A enzima Glucosamina-6-fosfato desaminase (GlcN6P desaminase) é envolvida na conversão reversível da D-glucosamina-6-fosfato (GlcN6P) em Fru6P e amônia, como parte do caminho metabólico de aminoaçúcares como fonte de energia celular. A enzima hexamérica (peso mol. 178200) exibe uma cooperatividade homotrópica intensa em direção à GlcN6P a qual é modulada alostericamente pelo ativador N-acetil-D-glucosamina 6-fosfato (GlcNAc6P). A GlcN6P desaminase foi cristalizada no grupo espacial R32, com parâmetros de rede a = b = 125.9 &#197 e c = 223.2 &#197 e um conjunto de dados à 2.1 &#197 de resolução foi coletado usando radiação de luz síncrotron (Horjales et ai., 1992). A procura no banco de dados de seqüências OWL não mostrou homologia significante com qualquer outra família de proteína, desta maneira a determinação da estrutura foi feita pela técnica de substituição isomórfica múltipla (MIR) a partir de dois derivados, um composto de platina, o K2PtCl4 e um complexo de mercúrio, o ácido mersálico. O mapa MIR a 3 &#197 de resolução mostrou contornos claros e utilizando técnicas de nivelamento de solvente (solvent flattening) estendeu-se as fases até 2.5 &#197. A enzima cristaliza-se com dois monômeros na unidade assimétrica. A densidade eletrônica final foi interpretada com o auxílio do programa gráfico \'O\', sendo possível determinar sem ambigüidade 230 dos 266 resíduos de cada monômero; a partir daí foram usados subseqüentes mapas de Fourier diferença para a localização de todos os outros resíduos. O refinamento do modelo foi feito utilizando o programa X-PLOR (Brünger, 1993), usando a rotina simulated annealing, obtendo o fator R final de 17.4% com 348 moléculas de água e quatro íons inorgânicos de fosfato. O enovelamento do monômero tem uma estrutura do tipo &#945/&#946 com uma folha-&#946 pregueada paralela central com sete fitas com topologia 4x, 1x, 1x, -3x, -1x, -1x, envolvida por ambos os lados por oito hélices-&#945 e uma hélice 310 com duas voltas. A sexta fita da folha-&#946 central tem um prolongamento no C-terminal que faz parte de uma segunda folha-&#946 antiparalela de três fitas com topologia 2, -1. O hexâmero tem uma simetria local 32, com dois trímeros empacotados frente-a-frente com uma rotação relativa de 15&#176 em tomo do eixo de ordem 3 e ligados por pontes salinas e algumas interações hidrofóbicas em tomo do eixo não cristalográfico de ordem 2. As moléculas de cada trímero formam um contato não usual de três resíduos Cis 219 próximo ao eixo de ordem três. Os complexos com ativador alostérico (GlcNAc6P) e inibidor competitivo (2-desoxi 2-amino glucitol 6-fosfato) foram co-cristalizados isomorficamente com a estrutura nativa. Os mapas Fourier diferença mostram claramente densidades para os ligantes, definindo sem ambigüidade o sítio ativo e alostérico. O refinamento dos complexos produziu a mesma conformação da proteína nativa, na margem de erro experimental. Os sítios alostéricos (seis) estão localizados na interface adjacente dos monômeros de cada trímero e os sítios ativos (ou catalíticos) no lado externo de cada monômero, no C-terminal da folha-&#946 central. O monômero tem uma topologia com enovelamento similar a um domínio de ligação de NAD, excluindo os segmentos de aminoácidos 1-35, 145-188 e 243-266. As estruturas dos complexos e da nativa estão em um estado alostérico R em concordância com o modelo MWC para um sistema do tipo K (Monod et al, 1965). Um mecanismo alostérico similar ao da GlcN6P desaminase é encontrado na enzima fosfofrutoquinase (Evans, 1981). Um mecanismo catalítico é proposto para a reação de isomerisação-desaminação da enzima GlcN6P desaminase a partir do mecanismo geral para aldose-cetona isomerases. / The enzyme Glucosamine-6-phosphate deaminase (GlcN6P deaminase) is involved in the reversible conversion of D-glucosamine-6-phosphate (GlcN6P) into Fru6P and ammonia. The hexameric enzyme (mol.wt.=178200) exhibits an intense homotropic co-operativity towards GlcN6P which is allosterically modulated by the activator N-acetyl-D-glucosamine 6-phosphate (GlcNAc6P). The GlcN6P deaminase was crystallized in space group R32, with cell parameters a=b= 125.9 &#197 and c = 223.2 &#197 and a native dataset was collected to 2.1 &#197 resolution at a synchrotron source (Horjales et al, 1992). A search of the OWL sequences database has shown no significant homology with any other known protein family. Therefore, the structure determination will have to be achieved through the Multiple Isomorphous Replacement technique from two isomorphous derivatives, a platinum compound K2PtCl4 and a mercury complex, mersalyl acid. The MIR map at 3 &#197 resolution showed clear molecular boundaries and solvent flattening techniques (Wang, 1985) were used to extend the phase set to 2.5 &#197. The final electron density map was interpreted with the aid of the graphic program \'O\'. The enzyme crystallizes with a dimmer in the asymmetric unit and 230 out of the total 266 residues of each crystallographically independent monomer could be unambiguously identified in the map. The remaining residues were located after subsequent difference Fourier maps. The refinement was made with program X-PLOR (Brunger, 1993), using the simulated annealing routine, obtained R=17.4 % with 348 water molecules and four inorganic phosphate ions. The monomer fold shows an &#945/&#946 structure with a central 7-stranded &#946-sheet with topology 4x, 1x, 1x, -3x, -1x, -1x, surrounded on both sides by eight &#945-helices and 2-turn 310 -helix. The sixth strand of the central &#946-sheet is common to a second 3-stranded anti-parallel &#946-sheet with topology 2, -1. The hexamer has local 32 symmetry, with two trimmers packed in a face-to-face arrangement with a relative rotation of 15&#176 around the 3-fold axis, and linked together by salt-bridge and some hydrophobic contacts. The molecules of each trimmer have extensive contacts and show an unusual feature of the three Cys219 residues closely clustered around the 3-fold axis. The complexes with allosteric activator (GlcNAc6P) and inhibitor (2-deoxy-2-amino glucitol 6-phosphate) were co-crystallized isomorphously with the native structure. The difference Fourier maps shows clear density for the ligands, unambiguously defining the active and allosteric sites. The complexes refinement produced the same conformation of the native, within experimental error. The allosteric sites are located at the interfaces of adjacent monomers from each trimer and the active sites (or catalytic) lie at the external side of each monomer, at the C-terminal end of the central parallel &#946-sheet. The monomer has a similar folding topology as a typical NAD binding domain, excluding the segments of aminoacids 135, 145-188 and 243-266. The native and complexes structures are at the allosteric state R concerted with MWC model for a K-system (Monod et al, 1965). A similar allosteric mechanism is found in the enzyme phosphofructokinase (Evans, 1981). A catalytic mechanism is proposed for the isomerisation-deamination reaction of the enzyme from general mechanism for aldo-keto isomerases.

The binding of HIV-1 Vif to the cellular cytidine deaminase Apobec3G and subsequent prevention of Apobec3G virion incorporation have recently been identified as critical steps for the successful completion of the HIV-1 viral life cycle. This interaction occurs in the cytoplasm where Vif complexes with Apobec3G and directs its degradation via the proteasome pathway or sequesters it away from the assembling virion, thereby preventing viral packaging of Apobec3G. While many recent studies have focused on several aspects of Vif interaction with Apobec3G, the subcellular localization of Vif and Apobec3G during the viral life cycle have not been fully considered. Inhibition of Apobec3G requires direct interaction of Vif with Apobec3G, which can only be achieved when both proteins are present in the same subcellular compartment. In this thesis, a unique approach was utilized to study the impact of Vif subcellular localization on Vif function. The question of whether localization could influence function was brought about during the course of studying a severely attenuated viral isolate from a long-term non-progressor who displayed a remarkable disease course. Initial observations indicated that this highly attenuated virus contained a mutant Vif protein that inhibited growth and replication. Upon further investigation, it was found that the Vif defect was atypical in that the mutant was fully functional in in vitro assays, but that it was aberrantly localized to the nucleus in the cell. This provided the basis for the study of Vif localization and its contribution to Vif function. In addition to the unique Vif mutant that was employed, while determining the localization and replication phenotypes of the differentially localized Vif proteins, a novel pathway for Vif function was defined. Copious publications have recently defined the mechanism for Vif inhibition of Apobec3G. Vif is able to recruit Apobec3G into a complex that is targeted for degradation by the proteasome. However, this directed degradation model did not fully explain the complete neutralization of Apobec3G observed in cell culture. Other recent works have proposed the existence of a second, complementary pathway for Vif function. This pathway is defined here as formation of an aggresome that prevents Apobec3G packaging by binding and sequestering Apobec3G in a perinuclear aggregate. This second mechanism is believed to work in parallel with the already defined directed degradation pathway to promote complete exclusion of Apobec3G from the virion. The data presented here provide insight into two areas of HIV research. First, the work on the naturally occurring Vif mutant isolated from a long-term non-progress or confirms the importance of Vif in in vivo pathogenesis and points to Vif as a potentially useful gene for manipulation in vaccine or therapy design due to its critical contributions to in vivo virus replication. Additionally, the work done to address the subcellular localization of Vif led to the proposal of a second pathway for Vif function. This could have implications in the field of basic Vif research in terms of completely understanding and defining the functions of Vif. Again, a more complete knowledge about Vif can help in the development of novel therapies aimed at disrupting Vif function and abrogating HIV-1 replication.

Magister Scientiae - MSc (Biotechnology) / The yearly increase of global population will result in a greater demand for crop production, but with the climates changes and a lack of available agricultural land it will become increasingly more difficult to provide sufficient crops to feed everyone adequately. Application of the PGPE has proven over the past researches to be able enhance growth of plants via various growth promoting mechanisms. To identify suitable growth promoting bacteria candidate, E. plantagineum plant was used to isolate endophytes from the root after surface sterilization. The isolates bacteria were used to inoculate Brassica napus L seeds. The effects of isolate's ability to promote growth were evaluated based on the certain growth parameters after 42 days in the green house. Isolate CP5 produced highest results in all growth parameter. Isolates CP5 was selected as potential candidate as significant improvement was shown by this isolate. This isolate was tested for the ability to produce ACC deaminase, solubilize phosphate, synthesize IAA and siderophore production. Furthermore isolate CP5 growth promotion abilities was tested on Brassica napus L under antimony stress. / 2021-08-31

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Metabolic syndrome (MetS) is a complex disorder represented by a set of cardiovascular risk factors, including endothelial dysfunction, oxidative stress and inflammation. Syzygium cumini has hypoglycemic, anti-inflammatory, antipyretic, hypolipidemic and antioxidant properties, besides antiviral and anticarcinogenic action. Considering the importance of MetS in the current economic and social context, morbidity and complications following this pathology, the aim of this study was to assess biochemical and inflammatory parameters in patients with MetS. Moreover, to check the effect of aqueous extract of Syzygium cumini (ASc), as well as its mechanism of action on the activity of the enzyme adenosine deaminase (ADA) and other biochemical parameters under hyperglycemic and oxidative conditions in vitro. To develop the first step of this work, were obtained samples of serum and whole blood of patients diagnosed with MetS (n=40) before initiating a physical activity program, in which we analyzed biochemical, oxidative and inflammatory parameters. The results showed an increase in ADA, acetylcholinesterase (AChE) and dipeptidyl peptidase IV (DPP-IV) activities in lymphocytes of patients with MetS. Further, in these patients, we observed an increase in the activity of butyrylcholinesterase (BuChE) and ˠ- glutamyltransferase (ˠ-GT) and in C-reactive protein (hsCRP) and nitric oxide (NOx) levels, as well as disturbances in antioxidant defenses and some correlations between the parameters analyzed were obtained. Thus, these results demonstrated that MetS affects the purinergic and cholinergic systems reflecting the inflammatory and immune status of these patients, besides altering the antioxidant defenses of the same. For in vitro determinations, first, erythrocytes (RBCs) from healthy individuals were used. ASc was able to prevent the increase in ADA activity caused by exposure of RBCs to hyperglycemic conditions for 2 hours. Also, ASc acted on the activity of ADA similarly caffeine and insulin, on the other hand, dipyridamole attenuated the effect of ASc by antagonizing its the effect or by competition with the extract. Thus, it can be suggested that the ASc act by influencing the metabolism of adenosine, and its effect is related to the presence of phenolic compounds and the antioxidant properties attributed to this plant. In the next step of this work , we observed an increase in the ADA activity and lipid peroxidation and decreased cell viability after exposure of lymphocytes from healthy subjects to 2,2 ' -azobis -2- amidinopropane dihydrochloride ( AAPH ) by 2 hours in vitro. ASc and gallic acid were able to reduce the ADA activity, but did not alter the lipid peroxidation caused by AAPH . The ASc increased cell viability and reduced the activity of the enzyme lactate dehydrogenase ( LDH ). ASc, by reducing the activity of ADA, may be increasing adenosine levels and helping to maintain the beneficial effects caused by the same, such as antioxidant, anti-inflammatory and antithrombotic actions. Furthermore, the results demonstrate the cytoprotective effect evidenced by the extract. We conclude that the changes found in patients with MetS are related to inflammatory and oxidative processes and may favor the prevention and control of this clinical situation Also, the protective effects demonstrated by ASc contribute to the understanding of the wide use of this plant and its therapeutic value in the treatment of various clinical conditions. / A Síndrome Metabólica (SMet) é um transtorno complexo representado por um conjunto de fatores de risco cardiovascular, entre eles a disfunção endotelial, o estresse oxidativo e a inflamação. O Syzygium cumini, conhecido popularmente como jambolão, é uma planta que apresenta propriedades hipoglicêmicas, antiinflamatórias, antipiréticas, hipolipidêmicas e antioxidantes, além de ação antiviral e anticarcinogência. Considerando a importância da SMet no contexto social e econômico atual, a morbidade e as complicações consequentes desta situação clínica, o objetivo deste estudo foi avaliar parâmetros bioquímicos e inflamatórios em pacientes com SMet. Além disso, verificar o efeito do extrato aquoso de Syzygium cumini (ASc), bem como o seu mecanismo de ação sobre a atividade da enzima Adenosina desaminase (ADA) e outros parâmetros bioquímicos sob condições hiperglicêmicas e oxidativas, in vitro. Para o desenvolvimento da primeira etapa deste trabalho, foram utilizadas amostras de soro e sangue total de pacientes com diagnóstico de SMet (40 pessoas) antes de iniciarem um programa de atividades físicas, nos quais foram analisados parâmetros bioquímicos, inflamatórios e oxidativos. Os resultados demonstraram um aumento na atividade das enzimas ADA, acetilcolinesterase (AChE) e dipeptidil peptidase IV (DPP-IV) em linfócitos de pacientes com SMet. Ainda, nesses pacientes, observou-se um aumento na atividade das enzimas Butirilcolinesterase (BuChE) e gama-glutamiltransferase (GGT), e nos níveis de proteína C reativa (PCR) e óxido nítrico (NOx), bem como alterações nas defesas antioxidantes e algumas correlações entre os parâmetros analisados foram obtidos. Assim, esses resultados demonstraram que a SMet afeta a atividade da ADA e o sistema colinérgico refletindo o estado imune e inflamatório desses pacientes, além de alterar as defesas antioxidantes dos mesmos. Para as análises in vitro, primeiramente, foram utilizados eritrócitos (RBCs) de indivíduos saudáveis. O ASc foi capaz de prevenir o aumento na atividade da ADA causado pela exposição dos RBCs a condições hiperglicêmicas por 2 horas. Ainda, o ASc atuou sobre a atividade da ADA de maneira semelhante a cafeína e a insulina; por outro lado, o dipiridamol atenuou o efeito do ASc por antagonizar o efeito do mesmo ou por competição com o extrato. Assim,pode-se sugerir que o ASc influencia no metabolismo da adenosina, além de seu efeito estar relacionado a presença de compostos fenólicos e às propriedades antioxidantes atribuídas a essa planta. Na etapa seguinte da realização deste trabalho, observou-se um aumento na atividade da ADA e na lipoperoxidação, e redução da viabilidade celular após a exposição de linfócitos de indivíduos saudáveis ao 2,2 -azobis (aminodipropano) dihidrocloreto (AAPH) por 2 horas, in vitro. O ASc e ácido gálico foram capazes de reduzir a atividade da ADA, mas não alteraram a lipoperoxidação causada pelo AAPH. O ASc aumentou a viabilidade celular e reduziu a atividade da enzima Lactato desidrogenase (LDH). O ASc ao reduzir a atividade da ADA, pode estar aumentando os níveis de adenosina e colaborando para a manutenção dos efeitos benéficos provocados pela mesma, como ações antioxidantes, antiinflamatórias e antitrombóticas. Além disso, os resultados demonstraram o efeito citoprotetor evidenciado pelo extrato. Podemos concluir que as alterações encontradas nos pacientes com SMet estão relacionadas aos processos inflamatórios e oxidativos e podem favorecer medidas de prevenção e controle desta situação clínica. Também, os efeitos protetores demonstrados pelo ASc contribuem para o entendimento da ampla utilização desta planta e de seu valor terapêutico no tratamento de diversas patologias clínicas.

Orientador: Luiz Carlos de Mattos / Banca: Antonio Carlos Pires / Banca: Haroldo Wilson Moreira / Resumo: O diabetes melito (DM) é um grupo heterogêneo de alterações metabólicas, caracterizado por hiperglicemia crônica, com alterações do metabolismo de carboidratos, ácidos graxos e proteínas. O diabetes melito tipo 2 (DMT2) é a forma mais comum dessa doença, acomentendo aproximadamente 90% dos indivíduos que apresentam DM. Caracteriza-se principalmente por modificações da ação e secreção de insulina, embora sua etiologia, genética e fisiopatologia específicas ainda não estejam completamente determinadas. Os pacientes com DMT2 apresentam maior risco de desenvolvimento de complicações macro e microvasculares. Estudos revelaram que a enzima conversora de angiotensina (ECA) e a adenosina deaminase (ADA) podem estar relacionadas ao desenvolvimento de DMT2 ou de suas complicações. Com base nesses dados, foram estudados os polimorfismos I/D do gene ECA e TaqI do gene ADA em 162 indivíduos com DMT2, e 160 indivíduos saudáveis. Segundo a Associação Americana de Diabetes, os indivíduos diabéticos que apresentam HDLc abaixo de 40mg/dl ou LDLc acima de 100 mg/dl ou triglicerídeos acima de 150 mg/dl apresentam maior risco de desenvolvimento de doenças cardiovasculares. Por isso, foram selecionados 81 indivíduos com essas características para compor o grupo de estudo de pacientes diabéticos com "risco de doença cardiovascular". Os polimorfismos foram avaliados por PCR e PCR-RFLP para os genes da ECA e ADA respectivamente. As frequências obtidas para o polimorfismo do gene ECA foram: pacientes diabéticos: I/I (19,1%); I/D (52,5%); D/D (28,4%); grupo controle I/I (12,5%); I/D (55,6%); D/D (31,9%) e grupo de diabéticos com riscos de doença cardiovascular I/I (16%); I/D (59,3%); D/D (24,7%). E para o gene ADA: em pacientes diabéticos ADA*1/*1 (89,31%); ADA*1/*2 (10,06%); ADA*2/*2 (0,63%); grupo controle ADA*1/*1 (91,25%); ADA*1/*2 (7,50%); ADA*2/*2 (1,25%); pacientes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Diabetes mellitus (DM) is a heterogeneous group of metabolic disorders characterized by chronic hyperglycemia, with changes in the carbohydrates, fatty acids and proteins metabolism. Diabetes mellitus type 2 (DMT2) is the most common form of this disease, that affect approximately 90% of people who have DM. It is characterized mainly by changes in the action and insulin secretion, although the etiology, genetics and specific pathophysiology of the disease is not yet completely determined. DMT2 patients have a higher risk of developing macro and microvascular complications. Studies have shown that angiotensin-converting enzyme (ACE) and adenosine deaminase (ADA) may be related to the development of DMT2 or its complications. Based on these data, we studied the polymorphisms I / D of the ACE gene and the polymorphism TaqI in the ADA gene, in 162 patients with DMT2, and 160 blood donors. According to the American Diabetes Association, people with diabetes who have HDL-C below 40 mg / dL or LDL-C above 100 mg / dl or triglycerides above 150 mg / dl are at increased risk of developing cardiovascular disease. Therefore, we selected 81 individuals with these characteristics to compose the study group of diabetic patients named "risk of cardiovascular disease ". The polymorphisms are evaluated by PCR for ACE gene and PCR-RFLP for the ADA Gene. The frequencies obtained for the ACE gene polymorphism were: diabetic patients: I / I (19.1%), I / D (52.5%) and D / D (28.4%), control group: I / I ( 12.5%) I / D (55.6%) and D / D (31.9%) and group of patients with cardiovascular disease risk: I / I (16%) I / D (59.3% ), D / D (24.7%). And for the ADA gene polymorphism: in diabetic patients: ADA * 1 / * 1 (89.31%); ADA * 1 / * 2 (10.06%), ADA * 2 / * 2 (0.63%); control group: ADA * 1 / * 1 (91.25%); ADA * 1 / * 2 (7.50%); ADA * 2 / * 2 (1.25%), and patients with cardiovascular risk:... (Complete abstract click electronic access below) / Mestre

A enzima Glucosamina-6-fosfato desaminase (GlcN6P desaminase) é envolvida na conversão reversível da D-glucosamina-6-fosfato (GlcN6P) em Fru6P e amônia, como parte do caminho metabólico de aminoaçúcares como fonte de energia celular. A enzima hexamérica (peso mol. 178200) exibe uma cooperatividade homotrópica intensa em direção à GlcN6P a qual é modulada alostericamente pelo ativador N-acetil-D-glucosamina 6-fosfato (GlcNAc6P). A GlcN6P desaminase foi cristalizada no grupo espacial R32, com parâmetros de rede a = b = 125.9 &#197 e c = 223.2 &#197 e um conjunto de dados à 2.1 &#197 de resolução foi coletado usando radiação de luz síncrotron (Horjales et ai., 1992). A procura no banco de dados de seqüências OWL não mostrou homologia significante com qualquer outra família de proteína, desta maneira a determinação da estrutura foi feita pela técnica de substituição isomórfica múltipla (MIR) a partir de dois derivados, um composto de platina, o K2PtCl4 e um complexo de mercúrio, o ácido mersálico. O mapa MIR a 3 &#197 de resolução mostrou contornos claros e utilizando técnicas de nivelamento de solvente (solvent flattening) estendeu-se as fases até 2.5 &#197. A enzima cristaliza-se com dois monômeros na unidade assimétrica. A densidade eletrônica final foi interpretada com o auxílio do programa gráfico \'O\', sendo possível determinar sem ambigüidade 230 dos 266 resíduos de cada monômero; a partir daí foram usados subseqüentes mapas de Fourier diferença para a localização de todos os outros resíduos. O refinamento do modelo foi feito utilizando o programa X-PLOR (Brünger, 1993), usando a rotina simulated annealing, obtendo o fator R final de 17.4% com 348 moléculas de água e quatro íons inorgânicos de fosfato. O enovelamento do monômero tem uma estrutura do tipo &#945/&#946 com uma folha-&#946 pregueada paralela central com sete fitas com topologia 4x, 1x, 1x, -3x, -1x, -1x, envolvida por ambos os lados por oito hélices-&#945 e uma hélice 310 com duas voltas. A sexta fita da folha-&#946 central tem um prolongamento no C-terminal que faz parte de uma segunda folha-&#946 antiparalela de três fitas com topologia 2, -1. O hexâmero tem uma simetria local 32, com dois trímeros empacotados frente-a-frente com uma rotação relativa de 15&#176 em tomo do eixo de ordem 3 e ligados por pontes salinas e algumas interações hidrofóbicas em tomo do eixo não cristalográfico de ordem 2. As moléculas de cada trímero formam um contato não usual de três resíduos Cis 219 próximo ao eixo de ordem três. Os complexos com ativador alostérico (GlcNAc6P) e inibidor competitivo (2-desoxi 2-amino glucitol 6-fosfato) foram co-cristalizados isomorficamente com a estrutura nativa. Os mapas Fourier diferença mostram claramente densidades para os ligantes, definindo sem ambigüidade o sítio ativo e alostérico. O refinamento dos complexos produziu a mesma conformação da proteína nativa, na margem de erro experimental. Os sítios alostéricos (seis) estão localizados na interface adjacente dos monômeros de cada trímero e os sítios ativos (ou catalíticos) no lado externo de cada monômero, no C-terminal da folha-&#946 central. O monômero tem uma topologia com enovelamento similar a um domínio de ligação de NAD, excluindo os segmentos de aminoácidos 1-35, 145-188 e 243-266. As estruturas dos complexos e da nativa estão em um estado alostérico R em concordância com o modelo MWC para um sistema do tipo K (Monod et al, 1965). Um mecanismo alostérico similar ao da GlcN6P desaminase é encontrado na enzima fosfofrutoquinase (Evans, 1981). Um mecanismo catalítico é proposto para a reação de isomerisação-desaminação da enzima GlcN6P desaminase a partir do mecanismo geral para aldose-cetona isomerases. / The enzyme Glucosamine-6-phosphate deaminase (GlcN6P deaminase) is involved in the reversible conversion of D-glucosamine-6-phosphate (GlcN6P) into Fru6P and ammonia. The hexameric enzyme (mol.wt.=178200) exhibits an intense homotropic co-operativity towards GlcN6P which is allosterically modulated by the activator N-acetyl-D-glucosamine 6-phosphate (GlcNAc6P). The GlcN6P deaminase was crystallized in space group R32, with cell parameters a=b= 125.9 &#197 and c = 223.2 &#197 and a native dataset was collected to 2.1 &#197 resolution at a synchrotron source (Horjales et al, 1992). A search of the OWL sequences database has shown no significant homology with any other known protein family. Therefore, the structure determination will have to be achieved through the Multiple Isomorphous Replacement technique from two isomorphous derivatives, a platinum compound K2PtCl4 and a mercury complex, mersalyl acid. The MIR map at 3 &#197 resolution showed clear molecular boundaries and solvent flattening techniques (Wang, 1985) were used to extend the phase set to 2.5 &#197. The final electron density map was interpreted with the aid of the graphic program \'O\'. The enzyme crystallizes with a dimmer in the asymmetric unit and 230 out of the total 266 residues of each crystallographically independent monomer could be unambiguously identified in the map. The remaining residues were located after subsequent difference Fourier maps. The refinement was made with program X-PLOR (Brunger, 1993), using the simulated annealing routine, obtained R=17.4 % with 348 water molecules and four inorganic phosphate ions. The monomer fold shows an &#945/&#946 structure with a central 7-stranded &#946-sheet with topology 4x, 1x, 1x, -3x, -1x, -1x, surrounded on both sides by eight &#945-helices and 2-turn 310 -helix. The sixth strand of the central &#946-sheet is common to a second 3-stranded anti-parallel &#946-sheet with topology 2, -1. The hexamer has local 32 symmetry, with two trimmers packed in a face-to-face arrangement with a relative rotation of 15&#176 around the 3-fold axis, and linked together by salt-bridge and some hydrophobic contacts. The molecules of each trimmer have extensive contacts and show an unusual feature of the three Cys219 residues closely clustered around the 3-fold axis. The complexes with allosteric activator (GlcNAc6P) and inhibitor (2-deoxy-2-amino glucitol 6-phosphate) were co-crystallized isomorphously with the native structure. The difference Fourier maps shows clear density for the ligands, unambiguously defining the active and allosteric sites. The complexes refinement produced the same conformation of the native, within experimental error. The allosteric sites are located at the interfaces of adjacent monomers from each trimer and the active sites (or catalytic) lie at the external side of each monomer, at the C-terminal end of the central parallel &#946-sheet. The monomer has a similar folding topology as a typical NAD binding domain, excluding the segments of aminoacids 135, 145-188 and 243-266. The native and complexes structures are at the allosteric state R concerted with MWC model for a K-system (Monod et al, 1965). A similar allosteric mechanism is found in the enzyme phosphofructokinase (Evans, 1981). A catalytic mechanism is proposed for the isomerisation-deamination reaction of the enzyme from general mechanism for aldo-keto isomerases.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O Clostridium difficile tem como principal fator de virulÃncia a toxina A (TxA), a qual provoca inflamaÃÃo e destruiÃÃo tecidual aguda em intestinos de animais experimentais e de pacientes com a doenÃa induzida por esta bactÃria. Em locais de injÃria tecidual, adenosina à produzida em altas concentraÃÃes, onde exerce uma sÃrie de efeitos antiinflamatÃrios, limitados por sua rÃpida degradaÃÃo pela enzima adenosina desaminase. O objetivo deste trabalho foi investigar o efeito da inibiÃÃo da enzima adenosina desaminase pelo EHNA (eritro-9-(2-hidrÃxi-3-nonil)-adenina) sobre a enterite induzida pela TxA do C. difficile em alÃa ileal de camundongos. Para isto, injetamos EHNA (90 &#956;mol/kg) ou PBS i.p. 30 minutos antes da administraÃÃo de TxA (10 a 100 &#956;g) ou PBS na alÃa ileal isolada. Os animais foram sacrificados 3 horas depois da induÃÃo da enterite e as alÃas foram retiradas para estudo. As razÃes peso/comprimento da alÃa e volume de secreÃÃo/comprimento da alÃa foram calculadas e amostras de tecido foram coletadas para histopatologia, dosagem de atividade de mieloperoxidase (MPO), dosagem de TNF-&#945;, IL-1&#946; e IL-10 por ELISA, imunohistoquÃmica para TNF-&#945;, IL-1&#946;, NOS induzÃvel e PTX3, e PCR para TNF-&#945;, IL-1&#946; e PTX3. A injeÃÃo de TxA (10 a 100 &#956;g) nas alÃas ileais aumentou significativamente (p<0,05) as razÃes peso/comprimento da alÃa e volume de secreÃÃo/comprimento da alÃa com resultados consistentes a partir de 50 &#956;g. A TxA promoveu significativa (p<0,05) destruiÃÃo tecidual, edema, infiltraÃÃo de cÃlulas inflamatÃrias, aumento das citocinas TNF-&#945; e IL-1&#946;, e elevaÃÃo de iNOS e PTX3. Todos esses parÃmetros foram significativamente revertidos com o uso do EHNA (p<0,05). Em adiÃÃo, a TxA nÃo alterou os nÃveis de IL-10 em relaÃÃo ao controle, mas o prÃ-tratamento com EHNA promoveu uma elevaÃÃo nos nÃveis desta citocina. Assim, concluÃmos que na enterite induzida pela TxA em camundongos o EHNA demonstrou um potente efeito antiinflamatÃrio, reduzindo consideravelmente a lesÃo tecidual, a migraÃÃo neutrofÃlica, a expressÃo e os nÃveis de citocinas prÃinflamatÃrias (TNF-&#945;, IL-1&#946;) e produzindo um aumento nos nÃveis de IL-10. AlÃm disso, a administraÃÃo de TxA induziu um aumento na expressÃo da proteÃna PTX3 e no nÃmero de cÃlulas imunomarcadas para iNOS no tecido ileal, ambos revertidos pelo EHNA / The main factor of virulence in Clostridium difficile is toxin A (TxA), which can induce inflammation and acute tissue injury in the bowels of animals and humans affected by this organism. The high concentration of adenosine generated upon injury produces a number of antiinflammatory effects limited by rapid degradation by adenosine deaminase. The objective of this study was to determine the effect of EHNA (erythro-9-(2-hydroxy-3-nonyl)-adenine) inhibition of adenosine deaminase upon TxA-induced ileal loop enteritis in mice. EHNA (90 &#956;mol/kg) or PBS was injected i.p. 30 minutes prior to TxA (10-100 &#956;g) or PBS instillation into the ligated ileal loop. The animals were euthanized 3 hours after enteritis induction and the ileal loops were retrieved for analysis. The weight/length ratio and the secretion volume/length ratio were calculated and tissue samples were submitted to histopathological study, myeloperoxidase assay (MPO), measurement of TNF-&#945;, IL-1&#946; and IL-10 levels with ELISA, immunohistochemical tests for TNF-&#945;, IL-1&#946;, inducible NOS and PTX3, and PCR assay for TNF-&#945;, IL-1&#946; and PTX3. The instillation of TxA (10-100 &#956;g) into the ileal loop significantly increased (p<0.05) the weight/length ratio and the secretion volume/length ratio with consistent results above 50 &#956;g. TxA induced a significant amount (p<0.05) of histological damage, edema and inflammatory cell infiltration and increased the production of TNF-&#945;, IL-1&#946;, iNOS and PTX3. All changes were significantly reverted by treatment with EHNA (p<0.05). Moreover, IL-10 levels remained unchanged in animals treated with TxA, but increased in animals receiving EHNA. In conclusion, in mice with TxA-induced enteritis EHNA produced considerable antiinflammatory effects, reducing tissue injury, neutrophil migration, the expression and levels of proinflammatory cytokines (TNF-&#945; and IL-1&#946;) and producing an increase in IL-10 levels. In addition, TxA instillation increased PTX3 expression and the number of cells immunolabeled for iNOS in the ileal tissue, both of which were reverted by EHNA

Le somnambulisme est un trouble du sommeil fréquent qui appartient à la famille des parasomnies NREM. Malgré des décennies de recherche, sa pathophysiologie reste peu comprise. Les études de familles et les études de jumeaux démontrent qu’une forte composante héréditaire est en jeu. Toutefois, très peu d’études moléculaires ont été menées afin d’identifier des gènes impliqués et il n’y a toujours pas de consensus quant au mode de transmission dans les familles. Cet ouvrage contient deux études distinctes qui tenteront de répondre à ces deux problèmes. L’objectif de la première étude était de déterminer si des variants génétiques dans le gène Adénosine désaminase (ADA) étaient enrichis dans la population somnambule en comparaison avec les dormeurs sains. Le gène entier a été séquencé chez 251 patients somnambules provenant de Montréal et de Montpellier ainsi que chez 94 sujets contrôles sans histoire personnelle ni familiale de somnambulisme. Aucun variant génétique n’était enrichi chez les patients somnambules en comparaison avec les dormeurs sains et les bases de données génétiques publiques. Dans la deuxième étude, le premier objectif était de déterminer le mode de transmission du somnambulisme chez 20 familles canadiennes-françaises. Le deuxième objectif était de mesurer le risque récurrent ainsi que le risque relatif pour la fratrie et les enfants des patients index. Dans notre cohorte, le somnambulisme se transmettait principalement selon un mode autosomal dominant à pénétrance réduite. Les risques récurrents pour les apparentés de premier degré étaient : à vie 0,48 à 0,56, durant l’enfance 0,43 à 0,56 et à l’âge adulte 0,14 à 0,35. Les risques relatifs pour les apparentés de premier degré étaient : à vie 6,96 à 8,12, durant l’enfance 1,48 à 4,06 et à l’âge adulte 4,67 à 11,67 supérieurs à la population générale. D’autres études moléculaires comme le séquençage de l’exome et les études de liaison génétique dans les familles seront nécessaires afin d’identifier de nouveaux gènes candidats qui pourront agir à titre de biomarqueurs. Cela permettrait de faciliter le diagnostic et ultimement développer des approches thérapeutiques ciblées. / Sleepwalking is a common sleep disorder and it belongs to the family of NREM parasomnias. Despite decades of research, its pathophysiology remains poorly understood. Family and twin studies show that a strong hereditary component is involved. However, very few molecular studies have been conducted to identify the genes involved and there is still no consensus on the mode of transmission in families. This Master’s thesis contains two separate studies which will attempt to address these two problems. The aim of the first study was to determine whether genetic variants in the Adenosine Deaminase (ADA) gene were enriched in the sleepwalking population compared to healthy sleepers. The entire gene was sequenced in 251 sleepwalking patients from Montreal and Montpellier as well as in 94 control subjects with no personal or family history of sleepwalking. No genetic variants were enriched in sleepwalking patients compared to healthy sleepers and public genetic databases. In the second study, the first objective was to determine the mode of transmission of sleepwalking in 20 French-Canadian families. The second objective was to measure the recurrence risk as well as the relative risk for siblings and children of index patients. In our cohort, sleepwalking was transmitted mainly in an autosomal dominant mode with reduced penetrance. The recurrence risks for first-degree relatives were: lifetime 0.48 to 0.56, in childhood 0.43 to 0.56, and in adulthood 0.14 to 0.35. The relative risks for first-degree relatives were: lifetime 6.96 to 8.12, in childhood 1.48 to 4.06 and in adulthood 4.67 to 11.67 higher than the general population. Further molecular studies, such as exome sequencing, and genetic linkage studies in families will be needed in order to identify new candidate genes that can act as biomarkers. This would allow the development of an independent test for the diagnosis and ultimately have implications for targeted therapeutic approaches.

Activation-induced cytidine deaminase (AID) is a key protein required for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. AID is induced in B cells during an immune response. Lack of AID or mutant form of AID causes immunodeficiency; e.g., various mutations in the C terminus of AID causes hyper IgM (HIGM2) syndrome in humans. The C terminal 10 amino acids of AID are required for CSR but not for SHM. During both CSR and SHM, AID deaminates dCs within Ig genes, converting them to dUs, which are then either replicated over, creating mutations, or excised by uracil DNA glycosylase (UNG), leading to DNA breaks in Ig switch regions. Also, the mismatch repair (MMR) heterodimer Msh2-Msh6 recognizes U:G mismatches resulting from AID activity and initiates MMR, which leads to increased switch region double strand breaks (DSBs). DSBs are essential intermediates of CSR; lack of UNG or MMR results in a reduction of DSBs and CSR. The DSBs created in the Sμ and one of the downstream S-regions during CSR are recombined by non-homologous end joining (NHEJ) to complete CSR. Available data suggest that AID is required not only for the deamination step of CSR, but also for one or more of the steps of CSR that are downstream of deamination step. This study investigates the role of C terminus of AID in CSR steps downstream of deamination. Using retroviral transduction into mouse splenic B cells, I show that AID binds cooperatively with UNG and Msh2-Msh6 to the Ig Sμ region, and this depends on the AID C terminus. I also show that the function of MMR during CSR depends on the AID C terminus. Surprisingly, the C terminus of AID is not required for Sμ or Sγ3 DSBs, suggesting its role in CSR occurs during repair and/or recombination of DSBs.

HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research. The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function. Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition. Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.