La phosphorylation du domaine C-terminal de l’ARN polymérase II permet à ce complexe protéique d’exécuter la transcription des gènes, en plus de coupler à la transcription des événements moléculaires comme la maturation des ARNm. Mes résultats montrent que même si cette phosphorylation suit un patron similaire à l’ensemble des gènes, il existe des exceptions pouvant être dues à des mécanismes alternatifs de phosphorylation du CTD. Le présent ouvrage s’intéresse également au rôle qu’occupe la variante d’histone H2A.Z dans l’organisation de la chromatine. Des études précédentes on montré que le positionnement de certains nucléosomes le long de l’ADN serait influencé par H2A.Z et aurait une influence sur la capacité de transcrire les gènes. Par une approche génomique utilisant les puces à ADN, j’ai cartographié l’impact de la délétion de H2A.Z sur la structure des nucléosomes. Enfin, des résultats intéressants sur la dynamique d’incorporation de H2A.Z à la chromatine ont été obtenus. / RNA Polymerase II is the molecular complex responsible for the transcription of class II genes. Proper transcription and associated events such as mRNA processing are thought to require the phosphorylation of its C-terminal domain. Here I show that this phosphorylation follows a similar pattern for most of the genes, althought some exceptions exist. These exceptions could be explained by alternative phosphorylation mechanisms. Also, this work provides data on how the variant histone H2A.Z influences chromatin structure. Previous studies have shown a role for H2A.Z in the positioning of some nucleosomes along the DNA, which would impact the ability to transcribe genes. Here I used a microarray technology to profile nucleosome positions in a genome-wide manner. My data provide further evidence that H2A.Z influences nucleosome positioning. Interesting results regarding the dynamics of H2A.Z incorporation into chromatin are also shown.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

Made available in DSpace on 2015-03-26T13:32:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 717763 bytes, checksum: e623d83648529a004b8aa2a3e4877433 (MD5) Previous issue date: 2008-12-05 / We considered a Bayesian analysis of first order autoregressive, AR(1), panel data model, using exact likelihood function, comparative analysis of prior distributions and predictive distributions of future observations. The methodology efficiency was evaluated by a simulation study using three prior, which were related to different Generalized Beta distributions: symmetric, asymmetric and flat prior. We applied the proposed methodology to microarray time series real data of HeLa cells. The forecast of gene expression in one future time showed high efficiency. / Considerou-se uma análise Bayesiana do modelo auto- regressivo de primeira ordem, AR(1), para dados em painel, de forma a utilizar a função de verossimilhança exata, a análise de comparação de distribuições a priori e a obtenção de distribuições preditivas de dados futuros. A eficiência da metodologia proposta foi avaliada mediante um estudo de simulação, no qual a distribuição Beta Generalizada foi usada para representar 3 diferentes prioris: simétrica, assimétrica e constante. Realizou-se uma aplicação em dados reais de expressão gênica temporal de células HeLa gerados por microarray. Os resultados mostraram alta eficiência na previsão da expressão gênica para um instante futuro.

Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-07-08T18:20:15Z No. of bitstreams: 1 cp124617.pdf: 1851616 bytes, checksum: 98299c340bd6b09b83e3fdf34f6adcf5 (MD5) / Approved for entry into archive by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-07-08T18:20:49Z (GMT) No. of bitstreams: 1 cp124617.pdf: 1851616 bytes, checksum: 98299c340bd6b09b83e3fdf34f6adcf5 (MD5) / Made available in DSpace on 2016-07-08T18:20:49Z (GMT). No. of bitstreams: 1 cp124617.pdf: 1851616 bytes, checksum: 98299c340bd6b09b83e3fdf34f6adcf5 (MD5) Previous issue date: 2009 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Associação Fundo de Incentivo à Psicofarmacologia (AFIP) / Introdução: Transplante alogênico de células tronco hematopoéticas (TCTH) é uma importante terapia para doenças hematológicas, mas o sucesso de uma porção de transplantes é limitado pela doença do enxerto versus hospedeiro(GVHD). Os fatores de risco conhecidos para GVHD agudo (aGVHD) não fornecem uma estimativa precisa do risco individual e não auxiliam na individualização da terapia. Até o momento, não existe método diagnóstico que permita predizer aGVHD. A identificação de pacientes que desenvolverão aGVHD poderia permitir a individualização da terapia para uns e evitaria imunossupressão intensa para outros. Objetivos: Revelar um classificador molecular preditivo de aGVHD. Descobrir genes diferencialmente expressos e analisar eventos precoces que desencadeiam aGVHD. Explorar categorias funcionais e tipos celulares relacionados ao desenvolvimento de aGVHD. Casuística e métodos: Foram isolados, amplificados, marcados e co-hidridados com lâminas de microarray contendo 22.000 sondas, amostras de RNA mensageiro de 89 pacientes submetidos a TCTH HLA-idêntico mieloablativo ou de toxicidade reduzida, obtido de células mononucleares periféricas durante a enxertia medular. Os pacientes foram divididos em grupo treino e grupo teste, um modo utilizado para construir um modelo que discrimine pacientes com e sem aGVHD, e para testar o modelo em amostras independentes. Os genes informativos foram selecionados utilizando recursive feature elimination, seguido de sete diferentes algoritmos de classificação multivariados para estabelecer o classificador molecular no grupo treino. Os genes diferencialmente expressos entre amostras de pacientes com e sem GVHD foram submetidos a análise de enriquecimento das vias funcionais e agrupados de acordo com o perfil de expressão com células e tecidos através do SymAtlas. Resultados: Encontramos um classificador molecular composto de 233 genes nas amostras do grupo treino, que foram selecionados baseados na mais precisa classificação. No grupo teste da amostra, cerca de 80% dos pacientes puderam ser classificados. Para estes pacientes, o classificador mostrou uma acurácia preditiva de 75% (sensibilidade de 71% e especificidade de 78%). Analisando a anotação funcional dos genes diferencialmente expressos, observamos que em pacientes que desenvolveram aGVHD, houve aumento de expressao de genes da resposta antimicrobiana, transporte de gases, metabolismo de hemoglobina, além das alarminas. Nestas amostras também observamos a diminuição da expressão da IL1 e outros genes envolvidos na via do NF-kB. Vários genes super-expressos no periodo precoce do aGVHD foram associados a células precursoras. Conclusões: Nossos resultados mostram que um classificador molecular é capaz de identificar pacientes sob alto risco de desenvolver aGVHD. Estabelecer métodos de diagnóstico preditivo para aGVHD é o primeiro passo para a individualização da estratégia terapêutica após TCTH. Além disso, os resultados da análise de enriquecimento funcional e a expressão de genes em diferentes populações celulares, sugerem que eventos precoces envolvendo múltiplas populações de células precursoras possam predefinir a interação futura entre o enxerto em desenvolvimento e o paciente. / Background: Allogeneic hematopoietic stem cell transplantation is an important last resort therapy for hematological diseases. Unfortunately, the success of a large proportion of these transplants is limited by graft-versus-host disease (GVHD). Currently known risk factors for acute GVHD (histoincompatibility, sex mismatch, older patients, previous pregnancies) do not provide a precise estimate of individual patient risk and do not help for individualization of the therapy. Early identification of those patients who will develop aGVHD may allow for individualized treatment, and also for the reduction of unnecessary treatment for those patients not at risk. Nowadays, however, there is no diagnostic method that allows prediction of aGVHD. Objectives: The goal of our study was to reveal a gene expression profile that would predict the occurrence of aGVHD. In addition, using enrichment of gene ontology categories, to analyze differentially expressed genes in order to better understand biology of the events preceding aGVHD. Material and methods: we collected blood samples from 89 recipients of myeloablative and reduced conditioning regimen HLA-identical sibling allogeneic hematopoietic stem cell transplants at the time of successful engraftment. We isolated total RNA from the peripheral blood mononuclear cells, amplified it, labeled, and co-hybridized to the microarray slides containing probes for 22,000 genes..The patients were divided into training and test groups, the former used to build a model discriminating patients with and without aGVHD and the latter - to test the model on independent samples. We selected the informative genes using “recursive feature elimination” method followed by seven different multivariate classification algorithms in order to establish a molecular classifier in the training set. Then we validated this new classifier in the test set of patients.. We found differentially expressed genes using T-test and accepted those with estimated false discovery rate below 10%. We have used Biobase Explain to find enrichment of functional groups among differentially expressed genes.Results: We found a molecular classifier comprised by 233 gene probes in the training set of samples which were selected based on the most accurate classification. In the test group of samples, we found that 80% of patients could be classified based on the concordance between classification methods as described above. For these patients, the classifier showed 75% of a predictive accuracy (71% of sensitivity and 78% of specificity). Analysis of functional annotations of differentially expressed genes showed that patients that developed acute GVHD have increased expression of antimicrobial genes, hemaglobin metabolism genes and alarmins. In these samples we also observed decreased expression of IL-1 and other genes involved in NF-kB activation. Several genes up-regulated before aGVHD were associated with multiple types of precursor cells. Conclusion: Our results show that molecular profiling is able to identify patients under high risk of acute GVHD at the time of engraftment. Establishing of a predictive diagnostic method for aGVHD is the first step of individualization of therapeutic strategy after hematopoetic stem cell transplantation. In addition, the results of functional enrichment analysis and expression in different cell populations suggest that early events during engrafment involving precursor cell populations might predefine results of interaction between stem cell allograft and patient body.

Os papilomavírus são pequenos vírus de DNA dupla-fita, não envelopados, mucoepiteliotrópicos, capazes de infectar inúmeros vertebrados superiores de maneira espécie-específica. A infecção por estes vírus está associada a uma série de desordens proliferativas que levam desde de a formação de verrugas comuns até a do carcinoma invasivo. Aproximadamente 200 tipos de papilomavírus humano (HPVs) foram identificados, sendo que cerca de 40 deles infectam o trato genital. Dentre estes, os chamados HPVs de alto-risco estão associados etiologicamente ao carcinoma de colo de útero, enquanto que os de baixo-risco estão relacionados às lesões epiteliais benignas. A infecção por HPVs de alto-risco é muito comum, no entanto, a maioria destas é transitória e somente uma pequena proporção de mulheres desenvolvem o carcinoma. Entretanto, algumas mulheres são incapazes de eliminar esta infecção, levando a persistência viral e o conseqüente desenvolvimento da neoplasia. Para que a infecção pelo HPV persista é necessário um mecanismo de escape ao sistema imune do hospedeiro. O mecanismo de escape à resposta imune inata parece ser característico da infecção pelo HPV, pois o ciclo infeccioso deste vírus não promove inflamação. A infecção por HPV promove a liberação de citocinas, tal como o fator de necrose tumoral (TNF). Esta citocina possui um potente efeito citostático em queratinócitos normais e imortalizados com HPV, enquanto que em queratinóctos imortalizados com HPV18 este efeito não é observado. Do mesmo modo, observamos que a expressão do oncogene E6 de HPV16 ou 18 é suficiente para promover resistência ao efeito antiproliferativo do TNF em culturas em monocamada e organotípica. A expressão aumentada e contínua destes ocogenes é sabidamente o principal evento favorável ao desenvolvimento do câncer de colo de útero. Estas proteínas são essenciais na indução da transformação celular, visto que interferem na regulação do ciclo celular e apoptose. O produto dos genes E6 e E7 se liga ao produto dos genes supressores de tumor p53 e pRb, respectivamente, levando a sua degradação pela via de proteólise dependente de ubiquitina. As bases moleculares desta resistência ao TNF ainda são pouco conhecidas. Neste estudo, comparamos o efeito desta citocina em queratinócitos normais e que expressam E6 ou E7. Observamos através de cDNA Microarray a expressão de um grupo de genes, entre eles TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 e MMP9, com expressão diferencial entre as células sensíveis e as resistentes ao TNF. / Papillomaviruses are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of higher vertebrates in a species-specific manner. Papillomavirus infections are associated to a series of proliferative disorders that range from common warts to invasive carcinomas. Almost 200 types of human papillomaviruses (HPVs) have been identified and approximately 40 of them infect the genital tract. Only the so-called high-risk HPV types mediate human carcinogenesis, whereas the low-risk HPVs have been linked to benign epithelial lesions. High-risk genital HPV infection is very common, and the majority of individuals clear their infection with time. However, a proportion of women cannot effectively clear the virus, and the persistence of a high-risk HPV is the major risk factor for the development of anogenital malignancies. To persist, HPV must escape the host immune system. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and virion release is not associated with inflammation. Furthermore, HPV infections promote cytokine release, as tumor necrosis factor-alpha (TNF). This cytokine has a potent cytostatic effect on normal and HPV16 immortalized keratinocytes, while it does not affect HPV18 immortalized keratinocytes proliferation. In addition, we have observed that expression of HPV 16 or 18 E7 oncogene is sufficient to overcome TNF antiproliferative effect in monolayer and organotypic cell cultures. The increased and sustained expression of HPV oncogenes, E6 and E7, is the main contributor to the development of cervical cancer. Both E6 and E7 proteins are essential to induce and maintain cellular transformation, due to their interference with cell-cycle and apoptosis regulation. The most manifest function of the E6 protein is to promote the degradation of p53, while E7 is known to bind to and promote the proteasomal degradation of the retinoblastoma tumor suppressor gene product, pRb, and its family members. The molecular basis of TNF resistance is not well understood. In this study we compared the effect of TNF between normal and HPV16 E6 or E7 expressing keratinocytes. We observed by cDNA Microarray the differential expression of a common set of genes in TNF-sensitive cell lines, including TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 and MMP9, that differs from those modulated in TNF-resistant cells.

A própolis é uma mistura resinosa complexa de várias substâncias coletada de plantas pelas abelhas. Ela tem atraído atenção devido à variedade de suas propriedades biológicas e terapêuticas. Diversos estudos têm mostrado a conexão existente entre a morte celular tipo-apoptose em fungos e importantes processos biológicos como desenvolvimento, envelhecimento, resposta a estresse e patogênese. Neste contexto, este projeto avaliou a atividade antifúngica da própolis, com o objetivo de ampliar os conhecimentos acerca das vias metabólicas de morte celular em fungos, como S. cerevisiae e C. albicans, e também em relação à utilização da própolis como uma terapia antifúngica mais efetiva. Inicialmente, utilizou-se S. cerevisiae com o objetivo de compreender como a própolis afeta fungos ao nível celular. Foi observado que ela é capaz de induzir uma resposta de morte celular apoptótica. No entanto, a exposição aumentada à própolis promove um correspondente aumento na resposta tipo necrose. Verificou-se ainda que o citocromo c, mas não a endonuclease G Nuc1p, está envolvido na morte celular mediada por própolis em S. cerevisiae. Também foi observado que o gene da metacaspase YCA1 é importante para a morte celular induzida por esta substância natural. Para elucidar as funções dos genes que poderiam ser necessários para a sensibilidade à própolis em eucariotos, realizou-se um screening da coleção completa com cerca de 4800 cepas haplóides com genes únicos deletados de S. cerevisiae. Foram identificadas 138 cepas que apresentaram diferentes graus de sensibilidade à própolis quando comparadas com a cepa do tipo selvagem correspondente. Na análise deste screening por biologia de sistemas e também através do perfil transcripcional de S. cerevisiae exposto à própolis, foram observados genes envolvidos na cadeia de transporte de elétrons mitocondrial, da acidificação vacuolar, da regulação negativa da transcrição do promotor da RNA polimerase II, da regulação da macroautofagia associada com a proteína alvo para vacúolo e da resposta celular à privação de nutrientes. Os estudos de validação indicaram que a sensibilidade da própolis é dependente da função mitocondrial e que a acidificação vacuolar e autofagia são importantes para a morte causada por própolis em leveduras. Para o fungo patogênico C. albicans, foi observado que a própolis induz uma morte celular do tipo necrose; observou-se ainda que o gene IPF4847 (homólogo para o gene da metacaspase YCA1 de S. cerevisiae) é importante para a morte celular mediada por própolis. Além disso, com o objetivo de tentar esclarecer algumas funções de genes que poderiam estar envolvidos na sensibilidade à própolis de C. albicans, 800 mutantes deletados de C. albicans foram escaneados e destes, 51 apresentaram maior sensibilidade a própolis quando comparados com as cepas do tipo selvagem correspondente. Vários genes observados em nosso \"screening\" estão envolvidos na transição dimórfica em C. albicans. Desta forma, foi realizado um ensaio a fim de se verificar o papel da própolis na inibição da transição dimórfica e foi observado que este composto inibe não só a transição dimórfica como também o crescimento de todos os morfotipos (levedura, hifa, e pseudohifa) de C. albicans. Desta maneira, o uso da própolis para o tratamento clinico da candidíase pode ter grande aplicação, principalmente por afetar um mecanismo importante para a patogenicidade deste fungo / Propolis is a complex mixture of several resinous substances which are collected thorn plants by bees. Propolis has attracted the attention of researchers because of its variety of biological and therapeutic properties. Studies have shown the connection between Propolis and apoptosis-like cell death in fungi and other important biological processes such as development, aging, stress response and pathogenesis. In this context, this project evaluated the antifungal activity of Propolis, With the aim of expanding the knowledge about the metabolic pathways of cell death in fungi such as S. cerevisiae and C. albicans, and also in relation to the use of propolis as an effective antifungal therapy. For this, initially we utilised S. cerevisiae as a model organism to study the genetics, cell biology and genomics that determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c, but not endonuclease G (Nuc1p), is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required Or propolis sensitivity in eukaryotes, the full collection of approximately 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular responses to starvation. Validation studies indicated that propolis sensitivity is dependent on mitochondrial function and. that vacuolar acidification and autophagy are important for S. cerevisiae cell death caused by propolis. For the pathogenic fungus C. albicans it was observed that propolis induces cell death like necrosis. It was also observed that the IPF4847 gene (homologous to the YCA1 metacaspase gene of S. cerevisiae) was also important for cell death mediated by propolis in C. albicans. Furthermore, aiming to clarify some of the functions of genes that could be involved in C. albicans sensitivity to propolis, 800 C. albicans deletion mutants were screened, and 51 showed greater sensitivity to propolis compared with the corresponding wild-type strains. Several genes found in our screening were involved in the dimorphic transition of C. albicans. Thus, an assay was performed in order to verify the role of propolis in the inhibition of the dimorphic switch. It was observed that propolis can inhibit the dimorphic transition and the growth of all its morphotypes (budding hyphal and pseudohyphal) in C. albicans. So, the use of propolis for clinical treatment of Candidiasis may have a wide application, principally by affecting an important mechanism for the pathogenicity of fungi

Camundongos AIRmax e AIRmin homozigotos para os alelos R e S do gene Slc11a1 foram avaliados para susceptibilidade à artrite induzida por pristane (PIA). A presença do alelo S aumentou a incidência e a gravidade nos AIRmax, sugerindo que o gene Slc11a1, ou outro próximo, esteja interagindo com os loci de resposta inflamatória na modulação de PIA. Para identificar estes loci foram realizados estudos de associação genótipo-fenótipo e de expressão gênica global. Os RNAs das patas dos animais foram isolados após 180 dias da indução por pristane. As análises de expressão gênica global foram realizadas usando a plataforma Codelink (36k genes), cujos resultados foram validados por PCR em tempo real. Os estudos de associação foram realizados através da análise de polimorfismo de microssatélites pelo programa MapManager. Foram identificadas duas regiões nos cromossomos 1 e 11. Um número grande de genes diferencialmente expressos foi verificado nos animais AIRmax SS cujos temas biológicos significativamente sobre-representados foram a resposta inflamatória e quimiotaxia. Os camundongos AIRmax SS também possuem uma ativação maior dos genes Ccl3, Ccl7, C3ar1, Il10, Stat3, Tirap, Trem 1, Trem 3, Mefv, Ptx3, Chi3l3 e Kras. Alguns desses genes co-localizam com regiões previamente mapeadas nos cromossomos 1 e 11. / AIRmax and AIRmin mice homozygous for Slc11a1 R and S allele were evaluated for pristane-induced arthritis (PIA) susceptibility. The presence of S allele increased the incidence and the arthritis severity in ARmax mice, suggesting that Slc11a1 or other closed-linked gene interacts with inflammatory loci to modulate PIA. In order to identify inflammatory modifier loci modulating experimental arthritis development, genotype-phenotype association studies and global gene expression analyses were performed. Mice received i.p. injections of pristane and the paw RNAs were isolated at day 180. Global gene expression analysis was performed on Codelink bioarrays (36k genes) and validated by real time PCR. The microsatellite polymorphism analyses were performed using MapManager program. Two regions on chromosomes 1 and 11 were identified. Higher number of differentially-expressed genes were detected in AIRmax SS subline, which significant over-represented biological themes were related to inflammatory response and chemotaxis. Susceptible AIRmax SS mice also display high up-regulation of Ccl3, Ccl7, C3ar1, Il10, Stat3, Tirap, Trem 1, Trem 3, Mefv, Ptx3, Chi3l3 e Kras genes. Some of them co-localize with previously identified regions mapped on chromosomes 1 and 11.

Målet med projektet är att undersöka om det går att effektivisera hur man undersöker människors gener. Detta görs genom att skapa ett program i Java. Resultatet är ett program som sorterar genotypdata från 1000 Genomes Project och utvärderar nyttan av att undersöka genotyper från flera individer samtidigt.

La bioingénierie de greffons endothéliaux cornéens est une des solutions réalistes en cours de développement dans quelques laboratoires pour palier au grand déséquilibre entre pénurie mondiale de dons de cornée et besoins immenses et croissant des populations. Cette véritable médecine régénérative consistera à injecter des cellules endothéliales (CEs) dans la chambre antérieure aux stades précoces des dystrophies endothéliales et, pour les stades avancées des pathologies endothéliales, à reconstruire in vitro des greffons endothéliaux composés d'un support transparent et biocompatible colonisé par une monocouche des CEs. Dans les 2 cas, la première étape obligatoire est la production de masse de CEs ou de "CE-like". Dans ce travail, nous avons exploré les différentes possibilités pour obtenir suffisamment de CEs fonctionnelles pour envisager des applications cliniques : 1- L'expansion de CEs natives prélevées sur des cornées de donneurs, ou culture primaire, se heurtent aux capacités prolifératives limitées des CEs in vitro. Un des prérequis étant la compréhension des mécanismes d'arrêt de la prolifération des CEs humaines, nous avons fait la synthèse bibliographique des connaissances sur leur cycle cellulaire et leur sénescence, et présentons 2 articles originaux: le premier utilise un microarray spécifique de 112 gènes de contrôle du cycle cellulaire pour comparer les profils transcriptionnels des CEs de 6 modèles biologiques comportant théoriquement un stimulus prolifératif croissant: in vivo, post mortem, organoculture, culture primaire confluente, culture primaire non confluente et lignée immortalisée. Nous identifions de nombreux acteurs impliqués dans l'arrêt du cycle, en particulier ceux impliquant une réponse à des dommages oxydatifs de l'ADN. Le second article explore les capacités prolifératives résiduelles des CEs de donneurs âgés de plus de 50 ans, sont les plus nombreux en Europe et donc les pourvoyeurs habituels de CEs pour la bioingénierie. Nous montrons qu'une optimisation des techniques de culture permet d'obtenir in vitro une mosaïque endothéliale de 2000 cellules/mm2. Enfin, un troisième article montre qu'un stimulus physique inattendu constitué d'un train d'impulsion électrique permet d'obtenir un très grand nombre de figures mitotiques mais uniquement dans des zones de faible DCE, démontrant encore une fois l'importance majeur de l'inhibition de contact dans l'arrêt prolifératif. 2- La sélection et l'expansion, par culture en sphère, des progéniteurs endothéliaux de la périphérie endothéliale pourrait être un moyen de contourner la sénescence de la majorité des CEs. La synthèse bibliographique montre que la plupart des travaux publiés émanent d'une seule équipe japonaise. Dans notre second article, nous avons montré l'existence de rares "label-retaining cells" dans les cornées de donneurs âgés, qui pourraient correspondre à ces progénieteurs. Nous avons aussi montré qu'il était possible d'obtenir des sphères avec ces donneurs. 3- La différenciation de cellules souches embryonnaires, mésenchymateuses ou des cellules pluripotentes induites est enfin la troisième voie qui pourrait permettre de produire des CEs en grand nombre. La méthode d'induction de la différenciation pourrait reproduire le schéma physiologique et désormais bien caractérisé de formation de l'endothélium à partir du mésenchyme périoculaire dérivé de la crête neurale et que nous rappelons au début de notre mémoire bibliographique. Nous rappelons également les méthodes utilisables pour caractériser les cellules obtenues: vérification de leur identité par immunomarquage d'un panel de protéines caractéristique (en absence de marqueur unique spécifique) et vérification de leur fonctionnalité par mesures de leurs capacités de pompage ionique, au minimum de façon indirecte sur chambre d'électrophysiologie de type Ussing, au mieux de façon directe en quantifiant la déturgescence d'une cornée humaine conservée dans le bioréacteur breveté du BiiGC / Corneal endothelial engineering is becoming a more and more realistic solution to restore vision from corneal edema. This method focus to regenerate corneal endothelium by direct injection of corneal endothelial cells (ECs) into patient anterior chamber at the early stage of endothelial dystrophies, or by grafting a transparent biocompatible material covered by a monolayer of ECs. These two techniques require both in vitro isolation and amplification of ECs or endothelial-like cells. In this thesis, different strategies to obtain a high quantity of functional ECs for clinical application are explored: 1- Due to the limit proliferative capacity of EC, the first strategy consists to analyze mechanisms implicated EC cell cycle arrest and then to optimize protocol for native EC isolation or for cell proliferation activation ex vivo. This is summarized in three publications. The first publication describes the cell cycle regulation by comparing transcriptional expression of 112 genes in 6 biological models of EC with different proliferative profile: in vivo, postmortem, organ-culture, confluent primary culture, non confluent primary culture and immortalized cell line. , The key molecular actors identified using the combining microarray analysis and gene ontology methods are consistent with previous findings about oxidative DNA damage mechanism. The second publication characterizes EC differentiation process and its impact on EC proliferative capacity in old donor corneas. Analyses of differentiation/progenitor markers and of proliferative capacity underline the differentiation process of EC from the centre to the peripheral corneal endothelium. Thereby, an optimized culture protocol was developed, allowing the formation of high-density monolayer (> 2000 cells/mm2) with stable endothelial morphology. We proved the possibility to make profit from a majority of old-donor cornea grafts invalidated for penetrating graft In the third publication, the activation of endothelial cell cycle by electric pulses directly in corneal graft was characterized. We confirm the activation of endothelial cell cycle at different phases but also the damage of tissue during electroporation. 2- Second strategy consists of the amplification of ECs from potential EC progenitors. Using sphere forming culture and a new method to detect slow-cycling cells, we demonstrate the existence of "young" ECs population with higher proliferative capacity in corneal periphery. The isolation of ECs by sphere formation is one possible step for ECs selection in vitro. 3- The differentiation of embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells into corneal endothelial cells is the third approach considered by our laboratory. The manipulation of stem cells differentiation would be based on the molecular mechanisms implicated in the formation of corneal endothelium from periocular mesenchymal cells described in the first part of the bibliography. Finally, in order to validate the quality of endothelial cell mass obtained, we revisited recent methods for the evaluation of corneal endothelial identity (immunolocalisation of specific markers), for the measurement of pump activity of cell monolayer (Ussing chamber, perfusion chamber) or directly in deswelled cornea using the bioreactor patented by the BiiGC laboratory