Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends upon survival in high bile salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how L. monocytogenes responds to bile salts under anaerobic conditions. The study found resistance to be strain specific and not dependent upon virulence. Changes in the expressed proteome were analyzed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. A general response among virulent and avirulent strains found significant alterations in intensity of cell wall associated proteins, DNA repair proteins, protein folding chaperones and oxidative response proteins. Strain viability was correlated with an initial osmotic stress response followed by strain specific proteins associated with biofilm formation in EGDe and a transmembrane efflux pump in F2365.

Naegleria fowleri are free-living ameboflagellates found in soil and freshwater habitats throughout the world that cause a fatal disease in humans called Primary Amoebic Meningoencephalitis (PAM). Mechanisms of host resistance or susceptibility to infection have not been fully elucidated, and possible treatment methods are still sub optimal. The disease is diagnosed using specific laboratory tests available in only a few laboratories in the United States. Because of the rarity of infection and difficulty in initial detection, more than often PAM is misdiagnosed. Therefore, it is very important to find causative marker for early detection of an infection. The purpose of this study is to create a proteomic signature map using two-dimensional gel electrophoresis (2-D gel) and recommend a subset of proteins that may be directly linked to the pathogenic state of N. fowleri. Replicates of 2-D Gels were created for both strains of N. fowleri and the proteomic templates from these gels were compared with each other. Scatter Plots were generated measuring the density of protein spots from 2-D gels being analyzed for each study. For each strains of N. fowleri, the 2-D gels from each study were compared within and compared between the two studies for reproducibility in data. The resulting correlation values for all of the Scatter Plots were greater or equal to 0.90. Finally, the representative proteomic template for axenically grown N. fowleri and mouse passaged N. fowleri were compared and the correlation value of 0.60 was observed. This confirmed our theory that these two strains or states of N. fowleri have very different protein expressions, and we were able to identify a subset of proteins, both over expressed and newly synthesized, that may be linked to the highly pathogenic state of N. fowleri.

DYRK1A is a protein kinase encoded by a gene implicated in Down syndrome pathogenesis. Loss of DYRK1A could promote oncogenic transformation. However, the regulation and substrates of DYRK1A are not fully understood. MudPIT proteomic analysis revealed novel DYRK1A interacting proteins with poorly characterized or even unknown functions. Therefore, the aim of this thesis was to understand the function of DYRK1A through the characterization of its interacting proteins. To achieve this aim, we established stable cell lines expressing these proteins and confirmed the interactions between DYRK1A and seven candidate binding partners. Furthermore, we found that all novel DYRK1A-interacting proteins also bind DCAF7, a previously reported DYRK1A-binding scaffold protein that binds to the N-terminus of DYRK1A. Using cyto-nuclear fractionation and immunostaining we found that DYRK1A-interacting proteins were present in different cellular compartments, suggesting that DYRK1A could play distinct roles in the cell depending on its localization. DYRK1A has been shown to regulate cell proliferation and actin cytoskeleton therefore we used cell proliferation assays and actin staining to determine the role of DYRK1A-interacting proteins in these processes. Here we report functional characterization of the interacting partners of DYRK1A and present cell-based models that will help to understand the function and regulation of this important protein kinase.

Multiple endocrine neoplasia syndrome type 1 (MEN1) is an autosomal dominantly inherited disease, which is described as an association of tumors mainly in endocrine organs, including pancreas and adrenal glands. Pancreatic neuroendocrine tumors (PNETs) are the most common cause of death in MEN1 patients. More than one third of the MEN1 patients also develop enlargement of the adrenals. MEN1 is caused by a germline mutation of MEN1 gene, a tumor suppressor gene that is located on the human chromosome 11. As noticed, the MEN1 related tumors often develop prior to inactivation of both wild type alleles, indicating MEN1 haploinsufficiency. In this thesis, I utilized a conventional Men1 mouse model that has the phenotype mimicking the human MEN 1 traits, in order to investigate MEN1 tumorigenesis in endocrine pancreas and adrenal glands.   The microvascular aberrations contributing to development and maintenance of PNETs were characterized. The increased vascular density of PNETs developed in the Men1 mice was paralleled by an early and extensive redistribution of pericytes within endocrine tissue. These morphological alterations were supported by fine-tuned variations in expression of several angiogenic regulators  (VEGF, FGF and PDGF) and were further potentiated by hypoxia. Vascular reactivity and blood perfusion of tumor arterioles were significantly altered in response to glucose and L-nitro-arginine methyl ester. Investigation of adrenals from10-month-old Men1 mice showed 681 proteins in mass spectrometry data sets, in which 52 proteins were commonly found in the Men1+/+ and Men1+/- adrenals, and the differential expression between the genotypes reached significant levels. Prdx3, catalyzing the reduction of oxidative stress to cell survival, is one of the overexpressed proteins. Some proteins belonging to the PPARα pathway, e.g. ACLY were also overexpressed. Subsequent microRNA (miRNA) profiling analysis of adrenals from the same age group revealed 31 miRNAs whose expression was significantly altered in comparison between the genotypes. The tumor suppressor miRNAs, miR-486, miR-330 and miR-214, were significantly downregulated in Men1+/- adrenals. The latter, miR-214, is known to inhibit ACLY expression. This finding was in concordance with the proteomic analysis. The oncogene miRNAs, miR-132 and miR-494, were significantly enhanced in the Men1+/- adrenals. Gene ontology analysis demonstrated overrepresentation of the miRNA-targeted genes that are involved in nucleic acid metabolism, vasculature development, angiogenesis, and transcription. Together, these finding after validation in humans may be exploited to improve MEN1 cancer treatment.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-05-18T17:10:51Z No. of bitstreams: 2 Dissertação - Alessandro Fernandes Vaz - 2014.pdf: 2674595 bytes, checksum: 4be2a3df9927e868ec83389dd6ab3365 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-05-18T17:13:28Z (GMT) No. of bitstreams: 2 Dissertação - Alessandro Fernandes Vaz - 2014.pdf: 2674595 bytes, checksum: 4be2a3df9927e868ec83389dd6ab3365 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-05-18T17:13:28Z (GMT). No. of bitstreams: 2 Dissertação - Alessandro Fernandes Vaz - 2014.pdf: 2674595 bytes, checksum: 4be2a3df9927e868ec83389dd6ab3365 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides spp. is the etiological agent of paracoccidioidomycosis, the most important endemic systemic mycosis in Latin America. Paracoccidioides spp. is a dimorphic fungus; mycelia is found in soil at temperatures below 25ºC, while in host tissues, and temperature around 36-37ºC the fungus takes the yeast form. Infection begins with the inhalation of conidia or mycelia propagules that upon reaching the host lungs differentiate into yeast, establishing disease. The morphological transition from mycelia-to-yeast is involved in the virulence of this pathogen and this aspect of morphogenesis deserves special attention due to its relevance to the fungal virulence. In the present study, we employed proteomic strategies using liquid chromatography coupled to mass spectrometry to evaluate the differential proteomic profile of cells ongoing transition from mycelia-to-yeast after 22 h of temperature shift from 22ºC to 36ºC (P. brasiliensis Pb-18 phylogenetic lineage S1). Nine hundred and ninety-one proteins were identified (350 in the mycelia, 288 in the transition and 353 in the yeast), and 251 were differentially regulated. The analysis of the functional categories to which those proteins belong provided us a comprehension on the metabolic reprogramming that occurs during the cell differentiation process, providing putative virulence factors. / Paracoccidioides spp. é o agente etiológico da paracoccidioidomicose, a mais importante micose sistêmica endêmica da América Latina. Paracoccidioides spp. é um fungo dimórfico, que apresenta alteração morfológica de acordo com a temperatura do ambiente. A forma miceliana é encontrada no solo a temperaturas inferiores a 25 ºC, enquanto que em tecidos do hospedeiro e sob temperaturas de 36-37 ºC o fungo assume forma leveduriforme. A infecção se inicia com a inalação de conídios presentes na forma filamentosa do fungo, os quais ao atingirem os pulmões se diferenciam na forma leveduriforme, estabelecendo a paracoccidioidomicose. A transição de micélio para levedura está envolvida na virulência desse patógeno e esse aspecto da morfogênese do fungo não está totalmente compreendido. No presente estudo, foi utilizada a cromatografia líquida acoplada à espectrometria de massas (nanoUPLC-MSE) para avaliar o perfil proteômico durante a transição de micélio para levedura após 22 h da mudança da temperatura de 22ºC para 36ºC (P. brasiliensis – isolado Pb18). Um total de 991 proteínas foram identificadas (350 em micélio, 288 na transição e 353 em levedrua) e 251 foram diferencialmente expressas durante o processo de diferenciação celular. Entre as proteínas que foram abundantes em micélio estão as subunidades da ATPase que participa na cadeia transportadora de elétrons e enzimas envolvidas na fermentação alcoólica. Proteínas relacionadas à via glicolítica e alguns potenciais fatores de virulência começam sua acumulação após 22 h do início da transição morfológica. Em levedura enzimas relacionadas com a glicólise e a beta-oxidação tiveram alta expressão. As análises das categorias funcionais a que essas proteínas pertencem, forneceram uma melhor compreensão da reorganização metabólica que ocorre durante a transição morfológica de micélio para levedura, incluindo possíveis fatores de virulência.

O flúor é comprovadamente um agente terapêutico contra a cárie dentária, porém, quando consumido de maneira inadequada pode causar reações indesejáveis, tanto crônicas como agudas. A proteômica é uma ferramenta que permite analisar o perfil proteico completo dos sistemas biológicos, permitindo um melhor entendimento da fisiologia normal do organismo, bem como dos mecanismos de doenças, descoberta de biomarcadores para detecção precoce de doenças, identificação de novas terapias e descobertas de fármacos. Assim, o presente estudo teve como trabalho realizar um estudo proteômico diferencial em amostras de tecido renal de ratos submetidos à intoxicação aguda por fluoreto. Para isso, 27 animais, receberam desde o desmame água deionizada e ração AIN-93 por 50 dias. Ao completarem 75 dias de vida, e, após jejum por 12 horas, os animais receberam via gástrica as seguintes doses agudas de F: 0 (controle), 50 e 100 mgF/Kg peso corporal. Após 2 horas foram, os animais foram anestesiados e tiveram o sangue e os rins coletados. Inicialmente foi realizada a dosagem de flúor no plasma e tecidos renais, após difusão facilitada por hexamethyldisiloxano. Em um segundo momento, as proteínas do tecido renal foram extraídas e submetidas à eletroforese bidimensional. Os géis obtidos foram analisados através do software ImageMaster 2D-platinum v. 7.0. Os spots que se apresentaram diferencialmente expressos foram submetidos à identificação por espectrometria de massas. As proteínas identificadas foram classificadas em 5 categorias funcionais. A categoria metabolismo e energia reuniu a maior parte das proteínas (40%), seguida pelas categorias de transporte e processos celulares com 20% e 13% respectivamente. Na categoria estrutura e organização estrutural, que reúne proteínas com funções relacionadas ao citoesqueleto e membrana celular, foram identificadas 17% das proteínas. Por fim, os 10% restantes das proteínas pertenciam à categoria vias de informação, que abriga proteínas envolvidas nos processos de síntese/degradação de DNA e RNA. Com base nos resultados obtidos pode se concluir que doses agudas de fluoreto alteram a expressão a nível renal de diversas proteínas relacionadas a distintos processos celulares. / Fluoride has been widely used in dentistry as a caries prophylactic agent. However, if consumed in high concentrations, it can cause side effects as dental fluorosis, skeletal fluorosis and, in extreme situations, death. The recent development of proteomic techniques has allowed the analysis of the entire protein profile of biological systems, contributing to the understanding of the normal physiology of the organism, as well as the mechanisms of diseases and the investigation of biomarkers for their early detection, identification of new therapies and drugs. This study used proteomic techniques to analyze the differential protein expression in kidney of rats submitted to acute fluoride treatments. Three groups of five 75-day-old Wistar rats received, by gastric gavage, the following single doses of fluoride: 0 (control), 50 and 100 mgF/Kg body weight. After the treatments, the animals were killed and the left kidney and plasma were collected for fluoride analysis. For proteomic analysis, the right kidney was collected. Fluoride in plasma and renal tissues was analyzed with the electrode, after hexamethyldisiloxanefacilitated diffusion. Proteins from kidney were profiled by two-dimensional gel electrophoresis. Gels from control and treated groups were digitalized using the ImageScanner III and analyzed with ImageMaster 2D Platinum version 7.0 for statistical differences (ANOVA, p <0.05). The proteins identified were classified into five functional categories. The category of metabolism and energy had the majority of the proteins (40%), followed by the categories of \"transport\" and cell processes, with 20% and 13% of the proteins, respectively. In the category \"structure and cell organization, which brings together proteins with functions related to the cytoskeleton and plasma membrane were identified 17% of the protein. Finally, 10% of the proteins belonged to the category \"information pathways\", which comprises the proteins involved in the processes of synthesis and degradation of DNA and RNA. Based on the results obtained, it can be concluded that acute doses of fluoride alters the expression of several proteins related to different cellular processes in the kidneys.

Enquanto alguns trabalhos relatam que a administração crônica de F causa resistência à insulina, estudo recente do nosso grupo revelou que em animais com diabetes previamente induzido por estreptozotocina, a administração crônica de baixas doses de F aumenta a sensibilidade à insulina, por interferir em vias metabólicas musculares e hepáticas. Entretanto, o diabetes induzido por estreptozotocina causa alterações metabólicas diferentes do diabetes tipo 1. Desta forma, seria interessante verificar se o aumento da sensibilidade à insulina induzido por baixas doses de F também ocorreria utilizando um modelo de diabetes que mimetiza melhor o diabetes tipo 1 (camundongos NOD non-obese diabetic). Com base no exposto, o presente estudo investigou, em camundongos NOD recémdesmamados e expostos cronicamente a doses de F na água de beber que simulam a ingestão de F pela água artificial ou naturalmente fluoretada, se ocorrem alterações relacionadas à resistência à insulina, bem como na expressão de proteínas hepáticas e musculares. Para tanto, 24 camundongos NOD (não obesos diabéticos), obtidos imediatamente após o desmame, foram divididos em 3 grupos, de acordo com a concentração de F presente na água de beber (0, 10 ou 50 ppm), que foi administrada por um período de 21 dias. Decorrido o período experimental, os animais foram eutanasiados, sendo coletado o sangue para análise de F (eletrodo íon específico), glicose (método da glicose-oxidase) e insulina (ELISA), bem como o fígado e músculo gastrocnêmio, para análise proteômica quantitativa livre de marcadores (software Protein Linx Global Service). Os dados foram analisados por ANOVA e teste de Tukey, ou teste de Kruskal-Wallis e teste de Dunn (p<0,05). Apenas o grupo tratado com 50 ppm F apresentou concentração plasmática de F significativamente maior que o controle. Os animais tratados com 10 ppm F tiveram glicemia significativamente menor que o grupo controle, mas não houve diferença significativa entre os grupos em relação à insulinemia. A % de função das células pancreáticas foi significativamente maior no grupo tratado com 10 ppm F, quando comparado aos demais. A análise proteômica revelou alterações no perfil proteômico tanto do tecido muscular quanto do hepático. No tecido muscular, o grupo de 10 ppmF apresentou, em relação ao controle, expressão aumentada de proteínas envolvidas no metabolismo energético. Já o grupo de 50 ppm F, em relação ao controle, apresentou expressão aumentada de proteínas relacionadas à contração muscular, diferenciação do tecido adiposo marrom e apoptose. Para o tecido hepático, também foi observada expressão aumentada no grupo submetido a 10 ppm F em relação ao controle de proteínas envolvidas no metabolismo energético e síntese proteica, com destaque ainda para o aumento de isoformas de Glutathione S transferase, bem como de Heat shock-related 70 kDa protein 2. No grupo submetido a 50 ppmF foi observada alteração de proteínas envolvidas no metabolismo de espécies reativas de oxigênio e metabolismo energético. O aumento na expressão de proteínas antioxidantes, mediante tratamento com a baixa concentração de F, pode ajudar a explicar a proteção contra o desenvolvimento do diabetes, o que deve ser comprovado em estudos mecanísticos futuros. / While some studies report that chronic administration of fluoride (F) causes insulin resistance, a recent study in our group found that in animals with diabetes previously induced by streptozotocin diabetes, chronic low-dose F administration increases insulin sensitivity by interfering with metabolic pathways in muscle and liver. However, streptozotocin-induced diabetes causes different metabolic changes from type 1 diabetes. Thus, it would be interesting to see whether increased insulin sensitivity induced by low doses of F would also occur using a diabetes model that better mimics type1 diabetes (NOD mice - non-obese diabetic). Based on the foregoing, the present study investigated in NOD mice chronically exposed to doses of F in drinking water that simulate the ingestion of F by artificial or naturally fluoridated water, if there are changes related to insulin resistance as well as in the expression of liver and muscle proteins. Twenty-four NOD mice, obtained immediately after weaning, were divided into three groups, according to the concentration of F present in the drinking water (0, 10 or 50 ppm), which was administered by a period of 21 days. After the experimental period, the animals were euthanized and the blood was collected for analysis of F (ionspecific electrode), glucose (glucose oxidase method) and insulin (ELISA), as well as the liver and gastrocnemius muscle, for quantitative proteomic analysis (Protein Linx Global Service software). Data were analyzed by ANOVA and Tukey\'s test, or Kruskal- Wallis and Dunn\'s test (p <0.05). Only the group treated with 50 ppm F had plasma F concentration significantly higher than the control group. Animals treated with 10 ppm F had significantly lower glycemia than the control group, but there was no significant difference between the groups in relation to insulinemia. The % of pancreatic -cell function was significantly higher in the group treated with 10 ppm F compared to the others. Proteomic analysis revealed changes in the proteomic profile of both muscle and hepatic tissue. In the muscle tissue, the group of 10 ppm presented, in relation to the control, increased expression of proteins involved in energy metabolism. The group of 50 ppm F, in relation to 7control, presented increased expression of proteins related to muscle contraction, differentiation of brown adipose tissue and apoptosis. For hepatic tissue, increased expression was also observed in the group of 10 ppm F in relation to the control of proteins involved in energetic metabolism and protein synthesis, with emphasis also on the increase of Glutahione S transferase isoforms as well as Heat shock-related 70 kDa protein 2. In the group treated with 50 ppm F proteins related to ROS metabolism and energetic metabolism were altered. Increased expression of antioxidant proteins by treatment with low F concentration may help explain protection against the development of diabetes, which should be demonstrated in future mechanistic studies.

Enquanto alguns trabalhos relatam que a administração crônica de F causa resistência à insulina, estudo recente do nosso grupo revelou que em animais com diabetes previamente induzido por estreptozotocina, a administração crônica de baixas doses de F aumenta a sensibilidade à insulina, por interferir em vias metabólicas musculares e hepáticas. Entretanto, o diabetes induzido por estreptozotocina causa alterações metabólicas diferentes do diabetes tipo 1. Desta forma, seria interessante verificar se o aumento da sensibilidade à insulina induzido por baixas doses de F também ocorreria utilizando um modelo de diabetes que mimetiza melhor o diabetes tipo 1 (camundongos NOD non-obese diabetic). Com base no exposto, o presente estudo investigou, em camundongos NOD recémdesmamados e expostos cronicamente a doses de F na água de beber que simulam a ingestão de F pela água artificial ou naturalmente fluoretada, se ocorrem alterações relacionadas à resistência à insulina, bem como na expressão de proteínas hepáticas e musculares. Para tanto, 24 camundongos NOD (não obesos diabéticos), obtidos imediatamente após o desmame, foram divididos em 3 grupos, de acordo com a concentração de F presente na água de beber (0, 10 ou 50 ppm), que foi administrada por um período de 21 dias. Decorrido o período experimental, os animais foram eutanasiados, sendo coletado o sangue para análise de F (eletrodo íon específico), glicose (método da glicose-oxidase) e insulina (ELISA), bem como o fígado e músculo gastrocnêmio, para análise proteômica quantitativa livre de marcadores (software Protein Linx Global Service). Os dados foram analisados por ANOVA e teste de Tukey, ou teste de Kruskal-Wallis e teste de Dunn (p<0,05). Apenas o grupo tratado com 50 ppm F apresentou concentração plasmática de F significativamente maior que o controle. Os animais tratados com 10 ppm F tiveram glicemia significativamente menor que o grupo controle, mas não houve diferença significativa entre os grupos em relação à insulinemia. A % de função das células pancreáticas foi significativamente maior no grupo tratado com 10 ppm F, quando comparado aos demais. A análise proteômica revelou alterações no perfil proteômico tanto do tecido muscular quanto do hepático. No tecido muscular, o grupo de 10 ppmF apresentou, em relação ao controle, expressão aumentada de proteínas envolvidas no metabolismo energético. Já o grupo de 50 ppm F, em relação ao controle, apresentou expressão aumentada de proteínas relacionadas à contração muscular, diferenciação do tecido adiposo marrom e apoptose. Para o tecido hepático, também foi observada expressão aumentada no grupo submetido a 10 ppm F em relação ao controle de proteínas envolvidas no metabolismo energético e síntese proteica, com destaque ainda para o aumento de isoformas de Glutathione S transferase, bem como de Heat shock-related 70 kDa protein 2. No grupo submetido a 50 ppmF foi observada alteração de proteínas envolvidas no metabolismo de espécies reativas de oxigênio e metabolismo energético. O aumento na expressão de proteínas antioxidantes, mediante tratamento com a baixa concentração de F, pode ajudar a explicar a proteção contra o desenvolvimento do diabetes, o que deve ser comprovado em estudos mecanísticos futuros. / While some studies report that chronic administration of fluoride (F) causes insulin resistance, a recent study in our group found that in animals with diabetes previously induced by streptozotocin diabetes, chronic low-dose F administration increases insulin sensitivity by interfering with metabolic pathways in muscle and liver. However, streptozotocin-induced diabetes causes different metabolic changes from type 1 diabetes. Thus, it would be interesting to see whether increased insulin sensitivity induced by low doses of F would also occur using a diabetes model that better mimics type1 diabetes (NOD mice - non-obese diabetic). Based on the foregoing, the present study investigated in NOD mice chronically exposed to doses of F in drinking water that simulate the ingestion of F by artificial or naturally fluoridated water, if there are changes related to insulin resistance as well as in the expression of liver and muscle proteins. Twenty-four NOD mice, obtained immediately after weaning, were divided into three groups, according to the concentration of F present in the drinking water (0, 10 or 50 ppm), which was administered by a period of 21 days. After the experimental period, the animals were euthanized and the blood was collected for analysis of F (ionspecific electrode), glucose (glucose oxidase method) and insulin (ELISA), as well as the liver and gastrocnemius muscle, for quantitative proteomic analysis (Protein Linx Global Service software). Data were analyzed by ANOVA and Tukey\'s test, or Kruskal- Wallis and Dunn\'s test (p <0.05). Only the group treated with 50 ppm F had plasma F concentration significantly higher than the control group. Animals treated with 10 ppm F had significantly lower glycemia than the control group, but there was no significant difference between the groups in relation to insulinemia. The % of pancreatic -cell function was significantly higher in the group treated with 10 ppm F compared to the others. Proteomic analysis revealed changes in the proteomic profile of both muscle and hepatic tissue. In the muscle tissue, the group of 10 ppm presented, in relation to the control, increased expression of proteins involved in energy metabolism. The group of 50 ppm F, in relation to 7control, presented increased expression of proteins related to muscle contraction, differentiation of brown adipose tissue and apoptosis. For hepatic tissue, increased expression was also observed in the group of 10 ppm F in relation to the control of proteins involved in energetic metabolism and protein synthesis, with emphasis also on the increase of Glutahione S transferase isoforms as well as Heat shock-related 70 kDa protein 2. In the group treated with 50 ppm F proteins related to ROS metabolism and energetic metabolism were altered. Increased expression of antioxidant proteins by treatment with low F concentration may help explain protection against the development of diabetes, which should be demonstrated in future mechanistic studies.

O recente desenvolvimento de técnicas proteômicas tem permitido a análise do perfil proteico completo dos sistemas biológicos, permitindo um melhor entendimento da fisiologia normal do organismo, bem como dos mecanismos de doenças, descoberta de biomarcadores para detecção precoce de doenças, identificação de novas terapias e descobertas de fármacos. No presente trabalho, a análise proteômica foi usada para auxiliar no entendimento dos mecanismos moleculares envolvidos na intoxicação induzida pelo fluoretono fígado de ratos e definir biomarcadores potenciais de toxicidade. Três grupos de ratos Wistar machos recém-desmamados (21 dias de vida) foram tratados ad libitum com água de beber contendo 0 (controle), 5 ou 50 mg/L de fluoreto, por 60 dias (n=6/grupo). Os animais foram eutanasiados e o fígado e o soro foram coletados. O fígado foi dividido em lobos, sendo que o esquerdo foi destinado à dosagem de fluoreto (eletrodo íon-sensível, após difusão facilitada por hexametildisiloxano), o direito à análise histológica (hematoxilina e eosina) e o mediano, à análise proteômica (eletroforese bidimensional associada a LC-MS/MS). Os dados foram analisados por ANOVA e teste de Tukey (p<0,05). Foi possível detectar uma dose-resposta em relação à ingestão para os níveis de fluoreto presentes no soro e no fígado houve diferença significativa entre os grupos que receberam 5 e 50 mg/L de fluoreto. A análise morfométrica histológica não revelou alterações nas estruturas celulares e exame o morfológico indicou inclusões lipídicas nos grupos 5 mg/L e 50 mg/L de fluoreto, mais intensas no último. A análise quantitativa de intensidade (software ImageMaster 2D-Platinum v 7.0), comparando a porcentagem de alteração de volume do spot protéico, revelou que 33, 44 e 29 spots aumentaram ou diminuíram sua expressão nos grupos controle X 5 mg/L, controle X 50 mg/L e 5 mg/L X 50 mg/L de fluoreto, respectivamente. Além disto, foram encontradas 18, 1 e 5 spots exclusivos nos grupos controle, 5 mg/L e 50 mg/L de fluoreto, respectivamente. Dentre os spots proteínas com expressão diferencial, foram identificadas 94 proteínas (72,3%). Em síntese, a exposição ao fluoreto alterou a expressão a nível hepático de proteínas pertencentes a todas as categorias funcionais, com predominância daquelas relacionadas ao metabolismo, sendo que as alterações mais pronunciadas foram observadas no grupo de 50 mg/L de fluoreto. Proteínas que tiveram sua expressão ausente mediante exposição ao fluoreto ou que tiveram sua expressão induzida por este elemento são potenciais biomarcadores para este tipo de exposição e devem ser melhor investigadas. Uma vez que se trata do primeiro trabalho envolvendo análise proteômica de fígado de animais expostos a diferentes doses de fluoreto, estes achados apontam importantes vias e processos celulares afetados no fígado pela exposição ao fluoreto, os quais devem ser melhor analisados em estudos futuros. / The recent development of proteomic techniques allowed the analysis of the whole proteomic profile of the biological systems, allowing the comprehension of the normal physiology, as well as of the mechanisms underlying diseases. It has also made easier the discovery of biomarkers of diseases, as well as the identification of new therapies and drugs. In the present study, proteomic analysis was used as a tool to help understanding the molecular mechanisms underlying chronic fluoride toxicity in rat liver and also to define potential biomarkers of toxicity. Three groups of weanling male Wistarrats (21 days) were treated ad libitum with drinking water containing 0 (control), 5 or 50 mg/L fluoride for 60 days (n=6/group). After euthanasia, liver and serum were collected. The liver was divided into lobes. The left lobe was used for fluoride analysis (ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion). The right lobe was used for histological analysis (hematoxylin and eosine) while the median was used for proteomic analysis (2D-PAGE associated with LC-MS/MS). Data were analysed by ANOVA and Tukeys test, (p<0.05). A dose-response relationship was observed for serum fluoride levels. In liver, a significant increase in fluoride levels was observed in the 50 mg/L group when compared with the 5 mg/L group. Morphometric analysis did not reveal alterations in the cellular structures. The morphological exam revealed macrovesicular lipid droplets in the 5 and 50 mg/L groups which were more intense in the later. Quantitative intensity analysis (software ImageMaster 2D-Platinum v 7.0) comparing the percentage of alteration of volume of protein spots revealed 33, 44 and 29 protein spots that had increase or decrease in expression in the groups control X 5 mg/L, control X 50 mg/L and 5 mg/L X 50 mg/L, respectively. In addition, 18, 1 and 5 protein spots were detected exclusively in groups control, 5 mg/L and 50 mg/L, respectively. From the differentially expressed spots, 94 proteins were satisfactorily identified (72.3%). Briefly, exposure to fluoride altered the expression of liver proteins belonging to all the functional categories, especially those related to metabolism. More pronounced alterations were seen for the 50 mg/L group. Proteins that were not detected upon exposure to fluoride or that had their expression induced in the presence of fluoride are potential biomarkers of toxicity and should be better investigated. Since this is the first study involving proteomic analysis of liver in rats exposed to different doses of fluoride, these findings indicate important pathways and cellular processes affected by exposure to this element that should be addressed in details in future studies.

A oxidação de proteínas é um fenômeno metabólico e a degradação de proteínas oxidativamente modificadas confere uma proteção para a célula, evitando acúmulo e a agregação das mesmas. A ineficiência na remoção destas proteínas está relacionada ao processo de envelhecimento e ao aparecimento de doenças neurodegenerativas. A unidade catalítica do proteassomo, denominada de 20S (PT20S), é a principal via de degradação de proteínas danificadas pela oxidação sem que haja gasto de ATP, acoplamento de subunidades regulatórias ou poli-ubiquitinação do substrato proteico. A unidade PT20 por sua vez, pode sofrer modificação pós-traducional chamada de S-glutationilação, que aumenta a velocidade degradação proteica por processo independente de poli-ubiquitinação. Em levedura (Saccharomyces cerevisiae), foram identificados apenas dois resíduos de Cys glutationilados, ambos na subunidade &#945;5 (&#945;5-76 e &#945;5-221). A S-glutationilação ocasiona a abertura da câmara catalítica e uma maior eficiência na degradação de proteínas. Mutações sítio-específicas foram realizadas nessas Cys pela substituição por Ser. As consequências estruturais e funcionais dessas mutações foram o aumento da frequência da conformação fechada da câmara catalítica no &#945;5-76S-PT20S e &#945;5-221S-PT20S. As linhagens que carregam essas mutações apresentaram menor tempo de vida cronológico. Uma dupla mutação randômica na subunidade &#945;5 (S35P / C221S) induziu a abertura da câmera catalítica do 20SPT e esta linhagem apresentou tempo de vida cronológico significativamente aumentado e , aumento na resistência ao estresse oxidativo em paralelo ao aumento da atividade catalítica do 20SPT. O objetivo neste projeto de pesquisa foi realizar uma análise proteômica quantitativa no extrato celular das linhagens mutantes, com o objetivo de identificar proteínas que possam estar relacionadas com a regulação da longevidade celular. Foram selecionadas as linhagens que carregam as mutações: &#945;5-76S e &#945;5-S35P/C221S uma vez que apresentaram expectativa de vida oposta em relação à linhagem selvagem, além de queda e aumento da frequência da conformação aberta da câmera catalítica, respectivamente. A partir da quantificação sem marcação (Label-free quantification), foram identificadas 723-1000 proteínas nas amostras das linhagens selvagem e mutantes. Dentre elas, destacam-se as proteínas 3-isopropilmalato isomerase e argininossuccinato sintase, envolvidas na síntese de leucina e arginina, respectivamente, aumentadas na linhagem mutante C76S e reduzidas na linhagem S35P/C221S. O metabolismo de ambos os aminoácidos está relacionado com a via de sinalização TOR que, por sua vez, está envolvida com o tempo de vida cronológico em levedura. / The protein oxidation is a metabolic phenomenon and the degradation of oxidatively modified proteins confers a protection to cell, avoiding accumulation and aggregation of these proteins. The inefficiency in the removal of these proteins is related to aging process and neurodegenerative diseases. The catalytic unit of the proteasome, named 20S (PT20S) is the main degradative pathway of oxidized proteins in an ATP-independent manner, without proteasomal regulatory units assembly or protein poliubiquitination. The PT20 unit undergoes a post-translational modification named S-glutationilation, which increases the protein degradation by the ATP-independent process. In yeast, only two Cys residues were identified glutationilated, both in the &#945;5 subunit (&#945;5-C76 e &#945;5-C221). The S-glutationilation causes opening of the catalytic chamber and higher efficiency of protein hydrolysis. Site-specific mutations were performed in those Cys residues by their replacement to Ser. The structural and functional consequences of mutations were the increasedfrequency of theclosed conformation of the catalytic chamber in the &#945;5-76S-PT20S and &#945;5-221S-PT20S. The strains carrying those mutations presented shorter chronological life span. A double random mutation in the &#945;5 subunit (S35P / C221S) induced the opening of 20SPT catalytic chamber together toextended chronological life span and, increased resistant to oxidative stress in parallel to increased catalytic activity of the 20SPT. The goal of this project was to perform a label-free quantitative proteomic analysis in the mutant strains to identify proteins that could be related with the regulation of cellularlifespan. From that quantification, 723 - 1000 proteins were identified in the samples of the wild-type and mutant strains. Among these proteins, 3-isopropylmalate isomerase and argininesuccinate sintase, involved in the leucine and arginine biosynthesis, respectively, were found increased in the C76S mutant strain and reduced in the S35P/C221S mutant strain. The metabolism of both amino acids is related with TOR signallingthat, in turn,modulates chronological lifespan in yeast