Synthesis of heat shock proteins (Hsps) in response to elevated temperatures and other denaturing agents (including UV, heavy metals, and energy depletion) is a common productive response of prokaryotic and eukaryotic cells. Therefore, increasing in production of heat shock protein has been considered a sign of cells under stress. The phylum Sipuncula comprises of about 300 species worldwide; they are bilaterally symmetrical, unsegmented, and deposit feeding marine worms common in the substrate of wetland. In this research, I studied the dominant species Sipunculus nudus and analyzed it proteomics to reveal how it responses to persistent organic pollution. In order to compare the difference between HSP70 and HSP90, Sipunculans were sampling from two wetland (Xiangshan and Wanggong) in Hsinchu and Changhua,, respectively. The results showed that the concentrations of PAHs and OCPs both in substrate and S. nudus tissues from Xiangshan were higher than Wanggong. The concentrations of PAHs and OCPs in both areas showed that the environmental- sediment levels were higher than the sediment inside the gut or the body tissues. Principal component analysis showed that the compositions in S. nudus from both area were similar; however PAHs compositions from the environmental sediment or the sediment of the gut were similar. When the concentrations of HSP70 and HSP90 between both areas were compared, HSP70 expressions did not significant differed in both areas, while HSP90 express was higher in the S. nudus from Xiangshan than Wanggong. For S. nudus Hsp90 was upregulated in highly polluted area (i.e. Xiangshan). I propose that tissue expression of HSP90 plays an important role in the survival of S. nudus, and detection of HSP90 may provide pollution information of the surrounding environment.

The elderly are at increased risk for developing psychiatric diseases, which include Alzheimer's disease, depression, anxiety and suicide. The probability of multiple disease comorbidity is also increased in the elderly. At the cellular level, the loss of protein homeostasis is often at the root of disease emergence, and thus the scientific community is searching for ways to help maintain this balance. A vast group of proteins that are paramount to balancing and counterbalancing protein levels is the molecular chaperone protein group, which has evolved a tremendous variety of functions in the cell. They aid in protein trafficking, folding, receptor signaling, neurotransmission, vesicle forming and fusion, protein degradation, and apoptosis, among other activities. Despite their best efforts, disease still ensues, but because of their vast number and multiple abilities, it may be possible to modulate these proteins as a way to treat and prevent disease. Chaperones are of particular interest in diseases of aging, because chaperone induction and effectiveness is reduced with age. In addition, many diseases of the elderly are brought on by aberrant protein accumulation, like Alzheimer's disease. As a result, the hypothesis of this dissertation is whether the modulation of molecular chaperones changes disease pathology. A molecular chaperone family that is important to protein degradation is the Hsp70 chaperone complex. Hsp70 proteins have specialized function depending on cell type and cellular compartment, but Hsp70 proteins are very important for protein synthesis and degradation. As a result, they are in a position to contribute to the regulation of proteins that become aberrant. In recent years scientific literature has indicated that compounds that inhibit the enzymatic ATP hydrolysis of these proteins promote tau degradation, which accumulates in Alzheimer's disease. Alzheimer's disease is the sixth leading cause of death in the U.S., it is a progressive neurodegenerative disease, and is caused by the aberrant accumulation of the amyloid beta and tau proteins. Here, we show that treatment with the Hsp70 inhibitor methylene blue, reduces tau, saves neurons, and restores cognition, in a mouse model of tau accumulation (rTg4510). Cognitive rescue occurred despite a severe tangle load, equal to control treated tau transgenic mice. This study shows that reducing soluble tau can restore cognition, reducing tangles is not necessarily to ameliorate cognition, and saving neurons is not sufficient to increase cognition if they are burdened with soluble tau. This work shows that methylene blue does not affect the the number of tau tangles in this model, as suggested by in vitro data. It also suggests that further work into the development of Hsp70 ATPase inhibitors may find success in alleviating the soluble tau burden found in Alzheimer's disease. The co-chaperone FKBP5 is also of extreme importance, not because it is essential, but because research has implicated this protein with a host of psychiatric diseases. Single nucleotide polymorphisms in this gene, which increase the levels of FKBP5, interact with averse traumatic events to enhance the likelihood of developing mood and anxiety disorders, including major depressive disorder, post-traumatic stress disorder, bipolar disorder, and suicide. Moreover, we have found that FKBP5 protein levels increase with age in the human brain, increasing the risk for the elderly of developing disease if exposed to traumatic stress. Here, we tested the hypothesis that FKBP5 negatively regulates resilient behavior. We found that FKBP5 levels increase with age in the wild type mouse brain, and that wild type mice display reduced resiliency with age. FKBP5-/- mice, on the other hand, show enhanced resiliency to stress at all ages tested, and are protected from aging-induced despair. At the molecular level, FKBP5 is a robust inhibitor of the glucocorticoid receptor, which is responsible for the shut-off of the hypothalamic-pituitary-adrenal axis. In addition, excess glucocorticoid levels in the blood is a robust marker of psychiatric disease. Consequently, FKBP5 may be causing disease through enhanced levels of glucocorticoids. FKBP5-/- mice display reduced corticosterone after stress. Moreover, corticosterone production increases with age, and FKBP5-/- mice are protected from this increase. These studies are the first to show that reducing the levels of FKBP5 is a promising therapeutic option for the treatment of mood disorders in the elderly, resiliency naturally declines with age due to FKBP5, corticosterone levels after stress rise due to FKBP5, and that the ablation of this gene increases resiliency and prevents aging- induced despair. As a whole, these data show that the modulation of chaperone proteins has the potential for developing new therapies for the treatment of psychiatric diseases of the elderly.

Hsp104 and ClpB are hexameric ATPases that resolubilize aggregated proteins in collaboration with the Hsp70 chaperone system. Hsp104/ClpB functionally interact only with their respective Hsp70 system and this specificity is mapped to the Hsp104/ClpB coiled-coil domain (CCD). We hypothesize that the interaction between Hsp70 and Hsp104/ClpB CCD stimulates nucleotide exchange and release of substrate from Hsp70. In the current study, the CCDs of E. coli ClpB and S. cerevisiae Hsp104 have been purified. Isolated domains are monomeric and well folded. They inhibit refolding of aggregated firefly luciferase in a species-specific manner. We found that the ATPase activity of E. coli DnaK is stimulated at low concentrations of the E. coli ClpB CCD but not by yeast Hsp104 CCD. However, in another bacterial system (Thermus thermophilus) we found that the ClpB CCD inhibits The ATPase activity of DnaK suggesting that the interaction may have different consequences in distinct chaperone networks.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Situações de estresse metabólico e térmico deflagram a expressão celular e liberação para o plasma, de proteínas de choque térmico da família de 70 kDa (HSP70) para desempenhar função de defesa-chave em um processo conhecido como resposta ao choque térmico ou ao estresse, que as caracterizam como proteína citoprotetora. Estudos indicam a ocorrência de HSP70 extracelular (eHSP70) na circulação por até 24 h após desafios como o exercício físico, cuja liberação dá-se por tecidos do território hepatoesplâncnico, em uma resposta mediada por receptores α-adrenérgicos. Esta liberação de HSP70 para o plasma é atenuada pela ingestão de glicose. Estas evidências levaram-nos a supor que a liberação de eHSP70 em resposta ao estresse possa ter um papel fisiológico na regulação da glicemia, a qual é afetada por estímulo simpático. Neste trabalho, os resultados dos experimentos in silico indicaram a possibilidade de ancoragem e interação estável entre a HSP70 e a molécula de insulina. Ensaios de imunoprecipitação de insulina com amostras de plasma de animais submetidos ao tratamento de insulina Regular e Detemir revelaram HSP70 coimunoprecipitada especialmente em situação de jejum severo. Parte destas amostras foi submetida à quantificação de glicemia, insulinemia e HSP72 circulante, cujos resultados evidenciam elevada concentração desta proteína de choque térmico por ocasião de hipoglicemia induzida por insulina. Parte dos estudos in vivo deste trabalho foram desenvolvidos com ratos submetidos ao choque térmico agudo (uma sessão de 15 minutos) com hipertermia induzida (41,5 °C) ou com injeção de HSP70 (HSPA1A) exógena, com grupos experimentais organizados em grupos Controle (C) e Choque Térmico (HS) e, em tempos experimentais: imediatamente (tempo zero), 6, 12 e 24 h pós-choque. Foi observada a ocorrência das formas constitutiva e induzível de eHSP70 no plasma de animais submetidos ao choque térmico com valor maior nos períodos de 12 e 24 h pós-choque. Nossos estudos mostram que, in vivo, a eHSP70 plasmática produz alteração na tolerância à glicose, com maior hiperglicemia, quando o teste é realizado 12 h após o choque térmico. Este efeito foi bloqueado pela administração (i.p.) de anticorpo anti-HSP70 e se mostrou independente da síntese de novo de HSP70. Ainda, a presença de eHSP70 plasmática (induzida por choque térmico ou administrada via endovenosa) causa menor captação de glicose pelo músculo esquelético. Ensaios com músculo isolado indicam que o efeito inibidor da eHSP70 sobre a captação de glicose é dependente de insulina. Apesar das evidências de que a ligação HSP70-insulina no plasma tenha implicação sobre a oferta de glicose, não se pode descartar também a possibilidade de que as ações da eHSP70 estejam ocorrendo pela sua ligação a receptores específicos, e/ou de que sejam parte de uma resposta ao estresse na qual estejam implicados os hormônios contrarregulatórios. Em suma, os resultados indicam que a eHSP70 plasmática é capaz de se ligar- à insulina, provocar tolerância alterada à glicose e diminuir a captação de glicose pelo músculo esquelético, aumentando a disponibilidade de glicose circulante. / Metabolically stressful situations trigger the expression and release of the 70 kDa (HSP70) as an essential protective response, in a process known as heat shock (HS) response or stress response. HSP70 is characterized as cytoprotector entities. Previous studies indicate the occurrence of extracellular HSP70 (eHSP70) in the circulation up to 24 h after stressful challenges, such as physical exercise, and that such eHSP70 liberation comes from hepatosplancnic tissues, in an 1-adrenergic dependent response, which is attenuated by glucose ingestion. The above findings led us to suppose that stress-elicited eHSP70 release could play a physiological role on glycemic control, which are affected by sympathetic stimuli. In this work, in silico experiments indicated the possibility of docking and stable interaction between HSP70 and the insulin molecule. Immunoprecipitation of plasma samples revealed that HSP70 co-immunoprecipitates with insulin, especially under severe fast. Part of these samples was subjected to quantification of glucose, insulin and circulating HSP72, whose results show high concentration of heat shock protein during severe hypoglycemia induced by insulin. Part of the in vivo studies from this work were also carried out in healthy rats submitted to an acute (15 min) HS (41.5 °C) session or in HSP70 (HSPA1A)-injected rats, assigned into control (C) and HS groups which were observed immediately and after 6, 12 and 24 h post-shock. Both the constitutive (predominantly) and the inducible forms of eHSP70 were observed in the plasma of heat shocked animals, being the times 12 and 24 h those in which the highest concentrations were noted. Our studies suggest that in vivo produces eHSP70 change in plasma glucose tolerance, greater hyperglycemia when glucose tolerance test is performed 12 h after heat shock, and this effect was abrogated by anti-HSP70 antibody administration (i.p.) and independent of de novo synthesis of HSP70. Moreover, the presence of eHSP70 (HS-induced or injected) in the plasma or in soleus muscle incubations elicits lower glucose uptake from the skeletal muscle. Isolated muscle studies indicate that eHSP70 inhibiting effect over glucose uptake is insulin-dependent. However, although evidence suggests a role for HSP70 binding to insulin over glucose offering, it cannot be discarded the possibility that eHSP70 actions are due to their binding to specific HSP70 membrane receptors within the muscle cell, and/or that are part of a stress response in which the counter-regulatory hormone are involved. In short, the results indicate that eHSP70 plasma is able to bind to insulin, causing impaired glucose tolerance and decrease glucose uptake by skeletal muscle, increasing blood glucose availability.

A proteína de choque térmico de 70kDa (HSP70) é uma chaperona fundamental nos processos de reparo celular, não apenas em condição de hipertermia como também em outras alterações metabólicas. O exercício físico é capaz de induzir a síntese de HSP70 uma vez que estimula o metabolismo oxidativo aumentando a produção de espécies reativas de oxigênio. O presente estudo teve como objetivo avaliar o perfil antropométrico e nutricional de mulheres atletas de handebol, além de investigar se existe correlação entre os níveis de estradiol e o estado redox com o imunoconteúdo de HSP70 em linfócitos do plasma, no decorrer de um ano de treinamento físico. O estudo iniciou com a participação de 47 jogadoras, sendo que trinta atletas (entre 12 e 24 anos) cumpriram todas as etapas e foram incluídas na análise. As coletas foram realizadas em três períodos distintos: início, metade (após 4 meses de treino) e final da temporada de treinos. As atletas foram divididas em dois grupos de acordo com os níveis de estradiol: Estradiol Baixo (EB) (≥30 pg·mL-1) e Estradiol Normal (EN) (30–330 pg·mL-1). Neste estudo não observamos diferença na atividade da superóxido dismutase (SOD), no conteúdo de proteínas oxidadas, nem nos níveis de HSP70 extracelular (eHSP70), em nenhum período do estudo. A atividade da catalase (CAT) aumentou na metade da temporada no grupo EN. Foram observados menores níveis de grupamentos tiós reduzidos (SH) no grupo EN e menores níveis de peroxidação lipídica (TBARS) na metade e no final da temporada em ambos os grupos, havendo uma correlação inversa significativa entre o consumo de vitamina C e os níveis de dano oxidativo a lipídios. O imunoconteúdo de HSP70 intracelular (iHSP70) no grupo EN aumentou na metade e no final da temporada, enquanto que no grupo EB o conteúdo de iHSP70 mostrou-se elevado desde o início da temporada. Nossos resultados sugerem que o estradiol plasmático pode ter um importante papel na proteção ao dano oxidativo, e que os níveis de iHSP70, proteína considerada um biomarcador de inflamação, pode ser alterado tanto pelo estradiol, quanto pelo estresse oxidativo induzido pelo exercício. / The heat shock protein of 70kDa (HSP70) is a chaperone essential for cellular repair processes, not only on the condition of hyperthermia, but also in other metabolic challenges. Physical exercise is capable to induce an increase in the HSP70 synthesis once it augments oxidative metabolism increasing production of reactive oxygen species. In the current study we investigated whether a relationship between estradiol levels and parameters related to the redox environment and the immunocontent of HSP70 in lymphocytes and plasma of women handball players throughout 1 year a year of physical training, as well as anthropometric profile and nutritional status. Forty-seven female handball athletes (12 – 24 years old) started participating in the study and thirty completed all stages of the research, and were included in the study. The samples were taken at three different periods: Season beginning, mead-season (after four months of training) and season end. The athletes were divided into two groups according to levels of estradiol: low estradiol (≥30 pg·mL-1) (LE) and normal estradiol (30–330 pg·mL-1) (NE). In this study no changes were detected in SOD activity, protein carbonyl and extracellular HSP70 (eHSP70) in any study period. Catalase (CAT) activity increased in the middle of the season in NE group. Lower levels of reduced thiol (SH) were observed in the NE group and lower levels of uric acid in both groups (LE and NE) at the end of the season. The levels of thiobarbituric acid reactive species (TBARS) were lower in the middle and end of the season in both groups, e found a significant inverse correlation between the consumption of vitamin C and lipid peroxidation (TBARS). The immunocontent of intracellular HSP70 (iHSP70) in the NE group increased in the middle and the end of the season, whereas in group LE, iHSP70 was higher since the beginning of the season. Our results suggest that estradiol plasma levels can play an important role throughout the exercise training season and that the iHSP70, a biomarker of inflammation, is affected by both estradiol and oxidative stress induced by exercise.

Situações de estresse metabólico e térmico deflagram a expressão celular e liberação para o plasma, de proteínas de choque térmico da família de 70 kDa (HSP70) para desempenhar função de defesa-chave em um processo conhecido como resposta ao choque térmico ou ao estresse, que as caracterizam como proteína citoprotetora. Estudos indicam a ocorrência de HSP70 extracelular (eHSP70) na circulação por até 24 h após desafios como o exercício físico, cuja liberação dá-se por tecidos do território hepatoesplâncnico, em uma resposta mediada por receptores α-adrenérgicos. Esta liberação de HSP70 para o plasma é atenuada pela ingestão de glicose. Estas evidências levaram-nos a supor que a liberação de eHSP70 em resposta ao estresse possa ter um papel fisiológico na regulação da glicemia, a qual é afetada por estímulo simpático. Neste trabalho, os resultados dos experimentos in silico indicaram a possibilidade de ancoragem e interação estável entre a HSP70 e a molécula de insulina. Ensaios de imunoprecipitação de insulina com amostras de plasma de animais submetidos ao tratamento de insulina Regular e Detemir revelaram HSP70 coimunoprecipitada especialmente em situação de jejum severo. Parte destas amostras foi submetida à quantificação de glicemia, insulinemia e HSP72 circulante, cujos resultados evidenciam elevada concentração desta proteína de choque térmico por ocasião de hipoglicemia induzida por insulina. Parte dos estudos in vivo deste trabalho foram desenvolvidos com ratos submetidos ao choque térmico agudo (uma sessão de 15 minutos) com hipertermia induzida (41,5 °C) ou com injeção de HSP70 (HSPA1A) exógena, com grupos experimentais organizados em grupos Controle (C) e Choque Térmico (HS) e, em tempos experimentais: imediatamente (tempo zero), 6, 12 e 24 h pós-choque. Foi observada a ocorrência das formas constitutiva e induzível de eHSP70 no plasma de animais submetidos ao choque térmico com valor maior nos períodos de 12 e 24 h pós-choque. Nossos estudos mostram que, in vivo, a eHSP70 plasmática produz alteração na tolerância à glicose, com maior hiperglicemia, quando o teste é realizado 12 h após o choque térmico. Este efeito foi bloqueado pela administração (i.p.) de anticorpo anti-HSP70 e se mostrou independente da síntese de novo de HSP70. Ainda, a presença de eHSP70 plasmática (induzida por choque térmico ou administrada via endovenosa) causa menor captação de glicose pelo músculo esquelético. Ensaios com músculo isolado indicam que o efeito inibidor da eHSP70 sobre a captação de glicose é dependente de insulina. Apesar das evidências de que a ligação HSP70-insulina no plasma tenha implicação sobre a oferta de glicose, não se pode descartar também a possibilidade de que as ações da eHSP70 estejam ocorrendo pela sua ligação a receptores específicos, e/ou de que sejam parte de uma resposta ao estresse na qual estejam implicados os hormônios contrarregulatórios. Em suma, os resultados indicam que a eHSP70 plasmática é capaz de se ligar- à insulina, provocar tolerância alterada à glicose e diminuir a captação de glicose pelo músculo esquelético, aumentando a disponibilidade de glicose circulante. / Metabolically stressful situations trigger the expression and release of the 70 kDa (HSP70) as an essential protective response, in a process known as heat shock (HS) response or stress response. HSP70 is characterized as cytoprotector entities. Previous studies indicate the occurrence of extracellular HSP70 (eHSP70) in the circulation up to 24 h after stressful challenges, such as physical exercise, and that such eHSP70 liberation comes from hepatosplancnic tissues, in an 1-adrenergic dependent response, which is attenuated by glucose ingestion. The above findings led us to suppose that stress-elicited eHSP70 release could play a physiological role on glycemic control, which are affected by sympathetic stimuli. In this work, in silico experiments indicated the possibility of docking and stable interaction between HSP70 and the insulin molecule. Immunoprecipitation of plasma samples revealed that HSP70 co-immunoprecipitates with insulin, especially under severe fast. Part of these samples was subjected to quantification of glucose, insulin and circulating HSP72, whose results show high concentration of heat shock protein during severe hypoglycemia induced by insulin. Part of the in vivo studies from this work were also carried out in healthy rats submitted to an acute (15 min) HS (41.5 °C) session or in HSP70 (HSPA1A)-injected rats, assigned into control (C) and HS groups which were observed immediately and after 6, 12 and 24 h post-shock. Both the constitutive (predominantly) and the inducible forms of eHSP70 were observed in the plasma of heat shocked animals, being the times 12 and 24 h those in which the highest concentrations were noted. Our studies suggest that in vivo produces eHSP70 change in plasma glucose tolerance, greater hyperglycemia when glucose tolerance test is performed 12 h after heat shock, and this effect was abrogated by anti-HSP70 antibody administration (i.p.) and independent of de novo synthesis of HSP70. Moreover, the presence of eHSP70 (HS-induced or injected) in the plasma or in soleus muscle incubations elicits lower glucose uptake from the skeletal muscle. Isolated muscle studies indicate that eHSP70 inhibiting effect over glucose uptake is insulin-dependent. However, although evidence suggests a role for HSP70 binding to insulin over glucose offering, it cannot be discarded the possibility that eHSP70 actions are due to their binding to specific HSP70 membrane receptors within the muscle cell, and/or that are part of a stress response in which the counter-regulatory hormone are involved. In short, the results indicate that eHSP70 plasma is able to bind to insulin, causing impaired glucose tolerance and decrease glucose uptake by skeletal muscle, increasing blood glucose availability.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-09T23:35:51Z No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-15T22:34:21Z (GMT) No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) / Made available in DSpace on 2016-08-15T22:34:21Z (GMT). No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) Previous issue date: 2015-04-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Nas Am?ricas, a infec??o por Leishmania tem como principal agente etiol?gico Leishmania infantum. Nos ?ltimos 30 anos o padr?o de distribui??o das leishmanioses tem mudado substancialmente e a doen?a tem apresentado um perfil emergente na periferia dos grandes centros urbanos. A infec??o por Leishmania pode evoluir com um amplo espectro cl?nico desde o acometimento da pele, mucosas e v?sceras. Dos indiv?duos infectados por L. infantum apenas 10% desenvolvem a doen?a, sabe-se que 90% da infec??o humana ? assintom?tica e diversos fatores est?o envolvidos no curso da infec??o. Os principais fatores envolvidos no desenvolvimento da doen?a s?o a resposta imune do hospedeiro, a esp?cie e o conte?do g?nico do parasita. O sequenciamento dos isolados de Leishmania poderia aumentar a compreens?o acerca da sintomatologia dos indiv?duos. Dessa forma, o objetivo desse estudo foi avaliar a diversidade gen?tica de cepas de Leishmania circulantes entre humanos, sintom?ticos e assintom?ticos e c?es de ?reas end?micas do Rio Grande do Norte. A variabilidade gen?tica de um grupo de amostras de humanos e caninos com a doen?a visceral, doen?a cut?nea e infec??o assintom?tica foi avaliado com os marcadores moleculares hsp70 e ITS1. O genoma completo de 20 isolados de Leishmania oriundos de humanos, sintom?ticos e assintom?ticos, e c?es foram sequenciados para avaliar a diversidade dessas amostras. Os fragmentos amplificados de hsp70 e ITS1 das amostras e foram analisados e montadas utilizando o pacote Phred/Phrap. Os dendogramas foram constru?dos aplicando o m?todo neighbor joining com 500 bootstraps, seguido das infer?ncias sobre as rela??es entre as variantes de Leishmania. As sequ?ncias dos 20 isolados brasileiros foram mapeadas com o genoma de refer?ncia Leishmania infantum JPCM5, usando o programa Bowtie2, com identifica??o de 36 contigs. As informa??es dos SNPs v?lidos foram utilizadas na PCA. Os SNPs foram visualizados pelo Geneious 7.1 e IGV. As anota??es do genoma foram ent?o transferidas para seus respectivos cromossomos e visualizadas utilizando o Geneious. As sequ?ncias consenso de todos os cromossomos (com m?nimo de 75% das reads com a mesma base) foram alinhadas usando Mauve. As ?rvores filogen?ticas foram calculadas de acordo com c?lculos de m?xima verossimilhan?a e modelos JTT. Como resultados obtivemos que hsp70 e ITS1 n?o foram capazes de definir variabilidade gen?tica entre os isolados de humanos e c?es; nem para os isolados de cultura e de sangue perif?rico, oriundos de um mesmo paciente.O sequenciamento gen?mico dos 20 isolados brasileiros revelou uma forte rela??o entre as cepas de Leishmania circulantes em no Rio Grande do Norte. Os isolados da Grande Natal de humanos e caninos permaneceram agrupados em todas as an?lises, sugerindo que existe proximidade genot?pica e geogr?fica entre os isolados. As amostras isoladas na d?cada de 1990 apresentaram uma maior diversidade genot?pica quando comparadas as amostras recentemente isoladas. De forma geral, n?o encontramos correla??o entre as formas cl?nicas sintom?ticas e assintom?ticas e o conte?do g?nico dos isolados brasileiros de Leishmania. / Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector?s species and the parasite?s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

A proteína de choque térmico de 70kDa (HSP70) é uma chaperona fundamental nos processos de reparo celular, não apenas em condição de hipertermia como também em outras alterações metabólicas. O exercício físico é capaz de induzir a síntese de HSP70 uma vez que estimula o metabolismo oxidativo aumentando a produção de espécies reativas de oxigênio. O presente estudo teve como objetivo avaliar o perfil antropométrico e nutricional de mulheres atletas de handebol, além de investigar se existe correlação entre os níveis de estradiol e o estado redox com o imunoconteúdo de HSP70 em linfócitos do plasma, no decorrer de um ano de treinamento físico. O estudo iniciou com a participação de 47 jogadoras, sendo que trinta atletas (entre 12 e 24 anos) cumpriram todas as etapas e foram incluídas na análise. As coletas foram realizadas em três períodos distintos: início, metade (após 4 meses de treino) e final da temporada de treinos. As atletas foram divididas em dois grupos de acordo com os níveis de estradiol: Estradiol Baixo (EB) (≥30 pg·mL-1) e Estradiol Normal (EN) (30–330 pg·mL-1). Neste estudo não observamos diferença na atividade da superóxido dismutase (SOD), no conteúdo de proteínas oxidadas, nem nos níveis de HSP70 extracelular (eHSP70), em nenhum período do estudo. A atividade da catalase (CAT) aumentou na metade da temporada no grupo EN. Foram observados menores níveis de grupamentos tiós reduzidos (SH) no grupo EN e menores níveis de peroxidação lipídica (TBARS) na metade e no final da temporada em ambos os grupos, havendo uma correlação inversa significativa entre o consumo de vitamina C e os níveis de dano oxidativo a lipídios. O imunoconteúdo de HSP70 intracelular (iHSP70) no grupo EN aumentou na metade e no final da temporada, enquanto que no grupo EB o conteúdo de iHSP70 mostrou-se elevado desde o início da temporada. Nossos resultados sugerem que o estradiol plasmático pode ter um importante papel na proteção ao dano oxidativo, e que os níveis de iHSP70, proteína considerada um biomarcador de inflamação, pode ser alterado tanto pelo estradiol, quanto pelo estresse oxidativo induzido pelo exercício. / The heat shock protein of 70kDa (HSP70) is a chaperone essential for cellular repair processes, not only on the condition of hyperthermia, but also in other metabolic challenges. Physical exercise is capable to induce an increase in the HSP70 synthesis once it augments oxidative metabolism increasing production of reactive oxygen species. In the current study we investigated whether a relationship between estradiol levels and parameters related to the redox environment and the immunocontent of HSP70 in lymphocytes and plasma of women handball players throughout 1 year a year of physical training, as well as anthropometric profile and nutritional status. Forty-seven female handball athletes (12 – 24 years old) started participating in the study and thirty completed all stages of the research, and were included in the study. The samples were taken at three different periods: Season beginning, mead-season (after four months of training) and season end. The athletes were divided into two groups according to levels of estradiol: low estradiol (≥30 pg·mL-1) (LE) and normal estradiol (30–330 pg·mL-1) (NE). In this study no changes were detected in SOD activity, protein carbonyl and extracellular HSP70 (eHSP70) in any study period. Catalase (CAT) activity increased in the middle of the season in NE group. Lower levels of reduced thiol (SH) were observed in the NE group and lower levels of uric acid in both groups (LE and NE) at the end of the season. The levels of thiobarbituric acid reactive species (TBARS) were lower in the middle and end of the season in both groups, e found a significant inverse correlation between the consumption of vitamin C and lipid peroxidation (TBARS). The immunocontent of intracellular HSP70 (iHSP70) in the NE group increased in the middle and the end of the season, whereas in group LE, iHSP70 was higher since the beginning of the season. Our results suggest that estradiol plasma levels can play an important role throughout the exercise training season and that the iHSP70, a biomarker of inflammation, is affected by both estradiol and oxidative stress induced by exercise.

A proteína de choque térmico de 70kDa (HSP70) é uma chaperona fundamental nos processos de reparo celular, não apenas em condição de hipertermia como também em outras alterações metabólicas. O exercício físico é capaz de induzir a síntese de HSP70 uma vez que estimula o metabolismo oxidativo aumentando a produção de espécies reativas de oxigênio. O presente estudo teve como objetivo avaliar o perfil antropométrico e nutricional de mulheres atletas de handebol, além de investigar se existe correlação entre os níveis de estradiol e o estado redox com o imunoconteúdo de HSP70 em linfócitos do plasma, no decorrer de um ano de treinamento físico. O estudo iniciou com a participação de 47 jogadoras, sendo que trinta atletas (entre 12 e 24 anos) cumpriram todas as etapas e foram incluídas na análise. As coletas foram realizadas em três períodos distintos: início, metade (após 4 meses de treino) e final da temporada de treinos. As atletas foram divididas em dois grupos de acordo com os níveis de estradiol: Estradiol Baixo (EB) (≥30 pg·mL-1) e Estradiol Normal (EN) (30–330 pg·mL-1). Neste estudo não observamos diferença na atividade da superóxido dismutase (SOD), no conteúdo de proteínas oxidadas, nem nos níveis de HSP70 extracelular (eHSP70), em nenhum período do estudo. A atividade da catalase (CAT) aumentou na metade da temporada no grupo EN. Foram observados menores níveis de grupamentos tiós reduzidos (SH) no grupo EN e menores níveis de peroxidação lipídica (TBARS) na metade e no final da temporada em ambos os grupos, havendo uma correlação inversa significativa entre o consumo de vitamina C e os níveis de dano oxidativo a lipídios. O imunoconteúdo de HSP70 intracelular (iHSP70) no grupo EN aumentou na metade e no final da temporada, enquanto que no grupo EB o conteúdo de iHSP70 mostrou-se elevado desde o início da temporada. Nossos resultados sugerem que o estradiol plasmático pode ter um importante papel na proteção ao dano oxidativo, e que os níveis de iHSP70, proteína considerada um biomarcador de inflamação, pode ser alterado tanto pelo estradiol, quanto pelo estresse oxidativo induzido pelo exercício. / The heat shock protein of 70kDa (HSP70) is a chaperone essential for cellular repair processes, not only on the condition of hyperthermia, but also in other metabolic challenges. Physical exercise is capable to induce an increase in the HSP70 synthesis once it augments oxidative metabolism increasing production of reactive oxygen species. In the current study we investigated whether a relationship between estradiol levels and parameters related to the redox environment and the immunocontent of HSP70 in lymphocytes and plasma of women handball players throughout 1 year a year of physical training, as well as anthropometric profile and nutritional status. Forty-seven female handball athletes (12 – 24 years old) started participating in the study and thirty completed all stages of the research, and were included in the study. The samples were taken at three different periods: Season beginning, mead-season (after four months of training) and season end. The athletes were divided into two groups according to levels of estradiol: low estradiol (≥30 pg·mL-1) (LE) and normal estradiol (30–330 pg·mL-1) (NE). In this study no changes were detected in SOD activity, protein carbonyl and extracellular HSP70 (eHSP70) in any study period. Catalase (CAT) activity increased in the middle of the season in NE group. Lower levels of reduced thiol (SH) were observed in the NE group and lower levels of uric acid in both groups (LE and NE) at the end of the season. The levels of thiobarbituric acid reactive species (TBARS) were lower in the middle and end of the season in both groups, e found a significant inverse correlation between the consumption of vitamin C and lipid peroxidation (TBARS). The immunocontent of intracellular HSP70 (iHSP70) in the NE group increased in the middle and the end of the season, whereas in group LE, iHSP70 was higher since the beginning of the season. Our results suggest that estradiol plasma levels can play an important role throughout the exercise training season and that the iHSP70, a biomarker of inflammation, is affected by both estradiol and oxidative stress induced by exercise.