could not gather any financial support since most funding contests
were already closed by the time he could prepare his application. During 2008 we will make
another, more timely, attempt to bring Jailson to Portugal and proceed with his post-graduate
education. Venceslau Sanches has also completed his graduation work during 2007. This work
entitled “Analysis of two ancestral informative markers in the island of Santiago (Cape
Verde)”was supervised by Sandra Beleza.
3.5 Graduate training
During 2007, the Margarida Coelho proceeded with the activities related to the second year of
her PhD work, by concentrating on the genetic characterization of populations from southern
Angola (see below).
We have hosted two PhD students from other laboratories, Felicia Gomez and Edvar Ehler, who
requested to visit our lab in the course of their work. Felícia Gomez, is a student from the
Hominid Paleobiology Doctoral Program at the George Washington University (USA), who is
currently working under the supervision of Prof. Sarah Tishkoff. Felicia’s visit was essentially
meant to revitalize an ongoing collaboration between Jorge Rocha and Sarah Tishkoff on the
study of patterns of nucleotide and haplotype variation at ICAM-1 (intercellular adhesion
molecule-1), a vascular endothelial receptor that is thought to play a role in malaria
susceptibility. Edvar Ehler is a PhD student from the Department of Anthropology of Charles
University (Czech Republic), who seek advice in data analysis for his work on the Vlach
population isolates of the Czech Republic.
Finally, Sílvio Saranga, a student from Mozambique that was co-supervised by Jorge Rocha and
Professor José Maia from the Faculty of Sports of Porto University, has successfully presented
his PhD thesis entitled “Genetic and environmental factors in somatic growth, adiposity
43
patterns, somatotype and physical fitness: a study in nuclear families from the rural region of
Calanga-Mozambique”.
3.6 Visits to other labs
As part of her collaboration with Prof. Mark Shriver, Sandra Beleza has visited the Department
of Anthropology of Penn State University, where she gathered and analysed data on admixture
mapping and the evolutionary history of pigmentation genes (see below). Sandra was also
invited to visit the labs of Profs. Greg Barsh and David Kingsley at Stanford University. During
this visit, a new collaboration was started with Prof. Barsh’s team.
Jorge Rocha has spent two months as a Visiting Professor at the Department of Human Genetics
of the University of Chicago, where he participated in the PhD course on “Human Genetic
Variation and Disease”, led by Profs. Anna Di Rienzo and Jonathan Pritchard. The visit to
Chicago was also useful to the develop the framework of future collaborations with Anna Di
Rienzo’s lab, including the co-supervision of João Oliveira PhD work. During his stay in
Chicago Jorge Rocha has worked on the modelling of selection and diffusion of adaptive
mutations with the help of Drs. John November and Graham Coop from the same Department (see
below).
3.7 Theses
3.7.1 PhD thesis
-
Genetic and environmental factors in somatic growth, adiposity patterns, somatotype and
physical fitness: a study in nuclear families from the rural region of CalangaMozambique”. Sílvio Saranga (supervised by Jorge Rocha and José Maia).
3.7.1 Graduation theses
-
Evolutionary history of the candidate gene for pigmentation TYRP1, in human
populations. Isabel Alves (supervised by Sandra Beleza and Jorge Rocha).
-
Evolutionary history of normal variation in skin colour: the case study of he KITLG
gene. Cláudia Martinho (supervised by Sandra Beleza).
3.8 Participation in Scientific Meetings
During 2007, group members participated in the following scientific meetings: annual meeting
of the American Association of Physical Anthropology; annual meeting of the American
Society of Human Genetics; annual meeting of the Society for Molecular Biology and
Evolution; 17th Congress of the Italian Association of Anthropology; annual meeting of the
Portuguese Society of Human Genetics.
3.8.1 Poster presentations
-
Coelho M, Rocha J, Beleza S (2007) On the edge of the Bantu expansions: patterns of
mt-DNA and Y-chromosome variation in southwestern Angola. 57th Annual Meeting of
the American Society of Human Genetics. October 2007. San Diego, USA.
44
-
Gomez F, Tomás G, Reed F, Tishkoff SA, Rocha J (2007) Patterns of nucleotide
diversity and potential signatures of natural selection at ICAM-1. 57th Annual Meeting
of the American Society of Human Genetics. October 2007. San Diego, USA.
-
Quillen EE, Beleza S, Parra EJ, Rocha J, Pereira RW, Shriver MD (2007) Iris color and
texture in admixed populations. Annual Meeting of the American Association of
Physical Anthropology. March 2007. Philadelphia, USA.
-
Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A (2007) Sequence
diversity at the proximal 14q32.1 SERPIN subcluster: evidence for
natural selection favoring the pseudogenization of SERPINA2. Annual Meeting of the
Society of Molecular Biology Evolution. Halifax, Canada.
3.8.2 Oral communications
-
Beleza S, Martinho C, Alves I, Parra E, Shriver M, Rocha J (2007) Patterns of
microsatellite variation within the Kit Ligand and Tyrosinase Related Protein-1 genes:
implications for the evolutionary history of skin pigmentation in human populations.
Annual Meeting of the American Society of Human Genetics. 7th Annual Meeting of the
American Society of Human Genetics. October 2007. San Diego, USA.
-
Coelho M, Alves C, Coia V, Luiselli D, Uselli A, Hagemeijer T, Amorim A, Destro- Bisol
G, Rocha J (2007) Ethnogenesis in the slave trade: the case study of São Tomé (Gulf of
Guinea). Annual Meeting of the American Association of Physical Anthropology. March
2007. Philadelphia, USA.
3.8.3 Key Lectures
-
Rocha J (2007) Slave trade and human genetic microdifferentiation. Opening plenary
lecture in the Molecular Anthropology Section of the 17th Congress of the Italian
Association of Anthropology.
3.9 Publications
3.9.1 Published papers
-
Batini C, Coia V, Battagia C, Rocha J, Pilkington MM, Spedini G, Comas D, DestroBisol G, Calafell F (2007) Phylogeography of the human mitochondrial L1c haplogroup:
genetic signatures of the prehistory of Central Africa. Molecular Phylogenetics and
Evolution 43: 635-644.
-
Miller C, Beleza S, Pollen A, Schluter D, Kittles R, Shriver MD, Kingsley DM (2007)
Cis-regulatory changes in Kit ligand expression and parallel evolution of pigmentation
changes in sticklebacks and humans. Cell 131: 1179-1189.
-
Saranga S, Maia J, Rocha J, Nhamtubo L, Prista A (2007) Crescimento somático na
população africana em idade escolar. Estado actual do conhecimento. Revista
Portuguesa de Saúde Pública 25: 85-99.
45
-
Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A (2007) Sequence
diversity at the proximal 14q32.1 SERPIN subcluster: evidence for natural selection
favouring the pseudogenization of SERPINA2. Molecular Biology and Evolution 24:
587-598.
3.9.2 Papers accepted for publication (in press)
-
Coelho M, Alves C, Coia V, Luiselli D, Uselli A, Hagemeijer T, Amorim A, Destro- Bisol
G, Rocha J. (2008) Human microevolution and the Atlantic slave trade: the case study of
São Tomé (Gulf of Guinea). Current Anthropology (in press).
-
Saranga S, Prista A, Nhatumbo L, Beunen G, Rocha J, Blangero S, Maia J (2007)
Heritabilities of somatotype components in a population from rural Mozambique. American
Journal of Human Biology (in press).
3.9.3 Papers submitted for publication
-
Azevedo M, Eriksson S, Mendes N, Serpa J, Figueiredo C, Resende LP, RuvoënClouet N, Haas R, Borén T, Le Pendu J, David L: Infection by Helicobacter pylori
expressing the BabA Adhesin is influenced by the Secretor phenotype
4. Major research activities
The major research activities developed in 2007 were related with the three major areas
identified in section 1.
4.1 Characterization of the genetic structure and history of African populations
The activities corresponding to this area of research were essentially developed by Margarida
Coelho who entered the second year of her PhD project during 2007. Margarida has now
concluded the data collection phase of her work on the genetic characterization of populations
from the Namibe desert in Southern Angola. This work, which is being prepared for submission,
is included in the project entitled On the edge of the Bantu expansions: inference of recent
population history with independently evolving haplotypic systems (PTDC/BIABDE/68999/2006), and uses samples collected by Jorge Rocha and Sandra Beleza in a previous
field trip that had the collaboration of the local health authorities and the provincial government
of Namibe.
Current studies on African genetic variation and the Bantu expansions are still hampered by
poor sampling in vast regions from sub-equatorial Africa. In Angola, the area beyond the
Cuanza river remains a major gap that prevents the complete understanding of the dramatic push
of Bantu peoples towards the arid regions of Southwest-Africa. The particular anthropological
importance of Namibe is due to a number of biocultural features that are crucial for unraveling
the population history underlying Bantu migrations in Africa over the past few thousand years.
First, the spread of Bantu peoples into the dry steppes of Southwest-Africa must have been
associated with a profound change in the agricultural lifestyle that had predominated in well
irrigated lands. Second, the presence in southern Angola of exclusively pastoral Bantu groups
sharing their mode of life with neighbor non-Bantu Khoikhoi groups poses intriguing questions
about the nature of the interactions between the vanguard of West Bantu speakers and the
autochthonous peoples from the desert. For example, it is still not known to which degree the
46
present pastoral specialization of some Bantu speakers was brought with them or was developed
in adaptation to a new environment by the adoption of most of the Khoikhoi way of life. Finally,
the fact that Southwest-Africa is cut off from the major pastoral areas in East Africa by a belt of
tse-tse infested areas where cattle cannot be kept is a major challenge in the reconstruction of
the migration routes that led to the emergence of a cattle-herding zone in that particular area.
In this work Margarida has studied the patterns Y-chromosome, mtDNA, and lactase genetic
variation in 359 individuals living in the Province of Namibe, most of them belonging to four
groups that speak languages from the West-Savannah Bantu branch: Umbundo, NyanekaNkhumbi, Nyemba and Herero/Kuvale. Presently, we are analyzing the data by addressing six
major issues: a) the relationship between Southwest Angola and other major areas of Africa; b)
the degree of interaction between the west and eastern streams of the Bantu expansions; c) the
extend of the differentiation between the various ethnic groups from Namibe; d) the levels of
admixture between Bantu and Khoisan groups; e) the possibility that typical East African
lactose tolerance mutations may have reached the groups from southern Angola that rely more
heavily on cattle raising; and f) the contribution of South-West Africa to the Slave trade to the
Americas. To address the first issue, we are applying admixture models (Bertorelle and
Excoffier, 1998) in which groups from South-West Angola are treated as hybrid populations
deriving variable proportions of their genetic heritage from different putative parental areas. So
far, estimates of the mtDNA/Y genetic contribution from different parental areas indicate that
the gene pool from Namibe is predominantly derived from West-Central and West Africa,
although a substantial contribution from non-Bantu East-Africans was also found in the mtDNA
pool, similarly to the Southeast Bantu groups that have been studied so far. These results
suggest that the western Bantu mtDNA pool is a mosaic, with a number of components of
distinct ancestries, including an important contribution from east African non-Bantu farmers. Ychromosome lineages appear to be geographically more homogeneous, with insignificant levels
of assimilation of non-Bantu lineages, suggesting a sex-biased, male driven model of dispersion.
Furthermore, we are using newly developed computational tools implementing likelihood based
approaches to infer key microevolutionary parameters (eg time of splitting, population sizes,
growth rates, and migration rates) and test alternative models of population history concerning
the interaction between the west and eastern streams of the Bantu expansion. Specifically, we
are testing alternative population histories related to the Isolation with Migration (IM) class of
models, in which pairs of populations that may vary in size diverge from a single ancestral
population and are allowed to exchange migrants (Hey and Nielsen, 2004). Our preliminary
results, obtained by comparing our own data with published data from Mozambique, are
particularly robust for the Y-chromosome. So far, these results suggest that both streams of the
Bantu expansion have split about 2000 years ago from a common source, but did not exchange
significant amounts of migrants after the initial separation, contrary to most conclusions from
the literature (Jobling et al. 2004), which are not based on explicit population history models.
Taken together, our observations indicate that the genetic resemblance between the Southwest
and Southeast Bantu populations is not a derived condition resulting from gene flow, but rather
an ancestral genetic property reflecting a recent common origin. Under this scenario, the EastAfrican mtDNA component observed in the Southwest would not result from migration of
Southeast groups, and would have more likely resulted from ancient East-West interactions that
preceded the push of Bantu peoples towards the south.
Another important aspect of our results is the finding that the most exclusively pastoral groups
from Namibe- Nyaneka-Nkhumbi and Herero- are very distinct from their neighbors in having
high levels of assimilation of Y chromosome (5-11,5%) and mtDNA (6,23-23%) Khoisan
lineages. Moreover, we have made the striking observation that the -14010C LCT mutation for
47
lactose tolerance, originally described in East-Africa cattle herders, is found at relatively high
frequencies (7-11,3%) in the Nyaneka-Nkhumbi and Herero pastoral groups, to which it is
virtually confined in southern Angola. The -14010C LCT mutation is part of a suit of lactose
tolerance mutations that have been recently described in East African pastoral populations and
seem to have reached high frequencies due to selection for the ability to digest milk in
adulthood (Tishkoff et al. 2007). Since these mutations are different from the -13910T mutation
that is found in lactose-tolerant individuals from Eurasia and from the Fulani group of WestAfrican herders, which were both previously studied by Margarida (Coelho et al. 2005), their
occurrence in East Africa provides a marked example of convergent evolution due to strong
selective pressures resulting from shared cultural traits (Tishkoff et al. 2007). The most
straightforward interpretation of our finding of the -14010C mutation in southern-Angolan is
that it was introduced into the Bantu herders by direct contact with non-Bantu Cushitic
pastoralists in East Africa from whom they might have acquired the cattle. In this case, our
observation could represent one of the first genetic evidences for a direct link between the
pastoral scenes from East and Southwest Africa. However, if we take into account the joint
occurrence of the -14010C mutation and the specific Khoisan lineages we can’t rule out the
intriguing possibility that this mutation was brought to Southwest Africa by Khoikhoi herders
originating in East Africa and was subsequently transferred to Bantu peoples that adopted the
Khoikhoi pastoral way of life. In any case, these results provide an interesting link to
Margarida’s former work on lactose tolerance (Coelho et al. 2005) and nicely illustrate the
interdependence between the study of the evolutionary history of particular traits and the
analysis of population structure, which constitute two major axes of our present research.
Still in the area of the genetic structure of the African populations we have finally received in
2007 the confirmation from the journal Current Anthropology that our work on the detailed
characterization of the genetic structure of the population of São Tomé was accepted for publication.
The work, which has been substantially developed by Margarida Coelho and Cíntia Alves,
results from a broad collaborative effort that includes the Population Genetics group at
IPATIMUP, led by Prof. António Amorim, the Universities of Rome1 and Bologna and the
Center for linguistics from the University of Lisbon. The work was also presented in an oral
communication the annual meeting of the American Association of Physical Anthropology (AAPA),
that was held in Philadelphia (USA) in March 2007.
4.2. Study of the evolutionary history of genes involved in human adaptation and/or disease
This area of activity may be divided in three parts. The first corresponds to the research project
Biocultural Adaptation: human adaptive responses to changes in subsistence economy and was
mainly developed by Jorge Rocha and João Oliveira, the student that received a grant from the
project. The second part is related to the activities of Susana Seixas, who begun to develop her
project entitled Looking for evidences of human adaptation in the proteolysis universe: the case
study of serine protease inhibitors. Finally, the third part consists of the activities related to the
undergraduate works of students Isabel Alves and Cláudia Martinho on the evolutionary history of
two pigmentation genes.
4.2.1. Studies on the evolutionary history of β-globin S mutation
Early studies of HBB*S using less than 15 restriction fragment length polymorphisms showed
that this allele is associated with four major haplotypes, each restricted to different areas in
Africa. However, the low resolution of these studies does not allow performing stringent tests of
the alternative hypotheses for the geographic segregation of HBB*S haplotypes. Presently, the
geographic distribution of HBB*S haplotype variation is thought to be either the result of
48
recurrent mutation in different geographic areas or the outcome of haplotype differentiation due
to recombination and/or gene conversion during the spread of a unique recent mutation.
Our goal was undertake an empirical characterization of the geographic structure of high
resolution haplotypes linked to the HBB*S in order to infer its history of selection and dispersal
in Sub-Saharan Africa.
In the year of 2007 João Oliveira concluded the data collection on the intra-allelic variation linked to
the S mutation of the β-globin gene (HBB*S). Overall he collected a total of 387 HBB*S alleleles
from 11 African countries and characterized the haplotype backgrounds linked to these alleles by
using 11 microsatellite loci located at different distances from the β-globin gene.
The data on the geographic structure of intra-haplotypic variation is presently being used to
discriminate between alternative scenarios for the evolution of this malaria-protective mutation.
A substantial part of Jorge Rocha’s stay at the University of Chicago was dedicated to outline
the conceptual and computational approach allowing the problem to be tackled within a
simulation framework. Presently, our strategy of data analysis relies on the use of computer
simulations to generate quantitative expectations for patterns of linked genetic variation based
on different evolutionary scenarios, followed by the comparison of these expectations against
the observed population genetics data. After returning from Chicago, Jorge Rocha has teamed
up with Prof. António Múrias, from the Center for Biodiversity Research (CIBIO) in the
University of Porto, to write a program that simulates the spread of haplotypes carrying the
advantageous alleles, using classic wave of advance models within stepping-stone frameworks
across arrays of populations. The parameters of the models to be tested include the initial
position of the mutation, population sizes and population growth rates, selection coefficients,
migration rates, recombination rates, mutation rates and allele ages.
By comparing the parameters of allele spread under different simulated scenarios to the
observed data, we are convinced that we were able to consistently reject most evolutionary
scenarios involving the generation of haplotype heterogeneity during the geographic spread of
the HBB*S. Since the alternative recurrent mutation scenario would involve the highly unlikely
occurrence of the same mutation at least 4 times, we are currently developing and testing a new
evolutionary hypothesis implying that the distribution of the HBB*S mutation resulted from
selection acting on standing variation. According to this hypothesis, HBB*S is an old mutation
(>11 000 years) that was spread into a highly structured framework of populations, with low
levels of gene flow, and rose locally in frequency only recently when malaria become a very
strong selective agent.
Although we still didn’t have conclusive results regarding this latter hypothesis, we are
especially pleased with the work on HBB*S, since it represents a considerable progress in our
ability to analyse and interpret data. It is the first time that we have developed a tool for data
analysis that was specifically tailored for the problem at hand, while being flexible enough to be
applied in other contexts. Moreover, this work represents our first attempt to deal quantitatively
with an evolutionary problem in a geographically explicit context. Finally, the work will be
included in João Pedro PhD workplan, which aims to further extend the study of the geographic
structure of haplotype variation to other advantageous variants.
49
4.2.2. Studies on the evolutionary of Serine Protease Inhibitors (SERPIN)
This area of research is essentially co-ordinated by Susana Seixas who begun her second posDoc grant in June 2007 in collaboration with Prof. Anna Di Rienzo from the Department of
Human Genetics of the University of Chicago. Meanwhile, Susana was selected for a job
position on the terms of the Program Ciência 2007 and begun to work on her project entitled
Searching for signatures of natural selection in the proteolysis universe: the case study of serine
protease inhibitors (SERPINs). This project has the collaboration of a young graduate
researcher (Zélia Ferreira) and one pos-Doc fellow (Nevyana Ivanova).
Previously, Susana had found a strong signature of natural selection favoring a 2kb deletion lying
in a genome region that was generally considered to be a pseudogene: SERPINA2 (Seixas et al.
2007). Based on this result and on preliminary evidence that mRNA from a non-deletion variant of
SERPINA2 was synthesized in different tissues, we hypothesized that natural selection is favoring
the active pseudogenization of this gene (Seixas et al. 2007).
Susana’s project builds upon this line of work and aims to study the evolutionary history of
genes involved in proteolytic reactions and to characterize the functional properties of variants
that can be considered potential targets for selection. During 2007, Susana has begun to set the
experimental framework for characterizing the functional activity of SERPINA2. So far, she has
finnished the amplification of SERPINA2 from a cDNA library and has successfully cloned
different variants of SERPINA2 into the pLenti6/V5 vector. The next step will be to make viral
constructs containing the SERPINA2, to infect animal cells and to select positive SERPINA2cell lines. This work is presently being done with the collaboration of the Cancer Genetics group
led by Dr. Raquel Seruca.
Another aspect of the Susana’s work involved the development of resequencing strategies to
evaluate potential signatures of selection that were previously found in three different SERPINs
through the analysis of the HapMap public database. In addition, Susana has performed
preliminary comparative genomics evaluations focused in these candidate regions and identified
a wide variety of evolutionary processes, including successive gene duplications,
pseudogenizations, neofunctionalizations, within SERPINBs from chromosome 18, and
structural rearrangements involving gene inversions and deletions in the WFDC cluster located
in chromosome 20.
4.2.3. Studies on the evolutionary history of skin pigmentation genes
During 2007 we have started the analysis of the evolutionary history of four pigmentation genes
(TYRP1, KITLG, SLC24A5 and SLC45A2), based on the analysis of microsatellite haplotype
diversity around these genes in an extended dataset of populations from West Africa, Europe
and East Asia. The aim of this work is to evaluate the age, geographic origin and major factors
influencing the current distribution of variants with functional effects on skin pigmentation.The
work is part of the project entitled Understanding the genetic architecture and evolution of human
pigmentary traits: admixture mapping studies in Cape Verde and is being developed with in
collaboration of Profs. Esteban Parra (University of Toronto, Canada) and Mark Shriver
(Pennsylvania State Universiy, USA).
Preliminary analyses of TYRP1 and KITLG, have been presented by Sandra Beleza in an oral
communication at the annual meeting of the American Society of Human Genetics (San Diego,
October 2007), and were the subject of the graduation theses of Isabel Alves and Cláudia
50
Martinho, two students from the Science Department of the University of Porto. So far, we have
characterised the levels of intra-allelic diversity of KITLG and TYRP1 lineages defined with tag
SNPs rs642742 and rs2733831, by analysing 4 linked microsatellites from a sample of African
and European populations.
The KIT ligand (KITLG) gene controls the proliferation, migration, differentiation and survival
of KIT receptor-expressing melanocytes. The Tyrosinase Related Protein 1 (TYRP1) is involved
in the synthesis of eumelanin from dopaquinone. By applying a variety of neutrality tests to
publicly available genomewide SNP data resources, in a previous collaborative study Sandra
and her colleagues have found that non-African populations had strong signals of positive
selection in a genomic region surrounding the KITLG gene (McEvoy et al. 2006). Among the
many adjacent SNPs in the KITLG region, rs642742 was selected as a candidate target for
selection because it shows extreme frequency differences between human populations,
including frequencies of >90% for the ancestral allele in Africans switching to >80% for the
derived allele in both Europeans and East Asians. Moreover, the derived rs642742 was found to
alter a noncoding sequence that is highly conserved in mammals and could affect KTLG
expression. Further evidence for a role of rs642742 was obtained by Sandra in an admixture
mapping study showing a significant association between rs642742 genotypes and skin
pigmentation (Miller et al. 2007; see below). The TYRP1 gene was also identified as a target for
positive selection (McEvoy et al. 2006). However, contrary to KITLG whose signal of selection
encompasses both European and Asian populations, selection in TYRP1 seems to have affected
only European populations. We have used the SNP rs2733831 to tag TYRP1 lineages because it
lies in a much conserved intronic region of the gene and displays high frequency differences
between Africans and Europeans, with a predominance of ancestral alleles in Africans (>95%)
and derived alleles in Europeans (>50%). So far, our results show that the derived lineage for
TYRP1 dates back to 15,400–26,100 years ago and retains a strong signal of selection consistent
with previous SNP haplotype data, as indicated by coalescent-based simulation tests that we
have specifically developed to deal with intra-allelic microsatellite variation under different
demographic scenarios. The age of the derived allele in rs642742, close to KITLG, was
estimated to be considerably older (31,080 years) in accordance to its more wide distribution
across European and Asian populations. However, unlike TYRP1, our neutrality test based on
microsatellite intra-allelic variation fails to confirm the signal of selection in KITLG detect by
using SNPs. Such dissociation between microsatellite and SNP data may be best explained by
assuming that KITLG underwent a much older selective episode than TYRP, which can no
longer be detected due the fast recover of intra-allelic variation associated with high
microsatellite mutation rates.
Taken together our data indicate that current pigmentary variation might have been shaped by
multiple selective episodes occurring over different time scales and/or under diverse selection
coefficients. We hope that our future work may help to disentangle specifically which and types
of selection have been important in determining pigmentary trait variation in different human
populations.
4.3 Characterization of admixed populations to study the genetic basis of complex
phenotypes
This area is currently led by Sandra Beleza, who had entered the second year of her post-doc
grant.
51
Sandra finally got financial support for her post-doc project in the second semester of 2007.
Two different grants were recommended for funding. The first grant, entitled Understanding the
genetic architecture and evolution of human pigmentary traits: admixture mapping studies in
Cape Verde. The second grant, entitled Using Admixed Populations to Study the Genetic Basis
of Obesity and Hypertension: the Case-Study of Cape Verde and of the Portuguese Population
from the Regions of Tejo, Sado and Guadiana River Basins was submitted to the pharmaceutical
company Sanofi-Aventis in March 2007. The project approved by Sanofi-Aventis aims to apply
admixture mapping strategies to evaluate the genetic and environmental relative contributions to
the obesity and hypertension phenotypic variation observed between population groups.
After receiving financial support, Sandra immediately started the field work related with both
projects, helped by Joana Campos, a FCT project granted student. They both visited Cape Verde
(Santiago Island) during the months of November and December 2007 to start the collection of
2000 unrelated individuals objectively measured for skin and eye pigmentation, hypertension
and obesity-related phenotypes (first aim of the project). The most important activities of this
type of sampling are: a) to perform sociodemographic questionnaires; b) to collect biological
material for DNA extraction; c) to collect phenotypic data (blood pressure, anthropometrical
measures related with obesity, skin reflectometry and eye pictures). During this first phase of
the fieldwork, 326 samples were collected in Cidade da Praia (an urban area) and 57 in the rural
village Ribeirão Manuel, district of Santa Catarina. The total number of individuals sampled
was 383. The sampling was undertaken with the participation and collaboration of three students
from the Instituto Superior de Educação (ISE), as part of the educational/formative component
of the project. Two of the students, Jailson Lopes and Venceslau Sanches had previously used
samples collected in a pilot phase of the project to make their graduation theses (see above;
section 3.4). The third student will now take advantage of the data collected in the field work to
elaborate her graduation thesis under the supervision of Sandra.
During 2007, Sandra had also the opportunity to actively collaborate in a study that
demonstrated that the KITLG gene is involved in parallel evolution of pigmentation in
vertebrates as different as humans and the stickleback fish (Miller et al. 2007). By using highresolution mapping and expression studies, this study shows that light gills and light ventruns in
stickleback fish map to a divergent regulatory allele at the KITLG gene, in which derived
regulatory alleles are also shared by light skin populations from Europe and Asia (see above).
Sandra’s collaboration helped to detect signals of positive selection in European and Asian
populations that map to large intergenic regions flanking KITLG. In addition, Sandra was
involved in the identification of the ancestry informative SNP rs642742 as a candidate target for
selection and in the demonstration by admixture mapping that rs642742 variation has a
significant effect in human skin colour. By showing that similar genetic mechanisms may
underlie rapid evolutionary change in fish and humans, this study provides a very nice
illustration on how model experimental organisms, population genetics and evolutionary theory
may be bonded to address both functional and historical aspects of genetic variation.
5. Work plan for 2008
As stated in the beginning of this report, 2007 was a turning point in the life of our group.
Thanks to the funding strategy that we had developed in the former year, we were able to finally
initiate research projects that will have a key role in unfolding the research interests of all group
members. This has allowed us to acquire a more viable dimension, and has increased the
prospects for group consolidation by favoring the stabilization of our research focus in three
clearly identifiable areas, which are closely interconnected. Therefore, our major priorities for
52
2008 are to proceed according to the major goals defined by the research projects we have won
(see Annex 1).
One aspect of our activity that has been repeatedly subjected to inquire is the extension of our
collaboration and interaction with other groups in IPATIMUP. We are fully aware that this is a
critical dimension in the life of any group, although we don’t think that such collaboration is as
unproductive as it might seem, especially when the small dimension of our group is taken into
account. After all, the publication of two papers in collaboration with the Cancer Genetics group
(Suriano et al. 2006, Seixas et al. 2007) the recent acceptance for publication of a paper in
collaboration with the Population Genetics group (Coelho et al.) and the submission of a paper
in collaboration with the Carcinogenesis group (Azevedo et al.), not to mention participation in
common projects (see section 3.2), clearly indicate that this is not an overlooked dimension of
our work. However, we recognise that in the future it is desirable to develop a closer link to
biomedical research by bridging the epistemological gap that separates our core interest
(evolutionary biology) from most of IPATMUP’s groups core field of research (functional
biology). What kind of approach should be followed to accomplish this goal? An obvious
strategy would be to re-orientate research interests towards subjects that are more overtly
connected with medical and clinical research. This approach seems to be straightforward and
may apparently lead to quick results. However, besides the disruptive aspects of switching
people’s dearest subjects of research, this option risks to overlook an important difficulty: as
recognised by Mayr (1961), evolutionary and functional biology are indeed two separate fields
that probably will never merge, although both need to be integrated to produce a coherent
explanation of the biological world. To fully understand diseases currently affecting human
populations it is necessary to understand population history and the evolutionary factors that
lead to present genetic variation. Similarly, to have a comprehensive view of evolution it is
indispensable to know something about the most intimate aspects of cell function and the
pathological consequences of functional disruption. However, it is our opinion that any
approximation between the two main areas of biology must be rooted in the acknowledgement
of the differences that separate them. Paradoxically, this may sometimes imply an even more
deep involvement in research subjects that are apparently far removed from clinical research.
However, we are convinced that, in the end, the knowledge gathered in such studies will be
useful to bridge gaps and establish collaborations. For example, it was our interest in natural
selection (Seixas et al. 2007) that made us become interested in performing functional assays of
protease inhibition in collaboration with Raquel Seruca’s group; it was our interest in phylogeny
that led us to collaborate with the same group in a classification scheme for inferring the
pathogenic significance of CDH1 mutations (Suriano et al. 2006) ; and it was our interest in
Cape Verde and admixed populations that led us to participate in a work dealing with gill
pigmentation in fish (Miller et al. 2007). It therefore seems that the best way to undertake
collaborations between functional and evolutionary biologists is to explore the usefulness and
variety of their specific approaches and not necessarily to unify their subjects of research.
6. Literature cited
Bertorelle G, Excoffier L (1998) Inferring admixture proportions from molecular data. Mol.
Biol. Evol. 15: 1298-1311.
Coelho M, Luiselli D, Bertorelle G, Lopes AI, Seixas S, Destro-Bisol G, Rocha J (2005)
Microsatellite variation and evolution of human lactase persitence. Hum. Genet. 117: 329-339.
53
Hey J, Nielsen R (2004) Multilocus methods for estimating population sizes, migration rates,
and divergence time, with applications to the divergence of Drosophila pseudobscura and D.
persimilis. Genetics 167: 747-760.
Jobling MA, Hurles ME, Tyler-Smith C (2004) Human Evolutionary Genetics: origins people &
disease. Garland Science.
Mayr E (1961) Cause and effect in biology. Science 134: 1501-1506
McEvoy B, Beleza S, Shriver M. 2006. The genetic architecture of normal variation in human
pigmentation: an evolutionary perspective and model. Hum. Mol. Genet. 15: R175-R181.
Miller C, Beleza S, Pollen A, Schluter D, Kittles R, Shriver MD, Kingsley DM (2007) Cisregulatory changes in Kit ligand expression and parallel evolution of pigmentation changes in
sticklebacks and humans. Cell 131: 1179-1189.
Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A (2007) Sequence diversity at
the proximal 14q32.1 SERPIN subcluster: evidence for natural selection favouring the
pseudogenization of SERPINA2. Mol. Biol. Evol. 24: 587-598.
Suriano G, Seixas S, Rocha J, Seruca R. 2006. E-cadherin germline missense mutations: a
clinical dilemma. How to infer their pathogenic significance in HDGC. J. Mol. Med 84: 10231031.
Tishkoff SA, Reed FA, Ranciaro A, Voight BF, Babitt CC, Silverman JS, Powell K, Mortensen
HM, Hirbo JB, Osman M, Ibrahim M, Omar SA, Lema G, Nyambo TB, Ghori J, Bumpstead S,
Pritchard JK, Wray GA, Deloukas P (2007) Convergent adaptation of human of lactase
persistence in Africa and Europe. Nat. Genet. 39: 31-40
54
POPULATION GENETICS
Group Leader: António Amorim, PhD
Staff members: Maria João Prata, PhD; Leonor Gusmão, PhD; Luísa Azevedo, PhD; Alexandra Lopes, PhD (postDoc); Sandra Martins, PhD (post-Doc); Ricardo Araújo PhD (post-Doc); Ana Goios, PhD student; Barbara van Asch,
PhD student; Elisabete Oliveira, PhD student; Filipe Pereira, PhD student; Iva Gomes, PhD student; Rita Quental,
PhD student; Sofia Quental, PhD student; Rui Pereira, PhD student; Verónica Gomes, PhD student, Inês Soares
PhD student; Nádia Pinto, PhD student; Cíntia Alves, chief technician; João Carneiro, MSc student; Sergio Costa,
MSc student; Vânia Pereira MSc student; Alfredo Gusmão, research grantee (BIC); Ana Moleirinho, research
grantee (BIC).
Objectives/Goals of the research activity
The group aims at understanding the origin and evolution of (mainly) human genetic diversity and their
consequences and applications, both normal and pathological (using autosomal, X and Y linked, as well as mtDNA
markers).
This requires the development of descriptive and analytical formal tools and techniques adequate to specific
genomic segments, in order to achieve the genetic characterisation of normal populations, their origins, phylogeny
and evolution, and disease susceptibility profiles.
The applications in which we concentrate our efforts are molecular diagnostics and forensics, but a line of research
involving the history, conservation and management of domesticates and laboratory animals as well as food quality
assessment is now established. Diagnostic tools for dairy cattle pathogens are under development.
Major achievements during 2007
According to the work plan formulated in 2006,
- We significantly contributed to the knowledge of the dynamics of normal and pathogenic mtDNA diversity, in
particular the variation among lab mouse lines, reporting the variation observed among the established lines [3],
establishing and validating their phylogeny and providing the first solid evidence of fast evolution of the coding
region
- On the distinction between nuclearly transferred segments of mtDNA (NUMTs) from genuine mtDNA, we have
proved that PCR techniques can be designed to rule out nuclear contamination as an artefact source [16]
- Contributing to the search for X and Y chromosome factors of infertility, we provided new methods and data for the
characterization of partial AZFc deletions of the Y chromosome with amplicon-specific sequence markers [17].
- On the development of molecular tools for genotyping Aspergillus strains, we have selected a panel of
microsatellites amenable to a single PCR multiplex reaction which is under final testing.
- The variation of PKLR gene in relation to malaria susceptibility was tested in Cape Verde samples of infected and
non-infected individuals and results are under analysis.
- Concerning the issues of timings and mechanisms of triplet repeat instability, we have clarified the cis-acting
factors promoting the CAG intergenerational instability in Machado-Joseph disease [18] and, furthermore
demonstrated – in contrast to common belief – an Asian origin for the worldwide-spread mutational event in this
disease [13]
- The genetic pool of the Portuguese dog “Cão de Gado Transmontano” was assessed through the development of
a multiplex autosomal STR kit, also employable in identification/parentage analyses of other dog breeds; results are
under final analyses. The coevolution of domesticates and their retrovirus was reported in [19]. The collaborative
exercise on dog mtDNA typing produced the first results (on blood saples) and has been extended to the analysis of
hairs
(http://www.gepisfg.org/ISFG/Castellano/Grupos_de_trabajo/Genetica_forense_no_humana/ejercicio2008.php).
- On the general topic of genetic polymorphisms of pharmacogenetic relevance, we have reported dataset relevant
for determining policy guidelines for cancer prevention and drug treatments in the Portuguese population [11]. The
55
progress on the research of genetic factors in the susceptibility to, and treatment of, paediatric acute lymphoblastic
leukaemia is partially described in [9]: we found evidence that the frequency of non- functional TPMT variants in
sub-Saharan Africa is higher than previously reported, implying therefore that sub-Saharan people are at greater
risk of toxicity when receiving non-individualized doses of thiopurines. We have performed a case-control study in
North Tunisia that led to the conclusion that some variants in folate-related genes are significantly associated with
bladder cancer [10].
- X chromosome markers were addressed in terms of population analysis (North Portugal [8], Spain [15], USA [7]
and three African populations [20]) and forensic applications as well in the development of new high throughput
typing technologies (a PCR based decaplex [7]).
- In order to better characterise the role of X-Y homologous genes in germ cell development we conducted an
extensive expression analysis of coding isoforms for several X-Y gene pairs in a panel of tissues and in biopsies of
infertile patients and found different expression patterns for X and Y chromosome transcripts for a highly conserved
ribosomal protein, one of the Y copies being expressed only in germ cells (manuscript under preparation).
- The progress on the research line focusing the genes involved in congenital disorders of glycosylation, in terms of
disease-causing mutations, establishment of orthologues, and phylogenetic relationships was reported in [4], where
the origin of the common mutations of PMM2 was addressed.
- A substantial progress was done on the identification of the molecular alterations responsible for the maple syrup
urine disease phenotype in the Portuguese patients thus contributing to enlarge the spectrum of MSUD mutations
and the knowledge of the pattern of geographical distribution (results under analysis).
- The unravelling of patterns of DNA sequence variation has been addressed through the analysis of mtDNA
secondary structures (a manuscript reporting the findings was submitted and is currently under revision) and the
inferences from comparative genomics on the role of compensatory mutations at OTC locus gained confirmation by
in vitro assays [14].
- Finally, under the more general population genetics objectives (understanding the origin and evolution of genetic
diversity) we achieved (i) a more detailed demographic history of Azores [23], Colombia [5], Brasil [6], North
Portugal [12] and São Tomé e Príncipe [2] through the study of Y chromosome defined lineages, (ii) the evaluation
of the degree of genetic substructure in urban populations [1], and we studied the associations between genetic
diversity and allergenic response [22] and characterized the genetic profile of thyroid cancer cell lines [21]
WORK PLAN FOR 2008
We intend to study
- The dynamics of normal and pathogenic mtDNA diversity, in particular the variation among SAM lab mouse lines
and their wild and domestic relatives; specifically we intend to apply the knowledge obtained in [3] on the coding
region polymorphic positions to the development of a diagnostic kit based on the SNaPshot technology.
- Expression of X and Y chromosome genes in male germ line in humans and in mouse models of infertility
- Protein analysis of two homeobox genes on the X and Y chromosome in human testis and their possible
contribution to infertility.
- A microsatellite based multiplex PCR for differentiation between Aspergillus fumigatus strains (final testing and
field evalution)
- PKLR gene in relation to malaria susceptibility within the context glycolytic enzymes and haemoglobin of genetic
diversity
- Timings and mechanisms of triplet repeat instability
- The genetic pools of Portuguese dog, pig, sheep and goat autochthonous breeds: history and management,
control, certification and traceability; evaluation of the results of the quality control exercise (http://www.gepisfg.org/ISFG/Castellano/Grupos_de_trabajo/Genetica_forense_no_humana/ejercicio2008.php)
- Pharmacogenetic variation, namely of folate metabolising and detoxifying genes in paediatric acute lymphoblastic
leukaemia.
- Validation of the newly defined X chromosome markers, population and mutation analysis (http://www.gepisfg.org/ISFG/Castellano/Grupos_de_trabajo/Cromosomas_sexuales/crx.php)
- Mutation dynamics of OTC gene and diagnostic improvement.
- The mutational spectrum of Portuguese maple syrup urine disease and mucopolysaccharidosis type IIIB and
detection of modifier genes
56
- Meta-patterns of DNA sequence variation: development of detecting algorithms and comparative genomics;
generalised kinship analysis and sequence comparison not requiring alignments
- Testing and validation of population/ancestry specific SNP markers
- Indel (insertion/deletion) markers: development of multiplex typing assay, population analysis and forensic
applications
- Population demographic histories through lineage and individuality markers, with special emphasis on Roma and
Sub-Saharan ethnic/geographic groups
- Molecular diagnostic tools for dairy cattle pathogens
PAPERS
1. TOSCANINI U, GUSMAO L, BERARDI G, AMORIM A, CARRACEDO A, SALAS A, RAIMONDI E (2007)
Testing for genetic structure in different urban Argentinian populations. Forensic Sci Int. 165(1):35-40.
2. TROVOADA MJ, TAVARES L, GUSMÃO L, ALVES C, ABADE A, AMORIM A, PRATA MJ (2007) Dissecting
the genetic history of São Tomé e Príncipe: a new window from Y-chromosome biallelic markers. Ann Hum Genet
71:77-85.
3. GOIOS A, PEREIRA L, BOGUE M, MACAULAY V, AMORIM A (2007) mtDNA phylogeny and evolution of
laboratory mouse strains. Genome Res.17 293-298
4. QUELHAS D, QUENTAL R, VILARINHO L, AMORIM A, AZEVEDO L. (2007) Congenital Disorder of
Glycosylation Type Ia: Searching for the Origin of Common Mutations in PMM2. Ann Hum Genet 71: 348-53
5. BUILES JJ, MARTINEZ B, GOMEZ A, CARABALLO L, ESPINAL C, AGUIRRE D, MONTOYA A, MORENO M,
AMORIM A, GUSMAO L, BRAVO ML (2007) Y chromosome STR haplotypes in the Caribbean city of Cartagena
(Colombia). Forensic Sci Int. 167: 62-69
6. DOMINGUES PM, GUSMAO L, DA SILVA DA, AMORIM A, PEREIRA RW, DE CARVALHO EF. (2007) SubSaharan Africa descendents in Rio de Janeiro (Brazil): population and mutational data for 12 Y-STR loci. Int J Legal
Med. 121: 198 - 203
7. GOMES I, PRINZ M, PEREIRA R, MEYERS C, MIKULASOVICH RS, AMORIM A, CARRACEDO A, GUSMAO
L (2007) Genetic analysis of three US population groups using an X-chromosomal STR decaplex. Int J Legal Med.
121: 198 – 203
8. PEREIRA R, GOMES I, AMORIM A, GUSMAO L (2007) Genetic diversity of 10 X chromosome STRs in
northern Portugal. Int J Legal Med. 121: 192 - 197
9. OLIVEIRA E, QUENTAL S, ALVES S, AMORIM A, PRATA MJ (2007) Do the distribution patterns of
polymorphisms at the thiopurine S-methyltransferase locus in sub-Saharan populations need revision? Hints from
Cabinda and Mozambique. Eur J Clin Pharmacol. 63(7):703-6.
10. OUERHANI S, OLIVEIRA E, MARRAKCHI R, BEN SLAMA MR, SFAXI M, AYED M, CHEBIL M, AMORIM A,
EL GAAIED AB, PRATA MJ. (2007) Methylenetetrahydrofolate reductase and methionine synthase polymorphisms
and risk of bladder cancer in a Tunisian population. Cancer Genet Cytogenet. 176(1): 48-53.
11. OLIVEIRA E, MARSH S, VAN BOOVEN DJ, VAN BOOVEN DJ, AMORIM A, PRATA MJ, MCLEOD HL (2007)
Pharmacogenetically relevant polymorphisms in Portugal. Pharmacogenomics 8 (7): 703-712.
12. ALVES C, GOMES V, PRATA MJ, AMORIM A, GUSMAO L (2007) Population data for Y-chromosome
haplotypes defined by 17 STRs (AmpFlSTR YFiler) in Portugal. Forensic Sci Int. 171: 250-255
13. MARTINS S, CALAFELL F, GASPAR C, WONG VC, SILVEIRA I, NICHOLSON GA, BRUNT ER,
TRANEBJAERG L, STEVANIN G, HSIEH M, SOONG BW, LOUREIRO L, DURR A, TSUJI S, WATANABE M,
JARDIM LB, GIUNTI P, RIESS O, RANUM LP, BRICE A, ROULEAU GA, COUTINHO P, AMORIM A, SEQUEIROS
J (2007) Asian origin for the worldwide-spread mutational event in Machado-Joseph disease. Arch Neurol.
64(10):1502-8.
14. SURIANO G, AZEVEDO L, NOVAIS M, BOSCOLO B, SERUCA R, AMORIM A, GHIBAUDI EM (2007) In vitro
demonstration of intra-locus compensation using the Ornithine transcarbamylase protein as model. Hum Mol Genet.
16(18):2209-14
15. ALER M, SANCHEZ-DIZ P, GOMES I, GISBERT M, CARRACEDO A, AMORIM A, GUSMAO L (2007) Genetic
data of 10 X-STRs in a Spanish population sample. Forensic Sci Int. 173: 193-196
16. GOIOS A, PRIETO L, AMORIM A, PEREIRA L (2007) Specificity of mtDNA-directed PCR-influence of NUclear
MTDNA insertion (NUMT) contamination in routine samples and techniques. Int J Legal Med. 2007 Sep 14; [Epub
ahead of print]
57
17. NAVARRO-COSTA P, PEREIRA L, ALVES C, GUSMAO L, PROENÇA C, MARQUES-VIDAL P, ROCHA T,
CORREIA SC, JORGE S, NEVES A, SOARES AP, NUNES J, CALHAZ-JORGE C, AMORIM A, PLANCHA CE,
GONCALVES J (2007) Characterizing partial AZFc deletions of the Y chromosome with amplicon-specific sequence
markers. BMC Genomics. 8(1):342 [Epub ahead of print]
18. MARTINS S, COUTINHO P, SILVEIRA I, GIUNTI P, JARDIM LB, CALAFELL F, SEQUEIROS J, AMORIM A
(2007) Cis-acting factors promoting the CAG intergenerational instability in Machado-Joseph disease. Am J Med
Genet B Neuropsychiatr Genet. 2007 Oct 19; [Epub ahead of print]
19. ARNAUD F, CAPORALE M, VARELA M, BIEK R, CHESSA B, ALBERTI A, GOLDER M, MURA M, ZHANG Y,
YU L, PEREIRA F, DEMARTINI J, LEYMASTER K, SPENCER T, PALMARINI M (2007) A Paradigm for Virus-Host
Coevolution: Sequential Counter-Adaptations Between Endogenous and Exogenous Retroviruses. PLoS
Pathogens. Nov 9; 3(11):e170
20. GOMES I, ALVES C, MAXZUD K, PEREIRA R, PRATA MJ, SÁNCHEZ-DIZ P, CARRACEDO A, AMORIM A
GUSMÃO L (2007): Analysis of 10 X-STRs in three African Populations. Forensic Sci Int: Genetics 1: 208-211.
21. MEIRELES AM, PRETO A, ROCHA AS, REBOCHO AP, MÁXIMO V, PEREIRA-CASTRO I, MOREIRA S,
FEIJÃO T, BOTELHO T, MARQUES R, TROVISCO V, CIRNES L, ALVES C, VELHO S, SOARES P, SOBRINHOSIMÕES M (2007) Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived cell
lines. Thyroid. Aug;17(8):707-15. PMID: 17725429 [PubMed - in process]
22. MARTÍNEZ B, BARRIOS K, VERGARA C, MERCADO D, JIMÉNEZ S, GUSMÃO L, CARABALLO L (2007) A
NOS1 gene polymorphism associated with asthma and specific immunoglobulin E response to mite allergens in a
Colombian population. Int Arch Allergy Imm 144:105-13
23. NETO D, MONTIEL R, BETTENCOURT C, SANTOS C, PRATA MJ, LIMA M (2007) The African contribution to
the present-day population of the Azores Islands (Portugal): Analysis of the Y chromosome haplogroup E. Am J
Hum Biol 19:854-60.
Books/ Book Chapters
1. AMORIM A (2007) Genetic markers: The interplay between concepts and technology in the Anthropological
scene. In Santos C, Lima M (eds) Recent Advances in Molecular Biology and Evolution: Applications to
Biological Anthropology, Research Signpost, Kerala.
2. SAMPAIO P, CORREIA A, GUSMÃO L, AMORIM A, PAIS C (2007) Sequence analysis reveals complex
mutational processes for allele length variation at two polymorphic microsatellite loci in Candida albicans. In:
MÉNDEZ-VILAS A (ed.) Communicating Current Research and Educational Topics and Trends in Applied
Microbiology. Microbiology Series nº1, Vol 1: 926-935. Formatex, Badajoz.
Prizes
Best poster presentation
Oliveira E, Gomes V, Quental S, Ferreira F, Alves S, Amorim A, Prata MJ “Pharmacogenetic Role of TPMT, ITPA
and Folate Pathway in Treatment of Pediatric Acute Lymphoblastic Leukaemia”
Portugaliae Genetica 10th Edition, Porto, 22-24 Mar 2007
PhDs
Finished
- Martins S “Evolutionary and epidemiological genetics of Machado-Joseph disease”, Faculty of Sciences,
University of Porto, IPATIMUP and Univ. Pompeu Fabra, Barcelona
Ongoing
- Pereira F “Development of uniparentally transmitted genetic markers for the characterization of male and
female gene pools of Portuguese small ruminants autochthonous breeds” Faculty of Sciences, University of
Porto, IPATIMUP, and Department of Genetics, Smurfit Institute, Trinity College, Dublin 2, Ireland. Fundação
para a Ciência e Tecnologia (SFRH/BD/19585/2004). Since October 2004
- Oliveira E “Study of Genetic Polimorphisms in Pediatric Acute Lymphoblastic Leukemia - Pharmacogenetic
Role in the Treatment and Relationship with susceptibility to the leukemogenic process” Faculty of Sciences,
University of Porto, IPATIMUP, and School of Medicine , Washington University in St. Louis. Fundação para a
Ciência e Tecnologia (SFRH/BD/17124/2004). Since November 2004.
58
-
-
-
-
-
-
-
Goios A “Dynamics of the mitochondrial genome – the moving boundary between normal and pathogenic
diversity” Faculty of Sciences, University of Porto, IPATIMUP and Department of Statistics, University of
Glasgow. Fundação para a Ciência e Tecnologia (SFRH/BD/16518/2004). Since November 2004.
Gomes I “X chromosome markers: genetic characterization, population analysis and forensic applications”
University Santiago de Compostela, IPATIMUP and CEGEN (National Genotyping Center of Spain). Fundação
para a Ciência e Tecnologia (SFRH/BD/21647/2005). Since November 2004.
Quental R “Congenital Disorders of Glycosylation (CDG): Enzymatic, Biochemical and Molecular Features”
Faculty of Sciences, University of Porto, IPATIMUP and Department of Human Genetics, Catholic University of
Leuven, Belgium. Fundação para a Ciência e Tecnologia (SFRH/BD/23657/2005). Since February 2006.
Quental S “Functional, Expression and Structural investigation of the mutational spectrum of Portuguese Maple
Syrup Urine Disease patients” Faculty of Sciences, University of Porto, IPATIMUP and Department of Human
Genetics, School of Medicine, Emory University, Atlanta, USA. Fundação para a Ciência e Tecnologia
(SFRH/BD/22685/2005). Since January 2006.
Van Asch B “Development of uniparentally transmitted genetic markers for the characterization of male and
female gene pools of Portuguese pig (Sus scrofa) autochthonous breeds: applications for the control,
certification and traceability of meat products” Faculty of Sciences, University of Porto, IPATIMUP and Modelo
Continente Hipermercados SA. Fundação para a Ciência e Tecnologia (SFRH/BDE/15581/2006). Since July
2006.
Pereira R “Bridging the gap between Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms
(SNPs)” Institute of Legal Medicine of the University of Santiago de Compostela and IPATIMUP. Fundação
para a Ciência e Tecnologia (SFRH/BD/30039/2006). Since January 2007.
Gomes V “Ethnicity and genetics in sub-Saharan Africa” Institute of Legal Medicine of the University of Santiago
de Compostela and IPATIMUP. Fundação para a Ciência e Tecnologia (SFRH/BD/36045/2007). Since
December 2008.
MScs
Finished
-
Pereira R “Marcadores do cromossoma X: perfil genétic da população do Norte de Portugal e aplicações
forenses”, Faculty of Medicine, University of Porto and IPATIMUP. 14/02/2007
Gusmão A “A comunidade cigana em Portugal: Padrão de Diversidade no Gene Pool Masculino Baseado em
Marcadores do Cromossoma Y”, Faculty of Sciences, University of Lisbon and IPATIMUP, 12/06/2007
Ongoing
- Vânia Pereira “Deciphering the origin of foci of high prevalence of hereditary anemias in Portugal:
epidemiological and evolutionary study”, University of Porto and IPATIMUP
- João Carneiro: “Forensic applications of mtDNA” University of Porto and IPATIMUP
- Sérgio Costa: “Portuguese autochthonous dog breeds - mtDNA diversity”, University of Porto and IPATIMUP
NATIONAL AND INTERNATIONAL COOPERATIONS
The group is currently engaged in various collaborative projects with
-
Dep. Statistics, Univ. Glasgow (mtDNA), Vincent Macaulay
Department of Pathology, Cambridge University (sex chromosomes biology), Nabeel A Affara
Faculdade de Medicina, UP (Aspergillus genotyping), Cidália Pina-Vaz
Hôpital Nôtre Dame, Montreal, Canada (Machado-Joseph disease), Guy Rouleau
Inst. Med. Legal, Univ. Santiago Compostela (forensic genetics), Angel Carracedo
Instituto de Genética Médica Jacinto de Magalhães / INSA (human genetic diseases), Laura Vilarinho
Instituto Nacional de Medicina Legal (forensic genetics and databases), Francisco Corte-Real
Instituto Nacional de Toxicologia y Ciencias Forenses, Madrid (forensic genetics), Antonio Alonso
Instituto Português de Arqueologia, Lisboa, Portugal (domesticates), Simon J.M. Davis
Laboratorio de Genetica Forense, Universidad de Valencia (forensic genetics), Mercedes Aler
59
-
New York City Office of Chief Medical Examiner, (forensic genetics), Mechthild Prinz
Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa, Israel
(Jewish populations) Doron Behar
UniGene, IBMC Porto (genetic diseases), Jorge Sequeiros
Univ Pompeu Fabra (population genetics modelling), Francesc Callafel
Univ. Oxford (gene function & evolution), Rosalind Harding
Univ. Tunis (population genetics), Houssein Khodjet, Lotfi Cherni
Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, USA (mtDNA),
David C. Samuels
Jackson Laboratory, Bar Harbor, USA (lab mouse genetics), Molly Bogue
PRICAI-Fundacion Favaloro, Buenos Aires, Argentina (population genetics), Eduardo Raimondi
Lab. Genes, Medellin, Colombia (population genetics), Mª Luisa Bravo
Lab. Imunogenetica, Univ. Cartagena, Colombia (population genetics), Luiz Caraballo
Lab. Diagnóstico DNA, Univ. Estadual Rio de Janeiro (population genetics), Eliseu Carvalho
Inst. Higiene e Medicina Tropical, Lisboa (malaria and PKLR polymorphisms), Virgílio do Rosário, Ana Paula
Arez
Institute of Integrative and Comparative Biology, Univ. Leeds (mtDNA), Martin Richards, Pedro Soares
Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, Scotland (domesticates),
Massimo Palmarini.
Faculty of Veterinary Medicine, Utrecht University, The Netherlands (domesticates) Johannes A. Lenstra.
Agriculture Research Institute of Mozambique, Department of Animal Sciences, Artificial Insemination Centre,
Maputo, Mozambique (domesticates), Sónia Maciel
Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aarhus, DK-8830
Tjele, Denmark (population models and fertility) Rodrigo Labouriau
Institute for Pharmacogenomics and Individualized Therapy – University of North Carolina, Chapel Hill NC, USA
– Howard McLeod
IPO – Porto, Serviço de Pediatria; Lucília Norton
IPO – Lisboa, Serviço de Pediatria; Mário Chagas
Hospital Pediátrico de Coimbra – Serviço de Hematologia; Maria Manuela Benedito e Maria Lurdes Maricato
Hospital S.João Porto – Serviço de Pediatria; Fátima Ferreira
Laboratório de Genética Humana e Médica, Universidade Federal do Pará, Brazil (population genetics), Sidney
Santos
Visiting researchers at IPATIMUP
Patrícia Machado, Inst. Higiene e Medicina Tropical, Lisboa, Portugal
Elizeu Fagundes de Carvalho, Universidade do Estado do Rio de Janeiro, Brazil
Juan José Builes, Lab. Genes, Medellin, Colombia
Inês Nogueiro. Departamento de Antropologia da Faculdade de Ciências e Tecnologia da Universidade de
Coimbra. Portugal
Francisca Gonzaga. Center of Research in Natural Resources (CIRN) and Department of Biology, University of the
Azores, Portugal
Visits / Courses Abroad
A Amorim: Centre de Recherche du Centre Hospitalier Universitaire Sainte-Justine, Montreal, Canada, 0911/11/2007
A Goios: Dep. Statistics, Univ. Glasgow (host: Vincent Macaulay), ?? 3-30 May
I Gomes: Oct. 2007 -Jan. 2008, Visiting Scientist at the Counterterrorism and Forensic Science Research Unit, FBI
academy, Virginia, USA.
S Martins: Center for the Study of Brain Diseases, CHUM Research Center, Notre Dame Hospital, Montreal,
Canada (host: Guy A. Rouleau), 8 Nov -14 Dec.
A Lopes: Jan. 2007-Jan. 2008, Visiting postdoctoral fellow at the Department of Pathology, University of Cambridge,
UK.
60
L Gusmão: Disciplina de Genética de populações. Programa de Pós-Graduação em Biologia, Área de concentração
em Biociências Nucleares e Ecologia. Instituto de Biologia Roberto Alcântara Gomes. Universidade do Estado do
Rio de Janeiro. Brazil, 1-9 March
Organization of Scientific Meetings
• Portugaliae Genetica 10th. edition, 22-24 March
Coordination of QC/QA cooperative exercises
• Colaborative exercise on canine mtDNA, Non-human Forensic Genetics Workgroup, António Amorim and
José Pestano Brito, with Barbara Van Asch; GEP- ISFG
• Colaborative exercise on X-chromosome STRs, Sex chromosomes Genetics Workgroup, Leonor Gusmão
and António Amorim, GEP-ISFG
National Projects
Lead by IPATIMUP
01-05-2003 - 01-05-2006
PI: MJ Prata
total budget: €56200
POCI/ANT/57037/2004 – “Estudo evolutivo e epidemiológico de focos de anemias hereditárias em Portugal”.
01-10-2005 - 01-10-2008
PI: MJ Prata
total budget: €30000
POCI/AFR/62242/2004 – “Etnias e genética na fronteira da rota de migração Bantu: o caso dos Karamojong”.
Ethnicity and genetics in the fringe of Bantu migration route: The Karamojong case
01-01-2005 - 01-01-2008
PI: L Gusmão total budget:
€30000
1931/2006 Programa Saúde XXI/Medida 1.1 “Registo da deficiência genética da metabolização de tiopurinas na
população portuguesa e prevenção da toxicidade em terapêuticas citostáticas”
01/05/ 2006 – 31/12/2007 PI: MJ Prata
total budget: €150000
PTDC/ANT/70413/2006 - "A comunidade cigana portuguesa: a história demográfica à luz dos padrões de
diversidade genética" PI: MJ Prata total budget: €77 000.
IPATIMUP as participant
POCI/SAU-ESP/55110/2004 “Avaliação da epidemiologia da malária na República de Cabo Verde”
01-07-2005 - 01-07-2008
PI: Ana Paula Martins dos Reis Arez (IHMT)
POCI/DES/62499/2004 - Aspectos genéticos da actividade física, aptidão física associada à saúde, sobrepeso e
obesidade. Um estudo em gémeos dos 6 aos 20 anos de idade.
PI: José António Ribeiro Maia (FCDEF, UP)
International Projects
Factores de riesgo en asma: genes relacionados con la remodelación bronquial. Instituto de Investigaciones
Inmunológicas, Universidad de Cartagena, Colombia.
Editorial Boards
Forensic Science International (A Amorim)
Forensic Science International: Genetics (L Gusmão)
Patents
Pereira F, van Asch B, Gusmão L
Process for animal species identification in samples with genetic material based on mitochondrial DNA size variation
Portuguese patent nº 104599 (pending)
PCT extension (filed 6th November 2006)
Invited talks
A Amorim - Population genetics and medicine; Centre de Recherche du Centre Hospitalier Universitaire SainteJustine, Montreal, Canada, 09/11/2007
L Gusmão - A genética forense em Portugal e na Europa. Universidade Federal do Pará. Belém. Brasil, 20 de Julho
de 2007
61
L Gusmão - Genética forense: Histórico e aplicações. I Encontro Internacional de Genética Forense. Universidade
Federal de Alagoas. Maceió, Alagoas, Brasil, 25-26 de Julho de 2007.
L Gusmão - Estrutura e objectivos do grupo espanhol e português da sociedade internacional de genética forense
(GEP-ISFG) – Exercícios colaborativos. I Encontro Internacional de Genética Forense. Universidade Federal de
Alagoas. Maceió, Alagoas, Brasil, 25-26 de Julho de 2007.
L Gusmão - Decaplex X-STRs. I Encontro Internacional de Genética Forense. Universidade Federal de Alagoas.
Maceió, Alagoas, Brasil, 25-26 de Julho de 2007.
Oral presentations
Lopes AM, Sargent CA, Ellis PJ and Affara NA. Expression of X/Y homologous genes in testis. “1st Florence-Utah
International Symposium - Genetics of Male Infertility” September 2007, Florence, Italy.
62
TUMOR EVOLUTION AND DEVELOPMENT
Group Leader
Luís Teixeira da Costa, PhD, Researcher at IPATIMUP
Staff members
Ana Branco – Undergraduate student
Ana Isabel Silva – Undergraduate student
Ângela Costa, BSc – PhD student
Elisabete Figueiredo, BSc – MSc student
Inês Barbosa,BSc (Through July 2007)
Isabel Castro, BSc – MSc Student
Joana Caria – Undergraduate student
Lúcia Tavares – Undergraduate student
Nuno Camboa, BSc – PhD student
Objectives/Goals of the research activity
Our main long term goal is to understand the molecular mechanisms underlying tumor evolution. Rather than
focusing on an organ or tissue-specific type of tumor, we intend to pursue that goal by using different models to try
to answer specific fundamental questions about tumor evolution and development. At the initial stage of “group
establishment”, this was translated into focusing on problems with which the PI had previous experience and that
could be tackled with generally available technical means. In the long term, the regular use of animal models is
crucial for us, and “obtaining access” to them has therefore been one of our main medium term goals.
Background and major achievements during 2006
1.Publications
Two research articles were published in 2007:
Macedo JE, Costa AM, Barbosa IA, Rebelo S, de Moura CS, da Costa LT, Hespanhol V.
Genetic alterations in lung cancer: assessing limitations to its routine clinical use.
Rev Port Pneumol. 2007 Jan-Feb;13(1):9-34.
Meireles AM, Preto A, Rocha AS, Rebocho AP, Máximo V, Pereira-Castro I, Moreira S, Feijão T, Botelho T,
Marques R, Trovisco V, Cirnes L, Alves C, Velho S, Soares P, Sobrinho-Simões M.
Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived cell lines.
Thyroid. 2007 Aug;17(8):707-15. (As a colaboration)
One MSc thesis was completed in 2007:
Isabel Pereira-Castro
Caracterização da P149: uma nova proteína humana que interage com membros da via Wnt
2.Training
Four undergraduate students completed their “Estágio de Licenciatura” in Biology in 2007: Ana Branco, Ana Isabel
Silva, Joana Caria and Lúcia Tavares
3. Ongoing research work
We continued and expanded our previous work on: a) the regulation of TCF4/β-catenin-mediated transcription in
intestinal development, homeostasis and tumorigenesis; b) Chromosomal instability. The following topics have been
explored:
- Involvement of Groucho family members (Grgs) in intestinal tumorigenesis (in colaboration with Profs Forrest
Spencer and Shannon Fisher, of The Johns Hopkins University). The TCF4 mutations causing reduced Grg-affinity
previously identified by us were combined to generate new TCF4 mutants that are being tested for their ability to
resist Grg-mediated transcription repression in mammalian cells, affect intestinal development or cause intestinal
tumorigenesis.
63
- Characterization of novel Grg4 splice forms. We have now shwon that several of the novel Grg4 splice forms
identified by us are unable to bind TCF4. Experiments are underway to determine whether they can dimerize with
the “canonical” Grg4 form and work as negative regulators of Grg-mediated transcription repression.
- Characterization and functional analysis of C149. We performed detailed mapping studies of the critical C149
domains for C149-TCF4 and C149-Grg and generated multiple point mutants in both the Btd and Zinc-finger
domains of the protein to assess role in those intearctions. We also generated mutants lacking other conserved
motifs in the protein we have identified to test their influence on C149’s subcellular localization or transcriptionrepression ability. Finally, we started to generate expression vectors to analyse C149’s role in development and
tumorigenesis in vivo.
- Mechanism of β-catenin regulation by Wnt signalling. We designed a multiple vectors with various forms of βcatenin that should allow us to test competing models of the mechanism of β-catenin regulation by Wnt signaling.
The majority of the vectors required, as well as many of the cell lines stably expressing the different proteins, have
already been generated and the remaining should be available in the next few months.
- p53’s role on chromosomal instability in cancer. We have generated cell lines to test our hypothesis that
inactivation of p53 has a positive effect on survival in chromosomally unstable cells.
- Chromosomal (in)stability in vertebrates. As part of our ongoing colaboration, we have now generated multiple
lines of zebrafish with “tagged” chromosomes that will be used in a variety of studies, led by Prof Forrest Spencer,
on the mechanisms of chromosomal instability in vertebrates.
Additionally, we have continued to colaborate, both with other IPATIMUP groups and with researchers abroad:
- Auto-regulation of CDX2 expression (Carcinogenesis group)
- Potential involvement of an H-Ras polymorphism in alternative splicing (Tumor Biology group)
- Genetic and functional testing of candidate breast cancer genes (Phil Buckhaults, University of South Carolina)
3. Development work
With the triple objective of improving technical training, reducing costs and promoting finatial stability, we have
committed, from the start to the development of – preferably novel – research tools. In 2007 we:
- Expanded our range of TA-cloning vectors.
- Developed an easy-to-use, transposon-based, high-efficiency method to generate stably transfected cell lines.
- Developed a novel Western-blot molecular weight marker
64
GENETIC DIVERSITY AND BIOINFORMATICS
Group Leader: Luísa Pereira
Staff members:
Sandra Manuela Gonçalves Oliveira - Master degree student in Mathematical Engineering, Faculty of Sciences,
University of Porto
Carla Alice Afonso - Master degree student in Legal Medicine, ICBAS – Instituto de Ciências Biomédicas Abel
Salazar
Verónica Cristina Neves da Nova Fernandes – Grant BI (initiation to research) in the project
PTDC/ANT/66275/2006; Master degree student in Biochemistry, University of Beira Interior
Marta Daniela Araújo da Costa – Grant BI in the project PTDC/AFR/71422/2006
Fernando Miguel Laranjeira Freitas – Master degree student in Network and Informatics Systems Engineering,
Faculty of Sciences, University of Porto
Objectives/Goals of the research activity
The basic aim of the group is 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 in a detailed characterisation of worldwide genetic diversity, for the uniparental markers, and in the
design of studies of complex phenotypes, namely fertility, longevity (recently established as a main research area in
the future I3S) and cancer (by collaborating with other IPATIMUP’s groups). New developments in biostatistics and
bioinformatics will be essential for an efficient evaluation of genetic diversity and mutation models in neutral and
pathological conditions, and we intend in the near future to experimentally test the theoretical inferences being
contributed by integrative computational analyses.
Background and major achievements during 2007:
The group was established in July 2006. The main achievements obtained in one and half year were:
(a) formation of new staff: especially students finishing graduation or initiating master degrees; in this initial phase of
consolidation of the group we invested only in projects of one year, in order to not compromise any student; the next
step will be apply for PhD and post-doc grants;
(b) funding: we were successful in getting funding for two projects submitted to the Portuguese Foundation of
Science (FCT) (100% successful rate);
(c) renovation and establishment of international and national collaborations (listed bellow), achieving to create a
multidisciplinary network;
(d) collection of population samples from North Africa (Morocco, Tunisia, Egypt and Libya), East Africa (Sudan,
Ethiopia and Somalia) and the Arabian Peninsula (Yemen, Oman, Saudi Arabia and United Arab Emirates), by
establishing collaborations with local research groups or groups conducting research projects in those geographic
areas.
Publications in 2007 reflect mainly previous research, namely in the field of male infertility, but a better image of the
group’s lines of research can be retrieved from accepted publications and oral communications, already conducted
with new collaborators and new staff, and included in the new research funded projects.
Work plan for 2008:
We are conducting the following lines of research:
. Applications to population genetics:
MtDNA and Y-chromosome are very useful tools to address many questions of human evolution, demography and
history. It is essential to have questions and hypotheses, archaeological/anthropological evidences, good population
samples and adequate statistical analyses. Our group has established a multidisciplinary network (geneticists,
archaeologists, anthropologists and statisticians), possesses good population samples and is addressing questions
that were not being addressed by other groups working in the same field, from IPATIMUP or other Portuguese
Institutions, being successful in achieving funding for its research. The questions that we are addressing are:
65
a) Improvement of the Out-of-Africa model, evaluating alternative migration routes, via Levant or via the Horn
of Africa.
b) Evaluation of the Neolithic genetic influence in the Great Mediterranean, with new data from North Africa
c) Evaluation of the trans-Saharan caravan slave trade from East Africa (mainly Sudan) to West Africa
(namely to Morocco).
. Applications to clinical genetics:
a) Evaluation of longevity in humans of African origin
. We already conducted complete mtDNA sequencing in 21 centenarians and 22 controls from Tunisia; data were
presented in two congresses and are being drafted for publication
. We initiated contacts with the Medical Faculty of the University of Mondlane in Mozambique and submitted the
project to the ethics committe.
. As suggested by the evaluators of the project, we are enlarging the initial aims to include a social perspective. A
master degree student in Sociology, University of Porto, Paula Maria Nunes Gonçalves da Silva, is initiating her
thesis research in the subject of how ageing is perceived in African and European populations.
b) Evaluation of longevity in North Portuguese. We will collaborate in a multidisciplinary project leaded by
Professor Henrique Almeida from IBMC. He and his collaborators organized the “2007 Link-age Meeting” in
Porto. They are collecting a big number (around 200) of blood samples from nonagenarians and
centenarians from North Portugal and will evaluate indices of ROS production and OXPHOS activity. We
are going to classify these samples in mtDNA haplogroups and choose some for complete mtDNA
sequencing. As far as we know, this will be the first study evaluating the relationship between mtDNA
haplogroups and ROS/OXPHOS indices in centenarians.
c) We are working in collaboration with IPATIMUP’s group “Cancer Biology”, namely with Valdemar Máximo,
on the relation between mtDNA mutations and cancer.
.Valdemar has studied Hürtle cell syndrome in some carcinomas, characterised by the accumulation of a huge
number of mitochondria, and compared it with the same kind of carcinomas but with the normal amount of
mitochondria. Hürtle cells display more mtDNA mutations, in accordance with its higher number of mitochondria. We
are now intending to develop a real-time PCR design to estimate mtDNA copy number, relatively to nuclear DNA, in
order to evaluate the following: 1) how much more mtDNA is present in Hürtle cells?; 2) is the higher number of
mtDNA mutations present in Hürtle cells in accordance with mtDNA mutation rate corrected by the number of
molecules?
. many studies being performed in cancer/mtDNA have disregarded phylogenetic information accumulated in
population genetics. These phylogenetic data can be informative not only as quality control (due to nonrecombination, some mutations are associated, constituting an haplogroup; the absence of those mutations in a
sample should be carefully checked), but also as evidence for a potential role in pathology (a mutation observed in
many population groups can hardly be expected to have a pathological effect). We are evaluating the construction of
an efficient clinical database for mtDNA mutations in cancer which integrates for the first time the big amount of
phylogenetic information being accumulated since 2000. This issue was explored in a small project of a student from
Bioinformatics’ degree (Escola Superior de Biotecnologia, Universidade Católica), supervised by Valdemar Máximo
and Luísa Pereira.
. Bioinformatics
a) Evaluation of the relationship between mtDNA binding energy and mutability. The computational work
being conducted in collaboration with David Samuels, from Virginia Bioinformatics Institute, led to several
conclusions, already presented in an oral communication and under preparation for publication. The major
conclusions were: 1) most tRNA’s are locals with low free energy and, so, prone to bubble opening; 2)
neutral polymorphisms do not localize on regions with lower binding energy; 3) pathogenic positions tend to
significantly locate in regions with higher probability of opening and this value of significance was higher
when analyzing only tRNA pathogenic mutations; 4) in general, binding energy seems to play a role in the
appearance of pathogenic mutations, especially in tRNAs, but not on the generation of neutral diversity.
We intend to design tests to experimentally evaluate the probability of bubble opening and its stability
between regions of the mtDNA: the tRNAs, the origins of replication (OH and OL) and other regions less
prone to open. The evaluation in the origins of replication will provide experimental insights to compare
between the asymmetric and the strand-coupled models of replication. The experimental tests can be
conducted by combining fluorescence correlation spectroscopy and fluorescence quenching in synthetic
66
DNA molecules; we will establish collaboration with a national or international group having the know-how
in spectroscopy.
Financing/projects:
1- 1/4/2006-31/03/2007 - “Y-chromosome phylogeography in Southeast Asia and the Pacific”; The British Academy;
PI: Martin Richards; Colaborators: Luísa Pereira, Jim Wilson; 43,783 £
2- 2006-2007 - “Genetic exchanges between Morocco and Portugal”, GRICES /CNRST; PIs: Luísa Pereira and
Nourdin Harich; Financing: missions
3- 2007 – “Characterisation of the genetic diversity in the Egyptian population from el-Hayez oásis”, GRICES/ASCR;
PIs: Luísa Pereira and Viktor Cerny; Financing: missions
4- 06/2007-05/2010: “Longevity in humans of African origins - a genetic approach”. FCT (PTDC/AFR/71422/2006)
PI: Luísa Pereira; Financing: 80,000 €
5- 06/2007-05/2010: “Refining the geographical origin and dispersal routes of early modern humans and early
farmers of the Greater Mediterranean with high-resolution genetic techniques”. FCT (PTDC/ANT/66275/2006); PI:
Luísa Pereira; Financing: 87,000 €
Theses – ongoing:
- co-supervision of PhD - Ana Goios: “Dynamics of the mitochondrial genome – the moving boundary between
normal and pathogenic diversity”; Faculty of Sciences, University of Porto, IPATIMUP and Department of Statistics,
University of Glasgow. Fundação para a Ciência e a Tecnologia (SFRH/BD/16518/2004). Since November 2004.
Theses – delivered:
- Master thesis in Mathematical Engineering – Sandra Manuela Gonçalves Oliveira: “Ferramentas informáticas para
a selecção e o alinhamento de genes de DNA mitocondrial – aplicação ao estudo de diversidade da ordem
Primatas” (“Informatic tools for selection and alignment of mtDNA genes – application to the study of diversity in
Primates”). Faculty of Sciences, University of Porto. Viva: 24-01-2008. Supervisor: Luísa Pereira; Co-Supervisor:
Gueorgui Smirnov; Department of Applied Mathematics Faculty of Sciences, University of Porto.
- Master thesis in Legal Medicine – Carla Alice Afonso: “Diversidade mitochondrial no Sudão: aplicações à genética
forense e à genética populacional” (“Mitochondrial DNA diversity in Sudan: population and forensic genetics
applications”). ICBAS – Instituto de Ciências Biomédicas Abel Salazar. Viva: 17-03-2008. Supervisor: Luísa Pereira.
Main international collaborations
Vincent Macaulay, Department of Statistics, University of Glasgow, UK
Martin Richards, Institute of Integrative & Comparative Biology, Faculty of Biological Sciences, University of Leeds,
UK
Doron Behar, Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa,
Israel
Farida Alshamali, Dubai Police General Headquarters
David C. Samuels, Virginia Bioinformatics Institute, Virginia, USA
Mathias Currat and Estella Poloni, Départment d’anthropology et d’ecologie, Université de Geneve, Switzerland
Viktor Cerny, Institute of Archaeology of the Academy of Sciences of the Czech Republic, Prague
João Zilhão, Department of Archaeology and Anthropology, University of Bristol
Nourdin Harich and Mostafa Kandil, University of El Jadida, Morocco
67
Main national collaborations
João Gonçalves, Leader of the group “Pathology of the sexual development”, Centre of Human Genetics, National
Institute of Health, Lisbon
Henrique Almeida, Leader of the group “Stress in Animals”, IBMC
Main IPATIMUP collaborations
Valdemar Máximo, Group of “Cancer Biology”
Publications in 2007
1- Goios A, Pereira L, Bogue M, Macaulay V, Amorim A (2007) MtDNA phylogeny and evolution of laboratory
mouse strains. Genome Res. 17: 293-298. (ISI 2006 Impact Factor: 10.256)
2- Pereira L, Gonçalves J, Franco-Duarte R, Silva J, Rocha T, Arnold C, Richards M, Macaulay V (2007) No
evidence for an mtDNA role in sperm motility: data from complete sequencing of asthenozoospermic males. Mol Biol
Evol. 24: 868-874. (ISI 2006 Impact Factor: 6.726)
3- Navarro-Costa P, Pereira L, Alves C, Gusmão L, Proença C, Marques-Vidal P, Rocha T, Correia SC, Jorge S,
Neves A, Soares AP, Nunes J, Calhaz-Jorge C, Amorim A, Plancha CE, Gonçalves J (2007) Sequence architecture
and deletion mechanisms vary widely in the partial AZFc deletion pool of the Y chromosome. BMC Genomics 8:342.
(ISI 2006 Impact Factor: 4.029)
Publications Accepted:
1- Pereira L, Gonçalves J, Bandelt H-J (2008) Mutation ‘C11994T’ in the mitochondrial ND4 gene is not a cause of
low sperm motility in Portugal. Fertil Steril. (ISI 2006 Impact Factor: 3.277)
2- Goios A, Prieto L, Amorim A, Pereira L (2008) Specificity of mtDNA-directed PCR – influence of NUMT
contamination in routine samples and techniques. Int. J. Legal Med. (ISI 2006 Impact Factor: 2.620)
3- Behar DM, Metspalu E, Kivisild T, Rosset S, Tzur S, Hadid Y, Yudkovsky G, Rosengarten D, Pereira L, Amorim
A, Kutuev I, Gurwitz D, Bonne-Tamir B, Villems R, Skorecki K (2008) Counting the founders: the matrilineal genetic
ancestry of the Jewish Diaspora. PLoS ONE.
4- Cerny V, Mulligan CJ, Ridl J, Zaloudkova M, Edens CM, Hajek M, Pereira L (2008) Regional differences in the
distribution of the sub-Saharan, West Eurasian and South Asian mtDNA lineages in Yemen. Am. J. Phys. Anthropol.
(ISI 2006 Impact Factor: 2.136)
5- Afonso C, Alshamali F, Pereira JB, Fernandes V, Costa M, Pereira L (2008) mtDNA diversity in Sudan (East
Africa). Forensic Sci. Int.: Genetics Supplement Series. 22st Congress of the International Society for Forensic
Genetics. Copenhagen, Denmark. 22-25/08.
6- Freitas F, Pereira L (2008) Heterogeneity in coding mtDNA mutation rates: implications in forensic genetics.
Forensic Sci. Int.: Genetics Supplement Series. 22st Congress of the International Society for Forensic Genetics.
Copenhagen, Denmark. 22-25/08.
7- Pamplona JP, Freitas F, Pereira L (2008) A worldwide database of autosomal markers used by the forensic
community. Forensic Sci. Int.: Genetics Supplement Series. 22st Congress of the International Society for Forensic
Genetics. Copenhagen, Denmark. 22-25/08.
8- Goios A, Gusmão L, Rocha M, Pereira L, Bogue M, Amorim A (2008) A SNaPshot multiplex kit for mtDNA
identification of mouse inbred strains. Forensic Sci. Int.: Genetics Supplement Series. 22st Congress of the
International Society for Forensic Genetics. Copenhagen, Denmark. 22-25/08.
68
Invited oral communications:
1- Pereira L (2007) mtDNA sequences and sperm motility. Institute of Integrative & Comparative Biology, Faculty of
Biological Sciences, University of Leeds, Leeds, United Kingdom. 9/05.
2- Pereira L (2007) Genes e artefactos na evolução humana. Sociedade de Antropologia e Etnologia. 24/11. (Genes
and artefacts in human evolution. Portuguese Society of Anthropology and Ethnology.)
Selected oral communications:
1- Fernandes V, Pereira JB, Costa M, Afonso C, Alshamali F, Pereira L (2007) On the track of the Out-of-Africa:
complete mtDNA sequencing of L3 haplotypes from Sudan. 11st Annual Meeting of the Portuguese Society of
Human Genetics. Porto, Portugal. 15-17/11.
2- Freitas F, Pereira L, Rocha R, Samuels DC (2007) Relationship between mtDNA binding energy and mutability in
human population. 2007 Link-age Meeting and IBMC Symposium. Porto, Portugal. 28/11-01/12.
69
PUBLIC HEALTH AND CANCER
(Department of Hygiene and Epidemiology, Porto University Medical School – IPATIMUP)
The Public Health group is a result of the cooperation between the Department of Hygiene and
Epidemiology from the Porto University Medical School (SHE-FMUP) and the IPATIMUP. It
aims the transfer of knowledge across different levels of scientific production – from bench side
to populations, and back – promoting the interaction between researchers with expertise on
molecular pathology, molecular and population genetics, and epidemiology. The SHE-FMUP
researchers are involved in this common project according to their expertise and research
interests.
In 2007, Nuno Lunet (Degree in Pharmaceutical Sciences, Master in Public Health, PhD in
Public Health), Bárbara Peleteiro (Degree in Biochemistry, Master in Epidemiology, PhD
student), Joana Bastos (Degree in Mathematics, Master in Probability and Statistics, PhD
student), Marta Pereira (Degree in Biochemistry, Master student) were involved in this
cooperation. The publications reflect the research that has been developed by these elements
at the SHE-FMUP, and in cooperation with other groups from IPATIMUP.
• Objectives/Goals of the research activity
Descriptive and analytic cancer epidemiology, specifically the fields of gastric cancer and
Helicobacter pylori infection, and systematic review and meta-analysis are our main research
activities. Taking into account ongoing projects and PhD work plans, we are engaged in
clarifying the role of H. pylori infection in the web of gastric cancer causation by identifying
which factors modulate the progression towards cancer in H. pylori-infected subjects, namely in
the Portuguese and in the Mozambican settings, and to explore the hypothesis of H. pylori
infection being a necessary cause for the occurrence of gastric cancer. We are also
determined to understand the dynamics of H. pylori infection in a life-course perspective by
identifying which factors contribute to the acquisition of H. pylori infection throughout life and
understanding how do the decline in the prevalence of H. pylori infection and the simultaneous
increase in the proportion of subjects acquiring the infection at older ages operate to explain
the long term trends in gastric cancer mortality, in high and low risk European countries.
• Major achievements during 2007
- Bárbara Peleteiro and Joana Bastos concluded their Master Thesis, respectively in
Epidemiology and in Probability and Statistics, and received a Grant from FCT to pursue
their PhD in Public Health (at the Porto University Medical School).
70
- Nine articles were published in Pubmed indexed papers and three in Portuguese papers
not indexed in Pubmed.
- The XVI edition of the Porto Cancer Meeting was organized by the group.
• Publications
Master theses
1. Peleteiro B. Risk factors for gastric intestinal metaplasia. MSc Thesis. University of
Porto, 2007.
Description:
The main aim was to identify factors that contribute to the occurrence of intestinal
metaplasia in H. pylori-infected individuals. The results obtained support a major role for
smoking as modulator of the carcinogenic effect of H. pylori infection, contributing to explain
geographical and individual differences in the risk of gastric cancer among the infected. In
addition, smoking and high-virulent H. pylori strains are differentially associated with the
complete and incomplete types of intestinal metaplasia, suggesting divergent pathways in
gastric carcinogenesis.
2. Bastos J. [Survival analysis. Application to patients with skin malignant melanoma.].
MSc Thesis. University of Lisbon, 2007.
Description:
The main aim was to describe the statistical approaches of survival analysis and present
methods used to convert continuous covariates in discrete, in survival studies conducted in
the context of the Cox model. The applications of the methods used were illustrated by the
statistical analysis of survival data from patients with skin malignant melanoma.
Book chapters
1. Lunet N, Peleteiro B, Bastos J, Barros H. Life course approaches to gastric cancer etiology:
current knowledge, potentials and promise. In: Tompkins MB, editor. Gastric Cancer
Research Trends. New York: Nova Publishers; 2007. p. 129-146.
71
Articles
1. Peleteiro B, Lunet N, Figueiredo C, Carneiro F, David L, Barros H. Smoking,
Helicobacter pylori virulence, and type of intestinal metaplasia in Portuguese males. Cancer
Epidemiol Biomarkers Prev 2007;16:322-6.
2. Peleteiro B. Lunet N, Santos Silva F, David L, Figueiredo C, Barros H. Short mucin 1
alleles are associated with infection with (low virulent) Helicobacter pylori strains [letter].
World J Gastroenterol 2007;13(12):1884-5.
3. Lunet N, Valbuena C, Lacerda-Vieira A, Lopes C, Lopes C, David L, Carneiro F, Barros
H. Fruit and vegetables consumption and gastric cancer by location and histological type:
case-control and meta-analysis. Eur J Cancer Prev 2007;16(4):312-327.
4. Padrão P, Lunet N, Santos AC, Barros H. Smoking, alcohol and dietary choices:
evidences from the Portuguese national health survey. BMC Public Health 2007;7:138.
5. Pinto M, Lunet N, Williams L, Barros H. Food and beverages billboard advertising is
frequent in Maputo, Mozambique [letter]. Food Nutr Bull 2007;28(3):365-6.
6. Lucas R, Lunet N, Carvalho R, Langa J, Muanantatha M, Nkunda LP, Barros H. Pattern
of medication use by students in a University from Maputo, Mozambique. Cad Saude
Publica 2007;23(12):2845-52.
7. Lunet N, Bastos J, Cumaio F, Silva P, Dias E, Barros H. Recall of drug utilization
depends on subtle structural questionnaire characteristics. Pharm World Sci 2007: DOI
10.1007/s11096-007-9161-8. [Epub ahead of print].
8. Pereira M, Azevedo A, Severo M, Barros H. Long-term stability of endogenous B-type
natriuretic peptide during storage at -20 degrees C for later measurement with Biosite
Triage assay. Clin Biochem. 2007 Oct;40(15):1104-7. Epub 2007 Jul 20.
9. Bastos J, Barros H, Lunet N. [Breast cancer mortality trend in Portugal (1955-2002)].
Acta Med Port 2007;20:139-44.
10. Lunet N, Bastos J, Peleteiro B. XVI Porto Cancer Meeting - Cancer Etiology: Bridging
Worlds. Arq Med 2007;21(3/4):115.
11. Bastos J, Rocha C. Análise de Sobrevivência: Métodos não paramétricos. Arq Med
2007; 21 (3/4): 111-14.
12. Pina FM, Figueiredo MG, Lunet N, Silva P, Silva A, Cruz F, Barros H. O eixo EGF e o
cancro da próstata: o transforming growth factor alfa (TGF-α) – experiência clínica. Acta
Urológica 2007;24(3):55-64.
• Ongoing common research projects
1. Gastric lesions in Mocambique and Portugal: the African Enigma (project funded by
Fundação Calouste Gulbenkian – FC-68697).
72
2. Environmental exposures and CDX2 expression in Helicobacter pylori-positive gastric cancer
(project funded by Fundação para a Ciência e Tecnologia - POCTI/SAUESP/61685/2004).
3. Risk of gastric cancer and its precursor lesions associated with salt consumption and
Helicobacter pylori infection (project funded by Agência Portuguesa de Segurança Alimentar).
• Organizations:
- Organization of the XVI Porto Cancer Meeting
The 16th edition of the Porto Cancer Meeting was organized by Nuno Lunet, Joana
Bastos and Bárbara Peleteiro. This year’s edition was entitled “Cancer Etiology:
Bridging Worlds”. As the past editions, the Meeting joined experts in distinct areas of
the research on cancer aetiology, aiming to bring closer researchers of different
awareness in the approach to the understanding of the causes of oncological diseases.
ACTIVITY PLANS FOR 2008
• Maintenance and improvement of the cooperation with other IPATIMUP groups through the
ongoing research projects and establishment of new projects
• Organization of statistics courses open to the IPATIMUP researchers
The main aim of this group is to transfer knowledge across different levels of scientific
production promoting the interaction between researchers with different expertises. The
creation of the Institute of Public Health of the University of Porto (ISPUP), in which the
members of this group will collaborate, will further increase the opportunities of interaction,
improving the ongoing partnership and allowing the establishment of new collaborations.
73
RELATÓRIO DE ACTIVIDADES DA UNIDADE DE EDUCAÇÃO CONTÍNUA E DIFUSÃO
CIENTÍFICA
A UNIDADE DE EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA do IPATIMUP (UECDC-IPATIMUP)
desenvolveu durante o ano de 2007 um conjunto de iniciativas em diversos domínios da promoção do
pensamento e cultura científica:
A- Projectos
1. Laboratório Aberto
O IPATIMUP inaugurou a 7/11/07 o Laboratório Aberto, destinado ao ensino experimental das ciências
no Centro de Recursos Educativos da Câmara Municipal do Porto. Até ao final de 2007 contou com
mais de 200 visitantes. O projecto é co-financiado pelo Ciência Viva e a Câmara Municipal do Porto.
Responsável: Luis Cirnes
Monitores: Dória Matias, Leandro Ribeiro e a Liliana Passos.
2. “Autolaboratório”
A
UECDC
do
IPATIMUP
encerrou
em
Dezembro
de
2007
a
execução
do
projecto
“AUTOLABORÁTORIO” (ref# CV / 138 / 2005) financiado pela Ciência Viva. Foram efectuadas 137
sessões em 29 escolas, que abrangeram uma população de 2786 alunos. Foi concluída a instalação do
website do projecto (www.autolaboratório.com). O projecto “Autolaboratório” participou ainda nas
seguintes iniciativas:
- Feira de Ciência da Escola Secundária Filipa de Vilhena (27 de Fevereiro de 2007)
- Semana da Ciência da Escola Secundária Oliveira do Douro (20 a 22 de Março de 2007)
- Mostra de Ciência no Pavilhão do Conhecimento (14 de Abril de 2007)
- Feira de Ciência do Colégio Horizonte (1 de Junho de 2007)
3. “A Magia da Ciência”
A UECDC-IPATIMUP deu continuidade ao projecto “A magia da Ciência – POCTI/DIV/2005/00061”
financiado pela FCT – Programa POCTI do III Quadro comunitário de apoio - Medida 3.1. - “Atribuição
de Financiamento para projectos de divulgação da cultura científica e tecnológica”. Foi finalizado o
desenvolvimento de um software – “MicE – Microscópio Educativo” que possibilitará às escolas uma
abordagem virtual à biologia celular.
4. “Despertar para a Ciência”
A UECDC-IPATIMUP iniciou a implementação do projecto “Despertar para a Ciência” aprovado pela
Agência Ciência Viva e com a parceria da Câmara Municipal da Trofa. Foi dado particular ênfase à
estruturação e produção de conteúdos para as sessões experimentais “EUREKA” cujos público-alvos
são alunos do 1ºciclo.
5. Projecto “EEA – Grants”
A UECDC-IPATIMUP iniciou a implementação do projecto “EEA - Grants”, nomeadamente a produção
de conteúdos para a actividade “Cancermobile”.
74
B- Programas
1. Rede de Residências: Experimentação Arte | Ciência e Tecnologia
A UECDC-IPATIMUP participou como centro de acolhimento, no programa “Rede de Residências:
Experimentação Arte | Ciência e Tecnologia” promovido pela Direcção Geral das Artes / Ministério da
Cultura e a Ciência Viva, recebendo a artista Sónia Moreira que de Outubro de 2007 a Janeiro de 2008
desenvolveu ao seu projecto “What we are and Who we are”. Os resultados do projecto foram
apresentados no dia 03 de Março de 2008 na FNAC do Porto durante o ciclo “Falar sobre Arte e
Ciência”.
2. Programa “Viver uma Escola Diferente”
Assinatura e arranque do protocolo com o Pelouro da Educação da Câmara Municipal do Porto Programa “Viver uma Escola Diferente” – proporcionou o ensino experimental das ciências na sala de
aulas de escolas (5) do primeiro ciclo. Janeiro 2007.
3. Programa “Ciência Viva em Férias”
O Programa “Ciência Viva em Férias” facultou a 30 alunos (28 portugueses e 2 espanhóis) um estágio
de duas semanas, que decorreu entre os meses de Junho a Setembro, 2007, integrado no o “Ciência
Viva”.
4. Segunda há Ciência.
O programa de visitas de alunos ao IPATIMUP, proporcionou a 76 alunos do Norte e centro do País o
contacto directo com os Investigadores do IPATIMUP. Janeiro a Junho de 2007.
5 – Estágio.
De 2 a 5 de Abril tivemos 5 alunos do 12º ano de Lisboa, a estagiar no IPATIMUP.
C- Conferências, Colóquios e Seminários
1. Colóquios sobre – Medicina e cancro - 2007
A UECDC-IPATIMUP em colaboração com a Fundação de Serralves organizou, a 18 e 31 de Outubro
na na Fundação de Serralves, o quarto Ciclo de Colóquios (2007) sobre Medicina e Cancro, este ano
subordinado ao tema “Cancro e biologia molecular do Cancro”.
2. XI Conferencia do Equinócio
A UECDC-IPATIMUP organizou a XI conferência do Equinócio intitulada “A lição de Fernando Gil”,
realizada a 9 de Outubro de 2006, com a coordenação da Prof. Doutor João Lobo Antunes e os
conferencistas Danièle Cohn, Maria Filomena Molder, Paulo Tunhas e Manuel Villaverde Cabral.
3. Palestras
A UECDC-IPATIMUP promoveu durante o ano de 2006, a realização de palestras sobre temas como a
Biologia, a Genética, o Cancro, em várias escolas secundárias (Esc. Sec. Sertã – Março 2007; Esc.
Sec. Gondomar – Outubro 2007; Esscola da Ponte Vila das Aves – Novembro 2007).
75
D- Actividades de Divulgação Científica
1. Escola das Ciências da Vida e da Saúde – Universidade Júnior
A UECDC-IPATIMUP participou na iniciativa “Escola das Ciências da Vida e da Saúde”, integrada no
programa “Universidade Júnior” promovido pela Universidade do Porto de 16 a 20 de Julho e de 03 a 07
de Setembro. Os alunos participantes, desenvolveram 2 projectos, um no âmbito da genética
("Genómica comparativa de diversas espécies de mamíferos através do estudo de sequencias
codificantes e não codificantes do DNA") e outro no âmbito da biologia celular ("Efeitos celulares de
mutações inactivantes em genes supressores tumorais").
2. “Semana da Ciência e da Tecnologia”
A UECDC-IPATIMUP na “Semana da Ciência e da Tecnologia”, de 20 a 24 de Novembro, o IPATIMUP
aderiu ao programa “Portas Abertas” do Ciência Viva, na qual os alunos que frequentaram o “Ciência
Viva em Férias – edição - 2006” puderam trazer amigos para visitar as instalações, bem como
esclarecer dúvidas com os investigadores.
3. “Dia do IPATIMUP”
No dia 22/02/07 fomos visitados pelas escolas secundárias de: Gafanha da Nazaré – 30 alunos, Sertã –
40 alunos e Tondela – 35 alunos.
4. Traz um amigo também
No dia Nacional da Cultura Cientifica, o IPATIMUP esteve de portas abertas para receber os alunos
que frequentaram o “Ciência Viva em Férias”. Intitulado -“ Traz um amigo também”. 24/11/2007.
Integrado com o Ciência Viva.
5. Participação no dia da Universidade
Participação no dia da Universidade do Porto, que decorreu no Pavilhão Rosa Mota – Porto. Conjunto
de experiências sobre os temas célula e ADN. Contou com a colaboração de alguns Investigadores.
Nos dias 15 a 18 de Março de 2007. Integrado com a Universidade do Porto.
E- Exposições
1. 5ª Mostra de Ciência, Ensino e Inovação da Universidade do Porto
A UECDC-IPATIMUP participou na “5ª Mostra de Ciência, Ensino e Inovação da Universidade do Porto”,
promovida pela Universidade do Porto, que decorreu de 15 a 18 de Março de 2007, no Pavilhão Rosa
Mota – Porto. O stand fez a divulgação das diversas vertentes de actividade do IPATIMUP
(Investigação, Formação, Diagnóstico e Divulgação), proporcionando ainda um conjunto de
experiências sobre os temas a célula e o ADN.
2. 2ª Semana da Educação da Câmara Municipal da Trofa
A UECDC-IPATIMUP participou na “2ª Semana da Educação da Câmara Municipal da Trofa”,
promovida pelo município da Trofa, que decorreu de 23 a 27 de Abril de 2007. O stand fez a divulgação
das diversas vertentes de actividade do IPATIMUP (Investigação, Formação, Diagnóstico e Divulgação),
proporcionando ainda um conjunto de experiências sobre os temas a célula e o ADN.
76
ÁREA: PROJECTOS/MULTIMÉDIA
Responsável: LUÍS Filipe Santos Silva
Em 2007 a UECDC-IPATIMUP continuou a promover a divulgação das conferências, colóquios e
seminários científicos, promovidos pelo IPATIMUP disponibilizando estes conteúdos sob forma de
podcasts alojados no iTunes e no site www.ipatimup-uecdc.com.
A. Podcasts de Conferências do IPATIMUP
1- 6th Porto Cancer Meeting “Cancer etiology: bridging worlds”
de 20 a 21 de Abril de 2007.
2- 10th Portugaliae Genética
de 22 a 24 de Março de 2007.
B. Podcasts de Seminários Científicos do IPATIMUP
3- “How much (or little!) do we understand about genomes?”
Elia Stupka, Consortium for Biomolecular Medicine, Trieste, Italy.
Conferência organizada a 17 de Outubro pelo IPATIMUP e pela Faculdade de Medicina do Porto.
4- “HDGC from families to populations.”
David Huntsman, Faculty of Medicine, Univ. British Columbia, Vancouver, Canada.
Conferência organizada a 15 de Outubro pelo IPATIMUP e pela Faculdade de Medicina do Porto.
5- “Cdx2 gene in intestinal repair and cancer.”
Jean Noel Freund, Inserm, Strasbourg, France.
Conferência organizada a 18 de Julho pelo IPATIMUP e pela Faculdade de Medicina do Porto.
6- “Molecular approaches for studying neurodegenerative diseases.”
Tiago Outeiro, IMM, Lisboa, Portugal.
Conferência organizada a 04 de Junho pelo IPATIMUP e pela Faculdade de Medicina do Porto.
7- “Integrin signalling and downstream effectors.”
João Relvas, Univ. of Zurich, Zurich,Switzerland.
Conferência organizada a 28 de Maio pelo IPATIMUP e pela Faculdade de Medicina do Porto.
8- “PTM – Biomarkers for cancer.”
Henrik Clausen, Univ. Copenhagen, Copenhagen, Denmark.
Conferência organizada a 12 de Março pelo IPATIMUP e pela Faculdade de Medicina do Porto.
9- “Molecular Tools.”
Ola Soderberg, Univ. Uppsala, Uppsala, Sweden.
Conferência organizada a 07 de Março pelo IPATIMUP e pela Faculdade de Medicina do Porto.
10- “Exploiting BH3- only proteins for effective cancer therapy.”
Dean Fennell, Queen’s Univ., Belfast, Northern Ireland.
Conferência organizada a 21 de Fevereiro pelo IPATIMUP e pela Faculdade de Medicina do Porto.
77
Equipa:
Jorge Oliveira (Tecnologias da Comunicação)
Rui Oliveira (Design gráfico)
Nuno Ribeiro (Programação multimédia e produção de conteúdos)
José Rui Fernandes (coordenador/web design)
78
SERVIÇO Á COMUNIDADE
UNIDADE DE PRESTAÇÃO DE SERVIÇOS (UPS)
Introdução
O principal objectivo da Unidade de Prestação de Serviços (UPS) em 2007 foi o de manter o Sistema de Gestão de
Qualidade definido desde 2003, de acordo com as normas propostas pelo CAP e manter esta Acreditação após a
segunda inspecção efectuada pelos inspectores da CAP “in loco”. Esta inspecção foi realizada em Março de 2007 e
a Acreditação foi mantida até Março de 2009, quando ocorrerá a terceira inspecção. O número total de exames da
Unidade foi de 13214. Como vem sucedendo há vários anos, continuámos a actuar como um centro de formação
profissional pós-graduado, tendo recebido, em 2007, 4 patologistas e 7 técnicos. Cinco destes técnicos estagiaram
por um período de 10 semanas cada um, no âmbito do protocolo estabelecido com a CESPU e ESTES-P.
1.
Recrutamento de pessoal:
•
Não houve alterações no quadro de pessoal.
2. Aquisição de Equipamento e Testes de Proficiência:
Com a finalidade de equipar o laboratório de rotina diagnostica, no âmbito do Programa de Acreditação da Unidade
via CAP, foram adquiridos os seguintes equipamentos e testes de proficiência.
Câmara Segurança biológica
Serra ossos com suporte
Ventilador para câmara biossegurança
Inspecção CAP
Testes de Proficiência do Colégio Americano de
Patologistas
UK-NEQAS
3. Estágios de internos:
Estágios
Nome
Mário Araujo Júnior,
Residente de
Anatomia Patológica da Universidade
Estadual do Rio de Janeiro, Brasil
Vanessa Carla Giatti, Residente de
Anatomia Patológica, Hospital do Servidor
Público Estadual, São Paulo, Brasil
Mara Patrícia Guilhermino de Andrade,
Médica Residente, Hospital do Servidor
Público Estadual, São Paulo, Brasil
Ellen
Caroline
Toledo
Nascimento,
Residente de Patologia,
FAMERP/
FUNFARME, São Paulo, Brasil
Período
02/01/2007 a
31/01/2007
05/01/2007 a
27/02/2007
01/05/2007 a
30/06/2007
01/05/2007 a
30/06/2007
Tipo de Estágio
Patologia Cirúrgica
e Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Situação Actual
Concluído
Concluído
Concluído
Concluído
4. Publicações com material da U.P.S.
Dufloth RM, Matos I, Schmitt F, Zeferino LC. Tissue microarrays for testing basal biomarkers in familial breast
cancer cases. Sao Paulo Med J 125: 226-230, 2007.
Paredes J, Lopes N, Milanezi F, Schmitt FC. P-cadherin and cytokeratin 5: useful adjunct markers to distinguish
basal-like ductal carcinomas in situ. Virchows Archives 450: 73-80, 2007.
79
Ricardo S, Milanezi F, Carvalho S, Leitão D, Schmitt F. HER2 evaluation through the novel rabbit monoclonal
antibody SP3 and CISH in tissue microarrays of invasive breast carcinoma. Journal of Clinical Pathology 60: 10011005, 2007.
Gomes AL, Bardales RH, Milanezi F, Reis RM, Schmitt F. Molecular analysis of c-Kit and PDGFRA in GISTs
diagnosed by EUS. American Journal of Clinical Pathology 127: 1-8, 2007.
Schmitt FC, Gomes AL, Milanezi F, Reis R, Bardales R. Mutations in gastrointestinal stromal tumors diagnosed by
endoscopic ultrasound-guided fine needle aspiration. Minerva Med 98: 385-388, 2007.
Longatto-Filho A, Costa SM, Milanezi F, Montruccoli D, Montruccoli GC, Baltazar F, Schmitt FC.
Immunohistochemical expression of VEGF-A and its ligands in non-neoplastic lesions of the breast samplingassisted by dynamic angiothermography. Oncol Rep 18: 1201-1206, 2007.
Lee AHS, Paish EC, Marchio C, Sapino A, Schmitt FC, Ellis IO, Reis-Filho JS. The expression of Wilm´s tumour1 and Ca125 in invasive micropapillary carcinoma of the breast. Histopathology 51: 824-828, 2007.
5.
Exames realizados na U.P.S.
Nº total de exames: 13.214
Captura híbrida: 123
Citologias ginecológicas: 7.299
Citologias não ginecológicas: 353
Citologias aspirativas: 2.305
Histológicos: 2.306 (1.152 Autópsias)
Histoquímicos: 80
Imuno-histoquímicos: 216 (inclui hepáticas com imuno)
Hibridização in situ: (Projecto ROCHE): 294
Imunofluorescência directa: 9
Relatório complementar: 31
Casos em consulta*: 198
•
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Os casos em consulta foram oriundos das seguintes instituições:
Afaf M. Elhag - The Laboratory Mafrag Hospital - Abu Dhabi - U.A.E.
Agostinho Sanches - Hospital Senhora de Oliveira Guimarães - Portugal
Albert Thiry - Centre Hospitalier Universitaire de Liège – Liège - Bélgica
Alberto Sáiz López - Hospital de Galdakao - Vizcaya - Spain
Ana Barroso – Centro Hospitalar de Vila Nova de Gaia – Porto - Portugal
Ana Catarino – Hospital da Luz – Lisboa - Portugal
Ana Paula Martins – Hospital Santa Cruz – Carnaxide – Lisboa – Portugal
Aurel Perren - Institut Fur Pathologie, Ismaninger – Munchen - Alemanha
B. Iglesias - Centro Médico POVIS - Vigo (Pontevedra) - Espanha
Bárbara Parente - Centro Hospitalar de Vila Nova de Gaia – Porto - Portugal
Begoña Sanromán Budiño - Hospital Universitario Gran Canaria Doctor Negrín - Espanha
Ben Davidson - Rikshospitalet - Radiumhospitalet Medical Center – Oslo - Noruega
Carla Carrilho – Hospital Central de Maputo - Moçambique
Carlos Prada Puentes - The Penine Acute Hospitals NHS Trust – Manchester – U.K.
Carmen Alberola - Hospital La Fe – Valencia - Espanha
Célia Fazzio - Laboratório Patologia - Hospital Base - São Paulo - Brasil
Christophe Girardet - Institut Central des Hôpitaux Valaisans – Sion - Suiça
Daniel Ferraciolo Brandão - Hospital das Clinicas da Faculdade De Medicina - São Paulo –
Brasil
Denis Larsimont - Départment Interhospitalier d' Anatomie Pathologie – Bruxelas - Bélgica
Élbio C. de Paula – GOIAS - Brasil
Eduardo De Miguel Herran - Hospital de Galdakao - Vizcaya - Espanha
80
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Eduardo Silva Ferreira – Porto - Portugal
Eduardo Studart - Laboratório Silvany Studart - Hospital Português – Salvador – Bahia - Brasil
Feriha Öz - Taksim – Istambul - Turquia
Fernanda Marcos – Centro Hospitalar de Lisboa Central – Hospital S. José – Lisboa - Portugal
Fernandez Flores - Clinica Ponferrada - Ponferrada – Leon - Espanha
Filomena Medeiros – Hospital Pulido Valente – Lisboa - Portugal
Fred Ellinger - Marilia – São Paulo - Brasil
Frédérique Tissier - Groupe Hospitalier Cochin - Saint-Vicente-de-Paul – Paris - França
Geneviève Belleannee - CHU - Hôpitaux de Bordeaux - França
Geneviève Fouilhoux - Departemente D' Anatomie et de Cytologie - Clermont- Ferrand - França
Hanifa Bouzourene - Centre Hospitalier Universitaire Vaudois – Lausanne - Suiça
Helena Garcia – CEDAP – Coimbra - Portugal
Inmaculada Barredo - Hospital de Galdakao - Vizcaya - Spain
Isabel Calhim – Centro Hospitalar do Porto – Hospital Geral Santo António – Porto - Portugal
J. P. Machayekhi - Centre D'Anatomie Pathologique et de Cytologie – Valence - França
Jean Louis Dargent - Institute Jules Bordet - Bélgica
Jera Jeruc - Institut Za Patologijo, Korytkova 2 – Ljubljana- Eslovénia
Jesús Pinto Blázquez- Hospital San Agustín – Asturias - Espanha
José Cameselle Teijeiro - Hospital Clínico Universitario - Santiago de Compostela - Espanha
José Ignacio Cortés Garcia – Hospital Garcia da Orta – Lisboa - Portugal
José Melo Cabral – Hospital Divino Espírito Santo – Ponta Delgada – Açores - Portugal
José Ramos Vizcaíno Vásquez - Centro Hospitalar do Porto – Hospital Geral Santo António –
Porto - Portugal
José Vílchez - Centro Hospitalar do Barlavento Algarvio, EPE – Algarve – Portugal
Juan Carlos Mellindez Barroso - Hospital Infante D. Pedro – Aveiro - Portugal
K. Sikand - Christie Hospital – Manchester – U.K.
Krystyna Kotanska-Groholt - The Norwegian Radium Hospital – Oslo - Noruega
Leire Andrés Alvarez - Hospital de Galdakao - Vizcaya - Spain
Lia Menasce - Christie Hospital – Manchester – U.K.
Lucília Gonçalves - Hospital Doutor Fernando Fonseca – Sintra - Portugal
Luiz Alberto Veronese - Instituto de Patologia de Araçatuba – São Paulo - Brasil
Manuela Vivario - Hopital Sainte- Therese – Bastogne - Bélgica
Mara Andrade - Neomater-Hospital – São Paulo - Brasil
Marcello Franco - UNIFESP/EPM - São Paulo - Brasil
Margarida Teixeira – Hospital S. Marcos – Braga - Portugal
Maria Fernanda Cunha – Hospital Santo André – Leiria - Portugal
Maria Helena Sousa Oliveira – Centro Hospitalar de Cascais – Lisboa - Portugal
Maria Isabel Lourenço – Hospital Pulido Valente – Lisboa - Portugal
Maria José Brito - Hospital Garcia da Orta, S.A. Almada - Portugal
Maria José Melo – Hospital CUF Descobertas – Lisboa - Portugal
Maria José Perez del Rio – Centro Hospitalar de Trás-os-Montes e Alto Douro – Vila Real Portugal
Maria João Martins – Hospitais da Universidade de Coimbra – Coimbra - Portugal
Maria Teresa Dias Carvalho - Hospital São Teotónio – Viseu - Portugal
Marie Triller - Anatomie Et Cytologie Pathologiques – Paris - França
Marise Amaral Rebouças Moreira - Laboratório Atalaia S/C ltda – Goiás - Brasil
Mary Toner - University Teaching Hospit. Trinity College Dublin - Irlanda
Mete Düren - Istanbul - TURKEY
Michèle Nicaise - Institut de Pathologie et de Génétique, Asbl – Gosselies - Bélgica
Moises Salgado Pedrosa - Centro Especializado em Anatomia Patológica – Minas Gerais Brasil
Muge Tuncyurek - Ege University Facult of Medicine - Izmir - Turkey
Mário R. Montemór Netto - Patologia Médica – Ponta Grossa - Brasil
Naoto Kuroda - Kochi Red Cross Hospital – Kochi - Japão
P. Anani - Laboratoire Argot Lab – Lausanne - Suiça
Paola Souza - Souza Anatomia Patológica - Brasil
Patrícia Sabino de Matos - FCM/ Unicamp – Campinas - Brasil
Paulo Roberto Grimaldi Oliveira – São Paulo - Brasil
81
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Pedro M. Torrabadella – CEDAP – Coimbra - Portugal
Pedro Monteiro - Centro Hospitalar Do Alto Ave, E.P.E. – Guimarães - Portugal
Peyker Temiz - Celal Bayar University Medical School – Manisa - Turquia
Pilar San Miguel - Hospital Povisa – Vigo – Espanha
R. Cunqueiro Sarmiento - Complexo Hospitalario Universitario de Vigo – Espanha
Ronald de Krijger - Erasmus MC - Holanda
San Miguel - Centro Médico POVI - Vigo - Espanha
Santiago Ramón Cajal - Hospital Vall d’Hebron – Barcelona - Portugal
Sofia Loureiro Santos – Hospital Garcia da Orta – Lisboa - Portugal
Sofia Neves – Centro Hospitalar de Vila Nova de Gaia – Porto - Portugal
T. Aramendi - Hospital de Móstoles – Madrid - Espanha
T. Rivera - Hospital de Móstoles – Madrid - Espanha
Umit Ince - Oruc Patoloji ve Sitoloji Lab. - Turquia
Yersu Kapran – Istanbul - Turquia
6.
Controle de Qualidade
•
•
•
•
•
Data de Instituição 19-01-1998
Membros
Prof. Fernando Schmitt
Drª Fernanda Milanezi
D. Susana Silva
•
•
Nº de Casos revistos em 2006 – 573 casos de patologia cirúrgica e citopatologia (não ginecológica)
Nº de Casos revistos em 2006 de citologias ginecológicas: 1970
O Sistema de Controle de Qualidade foi realizado através do sistema informático Sislab, cujos resultados estão
publicados no Manual da Qualidade da UPS-2006. Os principais achados comparativamente aos últimos 3 anos
foram:
Total nº de casos
Casos revistos
Tipo de Exames
Citologia aspirativa por agulha fina
Citologia não ginecológica
Histológico de biópsias
Histológico de peças cirúrgicas
Biópsia hepatica sem imuno-histoquímica
Biópsia hepatica com imuno-histoquímica
Biópsia gástrica com pesquisa de H. Pylori
Consultas
Citologia ginecológica a)
Imunofluorescência
2004
12.755
302
2005
13.573
311
2006
13.413
573
2007
13.214
670
2004
121
6
27
68
4
11
3
3
54a)
2005
164
6
24
91
2
11
9
3
N/A
1
2006
375
18
37
97
7
25
11
2
N/A
1
2007
399
62
81
101
3
14
5
3
N/A
2
a)
Desde Abril de 2004 os casos de citologia ginecológica foram avaliados separadamente de acordo com o
procedimento PR.MED.01
Valores de discordância:
Critério
Identificação do espécime, arquivo e macroscopia
Diagnóstico
Codificação
2004
2.1%
0.4%b)
0.9%
2005
2.6%
0.6%c)
0.6%
2006
1.7%
0.0%
0.2%
2007
0,45%
0.0%
0.0%
82
“Turn-around-time”
2004
2.8 dias
4.5 dias
4.9 dias
9.5 dias
3.2 dias
7.2 dias
6.9 dias
0.3 dias
14.7 dias
Citologia aspirativa por agulha fina
Citologia não ginecológica
Histológico de biópsias
Histológico de Peças cirúrgicas
Biópsia hepatica sem imuno-histoquímica
Biópsia hepatica com imuno-histoquímica
Biópsia gástrica com pesquisa de H. Pylori
Consultas
Citologia ginecológica a)
a)
2005
1.8 dias
4.7 dias
2.4 dias
3.2 dias
5.0 dias
5.2 dias
1.9 dias
N/A
2.4 dias
2006
0.8 dias
1.7 dias
1.3 dias
1.7 dias
3.8 dias
4.6 dias
1.5 dias
N/A
3 dias
2007
0.31 dias
1 dia
1.82 dias
2.2 dias
2 dias
4.25 dias
2 dias
N/A
1.7 dias
(Janeiro a Março)
Na análise do controle de qualidade de citologia ginecológica, os principais resultados foram os seguintes:
Total de casos
Casos revistos
Concordância entre
patologistas
e
citotécnico
Discordância entre
patologista
e
citotécnico
2004
5.920
1404
2005
8.952
2123
2006
8.780
1970
2007
7.299
1438
2004
1301
(92,6%)
2005
1962
(92,4%)
2006
1758
(89,2%)
2007
1313
(91,3%)
103
(7,4%)
161
(7,5%)
214
(10,8%)
125
(8,6%)
O número total de citologias consideradas não satisfatórias para a análise foi de 1,1% (1,4% em 2006), o que
representa uma diminuição conforme um dos objectivos propostos no último ano.
Ascus/Agus foi diagnosticado em 178 dos casos com uma relação ASCUS/Lesão de 3,2 (1.4 em 2006).
Para além da participação, com aprovação, em todos os testes de proficiência da CAP, continuámos a participar de
forma positiva nos programas de controlo de qualidade externo das técnicas de imuno-histoquímica do UKNEQAS e do Programa de Qualidade da Sociedade Brasileira de Patologia (PIQ). Todas estas actividades estão
registadas de acordo com o Procedimento Regulamentador PR MED-05 – Programas Externos de Educação e
Avaliação Contínua.
83
UNIDADE DE PRESTAÇÃO DE SERVIÇOS (UPSI)
1. Exames realizados
1.1
97
4
6
Nº de exames requeridos
Pedidos cancelados
Em curso (ainda por efectuar ou por falta de 1 ou mais colheitas)
1.2
Tipos de exame:
Caracterizações/Identificações genéticas
6
Paternidades
Com 1 pretenso pai, sem análise da mãe
Com 2 filhos
1.3
6
1
Com 1 pretenso pai e com análise da mãe
Com 2 filhos
Com 2 pretensos pais e com análise da mãe
Com 3 pretensos pais e com análise da mãe
Maternidades
Com 1 pretensa mãe, sem análise do pai
Com 2 filhos
59
3
8
1
Outros parentescos
Comparação de perfis genéticos (amostra/indivíduo)
3
6
2
2
Locais de requisição:
Local
Amarante
Arouca
Aveiro
Barcelos
Braga
Bragança
BRASIL (Minas Gerais)
Covilhã
ESPANHA (Madrid)
Gondomar
Horta
Leiria
Lisboa
Matosinhos
Oliveira de Azeméis
Penafiel
Ponta Delgada
Porto
Póvoa de Varzim
Santa Maria da Feira
Santo Tirso
São João da Madeira
Seixal
Vale de Cambra
Vila do Conde
Vila Nova de Cerveira
Vila Nova de Gaia
Vinhais
Total
Tribunais
0
2
0
0
1
4
0
0
0
0
0
0
0
0
0
9
0
0
1
6
1
1
0
1
5
0
19
1
51 (52,6%)
Particulares
1
0
1
1
0
1
1
1
1
1
1
2
16
1
1
0
1
12
0
0
1
0
1
0
0
1
0
1
46 (47,4%)
Total
1
2
1
1
1
5
1
1
1
1
1
2
16
1
1
9
1
12
1
6
2
1
1
1
5
1
19
2
97
84
1.4
Nº exames por requerente
Clientes
Tribunais:
Trib. Família e Menores de Vila Nova de Gaia
Trib. Judicial de Vila Nova de Gaia
Trib. Santo Tirso
Trib. Penafiel
Trib. Bragança
Trib. Vila do Conde
Trib. Arouca
Trib. Póvoa de Varzim
Trib. São João da Madeira
Trib. Santa Maria da Feira
Trib. Braga
Trib. Vinhais
Trib. Vale de Cambra
Total
18
1
1
9
4
5
2
1
1
6
1
1
1
Particulares
Clínica Dr. Joaquim Chaves - Lisboa
Internos (IPATIMUP)
Outros
15
3
28
Total
97
2. Publicações
2.1
Revistas Internacionais
2.1.1
Trovoada MJ, Tavares L, Gusmão L, Alves C, Abade A, Amorim A, Prata MJ (2007): Dissecting the Genetic
History of Sao Tome e Principe: A New Window from Y-Chromosome Biallelic Markers. Ann Hum Genet.
71: 77-85.
2.1.2
Gomes I, Alves C, Maxzud K, Pereira R, Prata MJ, Sánchez-Diz P, Carracedo A, Amorim A Gusmão L
(2007): Analysis of 10 X-STRs in three African Populations. Forensic Sci Int: Genetics 1: 208-211.
2.1.3
Alves C, Gomes I, Prata MJ, Amorim A, Gusmão L (2007): Population data for Y-chromosome haplotypes
defined by 17 STRs (AmpFlSTR YFiler) in Portugal. Forensic Sci Int 171: 250-255.
2.2
Revistas Nacionais
2.2.1
Maia J, Fernandes S, Amorim A, Alves C, Gusmão L, Pereira L (2007): Determinação da gemelaridade: do
questionário de Peeters aos micro-satélites aleatórios espalhados pelo DNA. Revista Portuguesa de
Ciências do Desporto (RPCD) vol. 7, nr. 2.
3. Actividades/Outros
3.1
Participação no Exercício de Controlo de Qualidade de 2007 (Paternidade e Forense) do Grupo Espanhol e
Português da International Society for Forensic Genetics, GEP-ISFG (Certificados em anexo).
3.2
Participação em Comités e Representações Internacionais:
- Leonor Gusmão
- Membro do Editorial Board da Forensic Science International: Genetics (Elsevier Science,
London. ISSN: 1872-4973).
85
- Secretária e membro da Direcção do GEP-ISFG desde Junho 2004;
- Nomeada Tesoureira e membro da Direcção da ISFG em 2007.
- António Amorim
- Presidente do GEP-ISFG desde Junho 2004
- Membro do Editorial Board da Forensic Science International (Elsevier Science,
London. ISSN: 0379-0738).
3.3
Organização no seio do GEP-ISFG de exercícios de controle de qualidade:
- Grupo de Trabalho de Cromossomas Sexuais - estudo colaborativo de taxas de mutação em marcadores
incluídos no YFiler kit com vista a dar continuidade ao já desenvolvido sobre taxas de mutação em
cromossoma Y – iniciado em 2006 e finalizado em 2007 (artigo científico submetido);
- Grupo de Trabalho de Cromossomas Sexuais - estudo de colaboração de STRs do cromossoma X com o
objectivo de se avaliar um multiplex (X-STR Decaplex, desenvolvido no IPATIMUP) – iniciado em 2006 e
a finalizar em 2008;
- Grupo de Trabalho de Genética Forense Não Humana - dois exercícios colaborativos:
- o 1º, iniciado em 2006, incidiu sobre DNA mitocondrial em mancha de sangue de Canis
familiaris;
- o 2º, extensão do 1º e a finalizar em 2008, incidiu sobre DNA mitocondrial em pêlo de Canis
familiaris.
3.4
Desenvolvimento e optimização de uma reacção PCR em multiplex de marcadores autossómicos (9
STRs), específica para a determinação de perfis genéticos em cães. Aplicação do método em cães da raça
“Cão de Gado Transmontano”, sua análise populacional, e apuramento de relações de parentesco
genético.
3.5
Desenvolvimento de técnicas/meios para a diversificação de serviços/clientes em 2007 incluiu:
- a genotipagem/testes de paternidade em cães;
- a aplicação de marcadores do cromossoma X (utilizando para o efeito o X-STR Decaplex) em exames de
parentesco genético onde outros tipos de marcadores genéticos não fornecem a informatividade
pretendida;
- a elaboração de protocolos com outros laboratórios, nomeadamente com o laboratório “Gene - Núcleo
de Genética Médica” em Belo Horizonte, Brasil, na prestação de serviços de análise de amostras/vestígios
por técnicas de DNA mitocondrial.
4. Plano para 2008
4.1 – Definição do nosso papel na elaboração/manutenção da Base de Dados Nacional de Perfis
Genéticos.
4.2 – Iniciar e desenvolver um programa de Acreditação do laboratório (de acordo com a NP EN ISO/IEC
17025), necessária à nossa participação na Base de Dados Nacional de Perfis Genéticos.
4.3 – Diversificar serviços e clientes e elaboração de protocolos, incluindo:
- divulgação dos testes de perfis genéticos em cães pelas associações caninas e clínicas
veterinárias (e público em geral);
- desenvolvimento e divulgação de produtos específicos de identificação genética (por exemplo,
certificados de perfis de DNA; certificados de linhagens masculina/feminina; validação genética
de genealogias).
86
UNIDADE DE PRESTAÇÃO DE SERVIÇOS DE SUSCEPTIBILIDADE GENÉTICA(UPSS)
Resumo
Tal como em 2006, o objectivo fundamental da UPSS para o ano 2007 foi a consolidação da
sua actividade como prestador de serviços. Este objectivo foi conseguido através:
1. Do aumento do número de testes de diagnóstico genético (mais 73% relativamente a
2006) realizados em áreas que consideramos chave para a unidade, como a
gastrenterologia, oncologia e cardiovascular;
2. Do estabelecimento e implementação de novos testes nas áreas cardiovascular e
oncologia.
Para 2008 a UPSS estabeleceu como principais objectivos:
1. Aumentar o número de testes de diagnóstico genético realizados em áreas que
consideramos chave para a unidade, como a oncologia e cardiovascular;
2. Dar continuidade ao programa de estabelecimento e implementação de novos testes.
Em 2008 iremos continuar a implemetação de testes na área da hipertensão e iniciar a
implementação de novos testes na área de neurologia;
Publicações com material da UPSS
1. Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval
A, Hamelin R, Machado JC, Schwartz S Jr, Carneiro F, Seruca R. KRAS and BRAF
oncogenic mutations in MSS colorectal carcinoma progression. Oncogene 2007;26:
158-63.
2. Corso G, Roviello F, Paredes J, Pedrazzani C, Novais M, Correia J, Marrelli D, Cirnes
L, Seruca R, Oliveira C, Suriano G. Characterization of the P373L E-cadherin germline
missense mutation and implication for clinical management. Eur J Surg Oncol.
2007;33:1061-7.
3. Roviello F, Corso G, Pedrazzani C, Marrelli D, De Falco G, Berardi A, Garosi L, Suriano
G, Vindigni C, De Stefano A, Leoncini L, Seruca R, Pinto E. Hereditary diffuse gastric
cancer and E-cadherin: description of the first germline mutation in an Italian family. Eur
J Surg Oncol. 2007;33:448-51.
4. Pinto M, Wu Y, Mensink RG, Cirnes L, Seruca R, Hofstra RM. Somatic mutations in
mismatch repair genes in sporadic gastric carcinomas are not a cause but a
consequence of the mutator phenotype. Cancer Genet Cytogenet. 2008 15;180:110-4.
5. Meireles AM, Preto A, Rocha AS, Rebocho AP, Máximo V, Pereira-Castro I, Moreira S,
Feijão T, Botelho T, Marques R, Trovisco V, Cirnes L, Alves C, Velho S, Soares P,
Sobrinho-Simões M. Molecular and genotypic characterization of human thyroid
follicular cell carcinoma-derived cell lines. Thyroid 2007;17:707-15.
6. Davalos V, Dopeso H, Velho S, Ferreira AM, Cirnes L, Díaz-Chico N, Bilbao C, Ramírez
R, Rodríguez G, Falcón O, León L, Niessen RC, Keller G, Dallenbach-Hellweg G, Espín
E, Armengol M, Plaja A, Perucho M, Imai K, Yamamoto H, Gebert JF, Díaz-Chico JC,
Hofstra RM, Woerner SM, Seruca R, Schwartz S Jr, Arango D. High EPHB2 mutation
rate in gastric but not endometrial tumors with microsatellite instability. Oncogene
2007;26:308-11.
87
Exames realizados na UPSS
2005
alfa1-Antitripsina
Braf
BRCA
caderina - E
CARD15/NOD2
C-KIT/PDGFRA
CYP21
DAVD
EGFR
Estenose Supravalvular
H. Pylori
Hipercolesterolemia familiar
hMLH1 e hMSH2
HPV
KRAS
Marfan
Miocardio não-compactado
Miocardiopatia Dilatada
Miocardiopatia hipertrófica
MSI
N-myc/1p
OTC
P53
polimorfismos pró-inflamatórios
Prader-Willi/Angelman
RET
Síndrome de Brugada
Síndrome QT-Longo
SMAD4
Tipagem de HLA
TPMT
Translocações
UBE3A
X-Frágil
Outros
Total
95
0
15
5
3
0
0
0
0
0
2
0
1
6
0
0
0
0
0
19
3
5
1
1
64
4
0
0
0
0
0
8
10
3
0
245
2006
132
8
13
12
1
5
0
0
20
1
7
0
1
3
0
4
0
4
14
22
3
15
1
0
100
12
13
2
1
3
0
2
9
4
0
412
2007
161
1
94
6
4
9
10
15
58
0
12
11
1
4
5
8
10
20
49
39
2
22
4
1
95
14
30
5
1
1
3
4
8
2
4
713
88
As figuras 1 e 2 representam a distribuição e o número total, respectivamente, de exames
realizados na UPSS por área médica. O facto de maior relevo é o claro aumento da actividade
da UPSS, independentemente da área considerada. Ainda assim, as áreas cardiovascular e
oncologia foram aquelas que mais contribuiram para este crescimento.
Fig. 1: Distribuição dos exames realizados pela UPSS por área médica.
Fig. 2: Número de exames realizados pela UPSS por área médica.
89
Novos exames implementados na UPSS
Área Cardiovascular
• Displasia arritmogénica ventricular direita - pesquisa de mutações nos genes PKP2,
DSP e DSG2
• Miocardio não-compactado - pesquisa de mutações nos genes TAZ, LDB3 e DTNA
Área Oncologia
•
Pesquisa de mutações do gene KRAS
Outros
•
Diagnóstico de caracterização molecular/quantificação da actividade enzimática de
TPMT
•
Deficiência em 21-Hidroxilase - pesquisa de mutações
90
RECÉM-DOUTORADOS
António Pedro Fonseca
Avaliou a relação entre estirpes de Pseudomonas aeruginosa
isoladas de doentes durante a sua estadia num Hospital Central
Português recorrendo ao uso de “fingerprinting” genómico, métodos
fenotípicos e informação epidemiológica dos isolados. Estudou os
padrões de associação entre parâmetros de virulência, resistência aos
antibióticos, serótipo e genótipo nos isolados clínicos de P. aeruginosa.
Investigou a influência de diferentes estados fisiológicos e
mudanças morfológicas induzidas por antibióticos nas capacidades de
adesão, mobilidade e nas propriedades da superfície nos isolados
representativos da população da P. aeruginosa, bem como o efeito da
tensão de corte e das subculturas, com mudanças morfológicas
induzidas pelos antibióticos nas capacidades de adesão e formação de
biofilme da P. aeruginosa.
Demonstrou um elevado nível de diversidade em todos os
isolados estudados, bem como constatou a possível ocorrência de
contaminação-cruzada, colonização-cruzada e infecção-cruzada o que
sugeriu que a população da P. aeruginosa apresenta características
epidémicas no Hospital em estudo. Não houve um “cluster” fenotípico
em particular associado à mesma origem, serótipo, padrão β-lactâmico,
mobilidade e grupo genómico ou linha clonal. Não obstante, cinco
estirpes representativas da população da P. aeruginosa deste Hospital,
seleccionadas na base da reduzida similaridade genómica, também se
encontraram dispersas nos “clusters” fenotípicos.
Concluiu que a adesão e a formação de biofilme são alvos
atractivos para novas estratégias antibacterianas, nomeadamente pelo
uso de concentrações subinibitórias de antibióticos que induzam
mudanças na morfologia, na mobilidade e nas propriedades da
superfície, que estão dependentes do fenótipo e do estado fisiológico da
P. aeruginosa. Para além disso, o modelo dinâmico contribuiu para uma
melhor simulação das condições in vivo da adesão e formação de
biofilme da P. aeruginosa com a morfologia alterada induzida pelos
antibióticos β-lactâmicos.
Biólogo e Mestre em Engenharia Biomédica, doutorou-se em Biologia Humana em 8 de Outubro de 2007 com a tese
“Virulence of Pseudomonas aeruginosa clinical isolates: adhesion and biofilm formation as targets for antimicrobial therapy”.
Actualmente é Professor Auxiliar da Faculdade de Medicina da Universidade do Porto e Investigador do IPATIMUP.
91
Cristina Nogueira
Caracterizou alterações genético- moleculares do melanoma primário e
metastático, utilizando modelos humanos e animais. Analisou a interacção funcional entre RAS activado e PTEN inactivado na iniciação e
progressão do melanoma. Identificou um amplicon onde se localizam
oncogenes relacionados com a patogénese molecular do melanoma
(CDK4, MDM2). Através de oncogenómica comparativa contribui para a
identificação do gene NEDD9 como gene associado à metastização do
melanoma (Cell 125:1269 – 1281, 2006).
Bióloga, desenvolveu actividades de investigação no Danna-Farber Cancer Institute/Harvard Medical School e doutorou-se em
Biologia Humana na Faculdade de Medicina do Porto com a Tese intitulada “Molecular Genetics of melanoma initiation and
progression”. Actualmente está a fazer um pós-doutoramento no MIT (Massachusetts Institute of Technology), em Boston.
92
Sandra Martins
Estudou os mecanismos mutacionais da repetição CAG do gene ATXN3,
responsável pela doença de Machado-Joseph, sugerindo (1) um mecanismo
“multi- step” (com passos mutacionais envolvendo várias repetições) para a
evolução dos alelos normais, (2) origens diferentes para os alelos
intermé¬dios, e
(3) padrões diferentes de instabilidade intergeracional entre mutações de
origens distintas, com as maiores expansões associadas
preferen¬cial¬men¬te à linhagem mais comum (linhagem “Joseph”).
Relativamente à história das mutações sugeriu (4) uma origem asiática, com
cerca de 6000 anos, da mutação dispersa mundialmente, com introduções
mais recentes de haplóti¬pos fundadores ocorridas sucessivamente na
Alemanha, em França e em Portugal; a origem da linhagem “Machado” terá
ocorrido na população portuguesa, há menos de 2000 anos.
Bióloga, doutorou-se em 9 de Julho de 2007 com a tese “Evolutionary and epidemiological genetics of Machado-Joseph
disese”. Actualmente está a fazer um pós-doutoramento no IPATIMUP, em colaboração com a Universidade de Montréal.
93
RESUMO DOS PROJECTOS
Fundação para a Ciência e Tecnologia
“O papel de novos mecanismos regulatórios na perda de expressão da caderina-E” – 2007-2010 – 36 meses – IR: Carla
Oliveira Português
“Análise Trans-Específica do Impacto Fisiológico de Genes Inactivados” – 2007-2010 – 36 meses – IR: Luísa Azevedo
“Fenótipos/genótipos secretor e Lewis na infecção por Helicobacter pylori e calicivirus” – 2007-2010 – 36 meses – IR: Leonor
David
“Estudo da daptação humana no universo proteolítico: o exemplo dos inibidores de proteases de serina” – 2007-2010 – 36
meses – IR: Susana Seixas
“A comunidade cigana portuguesa: a história demográfica à luz dos padrões de diversidade genética” – 2007-2010 – 36 meses
– IR: Maria João Prata
“Reavaliação com ferramentas genéticas de levada resolução das origens geográficas e das rotas de dispersão dos primeiros
homens modernos e dos primeiros agricultores da bacia mediterrânea” – 2007-2010 – 36 meses – IR: Luísa Pereira
“Longevidade em humanos de origem africana - uma abordagem genética” – 2007-2010 – 36 meses – IR: Luísa Pereira
“Manipulação, in vitro e in vivo, da expressão do Sialyl Lewis X e E-caderina nos tumores da mama da cadela” – 2007-2010 –
36 meses – IR: Fátima Gartner
“Caracterização molecular e funcional da via do mTOR no carcinoma papilar da tireóide” – 2007-2010 – 36 meses – IR: Ana
Sofia Rocha
“No cancro colorectal com instabilidade de microssatélites são os genes BRAF e KRAS novos marcadores de prognóstico e
novas ferramentas terapêutica?” – 2005-2008 – 36 meses – IR: Raquel Seruca
“Genes associados à metaplasia intestinal da mucosa gástrica (mucina MUC2 e fucosiltransferase FUT3): regulação da
transcrição e relavância para a adesão do Helicobacter pylori” – 2005-2008 – 36 meses – IR: Leonor David
“Análise funcional dos repressores da caderina-E (Slug, ZEB1 e E12/E47) em carcinomas do estômago” – 2005-2008 – 36
meses – IR: Fátima Carneiro
“P-caderina no Cancro da Mama: o que regula a sua expressão e qual o seu papel na invasão de células neoplásicas?” –
2005-2008 – 36 meses – IR: Fernando Schmitt
“Identificação de genes associados a glicosilação induzidos em células gástricas pela Helicobacter pylori: “Glicómica” – 20052007 – 30 meses – IR: Celso Reis
“Adaptação bio-cultural: respostas evolutivas humanas a alterações relevantes na economia de subsistência” – 2005-2008 – 36
meses – IR: Jorge Rocha
“Estudo evolutivo e epidemiológico de focos de anemias hereditárias em Portugal” – 2005-2008 – 36 meses – IR: Maria João
Prata
“Clarificação da importância do polimorfismo da mucina MUC1 na infecção por helicobacter pylori” – 2005-2008 – 36 meses –
IR: Luís Filipe Silva
“Papel da activação oncogénica do BRAF na carcinogénese da tireoide” – 2005-2008 – 36 meses – IR: Ana Paula Soares
“Efeitos da infecção por Helicobacter pylori em células epiteliais gástricas” – 2005-2008 – 36 meses – IR: Céu Figueiredo
94
“Identificação de mecanismos moleculares subjacentes ao desenvolvimento de cancro gástrico em famílias portadoras e nãoportadoras de mutações germinativas da caderina-E” – 2005-2008 – 36 meses – IR: Carla Oliveira
“Etnias e genética na fronteira da rota de migração Bantu: o caso dos Karamojong” – 2005-2008 – 36 meses – IR: Leonor
Gusmão
“Identificação de vias de sinalização envolvidas na regulação do Cdx2 em dois modelos humanos de diferenciação intestinal e
polipose juvenil” – 2005-2008 – 36 meses – IR: Raquel Almeida
“Caracterização molecular genómica e pós-genómica das vias de sinalização RAS/RAF/ERK e P13K/AKT em tumores
agressivos da Tireóide” – 2005-2008 – 36 meses – IR: Ginesa Rostan
“Tumores mamários de gata – Análise Patológica, Molecular e Citigenética” – 2005-2008 – 36 meses – IR: Fátima Gartner
“Caracterização biológica de tumores mamários mistos caninos: histogénese, progressão tumoral e alterações genéticas” –
2005-2008 – 36 meses – IR: Fátima Gartner
“Mutações germinativas da Caderina-E do tipo "missense" e carcinoma difuso hereditário do estômago: um modelo para a
identificação das vias de sinalização mediadas pela caderina-E fundamentais na invasão” - 2005-2008 – 36 meses – IR:
Gianpaolo Suriano
“Estudo da heterogeneidade fenotípica patológica usando a proteína ornitina transcarbamilase (OTC) como modelo” – não
estava – 36 meses – IR: Gianpaolo Suriano
“PYLORI L&EPS: Caracterização Estrutural de Lipopolissacarídeos e Exopolissacarídeos de Helicobacter pylori –
Estabelecimento das bases químicas e bioquímicas para o desenvolvimento de vacinas e para a compreensão dos
mecanismos de adesão” – 2005-2008 – 36 meses – IR: Céu Figueiredo e Celso Reis
“Avaliação da Epidemiologia da Malária na Republica de Cabo Verde” – 2006-2008 – 24 meses – IR: António Amorim
“Bombas de efluxo em mycobacterium tuberculosis: caracterização molecular dos mecanismos de efluxo e uso de inibidores de
efluxo como novos compostos anti-bacilares” – 2005-2008 – 36 meses – IR: Filipe Sansonety
Fundação para a Ciência e Tecnologia - PROJECTOS DE REEQUIPAMENTO
“Interacções genético-ambientais na carcinogénese humana com ênfase no cancro gástrico – 2006-2007 – 24 meses – IR:
Maria do Céu Figueiredo
“Espectometria de massa (MALDI TOF) para caracterização de proteínas e análise proteómica no norte de Portugal” – 24
meses – IR: Celso Reis
Agência de Inovação
“Mecanismos que interferem na invasão celular e sua aplicação a estudos de oncologia e diagnóstico pré-natal” – 2005-2008 –
36 meses – IR: Raquel Seruca
“Teste de susceptibilidade genética para determinação de risco de desenvolvimento de Doença de Crohn na população
portuguesa”, ADI, Programa Ideia – Promotor GENETEST– 2005-2008 – 36 meses – IR: José Carlos Machado
Ministério da Saúde
“Registo da Deficiência Genética da Metabolização de Tiopurinas na População Portuguesa e Prevenção Toxicidade em
Terapêuticas Citostáticas” – 2006-2008 – 24 meses – IR: Maria João Prata
Agência Portuguesa de Segurança Alimentar
“Avaliação do risco do consumo de sal e da infecção por Helicobacter pylori no desenvolvimento de carcinoma gástrico e das
suas lesões precursoras em Portugal” – 2005-2008 – 36 meses – IR: Manuel Sobrinho Simões
95
Fundação Calouste Gulbenkian
“Caracterização de factores biológicos e estilos de vida com impacto de ocorrência e na forma de apresentação do cancro –
“Lesões da mucosa gástrica em Moçambique e Portugal: “o enigma Africano” – 2005-2008 – 36 meses – IR: Leonor David
“Utilização clínica dos alvos genéticos do tabaco” – 2006-2009 – 36 meses – IR: Luís Teixeira Costa
“Helicobacter Pylori: da Biologia à clínica” – 2006-2008 – 24 meses – IR: Céu Figueiredo
“Irradiação por tinea capitis e risco de cancro” – 2006-2009 – 36 meses – IR: José Teixeira Gomes
“Prevention and early diagnosis of cancer and precancerous lesions of cervix, stomach, breast and thyroid" - co-financiamento
EFTA - 2007-2010 – 36 meses – IR: Manuel Sobrinho Simões
PROJECTOS EM COLABORAÇÃO COM A INDÚSTRIA FARMACÊUTICA
“Diminuição da resistência ao imatinib (mesilato) em modelos de linhas celulares de leucemia através da diminuição específica
de MDR1 com siRNAs” – iniciou 2004 – IR: Helena Vasconcelos
“Tumores Estrumais Gastrointestinais: Caracterização Clinico-Patológica e Imuno-Histoquímica e Identificação de Factores de
Agressividade Biológica” – iniciou 2002 - IR: José Manuel Lopes
“Instalação de uma base de dados e banco de tecidos de tumores estromais gastrointestinais e tumores neuroendócrinos
(Regist e REGENE)” – iniciou 2006 - IR: Paula Soares
“É a via do mTOR relevante na iniciação/progressão dos melanomas e pode ser um alvo terapêutico nesses tumores? – iniciou
2006 - IR: Paula Soares e José Manuel Lopes
“Papel biológico da activação do BRAF na carcinogénese do colon” – iniciou 2004 – IR: Paula Soares
P53 e Resistência a Quimoterapia no cancro da Mama – iniciou 1999 – IR: Fernando Schmitt
“Approaching basal-like breast carcinomas to target theraphy. A project combining the reinforcement of logistics facilities with
translational research” – 2006-2009 – 36 meses – IR: Fernando Schmitt, Manuel Sobrinho Simões e Fátima Carneiro
“Uso de populações mistas para estudo da base genética da obesidade e hipertensão: O caso de Cabo Verde e da população
portuguesa das bacias do Tejo, Sado e Rio Guadiana” – GenOHmix” – 2007-2010 – 36 meses – IR: Sandra Beleza
“Expressão da P-caderina: seu efeito nas metásteses do cancro da mama e angiogénese utilizando modelos animais in vivo” –
2007-2010 – 36 meses – IR: Fernando Schmitt
PROTOCOLOS DE COLABORAÇÃO COM EMPRESAS DE OUTROS SECTORES INDUSTRIAIS E DE SERVIÇOS
“Criação de um Observatório/Consultório de Risco de cancro familiar e Ambiental” – 2006-2008 – 24 meses – IR: Raquel
Seruca
“Desenvolvimento de estudos que identifiquem compostos com propriedades quimioprotectoras da carcinogênese ou com
propriedades inibidoras da invasão de células tumorais” – 2005-2008 – 36 meses – IR: Paula Soares
PROTOCOLOS DE COLABORAÇÃO COM ASSOCIAÇÕES
“Validação do papel xiap na resistencioa da leucemia mielógena aguda à quimioterapia” – 2005-2008 – 36 meses – IR: Helena
Vasconcelos
“Investigação genética sobre o cão de gado transmontano” – IR: António Amorim
“Cão de Castro Laboreiro – Uma raça a preservar” – 2007 - IR: António Amorim
CONCURSOS E/OU ENQUADRAMENTOS INTERNACIONAIS
“Prevention, diagnosis and molecular characterisation of mismatch repair defect-related hereditary cancers of digestive
systems” – 2006-2009 – 36 meses IR: Raquel Seruca
96
“Molecular mechanisms of biosynthesis of sialylated cancer-associated mucin carbohydrate antigens in gastric carcinoma” –
2005-2008 – 36 meses – IR: Celso Reis
“The role of chronic infections in the development of cancer” – 2006-2010 – 48 meses – IR: José Carlos Machado
“Evaluation and Control of Neglected Mucosal Enteric Infections in Childhood” – 2006-2010 – 48 meses – IR: José Carlos
Machado
“Mapping of genes predisposing to familial thyroid tumours” – 2006-2009 – 36 meses – IR: Valdemar Máximo
“Archives Tissues: Improving Molecular Medicine Research and Clinical Pratice” – 2007-2009 – 24 meses – IR: Fátima
Carneiro
“Impacts – archive’s tissues: improving molecular medicine research and clinical practice” – 2007 – 2009 – 24 meses – IR:
Fátima Carneiro
"Global RNAi approaches to unravel eukaryotic host functions that modulate bacterial infections - ERA-NET PathoGenoMics" –
2007-2010 - 36 meses – IR: Céu Figueiredo
“Parasite and host genetic diversity in Helicobacter infections - ERA-NET PathoGenoMics” – 2007-2010 – 36 meses – IR: Jorge
Rocha e José Carlos Machado
“O cancro e as Lesões Pré-Cancerosas do estômago na China” – 2007-2008 – 24 meses – IR: Manuel Sobrinho Simões
“Prevention and early diagnosis of cancer and precancerous lesions of cervix, stomach, breast and thyroid" – EEA Grants 2007-2010 – IR: Manuel Sobrinho Simões
PROJECTOS ESTRATÉGICOS
“Subsídio para a concretização das acções de colaboração com Instituições Norte-Americanas” (renovação anual) – Fundação
Luso Americana para o Desenvolvimento
AGENCIA NACIONAL CIENCIA VIVA
“Autolaboratório” – 2006-2008 – 24 meses – IR: Luís Filipe Santos Silva
“Laboratório Aberto” – 2006-2008 – 24 meses – IR: Luís Cirnes
"Despertar para a Ciência" – 2007-2009 – 24 meses – IR: Luís Filipe Santos Silva
97
TRABALHOS PUBLICADOS
1.
Akerman M, Alves VA, Bubendorf L, Colgan T, Itoh H, Kapila K, Katz R, Mitchell G, Mulvany
NJ, Nasuti JF, Ng WK, Osamura RY, Schalper J, Schmitt FC, Serizawa A, Verhest A, Vielh P.
How technology is reshaping the practice of nongynecologic cytology. Acta Cytol 51: 123-152,
2007
0.8
2.
Alazzouzi H, Suriano G, Guerra A, Plaja A, Espin E, Armengol M, Alhopuro P, Velho S,
Shinomura Y, Gonzalez-Aguilera JJ, Yamamoto H, Aaltonen LA, Moreno V, Capella G,
Peinado MA, Seruca R, Arango D, Schwartz S Jr. Tumour selection advantage of nondominant negative P53 mutations in homozygotic MDM2-SNP309 colorectal cancer cells. J
Med Genet 44:75-80, 2007.
5.0
3.
Aler M, Sánchez-Diz P, Gomes I, Gisbert M, Carracedo A, Amorim A, Gusmão L. Genetic data
of 10 X-STRs in a Spanish population sample. Forensic Sci Int. 173:193-6, 2007
4.
Alves C, Gomes V, Prata MJ, Amorim A, Gusmao L. Population data for Y-chromosome
haplotypes defined by 17 STRs (AmpFlSTR YFiler) in Portugal. Forensic Sci Int. 13:250-5,
2007
1.4
5.
Azevedo NF, Guimarães N, Figueiredo C, Keevil CW, Vieira MJ. A new model for the
transmission of Helicobacter pylori: role of environmental reservoirs as gene pools to increase
strain diversity. Crit Rev Microbiol 33:157-69, 2007
3.8
6.
Baltazar F, Longatto-Filho A, Pinheiro C, Moreira MA, Queiroz GS, Oton GJ, Fraga Junior A,
Ribeiro LF, Schmitt FC. Cyclooxigenase-2 and Epidermal Growth Factor Receptor
expressions in different histological subtypes of cervical carcinomas. Int J of Gynecol Pathol
26: 235-241, 2007
2.2
7.
Barroca H, Farinha NJ, Lobo A, Monteiro J, Lopes JM: Deep-seated congenital juvenile
xanthogranuloma: report of a case with emphasis on cytologic features. Acta Cytologica
51:473-476, 2007
0.8
8.
Batini C, Coia V, Battagia C, Rocha J, Pilkington MM, Spedini G, Comas D, Destro-Bisol G,
Calafell F. 2007. Phylogeography of the human mitochondrial L1c haplogroup: genetic
signatures of the prehistory of Central Africa. Mol Phylogenet Evol 43: 635-644, 2007
3.5
9.
Brito C, Escrevente C, Reis CA, Lee VM, Trojanowski JQ, Costa J. Increased levels of
fucosyltransferase IX and carbohydrate Lewis(x) adhesion determinant in human NT2N
neurons. J Neurosci Res. 85:1260-70, 2007.
3.5
10.
Bruggemann, M (Brueggemann, M.); White, H (White, H.); Gaulard, P (Gaulard, P.); GarciaSanz, R (Garcia-Sanz, R.); Gameiro, P (Gameiro, P.); Oeschger, S (Oeschger, S.); Jasani, B
(Jasani, B.); Ott, M (Ott, M.); Delsol, G (Delsol, G.); Orfao, A (Orfao, A.); Tiemann, M
(Tiemann, M.); Herbst, H (Herbst, H.); Langerak, AW (Langerak, A. W.); Spaargaren, M
(Spaargaren, M.); Moreau, E (Moreau, E.); Groenen, PJTA (Groenen, P. J. T. A.); Sambade,
C (Sambade, C.); Foroni, L (Foroni, L.); Carter, GI (Carter, G. I.); Hummel, M (Hummel, M.);
Bastard, C (Bastard, C.); Davi, F (Davi, F.); Delfau-Larue, MH (Delfau-Larue, M. -H.); Kneba,
M (Kneba, M.); van Dongen, JJM (van Dongen, J. J. M.); Beldjord, K (Beldjord, K.); Molina, TJ
(Molina, T. J.) Powerful strategy for polymerase chain reaction-based clonality assessment in
T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936. Leukemia
21 (2): 215-221, 2007
6.1
11.
Buffart TE, Carvalho B, Mons T, Reis RM, Moutinho C, Silva P, van Grieken NC, Vieth M,
Stolte M, van de Velde CJ, Schrock E, Matthaei A, Ylstra B, Carneiro F, Meijer GA: DNA copy
number profiles of gastric cancer precursor lesions. BMC Genomics 8:345, 2007
2.1
1.4
98
12.
Builes JJ, Martínez B, Gómez A, Caraballo L, Espinal C, Aguirre D, Montoya A, Moreno M,
Amorim A, Gusmão L, Bravo ML. Y chromosome STR haplotypes in the Caribbean city of
Cartagena (Colombia). Forensic Sci Int. 167:62-9, 2007
13.
Canedo P, Figueiredo C, Machado JC. After H. Pylori, genetic susceptibility to gastric
carcinoma revisited. Helicobacter 12: 45-49, 2007
2.5
14.
Canedo P, Machado JC. Genetic susceptibility to gastric carcinoma. Acta Endosc 37: 239247, 2007.
_
15.
Cardoso H, Machado AS, Figueira P, Teixeira AV, Veloso FT, Lopes JM; Multiple Myeloma
Presenting with Malabsorption. Digest Dis Sci 52:1851-1854, 2007.
1.4
16.
Carneiro F, Moutinho C, Pera G, Caldas C, Fenger C, Offerhaus J, Save V, Stenling R, Nesi
G, Mahlke U, Bläker H, Torrado J, Roukos D, Sabourin J-C, Boeing H, Palli D, Bueno-deMesquita HB, Overvad K, Bingham S, Clavel-Chapelon F, Lund E, Trichopoulou A, Manjer J,
Riboli E,Gonzalez CA: Pathology findings and validation of gastric and esophageal cancer
cases in a European cohort (EPIC/EUR-GAST). Scandinavian Journal of Gastroenterology
42:618-627, 2007.
1.9
17.
Cassali G, Gobbi H, Malm C, Schmitt F. Evaluation of accuracy of fine needle aspiration
cytology for diagnosis of canine mammary tumours: comparative features with human
tumours. Cytopathology 18: 191-196, 2007.
1.0
18.
Cassali GD, Salvador A, Freitas C, Dutra AP, Schmitt FC. DNA flow cytometry of canine
mammary tumors: comparative aspects with human breast tumors. Arq Bras Med Vet Zootec
59: 1163-1168, 2007.
_
19.
Castro Alves C, Rosivatz E, Schott C, Hollweck R, Becker I, Sarbia M, Carneiro F, Becker KF.
Slug is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in
the down-regulation of E-cadherin. J Pathol 211 507-15, 2007.
5.8
20.
Corso G, Roviello F, Paredes J, Pedrazzani C, Novais M, Correia J, Marrelli D, Cirnes L,
Seruca R, Oliveira C, Suriano G. Characterization of the P373L E-cadherin germline missense
mutation and implication for clinical management. Eur J Surg Oncol 33: 1061-7, 2007
1.9
21.
Costa BM, Ferreira P, Costa S, Canedo P, Oliveira P, Silva A, Pardal F, Suriano G, Machado
JC, Lopes JM, Reis RM: Association between functional EGF+61 polymorphism and glioma
risk. Clin Canc Res 13:2621-2626, 2007.
6.2
22.
Costa S, Pinto D, Pereira D, Rodrigues H, Cameselle-Teijeiro J, Medeiros R, Schmitt F. DNA
repair polymorphisms might contribute differentially on familial and sporadic breast cancer
susceptibility: a study on a Portuguese population. Breast Cancer Res Treat 103: 209-217,
2007.
4.7
23.
Cserni G, Bianchi S, Vezzosi V, Arisio R, Peterse JL, Sapino A, Castellano I, Drijkoningen M,
Kulka J, Eusebi V, Foschini MP, Bellocq JP, Marin C, Thorstenson S, Amendoeira I, ReinerConcin A, Decker T, Lacerda M, Figueiredo P. Validation of clinical prediction rules for a low
probability of nonsentinel and extensive lymph node involvement in breast cancer patients. Am
J Surg 194:288-93, 2007
2.1
24.
Cserni G, Bianchi S, Vezzosi V, Arisio R, Bori R, Peterse JL, Sapino A, Drijkoningen M, Kulka
J, Eusebi V, Foschini MP, Bellocq JP, Marin C, Thorstenson S, Amendoeira I, Reiner-Concin
A, Decker T. Sentinel lymph node biopsy and non-sentinel node involvement in special type
breast carcinomas with a good prognosis. Eur J Cancer 43:1407-14, 2007
4.2
25.
Cserni G, Bianchi S, Vezzosi V, Arisio R, Bori R, Peterse JL, Sapino A, Castellano I,
Drijkoningen M, Kulka J, Eusebi V, Foschini MP, Bellocq JP, Marin C, Thorstenson S,
Amendoeira I, Reiner-Concin A, Decker T, Lacerda M, Figueiredo P, Fejes G. Sentinel lymph
node biopsy in staging small (up to 15 mm) breast carcinomas. Results from a European
1.2
1.4
99
multi-institutional study. Pathol Oncol Res 13:5-14, 2007
26.
Davalos V, Dopeso H, Velho S, Ferreira AM, Cirnes L, Diaz-Chico N, Bilbao C, Ramirez R,
Rodriguez G, Falcon O, Leon L, Niessen RC, Keller G, Dallenbach-Hellweg G, Espin E,
Armengol M, Plaja A, Perucho M, Imai K, Yamamoto H, Gebert JF, Diaz-Chico JC, Hofstra
RM, Woerner SM, Seruca R, Schwartz S Jr, Arango D. High EPHB2 mutation rate in gastric
but not endometrial tumors with microsatellite instability. Oncogene. 26:308-11, 2007.
6.6
27.
do Vale A, Costa-Ramos C, Silva A, Silva DS, Gärtner F, dos Santos NM, Silva MT. Systemic
macrophage and neutrophil destruction by secondary necrosis induced by a bacterial exotoxin
in a Gram-negative septicaemia. Cell Microbiol 2007. 9:988-1003.
5.1
28.
Domingues PM, Gusmão L, da Silva DA, Amorim A, Pereira RW, de Carvalho EF.SubSaharan Africa descendents in Rio de Janeiro (Brazil): population and mutational data for 12
Y-STR loci. Int J Legal Med. 121;238-41, 2007
29.
Duarte M, Longatto-Filho A, Schmitt FC. Angiogenesis, haemostasis and câncer: new
paradigms old concerns. J Bras Patol Med Lab 43: 441-449, 2007.
_
30.
Dufloth RM, Matos I, Schmitt F, Zeferino LC. Tissue microarrays for testing basal biomarkers
in familial breast cancer cases. Sao Paulo Med J 125: 226-230, 2007.
_
31.
Ferreira AC, Gomes L, Maximo V, Amil J, Carneiro F, Machado JC, Tavarela-Veloso F. GRIM19 mutations are not associated with Chron’s disease. Inflamm Bowel Dis 1-2, 2007
_
32.
Ferreira JG, Cruz CD, Neves D, Pignatelli D. Increased extracellular signal regulated kinases
phosphorylation in the adrenal gland in response to chronic ACTH treatment. J Endocrinol.
192:647-58, 2007.
3.1
33.
Filho AL, Baltazar F, Bedrossian C, Michael C, Schmitt FC.Immunohistochemical expression
and distribution of VEGFR-3 in malignant mesothelioma. Diagn Cytopathol 35:786-91, 2007
0.8
34.
Fonseca AP, Correia P, Sousa JC, Tenreiro R. Association patterns of Pseudomonas
aeruginosa clinical isolates as revealed by virulence traits, antibiotic resistance, serotype and
genotype. FEMS Immunol Med Microbiol. 51:505-16, 2007
Fonseca AP, Sousa JC. Effect of antibiotic-induced morphological changes on surface
properties, motility and adhesion of nosocomial Pseudomonas aeruginosa strains under
different physiological states. J Appl Microbiol. 103:1828-37, 2007.
Fonseca AP, Sousa JC. Effect of shear stress on growth, adhesion and biofilm formation of
Pseudomonas aeruginosa with antibiotic-induced morphological changes. Int J Antimicrob
Agents. 30:236-41, 2007
Fonseca E, Eloy C, Sobrinho-Simões M. Tumeurs vésiculaires bien differenciées: Nouveaux
conceptes moleculaires? Nouveaux critères diagnostiques? Bulletin de la Division Française
de l’AIP nº 45: 20-24, 2007
Gil da Costa RM, Matos E, Rema A, Lopes C, Pires MA, Gärtner F. CD117 immunoexpression
in canine mast cell tumours: correlations with pathological variables and proliferation markers.
BMC Vet Res 3:19, 2007
_
39.
Gil da Costa RM, Rema A, Payo-Puente P, Gärtner F. Immunohistochemical characterization
of a sebaceous gland carcinoma in a gerbil (Meriones unguiculatus). J Comparative Pathol.
2007 137:130-132.
0.9
40.
Goios A, Pereira L, Bogue M, Macaulay V, Amorim A. mtDNA phylogeny and evolution of
laboratory mouse strains. Genome Res. 17:293-8, 2007.
10.3
41.
Gomes AL, Bardales RH, Milanezi F, Reis RM, Schmitt F. Molecular analysis of c-Kit and
PDGFRA in GISTs diagnosed by EUS. Am J Clin Pathol 127: 89-96, 2007.
2.9
42.
Gomes AL, Reis-Filho JS, Lopes JM, Martinho O, Lambros MB, Martins A, Schmitt F, Pardal
3.0
35.
36.
37.
38.
2.6
2.2
_
_
100
F, Reis RM: Molecular alterations of KIT oncogene in gliomas. Cell Oncol 29:399-408, 2007.
43.
Gomes I, Alves C, Maxzud K, Pereira R, Prata MJ, Sanchez-Diz P, Carracedo A, Amorim A,
Gusmão L. Analysis of 10 X-STRs in three African Populations. Forensic Sci Int Genetics 1:
208-211, 2007
44.
Gomes I, Prinz M, Pereira R, Meyers C, Mikulasovich RS, Amorim A, Carracedo A, Gusmao L.
Genetic analysis of three US population groups using an X-chromosomal STR decaplex. Int J
Legal Med. 121:198-203, 2007.
2.6
45.
Gouveia A, Pimenta A, Lopes JM: Quando e como operar os tumours estromais
gastrointestinais primários. Revista Portuguesa de Cirurgia 1: 49-52, 2007.
_
46.
Gray W, Bayer-Pietsch E, Chieco P, Cochand-Priollet B, Desai M, Drijkoningen M, Griffin M,
Hagmar B, Kapila K, Kloboves-Prevodnik V, Kobayashi T, Krogerus L, Majak B, Mihailovici M,
Olsewski W, Schenk U, Schmitt F, Shabalova I, Shapiro N, Smith J, Tani E, Totsch M, Vass L,
Wiener H, Herbert A. The future of cytopathology in Europe. Will the wider use of HPV testing
have an impact on the provision of cervical screening ? Cytopathology 18: 278-282, 2007.
1.0
47.
Guimarães N, Azevedo NF, Figueiredo C, Keevil CW, Vieira MJ. Development and application
of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric
biopsy specimens. J Clin Microbiol 45:3089-94, 2007.
3.4
48.
Holtkamp M, Schmitt FC, Buchheim K, Meierkord H.Temperature regulation is compromised in
experimental limbic status epilepticus. Brain Res. 1127:76-9, 2007
2.3
49.
Katz M, Amit I, Citri A, Shay T, Carvalho S, Lavi S, Milanezi F, Lyass L, Amariglio N, JacobHirsch J, Ben-Chetrit N, Tarcic G, Lindzen M, Avraham R, Liao YC, Trusk P, Lyass A, Rechavi
G, Spector NL, Lao SH, Schmitt F, Bacus SS, Yarden Y. A reciprocal tensin3-cten switch
mediates EGF-driven mammary cell migration. Nature Cell Biology 9: 961-969, 2007.
18.5
50.
Kaurah P, MacMillan A, Boyd N, Senz J, De Luca A, Chun N, Suriano G, Zaor S, Van Manen
L, Gilpin C, Nikkel S, Connolly-Wilson M, Weissman S, Rubinstein WS, Sebold C, Greenstein
R, Stroop J, Yim D, Panzini B, McKinnon W, Greenblatt M, Wirtzfeld D, Fontaine D, Coit D,
Yoon S, Chung D, Lauwers G, Pizzuti A, Vaccaro C, Redal MA, Oliveira C, Tischkowitz M,
Olschwang S, Gallinger S, Lynch H, Green J, Ford J, Pharoah P, Fernandez B, Huntsman D.
Founder and recurrent CDH1 mutations in families with hereditary diffuse gastric cancer.
JAMA. 297:2360-72, 2007.
23.2
51.
Langerak, AW (Langerak, A. W.); Molina, TJ (Molina, T. J.); Lavender, FL (Lavender, F. L.);
Pearson, D (Pearson, D.); Flohr, T (Flohr, T.); Sambade, C (Sambade, C.); Schuuring, E
(Schuuring, E.); Al Saati, T (Al Saati, T.); van Dongen, JJM (van Dongen, J. J. M.); van
Krieken, JHJM (Van Krieken, J. H. J. M.). Polymerase chain reaction-based clonality testing in
tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the
BIOMED-2 Concerted Action BMH4-CT98-3936 Leukemia 21 (2): 222-229, 2007
6.1
52.
Lee AHS, Paish EC, Marchio C, Sapino A, Schmitt FC, Ellis IO, Reis-Filho JS. The expression
of Wilm´s tumour-1 and Ca125 in invasive micropapillary carcinoma of the breast.
Histopathology 51: 824-828, 2007.
3.2
53.
Lima J, Feijão T, Ferreira da Silva A, Pereira-Castro I, Fernandez-Ballester G, Máximo V,
Herrero A, Serrano L, Sobrinho-Simões M, Garcia-Rostan G.High frequency of germline
succinate dehydrogenase mutations in sporadic cervical paragangliomas in northern Spain:
mitochondrial succinate dehydrogenase structure-function relationships and clinicalpathological correlations. J Clin Endocrinol Metab. 92:4853-64, 2007
5.8
54.
Longatto-Filho A, Baltazar F, Bedrossian C, Michael C, Schmitt FC. Immunohistochemical
expression and distribution of VEGFR-3 in malignant mesothelioma. Diagn Cytopathol 35:
786-791, 2007.
0.8
101
55.
Longatto-Filho A, Costa SM, Milanezi F, Montruccoli D, Montruccoli GC, Baltazar F, Schmitt
FC. Immunohistochemical expression of VEGF-A and its ligands in non-neoplastic lesions of
the breast sampling-assisted by dynamic angiothermography. Oncol Rep 18: 1201-1206,
2007.
1.6
56.
Longatto-Filho A, Oliveira TG, Pinheiro C, Carvalho MB, Curioni OA, Mercadante AM, Schmitt
FC, Gattas GJ. How useful is the assessment of lymphatic vascular density in oral carcinoma
prognosis? World J Surg Oncol 5: 140, 2007
_
57.
Longatto-Filho A, Pinheiro C, Pereira SM, Etlinger D, Moreira MA, Jubé LF, Queiroz GS,
Baltazar F, Schmitt FC. Lymphatic vessel density and epithelial D2-40 immunoreactivity in préinvasive and invasive lesions of the uterine cervix. Gynecol Oncol 107: 45-51, 2007.
2.3
58.
Longatto-Filho A, Schmitt FC. Gynecological Cytology: too old to be a pop star but too young
to die. Diagn Cytopathol 35: 672-673, 2007.
0.8
59.
Lopes JM, Gouveia A, Pimenta A: O papel da anatomia patológica no diagnóstico e
prognóstico dos GISTs. Rev Port Cir 1: 35-39, 2007.
_
60.
Lunet N, Valbuena C, Vieira AL, Lopes C, Lopes C, David L, Carneiro F, Barros H: Fruit and
vegetable consumption and gastric cancer by location and histological type: case-control and
meta-analysis. Eur J Cancer Prev 16:312-327, 2007
2.0
61.
Mendez MA, Pera G, Agudo A, Bueno-de-Mesquita HB, Palli D, Boeing H, Carneiro F, Berrino
F, Sacerdote C, Tumino R, Panico S, Berglund G, Manjer J, Johansson I, Stenling R, Martinez
C, Dorronsoro M, Barricarte A, Tormo MJ, Quiros JR, Allen N, Key TJ, Bingham S, Linseisen
J, Kaaks R, Overvad K, Jensen M, Olsen A, Tjonneland A, Peeters PH, Numans ME, Ocke
MC, Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Slimani N, Jenab M,
Ferrari P, Riboli E, Gonzalez CA: Cereal fiber intake may reduce risk of gastric
adenocarcinomas: The EPIC-EURGAST study. International Journal of Cancer 121: 16181623, 2007.
4.7
62.
Macedo Joana Espiga, M S Costa Ângela, A M Barbosa Inês, Rebelo Sandra, Souto de
Moura Conceição, Teixeira da Costa Luís, Hespanhol Venceslau. Alterações genéticas no
cancro do pulmão: Avaliação das limitações ao seu uso na rotina clínica Rev Port Pneumol.
13:9-34, 2007
_
63.
Martínez B, Barrios K, Vergara C, Mercado D, Jiménez S, Gusmão L, Caraballo L. A NOS1
gene polymorphism associated with asthma and specific immunoglobulin E response to mite
allergens in a Colombian population. Int Arch Allergy Imm 144:105-13, 2007
64.
Martins S, Calafell F, Gaspar C, Wong VCN, Silveira I, Nicholson GA, Brunt E, Tranebjaerg L,
Stevanin G, Hsieh M, Soong B-W, Loureiro L, Dürr A, Tsuji S, Watanabe M, Jardim L, Giunti
P, Riess O, Ranum LPW, Brice A, Rouleau GA, Coutinho P, Amorim A, Sequeiros J. The
worldwide spread mutational event in Machado-Joseph disease has an Asian origin . Arch
Neurol. 64:1502-8, 2007.
5.2
65.
Masciari S, Larsson N, Senz J, Boyd N, Kaurah P, Kandel MJ, Harris LN, Oliveira C,
Troussard A, Miron P,Tung N, Pinheiro HC, Collins L, Schnitt S, Garber JE, Huntsman D.
Germline E-Cadherin mutations in familial lobular breast cancer. J Med Genet J Med Genet.
44:726-3, 2007
5.1
66.
Mateus R, Seruca R, Machado JC, Keller G, Oliveira MJ, Suriano G, Luber B. EGFR regulates
RhoA-GTP dependent cell motility in E-cadherin mutant cells. Hum Mol Genet 16: 1639-1647,
2007.
8.1
67.
Matos AJ, Duarte S, Lopes C, Lopes JM, Gartner F: Splenic hamartomas in a dog. Vet Rec
161:308-310, 2007.
1.9
68.
Meireles AM, Preto A, Rocha AS, Rebocho AP, Máximo V, Pereira-Castro I, Moreira S, Feijão
T, Botelho T, Marques R, Trovisco V, Cirnes L, Alves C, Velho S, Soares P, Sobrinho-Simões
1.9
2.5
102
M. Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived
cell lines. Thyroid. 17:707-15, 2007.
69.
Miller C, Beleza S, Pollen A, Schluter D, Kittles R, Shriver MD, Kingsley DM. Cis-regulatory
changes in Kit ligand expression and parallel evolution of pigmentation changes in
sticklebacks and humans. Cell 131:1179-89
29.1
70.
Milne AN, Sitarz R, Carvalho R, Carneiro F, Offerhaus GJ. Early onset gastric cancer: on the
road to unraveling gastric carcinogenesis. Curr Mol Med. 2007 7:15-28, 2007.
4.9
71.
More H, Humar B, Weber W, Ward R, Christian A, Lintott C, Graziano F, Ruzzo AM, Acosta E,
Boman B, Harlan M, Ferreira P, Seruca R, Suriano G, Guilford P. Identification of seven novel
germline mutations in the human E-cadherin (CDH1) gene. Hum Mutat. 28(2):203, 2007
6.5
72.
Nagel G, Linseisen J, Boshuizen HC, Pera G, Del Giudice G, Westert GP, Bueno-de-Mesquita
HB, Allen NE, Key TJ, Numans ME, Peeters PH, Sieri S, Siman H, Berglund G, Hallmans G,
Stenling R, Martinez C, Arriola L, Barricarte A, Chirlaque MD, Quiros JR, Vineis P, Masala G,
Palli D, Panico S, Tumino R, Bingham S, Boeing H, Bergmann MM, Overvad K, BoutronRuault MC, Clavel-Chapelon F, Olsen A, Tjonneland A, Trichopoulou A, Bamia C, Soukara S,
Sabourin JC, Carneiro F, Slimani N, Jenab M, Norat T, Riboli E, Gonzalez CA. Socioeconomic
position and the risk of gastric and oesophageal cancer in the European Prospective
Investigation into Cancer and Nutrition (EPIC-EURGAST). International Journal of
Epidemiology 36:66-76, 2007.
5.2
73.
Navarro-Costa P, Pereira L, Alves C, Gusmao L, Proenca C, Marques-Vidal P, Rocha T,
Correia SC, Jorge S, Neves A, Soares AP, Nunes J, Calhaz-Jorge C, Amorim A, Plancha CE,
Goncalves J. Characterizing partial AZFc deletions of the Y chromosome with ampliconspecific sequence markers. BMC Genomics 8(1):342, 2007
4.0
74.
Neto D, Montiel R, Bettencourt C, Santos C, Prata MJ, Lima M. The African contribution to the
present-day population of the Azores Islands (Portugal): Analysis of the Y chromosome
haplogroup E. Am J Hum Biol. 19:854-69, 2007
1.7
75.
Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval A,
Hamelin R, Machado JC, Schwartz S Jr, Carneiro F, Seruca R. KRAS and BRAF oncogenic
mutations in MSS colorectal carcinoma progression. Oncogene 26: 158-163, 2007.
6.6
76.
Oliveira E, Marsh S, van Booven D, Amorim A, Prata M, McLeod H.Pharmacogenetically
relevant polymorphisms in Portugal. Pharmacogenomics. 8:703-712, 2007
3.6
77.
Oliveira E, Quental S, Alves S, Amorim A, Prata MJ. Do the distribution patterns of
polymorphisms at the thiopurine S-methyltransferase locus in sub-Saharan populations need
revision? Hints from Cabinda and Mozambique. Eur J Clin Pharmacol. 63:703-6, 2007
78.
Oliveira MJ, Costa AM, Costa AC, Figueiredo C. Helicobacter pylori induit l'invasion dês
cellules gastriques via un mécanisme qui nécessite le c-Met cellulaire et un système de
sécrétion bactérien de type IV fonctionnel. La Lettre de L’ Infectiologue Tome XXII – HorsSérie: 3-6, 2007.
_
79.
Ouerhani S, Oliveira E, Marrakchi R, Ben Slama MR, Sfaxi M, Ayed M, Chebil M, Amorim A, El
Gaaied AB, Prata MJ. Methylenetetrahydrofolate reductase and methionine synthase
polymorphisms and risk of bladder cancer in a Tunisian population. Cancer Genet Cytogenet.
176:48-53, 2007
1.5
80.
Palli D, Masala G, Del Giudice G, Plebani M, Basso D, Berti D, E Numans M, Ceroti M,
Peeters PH, de Mesquita HB, Buchner FL, Clavel-Chapelon F, Boutron-Ruault MC, Krogh V,
Saieva C, Vineis P, Panico S, Tumino R, Nyren O, Siman H, Berglund G, Hallmans G,
Sanchez MJ, Larranaga N, Barricarte A, Navarro C, Quiros JR, Key T, Allen N, Bingham S,
Khaw KT, Boeing H, Weikert C, Linseisen J, Nagel G, Overvad K, Thomsen RW, Tjonneland
A, Olsen A, Trichoupoulou A, Trichopoulos D, Arvaniti A, Pera G, Kaaks R, Jenab M, Ferrari
4.7
2.0
103
P, Nesi G, Carneiro F, Riboli E, Gonzalez CA: CagA+ Helicobacter pylori infection and gastric
cancer risk in the EPIC-EURGAST study. International Journal of Cancer 120: 859-867, 2007.
81.
Paredes J, Correia AL, Ribeiro AS, Albergaria A, Milanezi F, Schmitt FC. P-Cadherin
expression in breast câncer: a review. Breast Cancer Res 9: 214, 2007
4.2
82.
Paredes J, Correia AL, Ribeiro AS, Schmitt FC. Expression of p120-catenin isoforms
correlates with genomic and transcriptional phenotype of breast cancer cell lines. Cell Oncol
29:467-476, 2007.
2.9
83.
Paredes J, Lopes N, Milanezi F, Schmitt FC. P-cadherin and cytokeratin 5: useful adjunct
markers to distinguish basal-like ductal carcinomas in situ. Virchows Arch 450: 73-80, 2007.
2.3
84.
Peleteiro B, Lunet N, Figueiredo C, Carneiro F, David L, Barros H: Smoking, Helicobacter
pylori virulence, and type of intestinal metaplasia in Portuguese males. Cancer Epidemiol
Biomarkers Prev 16:322-326, 2007
4.3
85.
Peleteiro B, Lunet N, Santos-Silva F, David L, Figueiredo C, Barros H: Short mucin 1 alleles
are associated with low virulent H pylori strains infection. World J Gastroenterol 13:1885-1886,
2007
_
86.
Pereira L, Goncalves J, Franco-Duarte R, Silva J, Rocha T, Arnold C, Richards M, Macaulay
V. No evidence for an mtDNA role in sperm motility: data from complete sequencing of
asthenozoospermic males. Mol Biol Evol. 24:868-74, 2007.
6.7
87.
Pereira R, Gomes I, Amorim A, Gusmao L. Genetic diversity of 10 X chromosome STRs in
northern Portugal. Int J Legal Med. 121:192-7, 2007.
2.6
88.
Pimenta A, Preto J, Gouveia A, Fonseca E, Pimenta M.Mediastinal tuberculosis lymphadenitis
presenting as an esophageal intramural tumor: A very rare but important cause for dysphagia.
World J Gastroenterol 13: 6104-6108, 2007
_
89.
Pinho S, Marcos NT, Ferreira B, Carvalho AS, Oliveira MJ, Santos-Silva F, Harduin-Lepers A,
Reis CA. Biological significance of cancer-associated sialyl-Tn antigen: modulation of
malignant phenotype in gastric carcinoma cells. Cancer Lett. 249:157-70, 2007.
3.3
90.
Pinho SS, Matos AJ, Lopes C, Marcos NT, Carvalheira J, Reis CA, Gärtner F.SialylLewis x
expression in canine malignant mammary tumours: correlation with clinicopathological
features and E-Cadherin expression. BMC Cancer. 7:124, 2007.
2.4
91.
Pitteloud N, Zhang C, Pignatelli D, Li JD, Raivio T, Cole LW, Plummer L, Jacobson-Dickman
EE, Mellon PL, Zhou QY, Crowley WF Jr. Loss-of-function mutation in the prokineticin 2 gene
causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism. Proc
Natl Acad Sci U S A. 104:17447-52, 2007
10.5
92.
Pires MA, Gärtner F. CD117 immunoexpression in canine mast cell tumours: correlations with
pathological variables and proliferation markers. BMC Vet Res 3:19, 2007.
_
93.
Preto A, Moutinho C, Velho S, Oliveira C, Rebocho AP, Figueiredo J, Soares P, Lopes JM,
Seruca R: A subset of colorectal carcinomas express c-KIT protein independently of BRAF
and/or KRAS activation. Virch Archiv 450:619-626, 2007.
2.3
94.
Quelhas D, Quental R, Vilarinho L, Amorim A, Azevedo L.Congenital disorder of glycosylation
type Ia: searching for the origin of common mutations in PMM2. Ann Hum Genet. 71(Pt
3):348-53, 2007
2.7
95.
Resende C, Regalo G, Duraes C, Carneiro F, Machado JC: Genetic changes of CEBPA in
cancer: mutations or polymorphisms? Journal of Clinical Oncology 25:2493-2494, 2007.
13.6
104
96.
Ricardo S, Milanezi F, Carvalho S, Leitão D, Schmitt F. HER2 evaluation through the novel
rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinoma.
J Clin Pathol 60: 1001-1005, 2007.
2.2
97.
Rindi G, Kloppel G, Couvelard A, Komminoth P, Korner M, Lopes JM, McNicol AM, Nilsson O,
Perren A, Scarpa A, Scoazec JY, Wiedenmann B: TNM staging of midgut and hindgut (neuro)
endocrine tumors: a consensus proposal including a grading system. Virch Archiv 451:757762, 2007.
2.3
98.
Rocha AS, Marques R, Bento I, Soares R, Magalhães J, de Castro IV, Soares P. Thyroid
hormone receptor beta mutations in the 'hot-spot region' are rare events in thyroid carcinomas.
J Endocrinol 192:83-6, 2007.
3.1
99.
Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark-Mansson H. The role of
osteopontin in tumor progression and metastasis in breast cancer. Cancer Epidemiol
Biomarkers Prev 16: 1-11, 2007.
4.3
100.
Roviello F, Corso G, Pedrazzani C, Marrelli D, De Falco G, Berardi A, Garosi L, Suriano G,
Vindigni C, De Stefano A, Leoncini L, Seruca R, Pinto E. Hereditary diffuse gastric cancer and
E-cadherin: description of the first germline mutation in an Italian family. Eur J Surg Oncol.
33:448-51, 2007.
_
101.
Sarmento-Castro R, Horta A, Vasconcelos O, Coelho H, Mendez J, Tavares AP, Seabra J,
Duarte M, Chaves L, Fortes O, Recalde C, Ventura A, Pires N, Pinho L, Dias N, Carneiro
F.Impact of peginterferon alpha-2b and ribavirin treatment on liver tissue in patients with HCV
or HCV-HIV co-infection. J Infection 54: 609-616, 2007.
2.0
102.
Savage K, Lambros MBK, Robertson D, Jones RL, Jones C, Mackay A, James M, Hornick JL,
Pereira EM, Milanezi F, Fletcher CDM, Schmitt FC, Ashworth A, Reis-Filho JS. Caveolin 1 is
overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a
morphologic, ultrastructural, immunohistochemical and in situ hybridization analysis. Clin
Cancer Res 13: 90-101, 2007.
6.2
103.
Schmitt FC, Gomes AL, Milanezi F, Reis R, Bardales R. Mutations in gastrointestinal stromal
tumors diagnosed by endoscopic ultrasound-guided fine needle aspiration. Minerva Med 98:
385-388, 2007.
_
104.
Schmitt FC. Cells carry the clue for targeted treatment: a new horizont for cytopathology.
Cytopathology 18: 275-277, 2007.
1.0
105.
Schmitt FC. Is BRCA1 a possible predictor of response to neo-adjuvant chemotherapy ?
Hereditary Cancer in Clinical Practice 5: 136-137, 2007.
_
106.
Schmitt FC. Punção aspirativa por agulha fina da tireóide: a precisão e solidez de um método
diagnóstico. Rev Port Endocrinol Diabetes e Metabol 2: 5-7, 2007.
_
107.
Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A. Sequence diversity at the
proximal 14q32.1 SERPIN subcluster: evidence for natural selection favoring the
pseudogenization of SERPINA2. Mol Biol Evol. 24:587-98, 2007.
6.7
108.
Suriano G, L Azevedo, M Novais, B Boscolo, R Seruca, A Amorim, E M Ghibaudi. In vitro
demonstration of intra-locus compensation using the Ornithine transcarbamylase protein as
model. Hum Mol Genet. 16:2209-14, 2007.
8.1
109.
Svrcek M, El-Bchiri J, Chalastanis A, Capel E, Dumont S, Buhard O, Oliveira C, Seruca R,
Bossard C, Mosnier J-F, Berger F, Leteurtre E, Lavergne-Slove A, Chenard M-P, Hamelin R,
Cosnes J, Beaugerie L, Tiret E, Duval A, Fléjou J-F. Specific clinical and biological features
characterize inflammatory bowel disease.associated colorectal cancers showing microsatellite
13.6
105
instability. J Clin Oncol. 25:4231-8, 2007
110.
Toscanini U, Gusmão L, Berardi G, Amorim A, Carracedo A, Salas A, Raimondi E.Testing for
genetic structure in different urban Argentinian populations. Forensic Sci Int. 165(1):35-40,
2007
1.4
111.
Trovisco V, Soares P, Preto A, Castro P, Máximo V, Sobrinho-Simões M. Molecular genetics
of papillary thyroid carcinoma: great expectations. Arq Bras Endocrinol Metabol 51:643-53,
2007
_
112.
Trovoada MJ, Tavares L, Gusmão L, Alves C, Abade A, Amorim A, Prata MJ. Dissecting the
genetic history of São Tomé e Príncipe: a new window from Y-chromosome biallelic markers.
Ann Hum Genet. 71(Pt 1):77-85, 2007
113.
Turner NC, Reis-Filho JS, Russell AM, Springall RJ, Ryder K, Steele D, Savage K, Gillett CE,
Schmitt FC, Ashworth A, Tutt AN. BRCA1 dysfunction in sporadic basal-like breast cancer.
Oncogene 26: 2126-2132, 2007.
6.6
114.
Volante M, Collini P, Nikiforov YE, Sakamoto A, Kakudo K, Katoh R, Lloyd RV, LiVolsi VA,
Papotti M, Sobrinho-Simoes M, Bussolati G, Rosai J.Poorly differentiated thyroid carcinoma:
the Turin proposal for the use of uniform diagnostic criteria and an algorithmic diagnostic
approach. Am J Surg Pathol. 31:1256-64, 2007
4.1
115.
Vollset SE, Igland J, Jenab M, Fredriksen A, Meyer K, Eussen S, Gjessing HK, Ueland PM,
Pera G, Sala N, Agudo A, Capella G, Del Giudice G, Palli D, Boeing H, Weikert C, Bueno-deMesquita HB, Carneiro F, Pala V, Vineis P, Tumino R, Panico S, Berglund G, Manjer J,
Stenling R, Hallmans G, Martínez C, Dorronsoro M, Barricarte A, Navarro C, Quirós JR, Allen
N, Key TJ, Bingham S, Linseisen J, Kaaks R, Overvad K, Tjønneland A, Büchner FL, Peeters
PH, Numans ME, Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Slimani N,
Ferrari P, Riboli E, González CA: The association of gastric cancer risk with plasma folate,
cobalamin, and methylenetetrahydrofolate reductase polymorphisms in the European
Prospective Investigation into Cancer and Nutrition. Cancer Epidemiol Biomarkers Prev
16:2416-2424, 2007.
4.3
2.7
106
REVISTAS
CIENTÍFICAS
INTERNACIONAIS
COM MEMBROS DO
IPATIMUP
NOS SEUS
EDITORIAL BOARDS
Acta Cytologica (SCI PRINTERS & PUBL INC)
Advances in Anatomic Pathology (LIPPINCOTT WILLIAMS & WILKINS)
Archives of Pathology and Laboratory Medicine (COLLEGE OF AMERICAN
PATHOLOGISTS)
Breast Cancer Research (BIOMED CENTRAL LTD)
Cancer Genetics and Cytogenetics (ELSEVIER SCI IRELAND LTD)
Critical Review in Oncogenesis (BEGELL HOUSE, USA)
Current Diagnostic Pathology (CHURCHILL LIVINGSTONE)
Cytojournal (BIOMED CENTRAL)
Cytopathology (BLACKWELL PUBLISHING LTD)
Diagnostic Cytopathology (WILEY-LISS)
Endocrine Pathology (BLACKWELL PUBLISHING LTD)
European Journal of Cancer Prevention (LIPPINCOTT WILLIAMS & WILKINS)
Forensic Science International (ELSEVIER SCI IRELAND LTD)
Hereditary Cancer in Clinical Practice (UICC GLOBAL CANCER CONTROL)
Histopathology (BLACKWELL PUBLISHING LTD)
Human Biology (WAYNE STATE UNIVERSITY PRESS)
International Journal of Surgical Pathology (WESTMINSTER PUBL INC)
Journal of Cellular and Molecular Medicine (CAROL DAVILA UNIV PRESS)
Journal of Pathology (JON WILEY & SONS LTD)
The Open Pathology Journal (BENTHAM OPEN)
Pathology Research & Practice (URBAN & FISCHER VERLAG)
Seminars in Diagnostic Pathology (W B SAUNDERS CO)
Ultrastructural Pathology (TAYLOR & FRANCIS INC)
Virchows Archiv (SPRINGER)
107
NÚCLEO DE AMIGOS
DO
IPATIMUP
Allianz Portugal, SA
Amorim Inv. e Participações
Astrazeneca Produtos Farmacêuticos Lda
Banco BPI, SA
Bayer Portugal, SA
BES
Bial - Portela & C.ª, SA
Fundação Millennium BCP
GlaxoSmithKline Prod. Farmacêuticos, Lda
Ipsen Portugal - Produtos farmacêuticos, SA
Merck Sharp & Dohme, Lda.
Mota Engil, SGPS, SA
Novartis Farma - Prod. Farmacêuticos, SA
Olinveste SGPS
RAR - Sociedade de Controle (Holding), SA
Roche Farmacêutica Química, Lda
SAG GEST - Soluções Automóvel Globais, SA
Sanofi-Aventis,
Sonae SGPS, SA
Têxtil Manuel Gonçalves SA
Unicer
108