A ocitocina (OT) é um neuropeptídio hipotalâmico, que dentre suas funções na fêmea destaca-se a contração uterina durante o parto e a ejeção do leite. No entanto, estudos vêm demonstrando importantes funções endócrinas e parácrinas no trato reprodutivo masculino. Evidenciando a possível ação conjunta entre OT e a Globulina ligadora de hormônios sexuais (SHBG). Entretanto, em cães não existem informações disponíveis quanto sua atuação. Assim, estudos direcionados aos receptores de ocitocina (OTR) e SHBG e suas funções no sistema reprodutor masculino, mais especificamente na fisiologia espermática, são de suma importância para os conhecimentos da fisiologia reprodutiva para posterior aplicação em biotecnologias reprodutivas em pequenos animais e humanos, fomentando também novas perspectivas para a utilização terapêutica da ocitocina em enfermidades reprodutivas. Portanto, o objetivo deste estudo é verificar a expressão gênica e proteica do OTR e SHBG no testículo e epidídimo de cães, correlacionando tais dados com a qualidade espermática e dosagem de testosterona. Para tal, foram coletados testículos e epidídimos de 26 cães em idade reprodutiva (1 a 5 anos). Após a orquiectomia, foi realizada a coleta dos espermatozoides provenientes da cauda do epidídimo e então, as amostras foram analisadas quanto à motilidade computadorizada do sêmen (CASA), integridade de membrana plasmática (Eosina/Nigrosina), integridade de membrana acrossomal (Fast Green / Rosa Bengala) e atividade mitocondrial (3´3 Diaminobenzidine). A imunolocalização do OTR e SHBG foi realizada através de imunoistoquímica e imunofluorescência. E a análise de expressão gênica, através da Reação em cadeia da polimerase em tempo real (qRT PCR). E da expressão proteica, através do Western Blotting. Foram encontradas correlações significantes e positivas entre as expressões gênicas do OTR e do SHBG, tanto no testículo como no epidídimo. Além disto, a expressão do OTR no testículo correlacionou-se positivamente com espermatozoides com membrana acrossomal íntegra e negativamente com a porcentagem de células com baixa atividade mitocondrial. Já o SHBG do testículo, correlacionou-se positivamente com a concentração de espermatozoides, porcentagens de células com membrana plasmática e acrossomal íntegras, motilidade, motilidade progressiva e velocidade rápida, e negativamente com a porcentagem de células com baixa atividade mitocondrial. Por outro lado, no epidídimo, a expressão gênica do SHBG apresentou correlação positiva com a porcentagem de células com membrana plasmática íntegra e expressão proteica de SHBG no testículo. Quanto a expressão proteica, o OTR no testículo obteve correlação positiva com testosterona e negativa com atividade mitocondrial nula, já no epidídimo, ocorreu correlação positiva com integridade de membrana acrossomal e negativa também com atividade mitocondrial nula. Em relação ao SHBG, houve correlação positiva com a expressão gênica do SHBG no epidídimo, células normais e padrões de velocidade. E na imunoistoquímica foi possível observar a imunomarcação do OTR e SHBG na musculatura lisa e células de Leydig do testículo e OTR na musculatura lisa do epidídimo. No entanto, não houve imunomarcação do SHBG no epidídimo, assim como expressão proteica. Nossos resultados demonstraram que o OTR e SHBG são expressos nos testículos e epidídimos de cães e que estão relacionados a funções espermáticas importantes, sendo essenciais para o sucesso reprodutivo / Oxytocin (OT) is a hypothalamic neuropeptide that plays important and well known roles in the female such as uterine contraction during childbirth and milk ejection. Notwithstanding, studies have shown important endocrine and paracrine functions also in the male reproductive tract, highlighted by the possible joint action between OT and sex hormone-binding globulin (SHBG). In dogs, however, there is no information available with regards to the role of these hormones in the reproductive function. Thus, studies directed to oxytocin (OTR) and SHBG receptors and their functions in the male reproductive system, specifically with regards to sperm physiology. Such knowledge is essential to understand the reproductive physiology for the subsequent use in reproductive biotechnologies in small animals and humans, especially by providing new perspectives for the therapeutic use of oxytocin in reproductive disorders. Therefore, the aim of this study is to assess the gene and protein expression of OTR and SHBG in the testis and epididymis of dogs, correlating these data with sperm quality and testosterone dosage. To this end, testis and epididymis were collected from 26 dogs in reproductive age (1 to 5 years). After orchiectomy, collection of sperm from the cauda epididymis was carried out and then the samples were analyzed for computerized motility of semen (CASA), plasma membrane integrity (eosin / nigrosine), acrosome membrane integrity (Fast Green / rose Bengal) and mitochondrial activity (3\'3 Diaminobenzidine). The immunolocalization of OTR and SHBG was performed by immunohistochemistry and immunofluorescence. Gene expression analysis was performed by real time polymerase chain reaction (qRT - PCR). The protein expression was further assessed by Western Blotting. Significant positive correlations were found between the gene expressions of OTR and SHBG in both the testis and epididymis. Furthermore, the OTR expression in testis was positively correlated to sperm with intact acrosome membrane and negatively to the percentage of cells with low mitochondrial activity. On the other hand, testicular SHBG was positively correlated with sperm concentration, percentage of sperm with intact plasma membrane and acrosome, motility, progressive motility and the percentage of RAPID sperm. Also, negative correlation was found between testicular SHBG and the percentage of cells with low mitochondrial activity. Furthermore, in the epididymis, SHBG gene expression was positively correlated to the percentage of cells with intact plasma membrane and protein expression of SHBG in the testis. In relation to the protein expression, the OTR in the testis correlated positively with blood plasma testosterone and negatively with sperm with no mitochondrial activity. In the epididymis, OTR protein expression correlated positively with sperm showing intact acrosome and negatively with cells with no mitochondrial activity. With regards to SHBG proteins expression, there was a positive correlation to SHBG gene expression in the epididymis, normal cells and some patterns of sperm velocity. In the immunohistochemistry, we observed the OTR and SHBG immunostainings in the smooth muscle and Leydig cells of the testis while, in the epididymis, the OTR immunostaining could be observed only in the smooth muscle. Interestingly, there was no immunostaining or protein expression of SHBG in the epididymis. Our results demonstrated that OTR and SHBG are expressed in the testis and epididymis of dogs and are related to important sperm functions, essential for reproductive success

This diploma thesis deals with an experimental analysis of lubrication of total hip joint replacement using fluorescence microscopy, focusing on so-called soft bearing pairs. Experiments were realized using a hip joint simulator, which was modified and refined for fluorescence microscopy. The aim of this diploma thesis was to determine the effect of the individual components of the synovial fluid on the lubrication mechanisms. For this purpose, different model fluids were designed to represent the composition of proteins and other synovial fluid constituents to elucidate the effect of albumin, -globulin and hyaluronic acid on the resulting lubrication performance. The experiments were separate into two categories different in type of load, dynamic (representative walking) and combined (representing standing with short walk). The experimental results were time-dependent fluorescence intensity values, representing dimensionless film thickness parameters. The values were recorded on the graphs and supplemented with fluorescence images taken during the characteristic sections of the measurements. The measured data was subsequently confronted with previously published articles. It was found that the dominant constituent responsible form film thickness is albumin, while -globulin and hyaluronic acid forms thin stable layer enhancing adsorption ability of albumin which contributes to increase of film thickness eventually.

This diploma thesis deals with an experimental analysis of knee joint replacement lubrication. The experiments were realized at a knee joint simulator which can apply conditions according to certain standard and survey the phenomena by using fluorescence microscopy. The aim of thesis is to clarify the influence of particular components of synovial fluid on the lubrication process. The intensity of fluorescence expresses dimensionless parameter of a lubrication film thickness. There was a fundamental study with mineral oils before the experiments with the synovial fluid. The study allows to have a look at contact transformation during walk. Results are shown in graphs as dependency of intensity on time, including pictures showing phenomena in the contact zone. Experiment results show that protein -globulin creates a layer on the surface. There is albumin on the layer and it makes the lubricating film thicker. The protein interaction is supported by hyaluronic acid and fosfolipids which stabilizes the created structure. According to lubrication is behaviour of film related to a complex structure of synovial fluid. Thesis gives more information about behaviour of synovial fluid and can be used for future development of knee replacements.

This diploma thesis deals with an experimental analysis of lubricant film formation in hip joint replacements. The main objective is to clarify the effect of mean speed, slide-to-roll ratio, and material of femoral head on the development of film thickness, focusing on the role of particular constituents contained in model synovial fluid, such as albumin and -globulin. For this purpose, a model ball-on-disc configuration was applied, while the development of lubricant film was evaluated using the combination of fluorescent microscopy and optical interferometry. To better understand the process, coefficient of friction between implant surfaces was later investigated as well. The effect of material, slide-to-roll ratio, mean speed and model fluid composition was analysed. Results indicate that the main parameter, influencing the character of film formation, is slide-to-roll ratio. Under most conditions, the dominant constituent responsible for the film thickness development was albumin. Coefficient of friction is affected mainly by the material of implant. The thesis contains original scientific results extending the knowledge in the area of hip joint biotribology.

Dizertační práce se zabývá mechanismy mazání v náhradách kyčelního kloubu. Byla provedena systematická studie formování proteinového filmu při zahrnutí různých materiálů a provozních podmínek. Hlavní pozornost je přitom věnována roli jednotlivých proteinů obsažených v synoviální kapalině při současné přítomnosti dalších proteinů. Jelikož metody aplikované v předchozích studiích neumožňovaly separovat jednotlivé složky maziva, byla vyvinuta optická měřící metoda na principu fluorescenční mikroskopie. Z důvodu verifikace metody byly provedeny dvě nezávislé studie zaměřené na měření tloušťky mazacího filmu a dělení maziva na výstupu mazaného kontaktu. Z důvodu určitých limitací fluorescenční mikroskopie byla dále využita i metoda optické interferometrie, jejíž využití je ilustrováno ve studii zabývající se formováním mazacího filmu v náhradách kyčelního klubu při uvažování reálné konformity třecích povrchů. Závěrečná část práce představuje nový metodologický přístup založený na in situ pozorování kontaktní oblasti umožňující popsat roli jednotlivých proteinů ve vztahu k vývoji tloušťky mazacího filmu. Práce obsahuje originální výsledky, které přináší nové poznání v oblasti biotribologie náhrad kyčelního kloubu vedoucí k dalšímu vývoji implantátů při snaze zabránit jejich selhání v důsledku omezené životnosti.

INTRODUÇÃO: Manifestações auto-imunes são frequentes na infecção pelo vírus da hepatite C (VHC). Apesar da associação com doenças auto-imunes de tireóide (DAIT) ser controversa, sabe-se que distúrbios tireoidianos podem surgir ou piorar com tratamento com IFN e ribavirina. Os objetivos deste estudo foram avaliar a função tireoidiana em pacientes infectados pelo VHC, caracterizar distúrbios tireoidianos antes, durante e após tratamento com IFN e estudar as frequências dos genótipos dos polimorfismos do gene CTLA4, correlacionando-os com características clínicas e laboratoriais, presença de disfunção tireoidiana e evolução durante tratamento com IFN. MÉTODOS: Avaliação prospectiva de 112 indivíduos com infecção crônica pelo VHC, 30 tratados com IFN, e 183 controles. Realizaram-se avaliações clínica, hormonal e de auto-imunidade tireoidiana e ultra-sonografia de tireóide no início e durante tratamento. Avaliações de globulina transportadora de hormônios tireoidianos (TBG), de CXCL10 e de biópsia hepática foram feitas pré-tratamento. Análises dos polimorfismos do gene CTLA4 -318C>T, A49G e CT60 foram realizadas por PCR-RFLP e de AT(n) por análise de fragmento através de eletroforese capilar. RESULTADOS: A frequência de DAIT entre infectados por VHC não diferiu dos controles (10,7 vs 13,5%, p=0,585). Os limites de distribuição dos níveis de T3 (T3T) e T4 (T4T) totais foram superiores aos de referência (T3T 112-246 ng/dL; T4T 7,8-15,2 g/dL), assim como de TBG (17-47 mg/L). TBG correlacionou-se com T3T (r=0,654, p<0,001) e T4T (r=0,741, p<0,001). Heterogeneidade (p=0,027) e hipoecogenicidade de parênquima (p=0,002) foram mais frequentes nos pacientes com DAIT. Aumento de vascularização esteve presente em 49,2% dos infectados sem distúrbio tireoidiano. CXCL10 esteve aumentada nos infectados (p=0,006), mas não se relacionou com disfunção tireoidiana. Sua elevação correspondeu ao grau de atividade necro-inflamatória na biópsia hepática (p=0,006) e correlacionou-se com T3T (r=0,388, p=0,003), T4T (r=0,444, p=0,001) e TBG (r=0,551, p<0,001). Dezenove por cento dos pacientes desenvolveram tireoidites auto-imunes por IFN e 16% não auto-imunes. Em 14 pacientes sem alteração tireoidiana durante o uso de IFN, T3T diminuiu ao longo de 12 meses (p=0,038) concomitante à queda de ALT (p=0,055). T4T diminuiu com 3 (p=0,039) e 12 meses (p=0,008), T4 livre e TSH permaneceram estáveis. Encontrou-se maior frequência de oito repetições AT na região 3UTR do gene CTLA4 nos infectados por VHC (p=0,019). O alelo C do polimorfismo -318C>T esteve relacionado com infecção pelos genótipos 1 (p=0,020, OR 0,19) e 3 (p=0,008, OR 9,13), assim como o alelo G do polimorfismo A49G (p=0,002, OR 0,38 e p=0,004, OR 2,49, respectivamente). Não se identificou relação dos polimorfismos do gene CTLA4 com distúrbios tireoidianos, antes ou após tratamento com IFN. CONCLUSÕES: Não foi encontrada associação entre infecção por VHC e doenças tireoidianas. Indivíduos infectados por VHC têm maiores níveis de T3T e T4T, correlacionados com TBG. Aumento de CXCL10 não se associou com disfunção tireoidiana, mas se correlacionou com TBG, T3T e T4T. IFN provocou tireoidites auto-imunes e não auto-imunes, além de reduzir T3T e T4T coincidente com melhora de lesão hepática. Não se encontrou relação dos polimorfismos do gene CTLA4 com características clínicas e laboratoriais ou presença de disfunção tireoidiana prévia ou induzida por IFN / INTRODUCTION: Autoimmune disorders are frequent in patients infected by the hepatitis C virus (HCV). Although the association with autoimmune thyroid diseases (AITD) is controversial, thyroid disturbance could occur or worsen with IFN and ribavirin treatment. The aims of the study were evaluate thyroid function in HCV-infected patients, characterize thyroid disturbance prior and after IFN treatment and analyze the frequency of the genotypes of the polymorphisms of CTLA4 gene, and their relation to clinical and laboratorial features, presence of thyroid dysfunction and disturbance along IFN treatment. METHODS: Prospective evaluation of 112 chronically HCV-infected subjects, 30 treated with IFN, and 183 controls. Clinical, hormonal, thyroid autoimmunity and ultrasound exams were performed before and during treatment. Thyroxine-binding globulin (TBG), CXCL10 and hepatic biopsies were also evaluated before treatment. Analysis of polymorphisms of CTLA4 gene -318C>T, A49G and CT60 were made by PCR-RFLP and AT(n) polymorphism analysis by capillary electrophoresis in automatic sequencer. RESULTS: The frequency of AITD among HCV-infected subjects was similar to the rate among controls (10.7 vs 13.5%, p=0.585). Total T3 (T3T) and T4 (T4T) distributions were right shifted (T3T 112-246 ng/dL; T4T 7.8-15.2 g/dL), as was TBG (17-47 mg/L). TBG correlated to both T3T (r=0.654, p<0.001) and T4T (r=0.741, p<0.001). Thyroid heterogeneity (p=0.027) and hipoechogenicity (p=0.002) were associated with AITD and, most notably, increased vascularization was present in 49.2% of HCV-infected patients without thyroid disturbance. CXCL10 was higher in HCV-infected group (p=0.006) but was not related to thyroid dysfunction. Increase in CXCL10 levels were consistent with hepatic necroinflammatory activity (p=0.006) and correlated to T3T (r=0.388, p=0.003), T4T (r=0.444, p=0.001) and TBG (r=0.551, p<0.001). Nineteen percent of subjects treated with IFN presented autoimmune thyroiditis and 16% had non-autoimmune thyroiditis. In 14 subjects without IFN-induced thyroid dysfunction, T3T decreased along 12 months of follow-up (p=0.038) concomitant to ALT decrease (p=0.055). T4T decreased within 3 (p=0.039) and 12 months (p=0.008), while both free T4 and TSH remained stable. Eight AT repetitions in 3UTR site of the CTLA4 gene were more frequent among HCV-infected subjects. The C allele of -318C>T polymorphism was associated with genotype 1 (p=0.020, OR 0.19) and 3 infections (p=0.008, OR 9.13), similar to allele G of A49G polymorphism (p=0.002, OR 0.38 and p=0.004, OR 2.49, respectively). No association of the polymorphisms of CTLA4 gene and thyroid disorders, prior or induced by IFN treatment, was found. CONCLUSIONS: No association between HCV-infection and thyroid diseases was found. HCV-infected subjects had higher T3T and T4T which were correlated to TBG. Increased CXCL10 was not associated to thyroid dysfunction, but correlated to TBG, T3T and T4T. IFN induced autoimmune and non-autoimmune thyroiditis. IFN also reduced T3T and T4T levels commensurately with liver improvement. The polymorphisms of CTLA4 gene were not associated with clinical and laboratorial features or presence of thyroid dysfunction, prior or induced by IFN

Our lab has previously reported the identification of a novel endogenous 19-nor steroid, estradienolone (ED), in pregnant women that strongly bound to sex hormone binding globulin. Estrogen-receptor related receptors (ERRs), which have no known natural ligands, are a family of orphan receptors consisting of 3 isoforms: ERRalpha, ERRbeta and ERRgamma. The ERRs have been shown to actively modulate estrogenic responses, to play an essential role in pregnancy, and are implicated in breast cancer prognosis. My results show that ED acts as an antagonist of the ERRalpha confirming preliminary results obtained by our group. Studies of cellular responses demonstrate that ED has strong anti-mitogenic properties. ED inhibited the growth of both estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells in a dose-dependent manner but did not have any effects on the proliferation of the non-cancerous immortalized epithelial breast MCF-10A cells. The finding that ED inhibits proliferation of both ER negative and ER positive breast cancer cells, and regulate ERR transcriptional activity may have important ramifications in breast cancer therapy.

Made available in DSpace on 2015-07-22T20:50:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-28 / Introdução: As doenças cardiovasculares (DCVs) representam o principal grupo de causa de morte no Brasil, com destaque para doença arterial coronariana (DAC). O sexo masculino apresenta maior incidência e mortalidade por DAC que o feminino. Uma das explicações para o fato era o possível efeito deletério da Testosterona no sistema cardiovascular masculino e o efeito protetor do Estradiol no sistema cardiovascular feminino. Contudo, evidências recentes da literatura apontam para um efeito protetor ou neutro da Testosterona no aparelho cardiovascular masculino, enquanto níveis elevados de Estradiol nos homens estiveram correlacionados com maior morbidade e mortalidade por doenças cardiovasculares. Objetivos: Realizar uma revisão da literatura da relação existente entre Testosterona sérica e doença cardiovascular em homens adultos; Avaliar a relação existente entre Testosterona Total, Testosterona Biodisponível, Testosterona Livre, Índice de Andrógenos Livres (IAL), Globulina Ligadora de Esteróides Sexuais (SHBG), Estradiol, Índice de Estrógenos Livres (IEL), relação Estradiol / Testosterona e a relação IEL / IAL e doença arterial coronariana em homens adultos; Entender o papel da Globulina Ligadora de Esteróides Sexuais como novo componente as síndrome metabólica. Material e Métodos: A revisão da relação entre testosterona e doença cardiovascular foi realizada através da base de dados do PubMed com a utilização dos unitermos testosterona e doença cardiovascular; a avaliação da relação existente entre esteróides sexuais e DAC foi realizada através de um estudo de caso controle com homens adultos submetidos ao Cateterismo de Artérias Coronárias no Instituto Dante Pazzanese de Cardiologia; o entendimento do papel da SHBG como novo componente as síndrome metabólica através da análise dos dados obtidos do estudo “Estradiol but not Testosterone is Related to Coronary Artery Disease”. Resultados: Os resultados foram dispostos em 03 artigos, a saber: ARTIGO 1 “Testosterona Sérica e Doença Cardiovascular em Homens”; ARTIGO 2 “Estradiol but not Testosterone is Related to Coronary Artery Disease in Men”; ARTIGO 3 (preparando para a submissão) “Sex hormone binding globulin a novel component of metabolic syndrome”. Conclusões: Os estudos selecionados da literatura que avaliaram a relação entre testosterona e doença cardiovascular apresentavam pequeno número de participantes e amostras selecionadas, ornando necessário que novos estudos avaliem o papel da testosterona na DCV nos homens. Os achados apresentados sinalizam para uma correlação positiva entre níveis séricos de Estradiol e IEL com DAC. Foram observados efeitos neutros da testosterona total, testosterona biodisponível, testosterona livre, índice de andrógenos livres SHBG, relação Estradiol / Testosterona e relação IEL / IAL na incidência dessa patologia. Baixos níveis de SHBG parecem se correlacionar positivamente com os componentes da síndrome metabólica, sendo necessários novos estudos que avaliem esse parâmetro como novo componente desta Síndrome. / Introduction: Cardiovascular diseases (CVDs) represent the main cause of death in Brazil, and among them especially the coronary artery diseases (CADs). Men present higher incidence and mortality rates for CAD than women. One of the explanations for this fact may be the possibly deleterious effect of testosterone on the male cardiovascular system and the protective effect of estradiol on the female cardiovascular system. However, recent studies in the literature indicate that testosterone has an either protective or neutral effect on the male cardiovascular system, while high levels of estradiol in men have been correlated to higher rates of morbidity and mortality from cardiovascular diseases. Objectives: To carry out a review of the literature regarding the relationship between testosterone and cardiovascular disease in men, to evaluate the existing relationships among total testosterone, bioavailable testosterone, free testosterone, free androgen index (FAI), sex hormone binding globulin, estradiol, free estrogen index (FEI), estradiol/testosterone ratio and FEI/FAI ratio and coronary artery disease in men; to understand the role of the sex hormone binding globulin as a new component of the metabolic syndrome. Material and Methods: The review of the literature regarding the relationship between testosterone and cardiovascular disease was performed using the PubMed database and the keywords testosterone and cardiovascular disease. The relationship between sex steroids and CAD was evaluated by a case-control study performed on men submitted to coronary angiography at the Instituto Dante Pazzanese de Cardiologia. The role of the sex hormone binding globulin (SHBG) as a new component of the metabolic syndrome was evaluated using the data obtained by the study “Estradiol but not Testosterone is Related to Coronary Artery Disease”. Results: The results were presented in three articles, namely: ARTICLE 1 - “Serum Testosterone and Cardiovascular Disease in Men”; ARTICLE 2 - “Estradiol but not Testosterone is Related to Coronary Artery Disease in Men”; ARTICLE 3 - (being prepared for submission) - “Sex hormone binding globulin, the novel component of metabolic syndrome?”. Conclusions: The studies retrieved from the literature which evaluated the relationship between testosterone and cardiovascular disease presented small numbers of participants and selected samples, which indicated the need for further studies to evaluate the role of testosterone in CVD in men. The findings presented suggest a positive correlation between estradiol and FEI levels with CAD. A neutral effect of total testosterone, bioavailable testosterone, free testosterone, free androgen index, SHBG, estradiol/testosterone ratio and FEI/FAI ratio on the incidence of this pathology was observed. Low levels of SHBG seem to correlate positively with the components of the metabolic syndrome, but further studies are necessary to evaluate this parameter as a new component of this syndrome. / TEDE / BV UNIFESP: Teses e dissertações

INTRODUÇÃO: Manifestações auto-imunes são frequentes na infecção pelo vírus da hepatite C (VHC). Apesar da associação com doenças auto-imunes de tireóide (DAIT) ser controversa, sabe-se que distúrbios tireoidianos podem surgir ou piorar com tratamento com IFN e ribavirina. Os objetivos deste estudo foram avaliar a função tireoidiana em pacientes infectados pelo VHC, caracterizar distúrbios tireoidianos antes, durante e após tratamento com IFN e estudar as frequências dos genótipos dos polimorfismos do gene CTLA4, correlacionando-os com características clínicas e laboratoriais, presença de disfunção tireoidiana e evolução durante tratamento com IFN. MÉTODOS: Avaliação prospectiva de 112 indivíduos com infecção crônica pelo VHC, 30 tratados com IFN, e 183 controles. Realizaram-se avaliações clínica, hormonal e de auto-imunidade tireoidiana e ultra-sonografia de tireóide no início e durante tratamento. Avaliações de globulina transportadora de hormônios tireoidianos (TBG), de CXCL10 e de biópsia hepática foram feitas pré-tratamento. Análises dos polimorfismos do gene CTLA4 -318C>T, A49G e CT60 foram realizadas por PCR-RFLP e de AT(n) por análise de fragmento através de eletroforese capilar. RESULTADOS: A frequência de DAIT entre infectados por VHC não diferiu dos controles (10,7 vs 13,5%, p=0,585). Os limites de distribuição dos níveis de T3 (T3T) e T4 (T4T) totais foram superiores aos de referência (T3T 112-246 ng/dL; T4T 7,8-15,2 g/dL), assim como de TBG (17-47 mg/L). TBG correlacionou-se com T3T (r=0,654, p<0,001) e T4T (r=0,741, p<0,001). Heterogeneidade (p=0,027) e hipoecogenicidade de parênquima (p=0,002) foram mais frequentes nos pacientes com DAIT. Aumento de vascularização esteve presente em 49,2% dos infectados sem distúrbio tireoidiano. CXCL10 esteve aumentada nos infectados (p=0,006), mas não se relacionou com disfunção tireoidiana. Sua elevação correspondeu ao grau de atividade necro-inflamatória na biópsia hepática (p=0,006) e correlacionou-se com T3T (r=0,388, p=0,003), T4T (r=0,444, p=0,001) e TBG (r=0,551, p<0,001). Dezenove por cento dos pacientes desenvolveram tireoidites auto-imunes por IFN e 16% não auto-imunes. Em 14 pacientes sem alteração tireoidiana durante o uso de IFN, T3T diminuiu ao longo de 12 meses (p=0,038) concomitante à queda de ALT (p=0,055). T4T diminuiu com 3 (p=0,039) e 12 meses (p=0,008), T4 livre e TSH permaneceram estáveis. Encontrou-se maior frequência de oito repetições AT na região 3UTR do gene CTLA4 nos infectados por VHC (p=0,019). O alelo C do polimorfismo -318C>T esteve relacionado com infecção pelos genótipos 1 (p=0,020, OR 0,19) e 3 (p=0,008, OR 9,13), assim como o alelo G do polimorfismo A49G (p=0,002, OR 0,38 e p=0,004, OR 2,49, respectivamente). Não se identificou relação dos polimorfismos do gene CTLA4 com distúrbios tireoidianos, antes ou após tratamento com IFN. CONCLUSÕES: Não foi encontrada associação entre infecção por VHC e doenças tireoidianas. Indivíduos infectados por VHC têm maiores níveis de T3T e T4T, correlacionados com TBG. Aumento de CXCL10 não se associou com disfunção tireoidiana, mas se correlacionou com TBG, T3T e T4T. IFN provocou tireoidites auto-imunes e não auto-imunes, além de reduzir T3T e T4T coincidente com melhora de lesão hepática. Não se encontrou relação dos polimorfismos do gene CTLA4 com características clínicas e laboratoriais ou presença de disfunção tireoidiana prévia ou induzida por IFN / INTRODUCTION: Autoimmune disorders are frequent in patients infected by the hepatitis C virus (HCV). Although the association with autoimmune thyroid diseases (AITD) is controversial, thyroid disturbance could occur or worsen with IFN and ribavirin treatment. The aims of the study were evaluate thyroid function in HCV-infected patients, characterize thyroid disturbance prior and after IFN treatment and analyze the frequency of the genotypes of the polymorphisms of CTLA4 gene, and their relation to clinical and laboratorial features, presence of thyroid dysfunction and disturbance along IFN treatment. METHODS: Prospective evaluation of 112 chronically HCV-infected subjects, 30 treated with IFN, and 183 controls. Clinical, hormonal, thyroid autoimmunity and ultrasound exams were performed before and during treatment. Thyroxine-binding globulin (TBG), CXCL10 and hepatic biopsies were also evaluated before treatment. Analysis of polymorphisms of CTLA4 gene -318C>T, A49G and CT60 were made by PCR-RFLP and AT(n) polymorphism analysis by capillary electrophoresis in automatic sequencer. RESULTS: The frequency of AITD among HCV-infected subjects was similar to the rate among controls (10.7 vs 13.5%, p=0.585). Total T3 (T3T) and T4 (T4T) distributions were right shifted (T3T 112-246 ng/dL; T4T 7.8-15.2 g/dL), as was TBG (17-47 mg/L). TBG correlated to both T3T (r=0.654, p<0.001) and T4T (r=0.741, p<0.001). Thyroid heterogeneity (p=0.027) and hipoechogenicity (p=0.002) were associated with AITD and, most notably, increased vascularization was present in 49.2% of HCV-infected patients without thyroid disturbance. CXCL10 was higher in HCV-infected group (p=0.006) but was not related to thyroid dysfunction. Increase in CXCL10 levels were consistent with hepatic necroinflammatory activity (p=0.006) and correlated to T3T (r=0.388, p=0.003), T4T (r=0.444, p=0.001) and TBG (r=0.551, p<0.001). Nineteen percent of subjects treated with IFN presented autoimmune thyroiditis and 16% had non-autoimmune thyroiditis. In 14 subjects without IFN-induced thyroid dysfunction, T3T decreased along 12 months of follow-up (p=0.038) concomitant to ALT decrease (p=0.055). T4T decreased within 3 (p=0.039) and 12 months (p=0.008), while both free T4 and TSH remained stable. Eight AT repetitions in 3UTR site of the CTLA4 gene were more frequent among HCV-infected subjects. The C allele of -318C>T polymorphism was associated with genotype 1 (p=0.020, OR 0.19) and 3 infections (p=0.008, OR 9.13), similar to allele G of A49G polymorphism (p=0.002, OR 0.38 and p=0.004, OR 2.49, respectively). No association of the polymorphisms of CTLA4 gene and thyroid disorders, prior or induced by IFN treatment, was found. CONCLUSIONS: No association between HCV-infection and thyroid diseases was found. HCV-infected subjects had higher T3T and T4T which were correlated to TBG. Increased CXCL10 was not associated to thyroid dysfunction, but correlated to TBG, T3T and T4T. IFN induced autoimmune and non-autoimmune thyroiditis. IFN also reduced T3T and T4T levels commensurately with liver improvement. The polymorphisms of CTLA4 gene were not associated with clinical and laboratorial features or presence of thyroid dysfunction, prior or induced by IFN

Made available in DSpace on 2014-07-29T15:16:28Z (GMT). No. of bitstreams: 1 Dissertacao SantosKFD.pdf: 3452223 bytes, checksum: 425b2eff60093d958263e376587dc3c7 (MD5) Previous issue date: 2010-03-31 / The objective of this dissertation was to analyze quantitative and qualitatively the total storage protein content and its fractions albumin, globulin, prolamin and glutelin from the grain of 29 genotypes of the wild rice Oryza glumaepatula and 70 interspecific lines backcross 2 obtained from the cross O. sativa x O. glumaepatula. From these lines, 34 BC2F8 were obtained from the cross O. sativa BG 90-2 x O. glumaepatula RS-16 and 36 BC2F10 were obtained from the cross O. sativa CICA-8 x O. glumaepatula RS-16. From the analysis of variance for the total protein content and its fractions, it were found highly significant differences (P<0.01) between the wild genotypes and the interspecific lines. The average of total protein content of wild genotypes was 12.17%, whereas the lines of BG 90-2 x RS-16 showed an average of 7.05% and the lines of CICA-8 x RS-16 showed an average of 8.40%. The wild genotype BGA14280 showed the highest total protein content (14.94%). In the comparative analysis of interspecific lines and their parents, it were found five lines with higher total protein content (average of 10.95%), which was significantly superior to the cultivated parent BG 90-2 (10.0%) and CICA-8 (9.61%). However, these lines showed lower content in relation to the wild parent RS-16 (14.06%). In relation to the protein fractions, 40 interspecific lines showed higher values in relation to their parents, excepting in one occasion, where the wild parent RS-16 showed higher value to the glutelin fraction. Considering the 29 wild genotypes, it were found the highest contents of albumin, prolamin and glutelin, excepting to the globulin fraction, where the wild genotype was not significantly different from the cultivar BRS Bonança. The SDS-PAGE analyses of the total protein and the glutelin fraction showed a differential profile of &#945;-glutelins for the wild genotypes, emphasizing the BGA14232 genotype, which did not showed the &#945;-polypeptides commonly identified in the remaining genotypes. Considering the interspecific lines and their parents, it was found similar profile of total protein and &#945;-globulins, with differences in relation to the 40 kDa &#945;-glutelin, which was found just in the wild parent RS-16, indicating a differential expression in O. glumaepatula, since the O. sativa showed a 39 kDa &#945;-glutelin. In relation to albumin, globulin and prolamin fractions, the wild genotypes showed a different protein profile in comparison to the interspecific lines and the cultivated rice, probably due to the two crosses in direction to BG 90-2 and CICA-8, which were the recurrent parents during the interspecific lines development. The highest protein content found to the O. glumaepatula, the different protein profile and the finding of interspecific lines with higher protein content in relation to their cultivated parents clearly show the positive contribution of this species to the genetic breeding aiming the increase of the nutritional value for the grain of the cultivated rice in Brazil. / O objetivo desta dissertação foi analisar quantitativa e qualitativamente proteínas de reserva totais e as frações albumina, globulina, prolamina e glutelina presentes no grão de 29 genótipos de arroz silvestre Oryza glumaepatula e de 70 linhagens interespecíficas Retrocruzamento 2 desenvolvidas a partir do cruzamento O. sativa x O. glumaepatula. Destas linhagens, 34 (RC2F8) foram obtidas do cruzamento O. sativa BG90-2 x O. glumaepatula RS-16, e 36 (RC2F10) foram desenvolvidas a partir do cruzamento O. sativa CICA-8 x O. glumaepatula RS-16. Através da análise de variância para o teor de proteína total e frações protéicas, foram encontradas diferenças altamente significativas (P<0,01) entre os genótipos silvestres e entre as linhagens interespecíficas. O teor médio de proteína total dos genótipos silvestres foi de 12,17%, enquanto as linhagens do cruzamento BG90-2 x RS-16 apresentaram média de 7,05% e as linhagens do cruzamento CICA-8 x RS-16 apresentaram média de 8,40%. O genótipo silvestre BGA14280 destacou-se por apresentar o maior teor de proteína total (14,94%). Na análise comparativa envolvendo as linhagens interespecíficas e seus parentais, foram encontradas cinco linhagens com maior conteúdo de proteína total (teor médio de 10,95%), sendo significativamente superiores aos parentais cultivados BG 90-2 (10,0%) e CICA-8 (9,61%). No entanto, essas linhagens apresentaram teor inferior ao parental silvestre RS-16 (14,06%). Em relação às frações protéicas, 40 linhagens interespecíficas apresentaram valores mais elevados em relação aos seus parentais, exceto em uma ocasião, onde o parental silvestre RS-16 apresentou alto teor de glutelina. Entre os 29 genótipos silvestres, foram encontrados os maiores teores de albumina, prolamina e glutelina, exceto para a fração globulina, onde o genótipo silvestre BGA14162 não diferiu significativamente da cultivar BRS Bonança. A análise de SDS-PAGE da proteína total e da fração glutelina foi encontrada um perfil diferencial de &#945;-glutelinas entre os genótipos silvestres, com destaque para o genótipo BGA14232 que não apresentou &#945;-polipeptídeos comuns aos demais genótipos. Entre as linhagens interespecíficas e os seus parentais, foram encontrados perfis de proteína total e &#945;-glutelinas bastante similares, havendo diferença em relação a &#945;-glutelina de 40 kDa presentes apenas no parental silvestre RS-16, o que pode indicar uma constituição protéica diferencial em O. glumaepatula, já que a O. sativa apresentou &#945;-glutelinas de 39 kDa. Em relação às frações albumina, globulina e prolamina os genótipos silvestres apresentaram um perfil protéico diferenciado comparado às linhagens interespecíficas e o arroz cultivado, e a provável causa foram os dois cruzamentos em direção à BG90-2 e CICA-8, que foram os parentais recorrentes durante o desenvolvimento das linhagens interespecíficas. Os maiores teores de proteína encontrados para a espécie O. glumaepatula, os diferentes perfis protéicos e a identificação de linhagens interespecíficas com maior conteúdo protéico em relação aos parentais cultivados, deixam claro a contribuição favorável desta espécie para o melhoramento visando o aumento do valor nutricional do grão do arroz cultivado no Brasil.