Orientadores: Marcelo Menossi Teixeira, Renato Atílio Jorge / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T10:26:16Z (GMT). No. of bitstreams: 1 Mattiello_Lucia_D.pdf: 1855407 bytes, checksum: 309c252e258389c8f81e79e8fba49e74 (MD5) Previous issue date: 2010 / Resumo: A presença do alumínio (Al) em solos ácidos é o principal fator limitante da produtividade agrícola no Brasil e no mundo. A resposta desenvolvida pelas plantas contra o Al é complexa e a identificação de genes responsivos após a exposição ao íon através de técnicas de análise em larga escala, como microarrays, pode facilitar a sua compreensão. Este projeto possui como objetivo ampliar o conhecimento sobre a fisiologia e a regulação gênica de raízes e folhas utilizando genótipos contrastantes de milho (Cat100-6 (Al-tolerante) e S1587-17 (Al-sensível)) cultivadas em solo ácido com concentração fitotóxica de Al. As linhagens de milho Cat100-6 e S1587-17 foram cultivadas por um ou três dias em solo ácido (pH 4,1) ou solo corrigido com Ca(OH)2 (pH 5,5). O genótipo S1587-17 apresentou uma maior inibição do crescimento radicular, resultado este altamente correlacionado com a acumulação de Al nos ápices radiculares e deposição de calose. Os dados fisiológicos confirmam a discriminação entre as duas linhagens em solo, abrindo perspectivas para entender pela primeira vez a base molecular das alterações das plantas em condições próximas à realidade de campo. O transcriptoma de raízes possibilitou a identificação possíveis candidatos a tolerância ao Al. Adicionalmente, com um experimento de hidroponia separamos as variáveis pH e presença de Al, ambas condições diferenciais do tratamento com solo. Identificamos, entre os candidatos, genes responsivos pela presença do Al e não pela acidez delimitando assim os genes com possíveis papéis na tolerância ao Al presente no solo ácido a apenas três: retinol desidrogenase, um fator de transcrição WRKY e uma proteína desconhecida. Esses resultados permitem concluir que o cultivo em solo é diferencial em relação à hidroponia, e outros fatores que apenas presentes no substrato solo podem provocar a indução de alguns genes. Diversas vias metabólicas são afetadas na linhagem sensível pelo tratamento em solo ácido e podem estar envolvidas na inibição radicular como a produção de lignina, celulose e calose e a síntese de etileno e auxina. O mapeamento nos cromossomos dos genes identificados pelo experimento de microarray das raízes de milho permitiu a identificação de genes localizados dentro de QTLs de milho previamente descritos na literatura como responsáveis pelo fenótipo tolerante. Diante esse resultado, podemos especular o papel de genes como uma proteína ligadora de RNA, uma inibidora de proteases e ciclinas na tolerância ao Al contido no solo ácido. Pela primeira vez na literatura, o transcriptoma de folhas coletadas após três dias de cultivo em solo ácido ou solo corrigido foi obtido com o uso de microarrays da Affymetrix. Essa análise indicou profundas alterações na Cat100-6, em contraposição à ausência de alteração significativa nas folhas na S1587-17. Genes referentes à fotossíntese e a fotorrespiração foram regulados negativamente pelo tratamento em solo ácido no genótipo tolerante. Contudo, o ciclo do ácido cítrico está ativado indicando uma putativa participação da produção de ácidos orgânicos nas folhas na resposta ao Al / Abstract: The presence of aluminum (Al) is the main factor limiting crops yield in Brazil and worldwide. The plant responses developed against this ion are complex and the identification of responsive genes after exposure to the ion with the use of a large scale technique, such as microarrays, can facilitate its comprehension. This project aimed to amplify the knowledge about physiology and gene expression regulation of roots and leaves associated towards Al resistance using contrasting maize genotypes (Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive) cultivated in acid soil containing phytotoxic concentrations of Al. Maize lines Cat100-6 and S1587-17 were cultivated for one or three days in acid soil (pH 4,1) or limed soil with Ca(OH)2 (pH 5,5). The genotype S1587-17 presented a higher root growth inhibition, which is highly correlated with Al accumulation in the root apexes and callose deposition. The physiological data confirms the discrimination of the two maize lines cultivated in soil, opening perspective to understand for the first time the molecular bases of alterations in plants on a closer condition to the field. Transcriptome from roots made possible the identification of possible tolerance candidates and genes constitutively expressed genes in the tolerant line. Additionally, throw a hydroponic experiment we splited the variables pH and Al presence, both differential conditions between soil treatments. It was possible to identify, among the candidates, genes responsive in the presence of Al in acid soil rather than acidity limiting genes with a possible roles in Al present in the acid soil tolerance to only three: retinol dehydrogenase, the transcription factor WRKY and an unknown protein. These results allow the conclusion that the soil culture is different in relation to hydropony, and other factors present only in soil substrate could provoke the induction of some genes. Several metabolic pathways were affected in the sensitive line after acid soil growth and could be involved on root growth inhibition such as lignin, cellulose and callose production and ethylene and auxin synthesis. The mapping of the identified genes through the microarray experiments into the chromosomes allowed the identification of genes localized into maize QTLs previously reported in the literature as responsible for the tolerant phenotype. Facing these results, we can speculate the role of these genes such as a RNA binding protein, a protease inhibitor, and cyclines in the Al present in the acid soil tolerance. For the first time in literature, the transcriptome of leaves collected after three days in culture with acid soil or limed soil with the Affymetrix microarrays. This analysis indicated great alterations in Cat100-6, meanwhile S1587-17 showed no significative alteration. Genes related to photosynthesis and photorespiration were down-regulated due acid soil treatment in the tolerant genotype. However, citric acid cycle was activated indicating the putative partitipation of organic acids produced in the leaves in thr Al response / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

O emprego da metodologia de microarranjos no estudo do câncer tem permitido a identificação de genes com alterações em seus perfis de expressão, os quais estão direta ou indiretamente envolvidos na etiologia dessa doença. O crescente número de publicações de experimentos de expressão gênica em repositórios de dados de microarranjos demonstra que, em geral, a limitação não está na quantidade ou qualidade desses experimentos, mas no processamento desses dados. Dessa forma, há a necessidade de desenvolver metodologias de bioinformática que sejam capazes de analisar tais perfis de forma integrada, incluindo no contexto da análise, dados provenientes de outros experimentos. O desenvolvimento do câncer tem sido associado principalmente a distúrbios nos mecanismos de controle do ciclo celular, cujas vias são dependentes de uma maquinaria transcricional e de seus elementos regulatórios; entre estes últimos, os fatores de transcrição (FTs) têm sido estudados como potenciais alvos para a terapia molecular. No presente trabalho, um conjunto de dados de microarranjos realizados a partir de amostras de glioblastoma foi obtido em repositórios públicos (GEO e ArrayExpress). O teste estatístico SAM foi aplicado aos dados e os genes diferencialmente expressos (FDR 0,05), induzidos em glioblastoma (1.830 genes), foram submetidos a uma análise de associação a FTs (programa FatiGO+), a qual associou os FTs HNF1A, IRF7 e E2F (p 0,05) aos genes induzidos. Adicionalmente, a lista de genes foi submetida a uma análise de associação a um banco de dados de assinaturas transcricionais do software TBrowser, extraídas de diversos experimentos de microarranjos do GEO. Como resultado, 7.910 assinaturas foram associadas (p 0,01), sendo que as cem primeiras também foram relacionadas a FTs por meio de ferramentas disponibilizadas no próprio TBrowser. Os FTs E2F1 e E2F4 foram associados a mais de 80% das assinaturas analisadas. Dessa forma, ambas as análises apontaram o FT E2F, mais especificamente E2F1 e E2F4, como associados à lista inicial de genes fornecida pelo SAM. A expressão de E2F1 e E2F4 foi avaliada por meio da técnica de RT-PCR quantitativa em tempo real em amostras de RNA de sete linhagens de glioblastoma (T98, U251, U138, U87, U343, MO59J e MO59K). Interessantemente, ambos os genes foram apontados como superexpressos em todas as linhagens, a maioria com significância estatística. A família de FTs E2F apresenta papéis importantes no controle da proliferação celular e apoptose. Alguns membros atuam como oncogenes, e outros, como genes supressores de tumor, dependendo do contexto molecular nos quais se encontram. Muitos trabalhos da literatura têm apontado a importância desses FTs, especialmente E2F1, no processo de itumorigênese. Com base nos resultados obtidos no presente trabalho, o status de expressão (indução) de E2F1 e E2F4 é provavelmente associado ao glioblastoma. Esses FTs podem influenciar a expressão de uma série de genes cruciais, frequentemente alterados nessa doença, o que ainda requer um estudo mais detalhado, visando à validação desses FTs como biomarcadores, ou até mesmo como alvos moleculares que possam ser empregados no estabelecimento de novas modalidades de terapias. / The application of DNA microarrays in cancer research has provided the identification of abnormal expression profiles of a series of genes, which may be directly involved in the etiology of such disease. The increasing number of publications related to gene expression profile in microarray repositories has demonstrated that the limitation of such experiments is not associated with the quantity neither the quality of such experiments, but with data processing. Therefore, there is a great interest in the development of methodologies in bioinformatics that allow the integrated analysis of such profiles, including data set from other experiments. Cancer development has been associated mostly with changes in cell cycle control, whose intracellular pathways are dependent on the transcriptional machinery. Regulatory elements involved in transcription, which include transcriptional factors (TFs), can be potential targets for molecular therapy. In the present study, a set of microarray data achieved from glioblastoma samples were obtained from public repositories (GEO and ArrayExpress). Data were submitted to statistical analysis using SAM, and 1,830 up-regulated genes (FDR 0.05) were submitted to TFs association analysis (FatiGO+ program). Those genes were associated to the TFs HNF1A, IRF7 and E2F (p 0.05). Moreover, the set of genes was submitted to a transcription signature bank data association analysis using the software TBrowser, extracted from GEO, which provided a wide range of data sets from microarray experiments. The analysis led to 7,910 associated signatures (p 0.01); out of them, the first 100 were related to TFs according to the tools available in TBrowser. The TFs E2F1 and E2F4 were associated to more than 80% from the analyzed signatures. Therefore, both analysis suggested that the TF E2F, specifically E2F1 and E2F4, were associated to the initial gene set obtained by the SAM analysis. The expression of E2F1 and E2F4 was evaluated by quantitative real-time PCR using RNA samples from seven glioblastoma cell lines (T98, U251, U138, U87, U343, MO59J and MO59K). Interestingly, both genes were found up-regulated in all cell lines. The E2F TFs family members present important roles in cell proliferation control and apoptosis. Depending on the molecular context involved, some members act as oncogenes and others as tumor suppressor genes. A great number of studies has suggested the importance of such TFs , specially E2F1, in the tumorigenesis process. According to the present results, the expression status (induced) of E2F1 and E2F4 may be associated with glioblastoma. These TFs may influence the abnormal expression of important genes in cancer, even though detailed studies should be necessary in order to validate these TFs as biomarkers or molecular targets for therapeutical intervention.

Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:29:49Z (GMT). No. of bitstreams: 1 Lorencini_Marcio_D.pdf: 16348273 bytes, checksum: 4ed299b197892b0d3297e6e6a65d947c (MD5) Previous issue date: 2014 / Resumo: Com o aumento do tempo de vida da população humana muitas modalidades médicas, incluindo a dermatologia, deparam-se com uma revolução na forma de garantir saúde e qualidade de vida aos pacientes. Em contato com o ambiente externo, a pele representa um órgão no qual as mudanças com o envelhecimento causam danos funcionais, além de potencial impacto estético e psicossocial. A epiderme, camada mais externa da pele, constitui uma barreira seletiva com destacada capacidade de renovação e manutenção da homeostasia corporal. Entretanto, o entendimento de diversos mecanismos associados à fisiologia e envelhecimento da epiderme permanece como desafio para a comunidade científica. Com base nesse cenário, o objetivo do presente trabalho foi compreender o atual estado da arte no tema de envelhecimento da epiderme e realizar experimentos voltados para lacunas existentes, com foco na integração de aspectos clínicos, fisiológicos, morfológicos, celulares e moleculares. O capítulo de abertura descreve uma avaliação global de transcritos associados ao envelhecimento da epiderme humana, com a técnica de microarranjos de DNA e coleta não invasiva com fitas adesivas. O estudo indica características moleculares específicas do fotoenvelhecimento epidermal, com alterações relevantes e complementares a dados clínicos e morfológicos prévios, como modulação das vias de organização do citoesqueleto de actina e sinalização de cálcio, expressão gênica alterada de proteínas do envelope córneo, e avaliação de um painel segmentado por décadas de vida que sugere aspectos inéditos de regulação homeostática da epiderme, além de genes com modulação contínua ao longo das idades. O segundo capítulo compara o envelhecimento nas regiões folicular e interfolicular da epiderme. Como um sistema biológico de simples obtenção e fácil manuseio, os bulbos dos folículos pilosos representam uma fonte rica de material epidermal distinto, conforme evidencias na ampla modulação gênica diferenciada. O terceiro capítulo inclui uma avaliação in vitro do envelhecimento da epiderme, com queratinócitos de indivíduos de diferentes idades cultivados em monocamada e no modelo de pele equivalente. Os resultados evidenciam diferenças na expressão de marcadores moleculares de proliferação e diferenciação entre queratinócitos neonatais e adultos, mas não entre adultos de diferentes idades. Não houve diferença nas populações de células tronco, entretanto, observou-se aumento de células na fase proliferativa do ciclo celular em neonatos, assim como predominância de células na fase estacionária do ciclo celular em adultos mais velhos. Concluindo, os resultados obtidos no presente trabalho contribuem de forma significativa para o avanço do entendimento dos mecanismos moleculares afetados pelo avanço da idade da epiderme, possilitando a busca de novas alternativas no tratamento do envelhecimento cutâneo / Abstract: With the increase in lifetime of the human population many medical disciplines, including dermatology, are facing a revolution in the approaches to ensure healthcare and quality of life for patients. In contact with the external environment, the skin is an organ in which the changes of aging cause functional damage, in addition to potential aesthetic and psychosocial impact. Epidermis, the outermost skin layer, is a selective barrier with outstanding capacity for renewal and maintenance of the body homeostasis. However, the understanding of several mechanisms associated with skin physiology and aging remains a challenge for the scientific community. Considering this scenario, the objective of this work was to evaluate the state of the art knowledge on epidermal aging and to conduct experimental approaches to cover gaps that still exist on that theme, focusing on the integration of clinical, physiological, morphological, cellular and molecular aspects of epidermis aging. The opening chapter describes a study based on global transcriptional evaluation associated with aging of the human epidermis, using DNA microarrays and noninvasive tape stripping. This study reveals molecular characteristics specific of epidermal photoaging, with relevant findings complementary to previous clinical and morphological data, such as modulation of the actin cytoskeleton and calcium signaling pathways; altered gene expression of proteins of the cornified envelope; and evaluation of a segmented panel structured by decades of life, which suggests new aspects of homeostatic regulation in the epidermis and unvails genes with continuous modulation throughout different ages. The second chapter compares the gene expression patterns of the follicular and interfollicular regions of epidermis undergoing aging. As a biological system easily sampled and handled, the bulbs of plucked hair follicles represent a rich source of distinct epidermal material, as evidenced by the wide differential gene modulation that was detected. The third chapter includes an experimental in vitro evaluation of skin aging using keratinocytes isolated from individuals of different ages and cultured in monolayer and in skin equivalent models. Differences in the expression of proliferation and differentiation molecular markers between neonatal and adult keratinocytes were observed. No differences were found regarding the stem cell populations, however, neonates showed an increased percentage of cells in the proliferative phase of cell cycle, while older adults presented a predominance of cells in the stationary phase of cell cycle. The results herein presented provide novel insights on the molecular mechanisms affected by epidermal aging, enabling the search of new alternatives in the treatment of aging skin / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Orientador: Michel Georges Albert Vincentz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:00:26Z (GMT). No. of bitstreams: 1 Baptista_JulianaCristina_D.pdf: 14259949 bytes, checksum: d33465f0d6490d2c792f6ee701daa4bd (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Genetica Vegetal e Melhoramento / Doutora em Genética e Biologia Molecular

Orientador: Vera Lucia Gil da Silva Lopes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T00:02:33Z (GMT). No. of bitstreams: 1 Simioni_Milena_D.pdf: 16919327 bytes, checksum: fa845dbc925978762cb44d70536f9ec7 (MD5) Previous issue date: 2012 / Resumo: As fendas orofaciais típicas (FOT) são defeitos congênitos prevalentes que possuem múltiplas etiologias. Estudos populacionais apontaram diferentes genes relacionados às FOTs. Entretanto, em muitos casos, a etiologia permanece desconhecida. A investigação individualizada, utilizando em conjunto diferentes ferramentas laboratoriais, é uma abordagem mais complexa que pode contribuir na caracterização etiológica das FOTs. Assim, este estudo teve como objetivo investigar os fatores genéticos envolvidos na etiologia das FOTs em uma casuística composta por 23 indivíduos afetados, incluindo casos sindrômicos e não-sindrômicos, familiais e esporádicos. Participaram do grupo controle 20 indivíduos sem história familial de fenda em três gerações. Todos os indivíduos portadores de FOT foram previamente avaliados por um médico geneticista e por exame de cariótipo em linfócitos, sendo dois casos detectados com aberrações cromossômicas. A análise de alterações no número de cópias de segmentos de DNA (Copy Number Variation, CNVs) por hibridação genômica em arrays (aGH), baseada na comparação com grupo controle, apontou um novo gene de candidato para FOT, o gene TCEB3, e identificou uma duplicação no gene FGFR1. Em um paciente com fenda palatal submucosa e quadro sindrômico, a utilização da técnica de aGH, em conjunto à hibridação in situ fluorescente (FISH) e Multiplex Ligationdependent Probe Amplification (MLPA), possibilitou a caracterização da aberração cromossômica detectada pelo cariótipo. Em todos os portadores de FOT também foi realizado sequenciamento direto dos genes IRF6, FOXE1, GLI2, MSX2, SKI, SATB2, SPRY1, MSX1, FGF8, FGFR1; em um paciente específico, foi avaliado o gene P63. Alterações de sequencia inéditas foram encontradas nos genes FOXE1, MSX1, GLI2 e FGF8, assim como uma inserção no gene P63, cujos efeitos na proteína codificada deverão ser confirmados em estudos futuros. Os resultados deste trabalho exemplificam a diversidade de fatores genéticos envolvidos na etiologia das FOTs e o desenho de estudo utilizado mostrou a eficácia do uso concomitante de diferentes abordagens investigativas neste grupo de defeitos congênitos / Abstract: Typical oral cleft (TOC) is a prevalent and heterogeneous group of congenital defects with multiple etiologies, which remain unknown in several cases. Population studies detected several genes related to TOC. An individualized investigation, involving different laboratorial tools at same time, is an approach that can contribute on the etiological characterization of the TOC. The aim of this study was to investigate genetic factors involved on TOC in a sample composed by 23 individuals (syndromic and non-syndromic; familial and sporadic cases). The control group included 20 individuals without TOC in three generations. All patients were previously evaluated by clinical geneticists and performed karyotype test that showed chromosomal aberrations in two cases. Copy number variation (CNV) investigation by genomic hibridization in arrays (aGH), based in a comparative to control group data, detected a new candidate gene to TOC (TCEB3), and identified a duplication affecting FGFR1 gene. In one patient with syndromic form of submucous cleft palate, the use of aGH technique, together with Fluorescent in situ hibridization (FISH) and Multiplex ligation-dependent probe amplification (MLPA), characterized the chromosomal aberration previously detected by karyotype. Direct sequencing of IRF6, FOXE1, GLI2, MSX2, SKI, SATB2, SPRY1, MSX1, FGF8 and FGFR1 genes was performed in all individuals; in a specific case, P63 gene was investigated. New sequence alterations were found in FOXE1, MSX1, GLI2 e FGF8 genes, as well as an insertion in P63 gene, which effects will be verified in futures studies. In conclusion, results here described reflect the diversity of genetic factors involved in the etiology of TOC and the type of study show the efficiency of the use of different techniques in the etiological investigation of this congenital defect / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Orientador: Joyce Maria Annichino Bizzacchi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:55:26Z (GMT). No. of bitstreams: 1 Bassora_FernandaDutraSantiago_D.pdf: 2667928 bytes, checksum: 612538a2c5c4e4e33577d2303de3a9c1 (MD5) Previous issue date: 2012 / Resumo: A trombose venosa é definida como a oclusão de um vaso do sistema venoso. Três fatores básicos para a formação de um trombo no interior dos vasos são: alteração do fluxo sangüíneo, da parede vascular e/ou dos elementos sangüíneos. A trombose venosa e a embolia pulmonar, que ocorre como uma complicação subseqüente representa uma causa importante de morbidade e mortalidade em pacientes hospitalizados. A freqüência da trombose venosa foi estimada em aproximadamente 1/1000 na população em geral. Na maior parte dos casos existe uma tendência para trombose determinada pela presença de um fator causal herdado ou adquirida, ou pela interação desses fatores, bem como por variações genéticas que determinam alterações nos níveis das proteínas pró-coagulantes e anticoagulantes. Dados da literatura têm sugerido a associação de mecanismos inflamatórios com a fisiopatologia da trombose venosa profunda (TVP). Os monócitos, estimulados por citocinas ou endotoxinas, expressam fator tecidual, o maior indutor da coagulação sangüínea, e que também tem a função de sinalização para a mobilidade celular e vascular. Os leucócitos apresentam receptores capazes de ligar e ativar o fator X da coagulação, servindo como via alternativa para a formação de trombina. As plaquetas podem aderir ao endotélio intacto e através da liberação de mediadores e citocinas como interleucina (IL)-1 e Fator de necrose tumoral-a (TNF-a), induzindo a expressão de moléculas de adesão e fator tecidual pela célula endotelial. Com base nestes dados e considerando que a migração leucocitária é um dos principais eventos que caracterizam o processo inflamatório, justificamos a escolha de células mononucleares (monócitos e linfócitos) como células centrais do nosso estudo. Utilizando técnicas de separação de células mononucleares por centrifugação em gradiente de "Ficoll-Hypaque", extração do Ácido ribonucléico (RNA) total, hibridação em Ácido desoxiribonucléico complementar (cDNA) -Microarray, e validação usando a reação em cadeia da polimerase em tempo real quantitativo (qRT-PCR) avaliamos do perfil de expressão gênica de alguns mediadores inflamatórios nessas células e a possível relação com a trombose venosa. Neste trabalho, usando a tecnologia de Microarray encontramos 60 induzidos e 56 genes reprimidos diferencialmente expressos nos pacientes com TVP, estes genes que estavam relacionados à resposta imune, inflamação, proteólise e transcrição. Destes genes diferencialmente expressos, selecionamos nove relacionados com inflamação para validação usando a técnica de qRT-PCR. Destes, somente houve aumento de expressão do gene da caspase 4 (CASP4) nos pacientes com TVP, sendo que, esta diferença se manteve no subgrupo com TVP espontâneo. Neste mesmo subgrupo, também foi verificado o aumento da expressão no gene Elong factor 1, alpha 2(EEF1A2) / Abstract: Venous thrombosis is defined as a vessel occlusion of the venous system. Three basic factors for the formation of a thrombus inside the vessel are: changes in blood flow, vascular wall and / or blood elements. Venous thrombosis and pulmonary embolism, which occurs as a subsequent complication is a major cause of morbidity and mortality in hospitalized patients. The frequency of venous thrombosis was estimated to be approximately 1 / 1000 in the general population. In most cases there is a tendency for thrombosis determined by the presence of a causal factor inherited or acquired, or by the interaction of these factors, as well as genetic variations that determine changes in protein levels of procoagulants and anticoagulants. Literature data have suggested the association of inflammatory mechanisms in the pathophysiology of deep venous thrombosis (DVT). Monocytes, stimulated by cytokines or endotoxin, express tissue factor, the greater inducer of blood coagulation, and also have the function of signaling for cell motility and vascular. Leukocytes have receptors that can bind and activate coagulation factor X, serving as an alternative route for the formation of thrombin. Platelets can adhere to intact endothelium and through the release of mediators and cytokines such as interleukin (IL)-1 and Tumor necrosis factor-a (TNF-a), inducing expression of adhesion molecules and tissue factor by endothelial cells. Based on these data and considering that leukocyte migration is one of the main events that characterize the inflammatory process, we justify the choice of mononuclear cells (monocytes and lymphocytes) as the central cells of our study. Using techniques of separation of mononuclear cells by gradient centrifugation "Ficoll-Hypaque," extraction of total RNA, cDNA-Microarray hybridization, and validation using real time qPCR assessed the gene expression profile of some inflammatory mediators in these cells and the possible relationship with venous thrombosis. In this work using Microarray technology we found 60 genes upregulated and 56 downrelated differentially expressed in patients with DVT. Genes that were related to immune response, inflammation, proteolysis and transcription. Of these differentially expressed genes, we selected nine genes that were related with inflammation to validation using qRT- PCR technique. Just one of then, the caspase 4 (CASP4) genes was differentially increased in DVT patients, this increase kept in patients with spontaneous DVT and an increased of Elong factor 1, alpha 2 (EEF1A2) gene too / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The loss of genomic stability associated with genetic deterioration is one of the many important aspects for the development of carcinomas. The genome maintenance mechanisms (GMM) ensure the integrity of DNA and cell survival. They are composed of genetic pathways that include the cell cycle checkpoints, DNA repair and recombination, programmed cell death (apoptosis) and senescence. The purpose of the work is to investigate the activity of these pathways in genome maintenance in diseases related to the evolution of cancer. For this public data from microarrays obtained from the Gene Expression Omnibus (GEO) database were used. We analized the expression of proteins and genome maintenance pathways that are altered in pre-cancerous and cancerous tissues related to genomic instability and may lead to tumor progression. The results allowed a quantitative characterization of an anti-cancer barrier in the tumor evolution proposed in the literature. / A perda da estabilidade genômica associada com a deterioração genética é um dos muitos aspectos importantes na evolução dos carcinomas. São os mecanismos de manutenção do genoma (GMM) que garantem a integridade do DNA e a sobrevivência da célula. Eles são compostos por vias genéticas que incluem os pontos de controle do ciclo celular, o reparo e a recombinação do DNA, a morte celular programada (apoptose) e a senescência. O objetivo deste trabalho é investigar a atividade dessas vias e genes de manutenção do genoma em doenças relacionadas à evolução do câncer. Para isso foram usados dados públicos de microarranjos obtidos do banco de dados Gene Expression Omnibus (GEO). Analisamos a expressão das proteínas e vias de manutenção do genoma que estão alteradas em tecidos pré-cancerosos, cancerosos e tecidos relacionados com a instabilidade genômica e que poderão levar a uma progressão tumoral. Os resultados permitiram caracterizar quantitativamente uma barreira anticâncer na evolução tumoral proposta na literatura.

Abstract Cardiac hypertrophy provides an adaptive mechanism to maintain cardiac output in response to increased workload, and although initially beneficial, hypertrophy eventually leads to heart failure, a major cause of morbidity and mortality in Western countries. The hypertrophic response in cardiac myocytes is accompanied by e.g. activation of signal transduction pathways, such as mitogen-activated protein kinases (MAPKs), and complex changes in gene programming. The purpose of this study was to characterize gene expression patterns in experimental models of cardiac load by using high-throughput DNA microarray technologies. In the present study, changes in gene expression were evaluated in response to acute pressure overload and prolonged hypertension as well as during the development of left ventricular hypertrophy (LVH) and the transition to diastolic heart failure in an animal model of genetic hypertension, the spontaneously hypertensive rat (SHR). Increased expression of several immediate early genes was seen in response to acute hemodynamic overload in vivo. The transition from LVH to diastolic hypertensive heart failure was almost exclusively associated with changes in genes encoding extracellular matrix proteins and their regulatory processes showing the importance of progressive extracellular matrix remodeling. The effect of p38 MAPK activation on gene expression patterns in vivo was elucidated. Cardiac-specific overexpression of p38 MAPK resulted in upregulation of genes controlling cell division and inflammation as well as cell signaling and adhesion. Accordingly, the functional role of p38 MAPK was related to myocardial cell proliferation, inflammation and fibrosis. Finally, temporal analysis of mechanical stretch induced gene expression changes in neonatal rat cardiomyocyte cultures in vitro indicated that mechanical stretch induced complex gene expression profiles, demonstrating that both positive and negative regulators are involved in the hypertrophic process. Many novel stretch responsive genes were identified, and a subset of them may be putative downstream targets of p38 MAPK. In conclusion, in the present study a number of well-established gene expression changes of cardiac hypertrophy were observed and novel modulators associated with increased cardiac load, such as thrombospondin-4, were identified. The study provides a better understanding of molecular mechanisms associated with increased cardiac load, and may indicate potential targets for novel therapeutic interventions.

Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities.   The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods.   In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing  samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing.   In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value. / <p>QC 20170905</p>

With the influx of high-dimensional data there is an immediate need for statistical methods that are able to handle situations when the number of predictors greatly exceeds the number of samples. One such area of growth is in examining how environmental exposures to toxins impact the body long term. The cytokinesis-block micronucleus assay can measure the genotoxic effect of exposure as a count outcome. To investigate potential biomarkers, high-throughput assays that assess gene expression and methylation have been developed. It is of interest to identify biomarkers or molecular features that are associated with elevated micronuclei (MN) or nuclear bud (Nbud) frequency, measures of exposure to environmental toxins. Given our desire to model a count outcome (MN and Nbud frequency) using high-throughput genomic features as predictors, novel methods that can handle over-parameterized models need development. Overdispersion, when the variance of a count outcome is larger than its mean, is frequently observed with count response data. For situations where overdispersion is present, the negative binomial distribution is more appropriate. Furthermore, we expand the method to the longitudinal Poisson and longitudinal negative binomial settings for modeling a longitudinal or clustered outcome both when there is equidispersion and overdispersion. The method we have chosen to expand is the Generalized Monotone Incremental Forward Stagewise (GMIFS) method. We extend the GMIFS to the negative binomial distribution so it may be used to analyze a count outcome when both a high-dimensional predictor space and overdispersion are present. Our methods were compared to glmpath. We also extend the GMIFS to the longitudinal Poisson and longitudinal negative binomial distribution for analyzing a longitudinal outcome. Our methods were compared to glmmLasso and GLMMLasso. The developed methods were used to analyze two datasets, one from the Norwegian Mother and Child Cohort study and one from the breast cancer epigenomic study conducted by researchers at Virginia Commonwealth University. In both studies a count outcome measured exposure to potential genotoxins and either gene expression or high-throughput methylation data formed a high dimensional predictor space. Further, the breast cancer study was longitudinal such that outcomes and high-dimensional genomic features were collected at multiple time points during the study for each patient. Our goal is to identify biomarkers that are associated with elevated MN or NBud frequency. From the development of these methods, we hope to make available more comprehensive statistical models for analyzing count outcomes with high dimensional predictor spaces and either cross-sectional or longitudinal study designs.