Source: Masters Abstracts International, Volume: 46-05, page: 2669. Adviser: Jan M. Risser. Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 16, 2010). Includes bibliographical references.

Professional antigen presenting cells (APCs) represent an important link between the innate and adaptive immune system. Macrophages (MΦs) and dendritic cells (DCs) serve as sentinels in the periphery collecting samples from their environment and processing this information. These cells then present antigenic fragments to T cells in the context of self-MHC molecules. Although a clear role for both of these APCs in the stimulation of already activated or memory T cells has been established, the ability of MΦs to activate naive T cells is still unknown. In this thesis the ability of bone marrow-derived MΦs and DCs to prime naive CD8+ and CD4+ T cells was investigated. Using adoptively transferred transgenic CFSE-Iabeled P-14 T cells, specific for gp33 from lymphocytic choriomeningitis virus in the context of Db, we were able to demonstrate the ability of both MΦs and DCs to induce naive CD8+ T cells proliferation. Once primed by MΦs these T cells gained effector function as shown by interferon- γ (IFN-γ) production and in vivo cytolysis. In addition, immunization of wild type animals with gp33-pulsed MΦs, as well as DCs, led to greater than a 95% reduction in lymphocytic choriomeningitis virus titers. To rule out the role of cross-presentation in the observed priming, two models were used. In the first model, lethally irradiated F1 bxs chimeras reconstituted with either H-2s or H-2b bone marrow were used as host for the adoptive transfer experiments. Since the gp33 peptide binds to Db, the H-2s reconstituted animals should be unable to cross-present the peptide to the P-14 T cells. Using this model, we were able to clearly demonstrate the ability of MΦs to activate naive P-14 T cells to undergo division. Additional experiments, demonstrated that these MΦ primed T cells went on to develop into effector cells. Finally, the ability of the MΦ primed T cells to develop into functional memory cells was demonstrated. To confirm the chimera results, these experiments were repeated using β2 microglobulin deficient animals (whose cells don't express MHC I) as host in adoptive experiments. MΦs were able to stimulate the naive P-14 T cells to divide and gain effector function as demonstrated by the ability to produce IFN-γ. In contrast to the CD8 system, MΦ were poor stimulators of D011.10 CD4+ T cell proliferation. Additionally, D011.10 T cells stimulated by DCs were able to produce interleukin-2 (IL-2), IL-4, tumor necrosis factor and granulocyte-macrophage colony stimulating factor where as MΦ stimulated D011.10 T cells were only able to produce IL-2. In conclusion this body of work clearly demonstrates the in vivo ability of MΦ to stimulate CD8+ T cell proliferation, effector function, as well as the formation of functional CD8+ T cell memory. Whether or not the nature of the memory pools stimulated by the two APCs is exactly the same is still unknown and needs further investigation. The ability of APCs other than DCs to stimulate functional protective memory needs to be considered in the quest to design vaccines that offer broad-spectrum protection.

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and “licenses” the APCs to prime CD8+ T cell responses. While other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naïve CD8+ T cells. We find an approximately 50% reduction in expansion and cytokine production of TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization and are not protected from a tumor challenge. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. We also investigated whether CD40-CD40L interactions are involved in the generation, maintenance, and function of memory CD8+ T cells. Using a virus infection system as well as a dendritic cell immunization system, we show that the presence of CD40 on DCs and other host APCs influences the survival of activated effector cells and directly affects the number of memory CD8+ T cells that are formed. In addition, memory CD8+ T cell persistence is slightly impaired in the absence of CD40. However, CD40 is not required for reactivation of memory CD8+ T cells. It seems that CD40 signals during priming also contribute to memory CD8+ T cell programming but this function can be independent of CD4+T cells, similar to what we showed for primary responses. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of primary as well as memory CD8+ T cell responses.

La polyarthrite Rhumatoïde (PR) et le syndrome de Sjögren primitif (pSS) sont des deux maladies auto-immunes invalidantes pour lesquelles il n’existe à ce jour pas de traitement permettant la guérison des patients. Bien que les lymphocytes B soient impliqués dans le développement ces pathologies, l’existence de lymphocytes B capables de réguler l’inflammation est maintenant largement reconnue. Ces lymphocytes B régulateurs (Bregs) sont capables à la fois d’induire des lymphocytes T régulateurs et d’empêcher le développement de lymphocytes T pro-inflammatoires. In vivo, le transfert de ces Bregs est protecteur dans de nombreux modèles murins de maladies auto-immunes suggérant ainsi que promouvoir ces Bregs serait prometteur pour traiter les patients atteints de PR ou pSS. Cependant, il n’existe à ce jour pas de marqueur spécifique des Bregs, ce qui complique leur étude. Comme leurs fonctions régulatrices sont principalement médiées par l’IL-10, une cytokine anti-inflammatoire, les Bregs peuvent être définis par leur capacité à produire de l’IL-10 après activation par des composés bactériens, notamment CpG, motifs mimant l’ADN microbien. Ces Bregs sont appelés B10+. Toutefois, les fonctions de ces B10+ ne sont pas bien définies chez l’homme. Ainsi, nous ignorons si la seule production d’IL-10 est suffisante pour définir les Bregs.Cette thèse a donc pour premier objectif de définir les fonctions les B10+ stimulés par CpG chez les individus sains et chez les patients, afin de déterminer si la production d’IL-10 était un bon marqueur des Bregs. Nous avons montré que les B10+ induits par CpG étaient capables d’induire des lymphocytes T régulateurs (Tregs et Tr1) mais ne diminuaient pas la proportion de lymphocytes T pro-inflammatoires (Th1 et LT-TNFα+). Chez les patients PR et pSS, les B10+ induisaient des Tregs et Tr1 mais chez les patients PR, ils induisaient également des Th1. Ces résultats suggèrent que la production d’IL-10 seule ne suffit pas à récapituler toutes les fonctions régulatrices des Bregs, et que le stimulus CpG impacte probablement leur fonctions.Pour mieux comprendre les mécanismes contrôlant la production d’IL-10 des lymphocytes B et pouvoir proposer d’autres stimuli pour les générer, nous avons étudié l’effet de l’acétate, un composé produit par les bactéries commensales, sur la génération des B10+. L’acétate était capable d’induire des B10+ chez la souris et chez l’homme. D’autre part, nous avons également étudié le métabolisme cellulaire de ces B10+. Nous avons montré que la glutamine était un nutriment essentiel à la génération des B10+ et à leurs fonctions régulatrices. En définitive, ce travail a permis de définir les fonctions des B10+ induits par CpG, de caractériser leur métabolisme et de proposer de un stimulus alternatif pour générer les B10+. / Rheumatoid arthritis (RA) and primary Sjögren's syndrome (pSS) are two debilitating autoimmune diseases. There is currently no treatment to cure patients. Although B lymphocytes are involved in the development of these pathologies, the existence of B cells capable of regulating inflammation is now widely recognized. These regulatory B cells (Bregs) are able of both inducing regulatory T cells and preventing the development of pro-inflammatory T cells. In vivo, the transfer of these Bregs is protective in many mouse models of autoimmune diseases thus suggesting that promoting these Bregs would be promising for treating patients with RA or pSS. However, there is currently no specific marker for Bregs, which largely hinders their study. Since their regulatory functions are mainly mediated by IL-10, an anti-inflammatory cytokine, Bregs can be defined by their ability to produce IL-10 after activation by bacterial compounds, including CpG. Those Bregs are called B10+ cells. However, the functions of these B10+ cells are not well defined in humans. We do not know whether IL-10 production is a sufficient marker to define Bregs.The main goal of this thesis was to define CpG-induced B10+ cell functions in healthy individuals and patients, to determine whether IL-10 production was a good marker for Bregs. We showed that CpG-induced B10+ cells were able to induce regulatory T cells (Tregs and Tr1) but did not decrease the proportion of pro-inflammatory T cells (Th1 and LT-TNFα +). In RA and pSS patients, B10+ cells induced Tregs and Tr1 but in RA patients they also induced Th1. These results shows that IL-10 production alone does not recapitulate all the Breg functions and that CpG stimulation might impact their functions.To better understand the mechanisms controlling IL-10 production by B cells, and to be able to propose other stimuli to generate them, we studied the effect of acetate, a compound produced by commensal bacteria, on B10+ cells generation. Acetate was able to induce B10+ cells in mice and humans. Besides, we studied B10+ cell metabolism. We have showed that glutamine was an essential nutrient for B10+ cell generation and for their regulatory functions. This work has made it possible to define the CpG-induced B10+ cell functions, to characterize their metabolism and to propose an alternative stimulus to generate B10+ cells.

Les thérapies anti-tumorales se sont considérablement améliorées au cours de la dernière décennie. Toutefois, les traitements utilisés actuellement rencontrent d'importantes limitations, notamment dans le cas de cancers métastatiques, révélant l'urgence de développer de nouvelles approches. Ainsi, l'immunothérapie par transfert adoptif de cellules T représente une approche innovante particulièrement prometteuse. Son principe s'appuie sur l'injection de cellules T autologues spécifiques d'antigènes tumoraux, préalablement manipulées et amplifiées ex vivo, chez des patients rendus lymphopéniques par chimiothérapie et/ou radiothérapie. Toutefois, même si l'état lymphopénique est induit par ces 2 protocoles de conditionnements, leurs effets sur l'environnement de l'hôte ainsi que sur le devenir des cellules T greffées étaient, jusqu'à nos travaux, mal connus. Par le biais de modèles murins, nous avons pu démontrer que le devenir des cellules T diffère après transfert dans des souris irradiées ou traitées par chimiothérapie (Bu/Cy). Ainsi, après transfert dans des animaux irradiés, on observe une prolifération préférentielle des cellules T CD8, dépendante de l'IL-7, est observée alors qu'un transfert chez des souris traitées Bu/Cy se traduit par une prolifération rapide, indépendante de l'IL-7, des cellules T CD4. De plus, ces comportements sont associés à d'importantes modifications de l'environnement généré chez l'hôte. Plus spécifiquement, nous avons démontré, dans les organes lymphoïdes secondaires, que la localisation et la représentation des différentes sous-populations de cellules dendritiques présentes étaient différentiellement modulées par le type de conditionnement utilisé. Par ailleurs, l'élimination spécifique des cellules CD11c+ chez des souris traitées Bu/Cy était accompagnée d'une inhibition importante de la prolifération rapide des cellules T CD4 greffées. L'ensemble de nos travaux montrent que les traitements lymphopéniques génèrent des environnements distincts capables de moduler le devenir des cellules T greffées.Durant ma thèse, nous avons également abordé de façon originale un aspect novateur de l'environnement en étudiant le rôle potentiel des nutriments comme régulateurs métaboliques des fonctions effectrices des cellules T. La glutamine est l'acide aminé le plus abondant du plasma, pouvant contribuer aux besoins bionénergétiques et biosynthétiques des cellules T en prolifération. Nous avons démontré dans nos travaux qu'une carence en glutamine lors de l'activation de cellules T CD4 par leur TCR entrainait un délai dans l'activation de la voie mTOR, une réduction de la production intracellulaire d'ATP aux temps précoces et se traduisait par une diminution de la prolifération. De plus, ces conditions étaient associées à une augmentation de la conversion de cellules CD4 T naïves, via TGFβ, en cellules régulatrices Foxp3+ , y compris en condition de polarization Th1. Par contre, la carence en glutamine n'a pas inhibé la différenciation Th2. Les cellules T Foxp3+ ainsi générées en condition limitante de glutamine présentaient in vivo des fonctions suppressives aussi efficaces que celles des cellules régulatrices nTregs. En effet, elles ont la capacité de bloquer l'induction de la colite provoquée par la greffe de cellules T effectrices dans des souris Rag2-/- . Nos travaux démontrent ainsi que l'environnement métabolique peut être un régulateur clé de la différenciation des cellules T CD4. L'ensemble de mes travaux de thèse ont mis en évidence de nouveaux paramètres capables de potentiellement modifier la survie et la réactivité des cellules T greffées. / Anti-tumor therapies have improved significantly over the decade. However, the currently used treatments have important limitations, notably for metastatic cancers, and the development of new approaches is therefore a high priority. Adoptive T cell therapy (ACT) represents an innovative strategy that has shown much promise. This therapy is based on the infusion of tumor-specific T cells, which have been manipulated and expanded ex vivo, into patients who have been rendered lymphopenic by chemotherapy and/or irradiation. It is interesting to note that while lymphodepletion is attained by the vast majority of conditioning regimens, the effects of these protocols on the host environment and potentially, on the destiny of adoptively-transferred T cells had not been elucidated prior to the studies which we initiated. Using a murine model, we found that the fate of adoptively-transferred T cells differs markedly in mice rendered lymphopenic by sub-lethal irradiation as compared to a busulfan/cyclophosphamide (Bu/Cy) chemotherapy regimen. Irradiation-mediated lymphopenia resulted in a skewed IL-7-dependent proliferation of donor CD8+ T cells, whereas Bu/Cy treatment led to an increased IL-7-independent, rapid CD4+ T cell proliferation. These alterations in T cell proliferation were associated with striking changes in the host microenvironment. More specifically, we demonstrated that the proportion and localization of different dendritic cell (DC) subsets in lymphoid organs were differentially affected by the type of conditioning. Furthermore, we found that these DC controlled the rapid donor CD4+ T cell division detected in Bu/Cy-treated mice as depletion of CD11c+ DC inhibited this proliferation. Altogether, our studies demonstrate that lymphopenic regimens generate distinct host environments which modulate the fate of adoptively-transferred T cells. Durind my PhD, we also investigated an original and novel aspect of the microenvironement by studying the potential role of nutrients as metabolic regulators of T cell effector function. Glutamine is the most abundant amino acid in the plasma and contributes to the bioenergetic and biosynthetic requirements of proliferating T cells. Here, we demonstrated that activation of CD4+ T cells under glutamine-deprived conditions results in a delayed mTOR activation with reduced early ATP production and decreased proliferation. Moreover, these conditions resulted in the conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs). This de novo Treg differentiation occurred even under Th1-polarizing conditions and was TGFβ-dependent. Interestingly, glutamine deprivation did not inhibit Th2 differentiation. Importantly, these converted Foxp3+ T cells showed enhanced in vivo persistence and were highly suppressive, completely protecting Rag-deficient mice from the development of autoimmune inflammatory bowel disease as efficiently as natural-occuring Tregs. Thus, our data reveal the external metabolic environment to be a key regulator of a CD4 T lymphocyte's differentiation. Altogether, the data generated during my PhD provide new insights into the identification of parameters that can potentially alter the survival and reactivity of adoptively-transferred T cells.

TEIXEIRA, Fabrícia Martins. Marcadores de ativação de linfócitos T e de suas citocinas como ferramentas diagnósticas na hipersensibilidade alérgica a fármacos. 2012. 110 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Programa de Pós-Graduação em Biotecnologia, Rede Nordeste de Biotecnologia- Renorbio, Fortaleza-CE, 2012. / Submitted by demia Maia (demiamlm@gmail.com) on 2016-05-23T12:51:39Z No. of bitstreams: 1 2012_tese_fmteixeira.pdf: 12054646 bytes, checksum: 9a383e69304dd6755cbdf120633dadc1 (MD5) / Approved for entry into archive by demia Maia (demiamlm@gmail.com) on 2016-05-23T12:52:28Z (GMT) No. of bitstreams: 1 2012_tese_fmteixeira.pdf: 12054646 bytes, checksum: 9a383e69304dd6755cbdf120633dadc1 (MD5) / Made available in DSpace on 2016-05-23T12:52:28Z (GMT). No. of bitstreams: 1 2012_tese_fmteixeira.pdf: 12054646 bytes, checksum: 9a383e69304dd6755cbdf120633dadc1 (MD5) Previous issue date: 2012 / Drug allergy reactions represent one third of adverse drug reactions, and although they are infrequent, they present high rates of morbidity and mortality, revealing a major public health problem. The main challenges related to drug hypersensitivity result from its unpredictability, no animal model for research and individual variability with regard to drug metabolism. Drug allergy reactions are difficult to be diagnosed once there is a lack of laboratorial tests for their investigation. The present study aimed to establish some immunological in vitro methods for diagnosing drug allergy. Patients (n=20) attending a dermatology outpatient clinic, Hospital Universitario Walter Cantídio, Universidade Federal Ceara, with mucocutaneous and systemic manifestations due to drug hypersensitivity were investigated by clinical history, laboratory findings, and in vivo and in vitro tests. The lymphocyte activation markers, CD25 and CD69, were evaluated by flow cytometry on the peripheral blood mononuclear cells previously incubated with different concentrations of the suspected drug, and analysis of interferon γ and interleukin 5 was done in the culture supernatant by enzyme immunoassay. Eighteen patients were tested by skin tests; nine patients showed positive results to one or more drugs. Fifteen patients showed positivity for at least one of activation markers in response to the suspected drug. The markers CD69 and/or CD25 were expressed by T cells CD4+ and CD8+, both in immediate and delayed reactions. Comparing stimulation index of the markers between patients and healthy no allergic individuals, it was observed a significant difference for CD4+CD69+ in the three suspected drug concentrations and CD4+CD25+ only in the lower drug concentration. No significant differences were found for the cytokines IFN-γ and IL-5 between patients and healthy individuals. The detection of both activation markers CD69 and CD25 increased the diagnostic sensitivity of the test. The use of both markers represents a promising tool in drug allergy diagnosis. Nonetheless, this hypothesis needs to be confirmed with a greater number of patients and controls. / As reações alérgicas a fármacos representam um terço das reações adversas a medicamentos, e embora sejam pouco freqüentes, apresentam altas taxas de morbidade e mortalidade, revelando um importante problema de saúde pública. Os principais desafios relacionados com a hipersensibilidade a fármacos decorrem do fato de sua imprevisibilidade, de que não existe um modelo animal para pesquisa e devido à variabilidade individual no que diz respeito ao metabolismo do fármaco. As reações alérgicas a medicamentos são difíceis de serem diagnosticadas, uma vez que há carência de métodos laboratoriais para sua investigação. O presente estudo teve como objetivo estabelecer alguns métodos imunológicos in vitro para o diagnóstico de alergia a medicamentos. Vinte pacientes atendidos no Ambulatório de Dermatologia do Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, com manifestações muco-cutâneas e sistêmicas decorrentes de hipersensibilidade a fármacos foram investigados através de história clínica, exames laboratoriais in vivo e in vitro. Foram avaliados os marcadores de ativação de linfócitos CD25 e CD69 através de citometria de fluxo, em células mononucleares do sangue periférico previamente incubadas com diferentes concentrações do fármaco suspeito, e análise das citocinas interferon γ e interleucina 5 no sobrenadante da cultura através de teste imunoenzimático. Dezoito pacientes foram submetidos aos testes cutâneos, sendo que nove mostraram resultados positivos a um ou mais fármacos. Quinze pacientes apresentaram positividade para pelo menos um dos marcadores de ativação em resposta ao fármaco suspeito. Os marcadores CD69 e/ou CD25 foram expressos pelas células T CD4+ e CD8+, tanto em reações imediatas como nas não imediatas. A comparação dos índices de estimulação desses marcadores entre pacientes e indivíduos saudáveis não alérgicos, resultou em diferença significativa para CD4+CD69+ nas três concentrações do fármaco suspeito e para CD4+CD25+ apenas na menor concentração do fármaco suspeito. Nenhuma diferença significativa para as citocinas IFN-γ e IL-5 foi observada entre os pacientes e os indivíduos controles. A detecção de ambos os marcadores de ativação CD69 e CD25 aumentou a sensibilidade diagnóstica do teste. O uso combinado dos marcadores representa uma ferramenta promissora no diagnóstico laboratorial das reações alérgicas a medicamentos. Não obstante, essa hipótese deve ser confirmada com um número maior de pacientes e controles.

Rápidas adaptações endometriais ocorrem com a chegada do embrião no útero para criar um ambiente uterino receptivo, que é essencial para o desenvolvimento do concepto. As descrições dos eventos endometriais que ocorrem antes da chegada do embrião ao útero, são escassas. O objetivo deste experimento foi demonstrar mudanças na arquitetura e na estrutura do endométrio que ocorrem no dia 5 pós-ovulação em éguas cíclicas e inseminadas. Amostras de biopsia endometrial foram recolhidas em torno do dia 5 pós-ovulação em um grupo de 10 éguas reprodutivamente sadias, uma de cada corno, durante dois ciclos. No primeiro como ‘Cíclicas’ (n = 10) e no seguinte estas mesmas foram inseminadas para formar o grupo ‘Inseminadas’ (n = 10). As éguas inseminadas foram subdivididas em dois subgrupos: aquelas em que as amostras foram recolhidas antes do dia 5.5 e aquelas em que as amostras o foram depois do dia 5.5. As biopsias endometriais foram analisadas por microscopia de luz e microscopia eletrônica de varredura. A densidade, o diâmetro, o epitélio, o lúmen e a secreção glandular, foram submetidos à análise histomorfométrica. As células brancas foram avaliadas em uma contagem diferencial. As células ciliadas, poligonais com microvilosidades, e a presença de células planas ou ingurgitadas no epitélio, na microscopia eletrónica de varredura, foram analisados por uma contagem individual de cada um dos tipos celulares. Houve um aumento no diâmetro glandular, uma diminuição significativa nas células ciliadas e um aumento significativo na população de linfócitos nas éguas inseminadas, quando comparadas com as égua cíclicas. Na análise dos subgrupos de éguas inseminadas, não houve diferenças nas variáveis. Estes resultados levam a concluir que mudanças significativas ocorrem no endométrio no dia 5 pós ovulação, na égua inseminada. / Rapid endometrial adaptations occur as the embryo enters the uterus to create a receptive uterine environment, which is essential for the conceptus' development. Descriptions of endometrial events before the embryo reaches the uterus, are scant The aim of this experiment was to demonstrate architectural and structural changes in the endometrium on day 5 after ovulation in cyclic and inseminated mares. Endometrial biopsy samples were taken during day 5 post-ovulation from a group of 10 reproductively healthy mares, one from each uterine horn, during two cycles. The first one they were considered as ‘cyclic’ (n = 10), and in the following one, the same mares were inseminated to constitute the ‘inseminated’ group (n = 10). Inseminated mares were subdivided into two subgroups: those sampled before day 5.5 and those sampled after day 5.5. Biopsy samples were analyzed through optic microscopy and scanning electron microscopy. Glandular density, diameter, epithelial height, lumen, and secretion were analyzed through histomorphometry. White blood cells were evaluated through a differential cell count. Ciliated cells, micro-ciliated polygonal cells, and flat or protruded cells over the epithelium, on scanning electron microscopy, were analyzed through an individual cell type count. Inseminated mares presented an increase in glandular diameter, significant decrease in ciliated cell population, and a significant increase in lymphocyte population, compared to cyclic mares. No differences in any of the variables were detected between subgroups from inseminated mares. The results here presented lead to the conclusion that significant endometrial changes occur on day 5 post-ovulation in inseminated mares.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Epidemiological studies suggest that the imbalance in antioxidant enzymes activity genetically caused, increases the risk of metabolic disorders and chronic noncommunicable diseases. This is the case of polymorphism that occurs in an substitution of Alanine (A) by a Valine (V) at codon 16 of the gene for superoxide dismutase manganese-dependent (Ala16Val-SOD2). The A allele has a higher efficiency of catalysis than allele V. However, this efficiency increases the levels of H2O2 leading to a consequent oxidative imbalance that, in turn, increases the risk of cancer. Furthermore, the V allele that presents a smaller efficiecy SOD2 is associated with endothelial dysfunction and cardiometabolic. Therefore, Ala16VALSOD2 polymorphism serves as a model for in vitro analysis of differential responses to pro and antioxidants factors. The objective of this study was to investigate the in vitro response of lymphocytes culture with different genotypes (AA, VV and AV) from healthy subjects exposed to ultraviolet (UV) is a pro-oxidant factor. In lymphocyte not exposed and UV exposed it was analyzed the cytotoxic variables (cell viability, mitotic index), biomarkers of oxidative metabolism (lipid peroxidation, enzymatic activity of SOD, catalase, thiol groups, protein carbonylation, total polyphenols and ascorbic acid) and genotoxic effects (by Comet Assay and chromosomal instability). The results showed that cells carrying the AA genotype had higher cell viability and mitotic index. However, after UV exposure such viability was similar between genotypes. At baseline levels higher SOD and total polyphenol levels were observed in AA genotype when compared to the VV genotype. However, lymphocytes AA had higher rates of DNA damage in the presence of UV radiation. These results suggest that the polymorphism Ala16Val-SOD2 may differentially affect the toxicity factors such as UV radiation. / Estudos epidemiológicos sugerem que o desbalanço na atividade de enzimas antioxidantes, geneticamente causado, aumenta o risco de disfunções metabólicas e doenças crônicas não-transmissíveis. Este é o caso do polimorfismo em que ocorre uma troca da Alanina (A) por uma Valina (V) no códon 16 do gene da enzima superóxido dismutase dependente de manganês (Ala16Val-SOD2). O alelo A possui uma eficiência de catálise maior do que o alelo V. Entretanto, esta maior eficiência aumenta os níveis de H2O2 levando a um conseqüente desbalanço oxidativo que, por sua vez, aumenta o risco de neoplasias. Por outro lado, o alelo V que apresenta uma SOD2 menos eficiente está associado a disfunções endoteliais e cardiometabólicas. Portanto, este polimorfismo serve como modelo in vitro para análises de respostas diferenciais a agentes pró e antioxidantes. Sendo assim, o objetivo desse estudo foi investigar a resposta in vitro de cultura de linfócitos com diferentes genótipos (AA, VV e AV), provenientes de indivíduos saudáveis, expostas a radiação ultravioleta (UV) que é um fator pró-oxidante. Antes e após exposição, foram analisados os efeitos citotóxicos (viabilidade celular e índice mitótico), indicadores do metabolismo oxidativo (peroxidação lipídica, atividade enzimática da SOD, catalase, grupos tióis, polifenóis totais e de ácido ascórbico) e efeitos genotóxicos (dano de DNA pelo Ensaio Cometa e instabilidade cromossômica,). Os resultados mostraram que as células portadoras do genótipo AA apresentaram maior viabilidade celular e índice mitótico. Entretanto, após a exposição UV tal viabilidade foi similar entre os genótipos. Em condições basais níveis elevados de SOD e polifenóis totais plasmáticos foram observados no genótipo AA em relação ao genótipo VV. Porém, linfócitos AA apresentaram maior índice de danos no DNA na presença da radiação UV. Estes resultados sugerem que o polimorfismo Ala16Val- SOD2 pode afetar diferencialmente a toxicidade a fatores como a radiação UV.

Orientador: Sandra Aidar de Queiroz / Banca: Valter Udler Cromberg / Banca: Danísio Prado Munari / Resumo: O medo é uma característica comportamental importante em espécies domesticadas e pode ser incluído no programa de seleção, pois responde à seleção artificial e tem conseqüências importantes ao bem estar e desempenho das aves domésticas. A reação de medo pode ser avaliada pelo tempo de permanência em imobilidade tônica (IT), que é o período em que o animal fica em estado catatônico induzido manualmente pelo homem. Quanto menos tempo permanecer neste estado, menor é o medo do animal e mais adaptado este se mostra a viver em cativeiro. A característica IT é bem representativa do nível de medo do indivíduo e também pode estar relacionada com a relação heterófilo/linfócito (H/L). Assim, o objetivo deste trabalho foi determinar efeitos de ambiente e estimar parâmetros genéticos da característica tempo de permanência em IT de perdiz (Rhynchotus rufescens) criada no ambiente de cativeiro. A análise realizada pelo método dos quadrados mínimos revelou a influência da época de nascimento dentro de geração e o peso corporal, sendo que animais mais pesados permaneceram maior tempo em IT (b = 0,32 ± 0,14g, p<0,05). O método de máxima verossimilhança restrita possibilitou a estimação do coeficiente de herdabilidade da característica IT, apresentando valor igual a 0,29 evidenciando influência do ambiente sobre o tempo de permanência em IT. Contudo, a seleção pode ser eficiente para alterar as médias desta característica / Abstract: Fear is an important behavior trait in domesticated species and can be included in the selection program, and have important consequences to the welfare and performance of poultry. The fear reaction can be measured by the time spend in tonic immobility (TI), which is the period where the animal stays in catatonic state induced by human hand. The less time remaining in this state, smaller is the fear of the animal and it shows more adapted to living in captivity. The trait IT is well representative of the level of fear of the individual and may also be related to the heterophil to lymphocyte ratio (H/L). The objective of this study was to determine the effects of environment and estimate the genetic characteristic of tonic immobility time in Red-winged Tinamou (Rhynchotus rufescens) hosed in captivity environment. The method of least squares analysis resulted the influence of season within generation and body weight, whose heavier animals showed longer period in IT (b = 0.32 ± 0.14 g, p <0.05). The restricted maximum likelihood method was applied to estimate heritability of TI with value of 0.29 indicating environmental influence However, the selection can be effective to change the means of this trit / Mestre