Made available in DSpace on 2015-04-14T14:51:19Z (GMT). No. of bitstreams: 1 438710.pdf: 388887 bytes, checksum: 75275a00e6033933e4d572bb06d67de4 (MD5) Previous issue date: 2012-03-30 / Population data of seven short tandem repeat loci of the PowerPlex? CS7 were obtained from a sample of 401 individuals from Rio Grande do Sul (Southern Brazil). The loci are the two pentanucleotides STRs in the PowerPlex? 16 (PENTA D and PENTA E), an extra pentanucleotide (PENTA C), and four loci STR in the FFFL Fluorescent STR System (LPL, F13B, FES/FPS, and F13A01). Allele frequency and other forensically relevant statistics data were generated for these seven loci. All of the analyzed loci meet Hardy-Weinberg equilibrium expectations and no linkage disequilibrium were found, except for LPL locus. The observed and expected heterozygosity, power of discrimination and exclusion and polymorphic information content were calculated. The combined power of discrimination and combined power of exclusion were 0.999999987 and 0.998159054, respectively. Genetic distances between population from Rio Grande do Sul and other populations are presented. / Dados populacionais de sete loci Short Tandem Repeat do kit comercial PowerPlex? CS7 foram obtidos a partir de uma amostra de 401 indiv?duos do Rio Grande do Sul (sul do Brasil). Os loci s?o os dois pentanucleot?deos STRs do PowerPlex? 16 (PENTA D e PENTA E), um pentanucleot?deo extra (PENTA C), al?m dos quatro loci STR do kit comercial FFFL Fluorescent STR System (LPL, F13B, FES/FPS e F13A01). As frequ?ncias al?licas e outros dados estat?sticos de relev?ncia forense foram analisados. Todos os loci atenderam ?s expectativas de Hardy-Weinberg e n?o foram encontrados desequil?brios de liga??o, exceto para o locus LPL. A heterozigosidade observada e esperada, o poder de discrimina??o e exclus?o e o conte?do de informa??o polim?rfica foram calculados. O poder combinado de discrimina??o e o poder combinado de exclus?o foram de 0,999999987 e 0,998159054, respectivamente. As dist?ncias gen?ticas entre a popula??o do Rio Grande do Sul e outras popula??es foram apresentadas neste trabalho.

Made available in DSpace on 2015-04-14T14:51:19Z (GMT). No. of bitstreams: 1 439217.pdf: 703680 bytes, checksum: 20bc10f7a801c99b27dde05bf2a6dc66 (MD5) Previous issue date: 2012-03-03 / Tuberculosis (TB) remains a leading cause of death worldwide, due to the great adaptability of the bacillus, which can survive in various conditions inside and outside the human host. Previous studies showed evidence that polymorphisms in P2X7 receptor are e associated with increased risk of TB. The present study aimed to analyze the role of purinergic P2X7 receptor in M. tuberculosis infection and host interaction mechanisms in vitro and in vivo. The macrophage murine cell line RAW 264.7 was used for in vitro experiments. . Our results demonstrated that treatment of RAW 264.7 cells with ATP (3 and 5 mM) resulted in a statistically significant reduction of counting colony forming units (CFUs). Male wild-type C57BL/6 (wild-type) and P2X7 receptor KO (P2X7R-/-) mice (25 30 g) were used throughout this study for in vivo. Immunohistochemistry showed that the purinergic P2X7 receptor expression was found significantly augmented in the lungs of mice infected with M. tuberculosis. Furthermore, M. tuberculosis-infected P2X7R-/- mice showed an increase of M. tuberculosis burden in lung tissue, when compared to infected wild type mice. Infected mice showed a marked increase in the spleen weight, in comparison to non-infected animals, indicating the occurrence of splenomegaly. In P2X7R-/- spleens, we observed a significant decrease in the populations of Treg (CD4+Foxp3+), T cells (CD4+, CD8+CD25+ and CD4+CD25+), dendritic cells (CD11c+) and B220+ cells. However, a significant increase in CD11b+ cells was observed in P2X7R-/- mice, when compared to wild-type animals. In the lungs, P2X7R-/- M. tuberculosis-infected mice exhibited pulmonary infiltrates containing an increase of Treg cells (CD4+Foxp3+), T cells (CD4+ and CD8+) and a decrease in the B220+ cells, when compared with wild-type M. tuberculosis-infected mice. The findings observed in the present study provide novel evidence on the role of P2X7 receptors in the pathogenesis and control of tuberculosis. Whether selective agonists or antagonists of this receptor might be useful for improving TB complications remains a matter to be investigated. / A tuberculose (TB) continua sendo uma das principais causas de morte no mundo, devido ? grande capacidade de adapta??o do bacilo que pode sobreviver em diferentes condi??es dentro e fora do hospedeiro humano. Estudos pr?vios mostraram evid?ncias de que polimorfismos no receptor purin?rgico P2X7 est?o associados ao aumento da suscetibilidade ? TB. O presente estudo objetivou analisar o papel do receptor purin?rgico P2X7, na infec??o por M. tuberculosis em camundongos, e os poss?veis mecanismos de intera??o do receptor P2X7 com o hospedeiro, em modelos in vivo e in vitro. Nos experimentos para avalia??o da viabilidade da micobacteria intracelular in vitro foi utilizada a linhagem de macr?fagos murinos, RAW 264.7. Nossos resultados demonstraram que o tratamento das c?lulas RAW 264.7 com ATP (3 e 5 mM) resultou em uma redu??o estatisticamente significativa da contagem de unidades formadoras de col?nias (CFUs). Nos experimentos in vivo foram utilizados camundongos machos C57BL/6 (tipo selvagem) e camundongos knockout para o receptor P2X7 (P2X7R-/-) (25-30 g). Os resultados com imuno-histoqu?mica mostraram que a express?o do receptor purin?rgico P2X7 foi encontrada significativamente aumentada nos pulm?es de camundongos infectados com M. tuberculosis. Al?m disso, camundongos P2X7R-/- infectados com M. tuberculosis mostraram um aumento da carga da micobacteria no tecido pulmonar, quando comparado com camundongos do tipo selvagem infectados. Camundongos infectados mostraram um aumento marcante no peso do ba?o quando comparado com animais n?o infectados, indicando a ocorr?ncia de esplenomegalia. Em ba?os de camundongos P2X7R-/-, observou-se uma diminui??o significativa nas popula??es de Treg (CD4 + Foxp3 +), c?lulas T (CD4 +, CD8 + CD25 + e CD4 + CD25 +), c?lulas dendr?ticas (CD11c +) e c?lulas + B220. No entanto, houve um aumento significativo de c?lulas CD11b + em camundongos P2X7R-/-, quando comparados aos animais do tipo selvagem. Nos pulm?es, camundongos P2X7R-/- infectados com M. tuberculosis apresentaram infiltrado pulmonar contendo um aumento de c?lulas Treg (CD4 + Foxp3 +), c?lulas T (CD4 + e CD8 +) e uma diminui??o nas c?lulas + B220, quando comparado com camundongos do tipo selvagem infectados com M. tuberculosis. Portanto, os resultados observados no presente estudo fornecem novas evid?ncias sobre o papel dos receptores P2X7 na patog?nese e controle da tuberculose. O uso de agonistas ou antagonistas seletivos deste receptor como uma ferramenta terap?utica continua sendo uma quest?o a ser investigada.

Made available in DSpace on 2015-04-14T14:51:19Z (GMT). No. of bitstreams: 1 439219.pdf: 722099 bytes, checksum: 87b93549e65018c129416830692d4bfc (MD5) Previous issue date: 2012-03-30 / Stenotrophomonas maltophilia is an important opportunistic and emerging pathogen commonly related to nosocomial infections and found in different environmental sources, including hospital settings. This microorganism is recognized for presenting intrinsic resistance to a range of important antimicrobials as well as to acquire resistance by horizontal gene transfer, which reduces the effective options for the treatment of infections caused by this organism. Therefore, the aim of this study was to determine the presence of S. maltophilia in the nosocomial environment and characterize the resistance of the environmental isolates, as well as clinical isolates obtained in the same hospital. Then, environmental samples were collected in the general ICU and the Unified Health System hospitalization unit of the S?o Lucas Hospital, Porto Alegre, Brazil. In addition, 100 clinical isolates were sent by the Clinical Pathology Laboratory of the same hospital. All samples were analyzed using a specific protocol for S. maltophilia detection by PCR developed in this study targeting 23S rRNA gene. Subsequently, the antimicrobial resistance was evaluated against ceftazidime, chloramphenicol, levofloxacin, minociclin and trimethoprim/sulfamethoxazole (TMP/SMX). The presence of integrons was verified in all isolates and those that presented reduced susceptibility to TMP/SMX was evaluated the presence of sul1 and sul2 gene, as well as was determined the plasmid profile. All isolates were submitted to detection of smf-1 gene. Among the 936 samples collected in the nosocomial environment, S. maltophilia was identified in 28. High rates of susceptibility to minocycline, levofloxacin and chloramphenicol were observed, and all of the 19 isolates that presented reduced susceptibility to TMP/SMX carried the sul1 gene, 14 of them presented class 1 integron and nine isolates showed simultaneously sul1 and sul2. All isolates that carried the sul2 gene also presented the 7.3 kb plasmid. The smf-1 gene was detected in 31 S. maltophilia isolates. The presence of S. maltophilia in hospital environment and medical devices indicates the permanence of this microorganism in the nosocomial environment, what can constitute a risk to infection for other hospitalized patients. In addition, the data obtained in this study in relation to TMP/SMX susceptibility can suggest that the resistance to these drugs have the tendency to increase, especially due to resistance determinants to these drugs can be associated to mobile genetic elements, which may facilitate horizontal transfer and the spread of these resistance genes. / Stenotrophomonas maltophilia ? um importante pat?geno oportunista e emergente, comumente relacionado a infec??es nosocomiais e encontrado em diferentes locais, incluindo o ambiente hospitalar. Este microrganismo ? reconhecido por apresentar resist?ncia intr?nseca a uma gama importante de antimicrobianos, bem como por adquirir resist?ncia atrav?s de transfer?ncia g?nica horizontal, o que reduz as op??es efetivas para o tratamento de infec??es ocasionadas por este microrganismo. Desta forma, o objetivo deste estudo foi determinar a presen?a de S. maltophilia no ambiente hospitalar e caracterizar a resist?ncia a drogas antimicrobianas dos isolados ambientais, bem como dos isolados cl?nicos obtidos no mesmo hospital. Para tanto, amostras ambientais foram coletadas na UTI Geral e no andar referente ? interna??o pelo Sistema ?nico de Sa?de (SUS) do Hospital S?o Lucas, Porto Alegre, Brasil. Al?m disso, 100 isolados cl?nicos foram cedidos pelo Laborat?rio de Patologia Cl?nica do mesmo hospital. Todas as amostras foram analisadas utilizando um protocolo de detec??o espec?fica de S. maltophilia atrav?s de PCR desenvolvido neste estudo, tendo o gene RNAr 23S como alvo. Posteriormente, foi avaliada a resist?ncia dos isolados frente ? ceftazidima, cloranfenicol, levofloxacina, minociclina e trimetoprim/sulfametoxazol (TMP/SMX). A presen?a de integrons foi verificada em todos os isolados e naqueles com suscetibilidade reduzida a TMP/SMX foi avaliada a presen?a dos genes sul1 e sul2, bem como foi determinado o perfil plasmidial. Todos os isolados foram submetidos ? detec??o do gene smf-1. De um total de 936 amostras coletadas no ambiente hospitalar, S. maltophilia foi identificada em 28. Foram observadas elevadas taxas de suscetibilidade ? minociclina, levofloxacina e cloranfenicol e todos os 19 isolados que apresentaram suscetibilidade reduzida ? combina??o TMP/SMX carrearam o gene sul1, 14 destes apresentaram o integron de classe 1 e nove isolados apresentaram concomitantemente os genes sul1 e sul2. Todos os isolados que carrearam o gene sul2 apresentaram o plasm?deo de 7,3 kb. O gene smf-1 foi detectado em 31 isolados de S. maltophilia. A presen?a de S. maltophilia nos materiais e equipamentos hospitalares indica a perman?ncia destas bact?rias no ambiente hospitalar, podendo constituir risco para infec??o de outros pacientes internados. Al?m disso, os dados obtidos neste estudo em rela??o ? suscetibilidade ? TMP/SMX podem sugerir que a resist?ncia a esta combina??o de drogas possa estar em ascens?o, especialmente porque os determinantes de resist?ncia a estas drogas podem estar associados a elementos gen?ticos m?veis, o que facilitaria a transfer?ncia horizontal e a dissemina??o destes genes de resist?ncia.

Made available in DSpace on 2015-04-14T14:51:20Z (GMT). No. of bitstreams: 1 439240.pdf: 319940 bytes, checksum: 78e312c8115e9e5356969e795047faba (MD5) Previous issue date: 2012-02-27 / Although Parkinson s Disease (PD) is predominantly a movement disorder, the presence of cognitive problems related to frontal function, even in the earliest stages has been observed. These problems include attentional, executive and memory deficits. A study using an experimental paradigm developed to investigate the effects of different encoding instructions on contextual memory showed that, unlike healthy controls, PD patients could not reverse their contextual memory deficits and take any advantage of encoding instructions. However, it is unclear whether they have a memory deficit or executive dysfunction would be accountable for results. Attentional and executive deficits observed in PD patients have important implications for daily activities, mainly when patient perform concomitant cognitive and motor activities that are attention demanding. Catechol- O-methyltransferase (COMT) is an enzyme that degrades cortical dopamine. Some studies have examined the relationship between the COMT val158met polymorphism and executive function in PD patients and have found low enzyme activity, associated to met/met polymorphism- implying higher prefrontal dopamine levels- is related to worse performance. However, a recent study did not demonstrate a direct effect of COMT genotype on the executive performance. The present study aimed to characterize different cognitive parameters of patients with PD and to assess their relationship with the COMT polymorphism. In this study participated 18 patients with early disease stage PD and 18 healthy adults matched for age, gender and education. Patients were selected from a data bank of patients genotyped for COMT polymorphism. All participants completed the Wisconsin Card Sorting Test (WCST), the Stroop Color and Word Test, and dual tasking paradigm. In addition, patients completed a contextual memory paradigm. No effect of COMT val158met genotype on frontal function in patients with PD (executive function and contextual memory) was observed. A decrease in cognitive task performance under dual-tasking was observed in PD patients, by contrast to healthy controls. Relative dual-task cognitive cost was significantly greater for PD patients compared to controls in the arithmetic-walking condition. In conclusion, difficulties in performing a cognitive task while walking can be influenced by the executive/attentional load of the task. / Embora a Doen?a de Parkinson (DP) seja predominantemente um dist?rbio do movimento, a presen?a de altera??es cognitivas relacionadas ? fun??o frontal tem sido observada mesmo nos est?gios iniciais da doen?a. Estas altera??es incluem d?ficits atencionais, executivos e de mem?ria. Um estudo usando um paradigma experimental desenvolvido para investigar os efeitos de diferentes instru??es codificadoras na mem?ria contextual mostrou que, ao contr?rio dos idosos, pacientes com DP n?o s?o capazes de reverter seus d?ficits de mem?ria e n?o tiram nenhuma vantagem das instru??es codificadoras. Entretanto, ainda n?o est? claro se estes pacientes t?m um d?ficit de mem?ria ou se as disfun??es executivas seriam as respons?veis pelos resultados observados. D?ficits atencionais e executivos observados em pacientes com DP t?m implica??es importantes nas atividades di?rias, principalmente quando o paciente desempenha atividades concomitantes cognitivas e motoras que requerem aten??o. A catecol- O-metiltransferase (COMT) ? uma enzima que degrada a dopamina cortical. Alguns estudos t?m examinado a rela??o entre o polimorfismo val158met da COMT e fun??o executiva em pacientes com DP e t?m observado que a atividade reduzida da enzima, relacionada ao polimorfismo, que implica em maiores n?veis de dopamina no c?rtex pr?-frontal, est? associada a uma pior performance. Entretanto, um estudo recente n?o demonstrou um efeito direto do gen?tipo da COMT no desempenho executivo. O presente estudo teve por objetivo caracterizar diferentes par?metros cognitivos de pacientes com DP e verificar sua rela??o com o polimorfismo da COMT. Neste estudo participaram 18 pacientes em est?gios iniciais da DP e 18 adultos saud?veis, pareados por idade, g?nero e n?vel educacional. Os pacientes foram selecionados de um banco de pacientes genotipados para o polimorfismo da COMT. Todos os participantes completaram o Teste de Classifica??o de Cartas de Wisconsin, o Teste de Stroop e o paradigma para avalia??o da realiza??o de tarefas simult?neas. Adicionalmente, os pacientes com DP completaram o paradigma de mem?ria contextual. N?o foi observado efeito do polimorfismo val158met da COMT sobre a fun??o frontal (fun??o executiva e mem?ria contextual) dos pacientes com DP. Os pacientes apresentaram um decl?nio cognitivo na realiza??o das atividades simult?neas, ao contr?rio dos controles. O custo cognitivo associado ? realiza??o da tarefa aritm?tica juntamente ? caminhada foi significativamente maior para os pacientes com DP, em rela??o aos controles. Em conclus?o, as dificuldades em realizar uma atividade cognitiva e motora simultaneamente podem ser influenciadas pela carga executiva/atencional da atividade.

Made available in DSpace on 2015-04-14T14:51:20Z (GMT). No. of bitstreams: 1 439544.pdf: 1258585 bytes, checksum: 19aa18ec90c3c36b13cb69c6788c4a18 (MD5) Previous issue date: 2012-03-30 / Bipolar Disorder (BD) is a complex, multifactorial and chronic psychiatic illness. It is characterized by alternating cycles of mania and depression, with periods of remission or euthymia. Several studies have suggested a direct interaction between nervous, endocrine and immune systems in the pathophysiology of BD. An immune activation, as evidenced by increased plasma levels of proinflammatory cytokines, has been frequently reported in BD. However, the majority of the studies have mainly investigated mania and depression phases, and very few studies have been developed with euthymic patients. In this study, we investigated cellular and molecular mechanisms potentially involved in the inflammatory process in BD, including various lymphocytes subtypes and intracelular pathways of lymphocyte activation. Twentyseven euthymic female subjects with BD type I and 24 age- and sex-matched controls were recruited in this study. Lymphocytes were isolated and stimulated in vitro to assess Th1/Th17/Th2 cytokines (IL-2, IL-4, IL-5, IL-10, IL-17, IFN-ע and TNF-?) and expression of mitogen-activated protein kinases (MAPKs). The expression of MAPKs (p-oERK and p38), lymphocyte subtypes and cytokines were assessed by flow cytometry. All cytokines assessed were found elevated in bipolar disorder compared with healthy controls. In particu-lar, it was evidenced a bias to a Th1 inflammatory profile in BD. Interestingly, we observed a significant reduction (-56%) of regulatory T cells (CD4+CD25+Foxp3+) and expansion (43%) of CD8+ regulatory T cells (CD8+CD28-) in BD. The lymphocytes of BD patients showed an significant increase in p-ERK in relation to p-p38, indicating lymphocyte activation. Our data suggest that multiple molecular and cellular mechanisms contribute to the immunological imbalance observed in BD / O Transtorno Bipolar (TB) ? uma doen?a psiqui?trica cr?nica, complexa e multifatorial, caracterizada por ciclos alternados de mania e depress?o, intercalados com per?odos de remiss?o ou eutimia. Diversos estudos v?m demonstrando associa??es entre sistema nervoso, end?crino e imunol?gico na patofisiologia do TB. Uma ativa??o imune, evidenciada por n?veis plasm?ticos aumentados de citocinas pr?-inflamat?rias, tem sido freq?entemente relatada no TB. No entanto, a maioria dos estudos refere-se principalmente ?s fases de mania e depress?o, e poucos estudos foram desenvolvidos na fase de eutim?a. Neste estudo, investigamos mecanismos celulares e moleculares potencialmente envolvidos no fen?meno inflamat?rio no TB, incluindo diversos subtipos linfocit?rios e vias intracelulares de ativa??o linfocit?ria. Vinte e sete pacientes mulheres com TB I eut?micos e 24 controles saud?veis pareados por sexo e idade foram recrutadas neste estudo. Os linf?citos foram isolados e estimulados in vitro para avaliar as citocinas Th1/Th17/Th2 (IL-2, IL-4, IL-5, IL-10, IL-17, IFN-ע e TNF-?) e express?o de prote?nas cinases ativadas por mit?geno (MAPKs). A express?o das MAPKs (p-ERK e p-p38), subtipos linfocit?rios e citocinas foram avaliados por citometria de fluxo. Todas as citocinas avaliadas encontraram-se elevadas nos bipolares em compara??o com controles sau-d?veis. Em particular, foi evidenciado um vi?s para um perfil Th1 (inflamat?rio) no TB I. Interessantemente, observamos uma redu??o significativa (-56%) de c?lulas T regulat?rias (CD4+CD25+Foxp3+) e expans?o (43%) de c?lulas T CD8+ regulat?rias (CD8+CD28-) no TB. Os linf?citos dos pacientes bipolares apresentaram um aumento significativo de p-ERK em rela??o a p-p38, indicando uma ativa??o linfocit?ria. Concluindo, nossos dados sugerem que m?ltiplos mecanismos celulares e moleculares contribuem para um desequil?brio imune observado no TB

Made available in DSpace on 2015-04-14T14:51:20Z (GMT). No. of bitstreams: 1 440822.pdf: 1496271 bytes, checksum: ed1a67fe6439eb139b2edd2275228d18 (MD5) Previous issue date: 2012-01-27 / Cadmium is considered the seventh most dangerous substance in the environment and is classified as carcinogen type I, potentially affecting a wide range of living organisms, including humans. In addition to its wide systemic impact and potential lethality, cadmium has been associated to neurobehavioral defects that may also compromise animals ecological status and survival. Despite its potential impact, the comprehension of cellular and molecular mechanisms underlying cadmium deleterious effects on animals behavior is still scarce. This study aimed to evaluate the behavioral effects of cadmium for 1 or 7 days at environmentally relevant concentrations (10 μg/L, 100 μg/L and 1000 μg/L) on zebrafish and to analyze brain oxidative stress and apoptotic markers. Cadmium-exposed zebrafish exhibited a generalized increase in locomotor activity after 1 and 7 days of treatment at all doses in all parameters evaluated, including distance travelled in a 5-min. evaluation period, mean speed and mobile periods. This hyperlocomotory effect significantly compromised animals general performance in exploring a new environment, which was evident in all cadmium exposed animals decreased path efficiency and altered distribution on the water column. Additionally, our results confirmed previous reports of increased oxidative damage in fishes exposed to cadmium and specifically demonstrated higher levels of damaged proteins in brain samples of animals exposed to cadmium at 100 μg/L for 1 day and at 10 μg/L for 7 days when compared to their respective control groups. Lipid peroxidation was also significantly increased in animals brain after 1 day cadmium exposure at 100 μg/L. Real-Time PCR analysis of transcripts for p53 and bax were not altered after 1 day cadmium exposure, but significantly increased after 7 days. Our results present evidence of cadmium deleterious effects on zebrafish cognitive functions and raise attention to the fact that its manifestation appears already after a one day exposure to 10 μg/L, a concentration accepted by most international regulating agencies / O c?dmio ? considerado a s?tima subst?ncia mais perigosa presente no ambiente e ? classificado como carcinog?nico tipo I, potencialmente afetando uma grande quantidade de seres vivos, incluindo os humanos. Ademais, o c?dmio tem sido associado a defeitos neurocomportamentais que podem comprometer o status ecol?gico e a sobreviv?ncia de animais. Apesar do potencial impacto, a compreens?o dos mecanismos celulares e moleculares por tr?s de seus efeitos delet?rios no comportamento de animais ? ainda escassa. Este estudo teve por objetivo avaliar os efeitos comportamentais do c?dmio por 1 e 7 dias a concentra??es relevantes no ambiente (10 μg/L, 100 μg/L and 1000 μg/L) em zebrafish e analisar o estresse oxidativo e marcadores de apoptose no enc?falo. Animais expostos ao c?dmio apresentaram aumento na atividade locomotora ap?s 1 e 7 dias de tratamento em todas as concentra??es e par?metros avaliados, incluindo dist?ncia percorrida num per?odo de 5 minutos, velocidade m?dia e per?odos m?veis. A hiperlocomo??o afetou significativamente o desempenho dos animais em explorar um novo ambiente em todos os grupos tratados, evidenciado por uma diminui??o na efici?ncia de percurso e alterada distribui??o na coluna d ?gua. Adicionalmente, nossos resultados confirmaram estudos pr?vios sobre aumento no dano oxidativo em peixes quando expostos ao c?dmio e especialmente demonstraram n?veis mais elevados de dano a prote?nas em amostras de enc?falo em animais tratados a 100 μg/L por 1 dia e a 10 μg/L por 7 dias quando comparados aos seus respectivos controles. Lipoperoxida??o aumentou significativamente no enc?falo de animais expostos por 1 dia a 100 μg/L. An?lises dos marcadores p53 e bax por Real-time PCR apresentaram nenhuma altera??o ap?s 1 dia de exposi??o, mas significativamente aumentaram ap?s 7 dias. Nossos resultados apresentam evid?ncias dos efeitos delet?rios do c?dmio no comportamento de zebrafish e chama aten??o para o fato de que a manifesta??o de seus efeitos aparece a partir de 1 dia de exposi??o a 10 μg/L, uma concentra??o aceita por muitas ag?ncias de regulamenta??o internacional

Made available in DSpace on 2015-04-14T14:51:20Z (GMT). No. of bitstreams: 1 442001.pdf: 1823261 bytes, checksum: 10761416aa58c3d7680e6ff4e42ce20f (MD5) Previous issue date: 2012-08-23 / Microcystins (MCs) constitute a family of cyanobacterial toxins, with more than 80 variants. These toxins are able to induce hepatotoxicity in several organisms, mainly through the inhibition of protein phosphatases PP1 and PP2A and oxidative stress generation. Recent evidence shows that MCs can either accumulate in brain or alter behavior patterns of fish species. Thus, this thesis aimed to study the effects of MC-LR (with the variable amino acids leucine (L) and arginine (R)) exposure on biochemical parameters in zebrafish, emphasizing the cholinergic and purinergic signaling, as well as to evaluate the behavioral patterns and whole-body cortisol levels. In vivo studies showed that 100 μg/L MC-LR for 24 h led to a significant increase in the AChE activity (27%) when zebrafish were exposed to the toxin dissolved in water, but did not cause any significant changes when injected intraperitoneally. In addition, semiquantitative RT-PCR analysis demonstrated that 100 μg/L MC-LR exposure also increased ache mRNA levels in zebrafish brain. The in vitro assays did not reveal any significant changes in AChE activity. We also assessed behavioral patterns and whole-body cortisol levels of adult zebrafish exposed to cell culture of the microcystin-producing cyanobacterium Microcystis aeruginosa. MC-LR exposure (100 μg/L) decreased by 63% the distance traveled and increased threefold the immobility time when compared to the control group. Interestingly, no significant alterations in the number of line crossings were found at the same MC-LR concentration and time of exposure. When animals were exposed to 50 and 100 μg/L, MC-LR promoted a significant increase (around 93%) in the time spent in the bottom portion of the tank, suggesting an anxiogenic effect. In addition, the results also showed that none of the MC-LR concentrations tested promoted significant alterations in absolute turn angle, path efficiency, social behavior, or whole-body cortisol level. Moreover, we evaluated the acute effects of different concentrations of MC-LR on NTPDases (nucleoside triphosphate diphosphohydrolases) and 5 - nucleotidase in adult zebrafish (Danio rerio) brain membranes. The results have shown no significant changes in ATP, ADP and AMP hydrolysis in zebrafish brain membranes. MC-LR in vitro also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidases activity in the tested conditions. Taken together these findings provide the first evidence that brain AChE is another potential target for MCs and that MC-LR exposure significantly impairs animal's exploratory performance. Nevertheless, further studies including long-time exposure should be performed in order to achieve a better understanding about MCLR toxicity mechanisms in the central nervous system / Microcistinas (MCs) constituem uma fam?lia de toxinas, com mais de 80 variantes. Estas toxinas s?o capazes de induzir hepatotoxicidade em diversos organismos, principalmente atrav?s da inibi??o das fosfatases PP1 e PP2A e gera??o de estresse oxidativo. Evid?ncias recentes mostram que MCs podem acumular no c?rebro e alterar padr?es comportamentais em diferentes esp?cies de peixes. Portanto, a presente tese teve por objetivo estudar os efeitos da exposi??o ? MC-LR (com os amino?cidos vari?veis leucina (L) e arginina (R)) sobre par?metros bioqu?micos em zebrafish, enfatizando os sistemas colin?rgicos e purin?rgicos, bem como, avaliar padr?es comportamentais e n?veis de cortisol corporal. Resultados do estudo in vivo mostraram que a exposi??o a 100 μg/L de MC-LR durante 24 h causaram um aumento significativo na atividade da AChE (27%) quando zebrafish foi exposto ? toxina dissolvida em ?gua; por?m, a toxina n?o causou mudan?as significativas quando injetada intraperitonealmente. Al?m disso, a an?lise semiquantitativa de RT-PCR demonstrou que a exposi??o ? 100 μg/L de MC-LR tamb?m aumentou os n?veis de RNAm da ache em c?rebro de zebrafish. Os ensaios in vitro n?o revelaram nenhuma altera??o significativa na atividade da AChE. N?s tamb?m avaliamos padr?es comportamentais e n?veis de cortisol corporal de zebrafish adultos expostos ? cultura de c?lulas de Microcystis aeruginosa produtoras de MC-LR. A exposi??o ? MC-LR (100 μg/L) diminuiu em 63% a dist?ncia viajada e aumentou tr?s vezes o tempo de imobilidade quando comparado ao grupo controle. Interessantemente, n?o houve altera??o no n?mero de linhas cruzadas na mesma concentra??o e tempo de exposi??o ? MC-LR. Quando animais foram expostos a 50 e 100 μg/L, a MCLR promoveu um aumento significante (aproximadamente 93%) no tempo gasto na por??o inferior do aqu?rio teste, sugerindo um efeito ansiog?nico. Adicionalmente, os resultados tamb?m mostraram que nenhuma das concentra??es de MC-LR testadas promoveu altera??es significativas nas mudan?as de ?ngulo, efici?ncia de rota e intera??o social ou, no n?vel de cortisol corporal total. Al?m disso, tamb?m foi avaliado o efeito de diferentes concentra??es de MC-LR na atividade das NTPDases (nucleos?deo trifosfato difosfoidrolase) e 5 nucleotidase em membranas cerebrais de zebrafish (Danio rerio) adultos. Os resultados mostraram que n?o houve altera??o nas hidr?lises de ATP, ADP e AMP. Nos experimentos in vitro tamb?m n?o houve altera??o nas hidr?lises dos nucleot?deos nas concentra??es testadas. Estes achados mostram que exposi??o aguda ? MC-LR n?o modulou a atividade das ectonucleotidases nas condi??es testadas. Ademais, fornecem a primeira evid?ncia que a AChE cerebral ? um outro alvo potencial das MCs e que exposi??o ? MC-LR prejudica significativamente a performance explorat?ria do animal. Estudos futuros incluindo exposi??o de longo prazo dever ser feitos para um melhor entendimento dos mecanismos de toxicidade das MCs no Sistema Nervoso Central

Made available in DSpace on 2015-04-14T14:51:21Z (GMT). No. of bitstreams: 1 442629.pdf: 4545565 bytes, checksum: 75785599771804ca3f6535bc606e1454 (MD5) Previous issue date: 2012-08-09 / The clinical depression treatment faces serious obstacles as the disease mechanism is not fully elucidated. In addition, there are no effective means to predict and prevent depression as well as any biological method of diagnosis. The use of antidepressants is still the basis of the treatments for depression. Lithium has been used clinically as effective drug to treat all phases of bipolar disorder, including major depression. The Selective serotonin re-uptake inhibitors (SSRIs), such as fluoxetine and citalopram, and Tricyclic antidepressant (TCA) as clomipramine are drugs constantly used for depression treatment. Recent evidence has shown an involvement of adenosine and its receptors in the pathophysiology of depression. ATP can be stored and co-released with other neurotransmitters like serotonin and can be hydrolyzed by a cell-surface enzyme family known as ectonucleotidases. Among these members, we highlight the nucleoside triphosphate diphosphohtdrolases (NTPDases) and ecto-5'-nucleotidase. They are able to control the availability of ligands such as ATP and adenosine to its specific receptors. Adenosine deaminase (ADA) can promote the hydrolytic deamination of adenosine to inosine, modulating the extracellulr levels of this neuromodulator. In cholinergic signaling, after its release, acetylcholine (ACh) promotes the activation of specific muscarinic or nicotinic receptors and thus, it promotes diverse cellular responses. ACh is hydrolyzed by acetylcholinesterase (AChE) in acetate and choline in synaptic cleft. The zebrafish has been used in research behavioral neuroscience and is also a choice model for elucidating the development and function of neuronal circuitry. Considering the cholinergic and purinergic signaling are important participation in the CNS and these neurotransmitter pathways have been identified and characterized in zebrafish, the objective of this study was to evaluate the effect of antidepressants on ectonucleotidases, ADA and ACh activities, which are essential enzymes in the modulation of these signaling pathways in the zebrafish brain. We evaluated the ex vivo effects of fluoxetine (1-10 ?M), clomipramine (1-10 ?M), citalopram (70-300 ?M) on ectonucleotidases and ADA activities. It has been also analyzed the in vitro (1 to 1000 ?M) and ex vivo (1 to 10mg/L) effect of lithium on ectonucleotidases and AChE activities and gene expression. There was a significant inhibition of ADP hydrolysis after ex vivo exposure to lithium at 5 and 10 mg/L, whereas an inhibitory effect was observed for AMP hydrolysis only at 10 mg/L. The same treatment decreased the AChE activity in a concentration of 10mg/L. Lithium did not induce significant changes in the analysis of gene expression patterns in the concentrations tested. In vivo treatment, there were no significant changes inectonucleotidases and AChE activities. Treatment with clomipramine promotes an inhibition ecto-5'-nucleotidase activities at the concentration of 5?M when compared to the control group. For ADA activity, we also observed a significant inhibition in the treatment with clomipramine at concentrations of 5 and 10 ?M in membrane fractions of zebrafish brain. However, treatment with fluoxetine and citalopram did not alter ectonucleotidases and ADA activities in the zebrafish brain. Our findings may contribute to a better understanding of pharmacology of antidepressants and their interaction with the cholinergic and purinergic neurotransmission. / O tratamento cl?nico da depress?o enfrenta s?rios obst?culos j? que o mecanismo da doen?a n?o ? totalmente elucidado. Al?m disso, n?o existem meios eficazes para prever e prevenir a depress?o bem como nenhum m?todo biol?gico de diagn?stico. O uso de f?rmacos antidepressivos ainda ? a base dos tratamentos para depress?o. O l?tio tem sido usado clinicamente como f?rmaco eficaz para tratar todas as fases do transtorno bipolar, incluindo depress?o aguda. Os inibidores seletivos da recapta??o de serotonina (ISRSs), como fluoxetina e citalopram, e os antidepressivos tric?clicos (TCA), como a clomipramina, s?o f?rmacos constantemente utilizados para o tratamento da depress?o. Evid?ncias recentes mostram o envolvimento da adenosina e seus receptores na patofisiologia da depress?o. O ATP pode ser armazenado e co-liberado, juntamente com outros neurotransmissores, como serotonina e noradrenalina, e pode ser hidrolisado at? adenosina por uma fam?lia de enzimas de superf?cie celular, conhecidas como ectonucleotidases. Dentre elas, destacam-se as Nucleos?deo Trifosfato Difosfoidrolases (NTPDases) e a ecto-5?-nucleotidase, capazes de controlar a disponibilidade de ligantes como ATP e adenosina aos seus receptores espec?ficos. A Adenosina desaminase (ADA) pode promover a desamina??o hidrol?tica da adenosina em inosina, modulando os n?veis extracelulares deste neuromodulador. Na sinaliza??o colin?rgica, a acetilcolina (ACh), ap?s liberada, promove a ativa??o de receptores muscar?nicos ou nicot?nicos, e desta maneira a ACh promove diversas respostas celulares. A ACh que permanece na fenda sin?ptica ? hidrolisada pela acetilcolinesterase (AChE) em acetato e colina. O peixe-zebra tem sido utilizado na pesquisa da neuroci?ncia comportamental, sendo tamb?m um modelo de escolha para elucidar o desenvolvimento e a fun??o do circuito neuronal. Considerando que as sinaliza??es purin?rgica e colin?rgica t?m importante participa??o no sistema nervoso central e que essas vias de neurotransmiss?o j? est?o caracterizadas em peixe-zebra, o objetivo desse estudo foi avaliar o efeito de f?rmacos antidepressivos na atividade das ectonucleotidases, ADA e AChE, enzimas essenciais na modula??o destas vias de sinaliza??o em c?rebro de peixe-zebra. Foram avaliados os efeitos ex vivo da fluoxetina (1-10 ?M), clomipramina (1-10 ?M) e citalopram (70-300 ?M) na atividade das ectonucleotidases e ADA. Foi analisado tamb?m o efeito in vitro (1 a 1000 ?M) e ex vivo (1-10mg/L) do l?tio sobre a atividade e express?o g?nica das ectonucleotidases e AChE.A exposi??o ao l?tio inibiu a hidr?lise de ADP nas concentra??es de 5 e 10mg/L e inibiu a hidr?lise de AMP na concentra??o de 10mg/L quando comparado ao grupo controle. Este mesmo tratamento diminuiu a atividade da AChE na concentra??o de 10mg/L.O l?tio n?o induziu altera??es significativas na an?lise do padr?o de express?o g?nica. No tratamento in vitro, n?o foram observadas altera??es na atividade das ectonucleotidases e AChE. O tratamento com a clomipramina mostrou uma inibi??o na atividade da ecto-5?-nucleotidase na concentra??o de 5 ?M quando comparado ao grupo controle. Na atividade da ADA tamb?m observamos uma inibi??o significativa no tratamento com a clomipramina nas concentra??es de 5 e 10 ?M em fra??es de membrana de c?rebro de peixe-zebra. Entretanto, o tratamento com fluoxetina e citalopram n?o alterou a atividade das ectonucleotidases e ADA no c?rebro do peixe-zebra. Nossos resultados podem contribuir para uma melhor compreens?o da farmacologia dos f?rmacos antidepressivos e a sua intera??o com a neurotransmiss?o colin?rgica e purin?rgica.

Made available in DSpace on 2015-04-14T14:51:21Z (GMT). No. of bitstreams: 1 442974.pdf: 1168003 bytes, checksum: 61a64ae8027da7ef2e5ef378d8f25eb3 (MD5) Previous issue date: 2011-07-05 / Breast cancer is an important cause of morbidity and mortality in Brazil and worldwide. New therapeutic strategies have been proposed and one of them is the use of cell therapy with dendritic cells (DCs), however, the literature regarding the real benefit of this approach shows great variability and inability to produce a lasting immunity. In this paper we made a detailed characterization of monocyte-derived dendritic cells (MoDCs) of patients with breast cancer and monocytes that originated them. A lower yield of MoDCs was obtained from patients when compared to age matched healthy controls. Patient MoDCs exhibited decreased expressions of maturation and activation markers. Furthermore, cultures of MoDCs of patients had significantly elevated levels of spontaneous production of IL-6, which is consistent with a pro-tumor phenotype. These differences in molecule expression and cytokine production led us to postulate that the signaling pathways and / or the expression of toll like receptors (TLRs) could be altered. A decrease of TLR9 and TLR2 and an increase in the expression of NFkBp50 was found in MoDCs of patients without stimulation. After stimulation with LPS and CPG the patients did not upregulate expression of MyD88, suggesting a downregulation of the signaling pathways activated by these molecular patterns. The number of monocytes was also were decreased in patients, showing a reduced expression of GM-CSF receptors compared to monocytes of healthy controls. Cytokine production by monocytes from cancer patients was also altered, with an increased production of IL-6, IL-4 and IL-10. TLR2 and TLR9 expression was downregulated in monocytes of patients. Together these data show that monocytes are already altered in patients with cancer, and that will influence the phenotype of DCs differentiated from them. Tumor burden seems to induce a tolerogenic and pro-tumoral phenotype in patients?cells. This finding is important for the development of DC-based cancer immunotherapy. / A neoplasia mam?ria ? uma causa importante de morbidade e mortalidade no Brasil e no mundo. Novas estrat?gias terap?uticas vem sendo propostas e uma delas ? a utiliza??o de terapia celular com c?lulas dendr?ticas (DCs), entretanto, os dados da literatura em rela??o ao real benef?cio desta abordagem apresentam grande variabilidade e inabilidade em produzir uma imunidade duradoura. No presente trabalho fizemos uma caracteriza??o detalhada das c?lulas dendr?ticas derivadas de mon?citos (MoDCs) das pacientes com c?ncer de mama e dos mon?citos que as originaram. As MoDCs das pacientes s?o obtidas com um menor rendimento quando comparadas a controles saud?veis pareados por idade, e exibem uma diminui??o nos marcadores de matura??o e ativa??o. Al?m disso, as culturas de MoDCs das pacientes apresentaram altos n?veis de IL-6, o que ? compat?vel com um fen?tipo pr?-tumoral. Essas diferen?as na produ??o de citocinas nos levou a postular que as vias de sinaliza??o e/ou a express?o de toll like receptors (TLRs) poderiam estar alteradas. Observamos uma diminui??o de TLR9 e TLR2 e um aumento na express?o de NFkBp50 nas MoDCs das pacientes, sem est?mulo. Ap?s est?mulo com LPS e CPG as pacientes n?o aumentaram a express?o de MyD88, sugerindo uma diminui??o Ada via de sinaliza??o da resposta a esses padr?es moleculares. Quando analisamos os mon?citos, eles tamb?m estavam diminu?dos nas pacientes, e apresentavam uma express?o de receptores para GM-CSF inferior a dos controles saud?veis. A produ??o de citocinas pelos mon?citos das pacientes com c?ncer tamb?m estava alterada com uma produ??o aumentada de IL-6, IL-4 e IL-10. A frequ?ncia de TLR9 e TLR2 estava diminu?da nos mon?citos das pacientes. Esses dados conjuntamente demonstram que os mon?citos j? est?o alterados nas pacientes com c?ncer, assim como as DCs diferenciadas a partir deles. O crescimento tumoral parece induzir um fen?tipo de toler?ncia nestas c?lulas. Esse dado ? de fundamental import?ncia para o desenvolvimento de imunoterapia baseada em DCs.

Made available in DSpace on 2015-04-14T14:51:22Z (GMT). No. of bitstreams: 1 444377.pdf: 376897 bytes, checksum: 468537059cc3bd01ae14fdd8d9e9222a (MD5) Previous issue date: 2012-04-27 / Rhipicephalus microplus is a tick that parasite bovines, damaging the milk and meat production, and constituting a vector for agents of various diseases. R. microplus cause serious losses in countries that depend on cattle production, and the control methods used are based on the use of acaricides, which present high cost and contaminate milk and meat, generating impacts on the environment. Therefore, new methods of control, as vaccines, have been suggested. Anti-parasite protective antigens can be divided into two groups: exposed, which interact with host immune system, and concealed, that do not interact with host immune system. Paramyosin (PRM) is a protein present in invertebrates primarily characterized by muscle function, but in various parasites it has been localized in nonmuscle regions, suggesting that it may also perform functions involved in the evasion of the host immune system. The Taenia solium paramyosin inhibits in vitro the classical pathway of complement system by binding to C1, and R. microplus paramyosin (RmPRM) has been shown to bind immunoglobulins. This study aimed to evaluate the recognition of RmPRM by the sera of animals infected with R. microplus and determine the levels of paramyosin gene expression in tissues and different developmental stages of the tick, as well as to analyze the anti-complement activity of the recombinant protein. The results showed that sera from naturally infected Bos indicus and B. taurus were able to recognize the recombinant PRM. Antibodies levels found among the bovines have considerable variation, but with higher titers predominating among B. indicus individuals. The RmPRM gene transcription was detected in most tissues, organs, eggs and larvae tested, with higher levels of expression found in the fat body, an organ without muscle tissue prominence. Furthermore, this study shows that RmPRM is able to inhibit the complement system, indicating that that this protein should be an important component for the parasite survival, possibly involved in modulation of the host immune system. Corroborating other studies in parasites, these results suggest that RmPRM performs other functions than those classically described in the musculature. / Rhipicephalus microplus ? um carrapato que parasita bovinos, prejudicando a produ??o de leite e carne, al?m de ser um vetor para agentes de diversas doen?as. O R. microplus causa s?rios preju?zos em pa?ses que dependem da pecu?ria, e os m?todos de controle utilizados s?o baseados no uso de acaricidas, os quais apresentam alto custo e contaminam o leite e a carne, al?m de causarem impactos ao meio ambiente. Desta forma, novos m?todos de controle, como o desenvolvimento de vacinas, t?m sido sugeridos. Ant?genos protetores ao gado t?m sido divididos em dois grupos: expostos, que interagem com o sistema imune do hospedeiro, e ocultos, que n?o interagem com o sistema imune do hospedeiro. A paramiosina (PRM) ? uma prote?na presente em invertebrados primariamente caracterizada pela sua fun??o muscular, por?m em diversos parasitos tem sido localizada em regi?es n?o musculares, sugerindo que a mesma tamb?m possa apresentar fun??es envolvidas com a evas?o do sistema imune do hospedeiro. A PRM de Taenia solium inibe in vitro a via cl?ssica do sistema complemento, por meio do bloqueio da fun??o de C1, e a de R. microplus liga a por??o Fc de imunoglobulinas. Este estudo teve por objetivo avaliar o reconhecimento da paramiosina de R. microplus (RmPRM) por bovinos infectados com o carrapato e determinar os n?veis de express?o do gene da paramiosina em tecidos e diferentes est?gios de desenvolvimento do carrapato, tendo sido tamb?m avaliada a atividade anti-complemento da prote?na recombinante. Os resultados mostraram que o soro de bovinos Bos indicus e B. taurus naturalmente infectados foram capazes de reconhecer paramiosina recombinante. Os n?veis de anticorpos encontrados entre os bovinos apresentaram varia??es consider?veis, por?m com os maiores t?tulos predominando entre indiv?duos B. indicus. A transcri??o do gene da RmPRM foi constatada na maioria dos tecidos, ?rg?os, ovos e larvas testados, com maior n?vel de express?o encontrado no corpo gorduroso, um ?rg?o sem predomin?ncia de tecido muscular. Al?m disso, este trabalho mostra que a RmPRM ? capaz de inibir o sistema complemento, indicando que esta prote?na deve ser um importante componente para a sobreviv?ncia do parasito, possivelmente envolvida na modula??o do sistema imune do hospedeiro. Corroborando com outros trabalhos em parasitos, estes resultados sugerem que a RmPRM desempenha outras fun??es al?m das classicamente descritas na musculatura.