L’objectif de ce projet de thèse est d’étudier et caractériser les mécanismes impliqués dans la bascule respiro-fermentaire chez des cellules eucaryotes dotées d’un métabolisme mitochondrial. Les cellules eucaryotes ont des besoins différents en oxygène pour la production d’énergie et leur survie dans un environnement donnée. Elles sont qualifiées de type aérobie stricte lorsque la présence d’oxygène leur est nécessaire ou aéro-anaérobie facultatif dans le cas où l’oxygène n’est pas indispensable à la production d’énergie. La levure Yarrowia lipolytica a été choisie comme modèle d’étude de par sa particularité à être un micro-organisme aérobie strict avec une grande capacité d’accumuler de lipides et de production d’acides organiques. Les études expérimentales et analytiques, par l’emploi de méthodes mathématiques de modélisation du métabolisme, ont permis d’identifier des contraintes métaboliques impliquées dans la transition respiro-fermentaire chez cette levure au métabolisme énergétique oxydatif. La production de l’acide citrique par Y. lipolytica, déjà rapportée dans la littérature, a été choisi comme un marqueur de cette transition respiro-fermentaire. Nous avons découvert que l’inhibition de la protéine oxydase alternative (AOX), impliquée dans la respiration mitochondriale, par la molécule n-propyl gallate (nPG) permet d’améliorer le rendement de production d’acide citrique par fermentation du glucose dans une culture de Y. lipolytica. Ces résultats montrent que la nPG, déjà utilisée dans l’industrie agro-alimentaire et pharmaceutique en tant que conservateur joue sur la bascule respiro-fermentaire par inhibition de la consommation d’oxygène et stimule ainsi la production d’acide citrique. La modélisation du réseau métabolique de Y. lipolytica, décrit à l’échelle du genome, par dynamic Flux Balance Analysis (dFBA) a permis d’identifier l’accumulation des espèces oxydantes dites ROS (Reactive Oxygen Species) comme un levier majeur de la bascule respiro-fermentaire et donc de la production d’acide citrique chez la levure Y. lipolytica. De plus, nos résultats préliminaires montrent que l’oxydation des lipides accumulés par Y. lipolytica pourrait être à l’origine de la génération des ROS. Cette étude doit être approfondie expérimentalement et constitue un apport important pour l’industrie agro-alimentaire et pharmaceutique.Mots clés : Bascule respiro-fermentaire, Acide citrique, lipides, Yarrowia lipolytica, n-propyl gallate, Reactive Oxygen Species, modélisation, dynamic Flux Balance Analysis / The main goal of this thesis project is to study and characterize mechanisms involved in respiratory to fermentative shift in eukaryotic cells endowed with mitochondrial metabolism. Eukaryotic cells have different oxygen requirements for energy production and survival in a given environment. They are described as strict aerobic when the presence of oxygen is necessary or optional aero-anaerobic in when oxygen is not essential for energy production. The yeast Yarrowia lipolytica was chosen as our study model thanks to its particularity since it is a strict aerobic microorganism with a high capacity to accumulate lipids and to produce organic acids. Experimental and analytical studies, using mathematical methods for modeling cell metabolism, allowed us to identify metabolic constraints involved in respiratory to fermentative transition in this yeast showing oxidative energy metabolism. Production of citric acid by Y. lipolytica, already reported in the literature, has been chosen as a marker for this in respiratory to fermentative shift. We found that the inhibition of the alternative oxidase protein (AOX) involved in mitochondrial respiration, by adding n-Propyl gallate (nPG) molecule improves the yield of citric acid production by fermentation of glucose in a Y. lipolytica culture. These results show that nPG, already used in food and pharmaceutical industry as a preservative, plays on respiratory to fermentative balance by inhibition of oxygen consumption and thus stimulates the production of citric acid. Modeling of the metabolic network of Y. lipolytica, described at genome-scale, by dynamic Flux Balance Analysis (FBA) has identified the accumulation of intracellular ROS (Reactive Oxygen Species) species as major levers for respiratory to fermentative shift and therefore the production of citric acid by Y. lipolytica. Therefore, our preliminary results show that oxidation of lipids accumulated by Y. lipolytica could be involved in generation of ROS species. This study must be experimentally deepened and constitutes an important contribution for the agri-food and pharmaceutical industry.Key words: Respiratory to fermentative shift, Citric acid, lipids, Yarrowia lipolytica, n-Propyl gallate, Reactive Oxygen Species, modeling, dynamic Flux Balance Analysis

Afin de mettre en évidence le phénomène de localisation d'énergie dans les protéines, un modèle utilisant les concepts de la Physique non-linéaire a été développé. Il permet, via l'utilisation d'un potentiel de type FPU et d'une dissipation placée sur la surface, de faire apparaître des breathers chaotiques dans certaines enzymes. Ces breathers ont une durée de vie importante par rapport aux échelles de temps caractéristiques du système. Ils sont localisés sur un seul résidu, toujours situé dans une région rigide de la protéine. Cela nous a conduit à l'hypothèse d'un lien possible entre la fonction catalytique, les propriétés locales de structure des enzymes et les localisations d'énergie. Plus précisément, l'activation d'un breather chaotique lors d'une réaction enzymatique au niveau des sites catalytiques pourrait permettre à la protéine de stocker de l'énergie pendant de longues durées.

Thesis advisor: Thomas C. Chiles / Normal B lymphocytes require extrinsic factors to grow and proliferate. Surface receptors (e.g., B-cell antigen receptor, BCR) function, in part, to link growth factors to signal transduction/metabolic pathways and the cell cycle machinery. Accumulating evidence indicates that signal transduction-dependent changes in both glucose energy metabolism and de novo transcription of the D-type cyclin-cdk4/6 pathway are necessary for quiescent B-lymphocytes to enter G1-phase of the cell cycle and grow. B cell growth represents a critical checkpoint for subsequent proliferation and clonal expansion of antigen-specific lymphocytes. On the former, we have shown earlier that acquisition of extracellular glucose and metabolism via the glycolytic pathway is required for conventional splenic B-2 lymphocytes to grow (i.e., increase cell size and mass) in response to antigen challenge; however, the metabolic fate and biological significance of glucose-derived carbons are unknown. Here, we show that in response to BCR ligation, glucose carbon flow is directed into a de novo lipogenic pathway that is regulated, in part, via phosphoinositide-3 kinase (PI-3K)-dependent activation of ATP citrate lyase (ACL), a key rate-limiting enzyme in de novo lipogenesis. Inhibition of ACL results in a loss of B-cell growth and cell viability. Regarding the latter point, the B-1a lymphocyte subset expresses cyclins D2 and D3 that are transiently expressed in a non-overlapping manner, notably cyclin D3 expression immediately precedes the G1/S phase transition, suggesting distinct functions for these D-type cyclins in B-1a lymphocyte G0-to-S phase progression. We show herein that murine B-1a cells deficient in cyclin D3 proliferate normally in response to extracellular stimuli, in part, due to a compensatory sustained up-regulation of cyclin D2. In keeping with this, human diffuse large B-cell lymphoma (DLBCL) represents a malignant clonal expansion of B cells characterized by several subsets, including germinal center (GC) and activated B-cell (ABC) types. Here, we show that the GC-type LY18 human DLBCL exhibits constitutive expression of cyclin D3, but not cyclins D1 and D2. Targeting of cyclin D3-holoenzyme complexes with cell permeable chemical- and peptide-based cdk4 inhibitors results in G1-phase arrest and apoptosis via a pathway that involves inhibition of pRb phosphorylation. By contrast, endogenous knock down of cyclin D3 with siRNA did not induce growth arrest or apoptosis, in part, due to redundancy with cyclin E. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

O interesse na aplicação de embalagens ativas para conservação de alimentos tem aumentado, bem como uma maior demanda por substâncias antimicrobianas naturais, devido à maior consciência dos consumidores quanto aos potenciais riscos à saúde ocasionados pelo consumo de compostos sintéticos. Entre os aditivos naturais, a própolis, por suas propriedades antibacteriana, antioxidante e antifúngica, tem despertado o interesse dos pesquisadores. Portanto, o objetivo deste trabalho foi investigar o efeito da adição do extrato etanólico de própolis (EEP) em filmes à base de gelatina plastificados com citrato de acetiltributila (CA) ou sorbitol (S), nas propriedades funcionais (propriedades mecânicas, solubilidade, permeabilidade ao vapor de água, parâmetros de cor e opacidade) e a atividade antimicrobiana dos filmes contra Staphylococcus aureus. Para a produção do EEP, 30g de resina de própolis (tipo 12) foram misturadas com 100mL de álcool etílico 80%, e a solução foi mantida sob agitação mecânica (500rpm, 30minutos) a 50ºC, e filtrada após 24horas sob refrigeração. Os filmes foram produzidos por casting com 2g de gelatina/100g de solução filmogênica, 30g de CA ou S/100g de gelatina, 35g de lecitina/100g de plastificante e o EEP nas concentrações de 0, 5, 40 ou 200g/100g de gelatina, e avaliados quanto às suas propriedades mecânicas (tração e perfuração), solubilidade em água (Sol), cor e opacidade, permeabilidade ao vapor de água (PVA), espectroscopia de infravermelho com transformada de Fourier (FTIR) e microscopia eletrônica de varredura, após acondicionamento (5 dias, UR = 58%, T = 25ºC). A atividade antimicrobiana dos filmes contra Staphylococcus aureus foi analisada pelo método da difusão em ágar. A incorporação de EEP causou variação na tensão (T) dos filmes com CA (58,7 a 66,4MPa), mas afetou mais intensamente os filmes com S (31,7 a 50,4MPa). Quanto à elongação (%), os filmes (CA e S) também apresentaram variações. Na perfuração, verificou-se que a adição de EEP não afetou significativamente a força máxima para ambos os plastificantes. Para a solubilidade em água, o filme com CA apresentou aumento significativo (Sol = 18,6%) para 200% de EEP, contudo, os filmes com S praticamente não variaram. Quanto à cor, para ambos os plastificantes estudados (CA e S) o aumento da concentração de EEP promoveu alterações significativas de L*, a*, b*, ΔE* e opacidade. As análises de PVA dos filmes (CA e S) mostraram que o aumento na concentração de EEP aumentou a propriedade de barreira dos filmes. Segundo a análise de FTIR, os filmes aditivados não apresentaram mudanças estruturais em relação ao controle, o que sugere que o EEP encontra-se disperso na matriz. As micrografias mostraram que a adição de EEP causou alterações, principalmente, nos filmes plastificados com S. A atividade contra S. aureus foi observada para os filmes (CA e S) com 40 e 200% de EEP, com aumento significativo do diâmetro de inibição em função do aumento da concentração de EEP. Os filmes mantiveram sua atividade antimicrobiana durante 177 dias de armazenamento. Estes resultados demonstraram a capacidade antimicrobiana dos filmes de gelatina aditivados com própolis, bem como sua estabilidade em função do tempo. / The interest in the use of active packaging for food conservation has increased, along with a higher demand for natural antimicrobial substances, due to higher consumer awareness of the potential health risks caused by synthetic compounds consumption. Among natural additives, propolis, has awaken the interest of the researchers for its antibacterial, antioxidant and antifungal properties. Thus, the aim of this work was to investigate the effect of the addition of an ethanolic propolis extract (EPE) to gelatin-based films plasticized with acetyltributyl citrate (AC) or sorbitol (S), on the functional properties (mechanical properties, solubility, water vapor permeability, color parameters and opacity) and the antimicrobial activity of the films against Staphylococcus aureus. For the EPE production, 30g for the propolis resin (type 12) were mixed with 100mL ethyl alcohol (80%), and the solution was maintained under mechanical stirring (500rpm, 30minutes) at 50°C, and filtered after 24hours under refrigeration. The films were produced by casting, using 2g of gelatin/100g of film forming solution, 30g of AC or S/100 g of gelatin, 35g of lecithin/100g of plasticizer and EPE in the concentrations of 0, 5, 40 or 200g/100g of gelatin, and evaluated for their mechanical properties (traction and puncture tests), water solubility (Sol), color and opacity, water vapor permeability (WVP), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy, after storage (5 days, relative humidity = 58%, temperature = 25°C). The antimicrobial activity of the films against Staphylococcus aureus was analyzed by agar diffusion method. The EPE addition caused variation of the tensile strength (T) of the films with AC (58.7 a 66.4MPa), but affected more intensely the films with S (31.7 a 50.4MPa). The films (AC and S) also presented variations for the elongation (%). For the puncture force, the addition did not affect significantly the maximum force for any of the plasticizers. For the water solubility, the films with AC presented significant increase (Sol = 18,6%) for 200% of EPE, however the films with S did not vary. For both plasticizers studied (AC and S), the increase of EPE concentration promoted significant changes of the color parameters, L*, a*, b*, ΔE* and opacity. The WVP analysis of the films (AC and S) showed that the increase of the EPE concentration increased the barrier property of the films. According to FTIR analysis, the EPE addition in the films did not present structural changes compared to the control, suggesting that the EPE was disperse in the protein matrix. The micrographs showed that the EPE addition caused changes mainly in films plasticized with S. The activity against S. aureus was observed for the films containing 40 and 200% of EPE, with significant increase of inhibition diameter with increase of EPE concentration. The films kept their antimicrobial activities during 177 days of storage. These results demonstrated the antimicrobial capacity of gelatin-based films added of propolis, and their stability in time.

O gene TrCit do fungo filamentoso Trichoderma reesei codifica a proteína citrato sintase, uma enzima chave do ciclo de Krebs. Análise da região 5´ upstream de TrCit mostra que o gene está adjacente ao gene TrIcl (que codifica a proteína isocitrato liase, uma enzima do ciclo de glioxalato), em uma orientação cabeça-cabeça. A região promotora intergênica de 647 pb rica em G + C, apresenta uma ilha CpG, seqüência INR, caixas GC, caixas CAAT, sítios de ligação para diversos fatores de transcrição e é isenta de caixa TATA. O gene TrCit de 1573 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1422 pb produz uma proteína de 474 aminoácidos, com um peso molecular estimado de 52,3 kD. O gene TrIcl de 1880 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1788 pb produz uma proteína de 596 aminoácidos, com um peso molecular estimado de 65,4 kD. A atividade transcricional da região promotora foi analisada utilizando como repórter o gene de higromicina B fosfotransferase (hph). Uma região funcional necessária à transcrição de ambos os genes foi identificada na região central do promotor e contém uma caixa GC que liga o putativo fator de transcrição Sp1 de T. reesei (TrZnFSp1). O gene do putativo fator de transcrição “zinc-finger" TrZnFSp1 de 1500 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1344 pb produz uma proteína de 448 aminoácidos, com um peso molecular estimado de 48,4 kD. Os resultados mostram que ambos os genes são transcritos de forma divergente a partir de um promotor bidirecional que compartilha na região central uma caixa GC, necessária para a transcrição de ambos os genes. / The TrCit gene from the filamentous fungus Trichoderma reesei codes for the citrate synthase protein, a key enzyme in the Krebs cycle. Analysis of TrCit 5’ upstream region showed that it is adjacent to the TrIcl gene that codes for isocitrate lyase protein, an enzyme involved in the glyoxylate cycle. Both genes, on a head-to-head orientation, are separated by an intergenic GC-rich and TATA-less promoter region of 647 base pairs. This bidirectional promoter has diverse cis regulatory elements: a CpG island, two INR sequences, GC boxes, CAAT boxes and several putative interaction sites for different transcription factors. The TrCit gene, 1,573-base pair-long, has an open reading frame of 1,422 base pairs interrupted by two introns. The gene codes for a protein with an estimated molecular weight of 52.3 kD. The TrIcl gene, 1,880-base pair-long, contains 3 exons and 2 introns and a putative coding sequence of 1,788 base pairs. The estimated molecular weight of TrICL is 65.4 kD. he transcriptional activity of the intergenic promoter region was analyzed using hygromicin B phosphotransferase (hph) as a reporter gene. A functional region required for the transcription of both genes was identified in the centre of this promoter. It has a GC box that interacts with a putative transcription factor Sp1 from T. reesei (TrZnFSp1). The results presented in this work show that both genes are divergently transcribed from a bidirectional promoter that shares an essential central GC box.

OBJETIVOS: O aumento da expectativa de vida tem gerado um envelhecimento populacional global, fazendo com que a proporção de indivíduos com mais de 60 anos de idade cresça mais rapidamente do que as demais faixas etárias. A incidência de litíase urinária em indivíduos idosos tem aumentado nos últimos anos, principalmente em nações industrializadas. Aspectos particulares do envelhecimento orgânico aumentam a morbidade associada à litíase urinária em idosos, tornando a prevenção e o tratamento clínico ainda mais relevantes nessa faixa etária. Nosso objetivo é analisar a avaliação metabólica de homens idosos portadores de cálculos renais. MATERIAIS E MÉTODOS: Realizamos um estudo clínico tipo caso-controle. Os critérios de inclusão foram: indivíduos do sexo masculino com mais de 60 anos de idade, com antecedente de cólica renal ou diagnóstico incidental de litíase urinária após os 60 anos (grupo caso); no grupo controle foram incluídos pacientes da mesma faixa etária sem antecedente de cólica renal ou diagnóstico incidental de litíase renal. Todos os participantes foram submetidos à anamnese e aqueles selecionados realizaram perfil metabólico para diagnóstico de litíase urinária: dosagem sérica de cálcio total, cálcio ionizado, ácido úrico, fósforo, glicemia, uréia, creatinina e paratormônio (PTH); coleta de urina para urocultura e pH urinário, e amostras de urina de 24 horas para quantificação do volume e dosagem de cálcio, citrato, creatinina, ácido úrico e sódio. Foram também submetidos à radiografia simples de abdome e ultrassonografia do aparelho urinário. Os indivíduos do grupo caso realizaram dois perfis metabólicos completos de urina de 24 horas, enquanto os do grupo controle somente um perfil. Os resultados foram submetidos à análise estatística. RESULTADOS: Cento e dez indivíduos foram convocados e, após aplicação dos critérios de inclusão, 70 foram selecionados. Cinquenta e um indivíduos concluíram a investigação clínica, sendo 25 no grupo caso e 26 no controle. Cinquenta e seis por cento dos pacientes do grupo caso apresentaram hipocitratúria comparados a 15,4% do grupo controle (p=0,002). A determinação de sódio em urina de 24 horas também apresentou diferença estatisticamente significativa entre casos e controles: 64% versus 30,8%, respectivamente (p=0,017). Estes resultados foram submetidos à análise de regressão logística univariada e múltipla, respectivamente, e mantiveram seus níveis de significância. CONCLUSÃO: A hipocitratúria e a hipernatriúria são os principais distúrbios metabólicos apresentados por indivíduos idosos do sexo masculino portadores de cálculo urinário / PURPOSES: Rise in life expectancy has caused a global populational ageing and people older than 60-years have increased more than any other age group. The incidence of urinary lithiasis in aging people has increased during the last years, mainly in developed nations. Some aspects concerning organic ageing increase morbidity related to urolithiasis in older individuals making prevention and medical management of urinary stones relevant in this age group. Our objective is to evaluate metabolic parameters in men older than 60 years with urinary stones. MATERIALS AND METHODS: A case-control study was performed. Inclusion criteria were: men older than 60- years old with renal pain episodes or incidental diagnosis of urinary lithiasis beginning after 60-years old (case arm). Control group was constituted by patients older than 60 years without renal colic past or diagnosis of urolithiasis. Patients were recruited from a database from the Urologic Clinic at University of São Paulo Medical School Hospital. Each individual was submitted to anamnesis and those selected underwent a metabolic evaluation for urinary stones: serum dosages of total and ionized calcium, uric acid, phosphorus, glucose, urea, creatinine and parathyroid hormone (PTH); urine culture and urinary pH. Twenty four hour urine samples were obtained for volume quantification and for calcium, citrate, creatinine, uric acid and sodium dosages. An abdominal x-ray and ultrasonography were performed in all patients. Case arm patients underwent two complete metabolic urinary investigations while control arm individuals to only one. All results were submitted to statistical analysis. RESULTS: One hundred and ten individuals were called up for initial evaluation and 70 were selected. Fifty-one individuals concluded the whole clinical investigation: 25 in the case arm and 26 in the control arm. Hypocitraturia was present in 56% of case arm patients and 15,4% of the control arm patients (p=0,002). Hypernatriuria in 24-hour urine samples was found in 64% of case arm patients and in 30,8% of control arm patients (p=0,017). These results were submitted to univariate and multiple logistic regressions and maintained their levels of significancy. CONCLUSION: Hypocitraturia and hypernatriuria are the main metabolic disorders among aging men with urolithiasis

The production of magnetic materials is of great interest for use in the micro-fabrication industry. In particular, Permalloy (Ni80Fe20) is used in the production of micro-electromechanical systems (MEMS) due to its favourable magnetic properties (high relative permeability, low coercivity and high magnetic saturation). This leads to applications in devices such as inductors, transformers and micro-actuators. The electrodeposition of NiFe is also of fundamental electrochemical interest, as there is anomalous thermodynamic behaviour, with the less noble (iron) metal depositing preferentially to the more noble (nickel) metal. To enable consistent alloy deposition nickel and nickel-iron baths are currently almost exclusively based on boric acid. Boric acid has an important role in the deposition of NiFe films but its role(s) in the electro-deposition mechanism is (are) not wholly understood. Recently (2011) boric acid has been identified as a “substance of very high concern” based on the criteria established by EU chemical regulation, REACH. In anticipation of increased regulation an alternative was sought to provide a benign alternative to boric acid in the NiFe plating bath suitable for use in micro-fabrication. Initial work was performed to benchmark the performance of existing boric acid based electro-deposition baths. Cyclic voltammetry was performed, which demonstrated the deposition of nickel and nickel-iron from boric acid baths. Coulombic efficiencies up to 93 % were measured for the deposition of nickel using the electrochemical quartz crystal microbalance (EQCM) on platinum electrodes. For nickel-iron deposition control of the film composition was demonstrated on copper electrodes through varying the iron (II) concentration, current density and temperature. A citrate bath for the deposition of nickel-iron was then developed and characterised. Cyclic voltammetry was performed in these citrate baths demonstrating the deposition of nickel and nickel-iron. Optimal conditions for depositing Ni80Fe20 were demonstrated to be an elevated temperature (60 °C) with a current density of 20 mA cm-2 and a pH of 3. Using the EQCM the efficiency for nickel deposition was measured to be > 80 %. The effects of sodium saccharin and sodium dodecyl sulfate as additives were investigated; these were shown to influence morphology but not the coulombic efficiency. Decreasing the pH was shown to lower the efficiency of nickel deposition from the citrate bath. Comparisons of key properties were made between NiFe films deposited from a boric acid bath and the citrate bath developed in this work. Test structures were used to compare the strain in the films; no significant difference was found. For 2.2 μm thick Ni80Fe20 films the sheet resistance was measured using Greek cross structures as 0.078 ± 0.004 Ω/square for films deposited from the boric acid bath and 0.090 ± 0.006 Ω/square from the citrate bath. The magnetic saturation, Ms, was measured as 895 ± 66 emu cm-3 for deposits from the boric acid bath and 923 ± 111 emu cm-3 from the citrate bath. These again show no significant difference in these values within experimental error. Coercivities for these films were measured to be between 20 and 120 A m-1. In combination, this work demonstrates the development and characterisation of a new citrate based electrodeposition bath for nickel and nickel-iron. Similar chemical, electrical, mechanical and magnetic properties were found from films deposited from both baths, thus demonstrating the suitability of the citrate bath for the deposition of nickel-iron films in microfabrication.

A necessidade de buscar novos tratamento para a Insuficiência Cardíaca (IC) crônica levanta o questionamento da eficácia e segurança de drogas que não foram adequadamente testadas ou que ainda não tiveram sua eficácia aceita pela comunidade científica. O sildenafil é um vasodilatador com potencial eficácia na redução da pressão sistólica da artéria pulmonar (PSAP), mas com pequenos estudos e sem demonstração de impacto em desfechos duros. Os diuréticos de alça são utilizados rotineiramente em pacientes com IC sem sinais de congestão e tal prática não está recomendada nas diretrizes terapêuticas, desconhecemos sua eficácia e segurança neste cenário. No intuito de elucidar estas questões, foram desenvolvidos I) revisão sistemática com metanálise para estudo uso de sildenafil. II) revisão sistemática com metanálise para estudo uso de diurético de alça, III) Delineamento e execução em andamento de ensaio clínico randomizado multicêntrico testando a retirada de diurético de alça. I e II) Métodos e resultados: Ambas revisões sistemáticas foram realizadas no Pubmed, Embase e Cochrane, e termos relacionados à insuficiência cardíaca crônica diurético de alça e sildenafil foram utilizados, respectivamente. Após avaliação de texto completo, apenas estudos em humanos foram incluídos na metanálise. A droga sildenafil foi avaliada em 9 estudos randomizados contra placebo e demonstrou redução de hospitalização (RR 0.29, 95% C.I 0.11 to 0.78) e melhora progressiva em parâmetros funcionais e hemodinâmicos O uso de diurético de alça foi testado em 7 ensaios clínicos e não mostrou significância em piora da função renal, distúrbio eletrolítico e mudança de peso. III) Métodos e resultados: Em um estudo duplo-cego randomizado, de não inferioridade, multicêntrico compara-se o a segurança e tolerabilidade da retirada de furosemida de pacientes com IC crônica e estável com disfunção ventricular. Com início da coleta em setembro de 2015, até o momento 96 pacientes foram randomizados. Conclusão: Quanto ao sildenafil, já temos evidências que apontam para um efeito benéfico e progressivo na melhora da capacidade funcional, perfil hemodinâmico e redução de hospitalização em pacientes com IC com disfunção ventricular e pressão da artéria pulmonar elevada A recomendação para uso de diurético de alça em pacientes estáveis com IC permanece uma incógnita e o ensaio clínico em andamento nos trará uma resposta de importante impacto clínico na tomada de decisão para manutenção do uso de diurético. / The challenges and promises of new treatments for chronic heart failure (CHF) raises the question of the efficacy and safety of drugs that have not been properly tested or that have not yet had their efficacy accepted by the scientific community. Sildenafil is a vasodilator with potential efficacy in reducing pulmonary artery systolic pressure (PSAP), but with small studies and no demonstration of impact on hard outcomes. Routinely, Loop diuretics are used in patients with HF without signs of congestion and such practice is not recommended in the therapeutic guidelines, we do not know its efficacy and safety in this scenario. In order to elucidate these questions, I) systematic review with meta-analysis were developed to study the use of sildenafil. II) systematic review with meta-analysis to study the use of loop diuretics, III) Design and execution in progress of a multicenter randomized clinical trial testing for loop diuretic withdrawal. I and II) Methods and results: Both systematic reviews were performed in PubMed, Embase and Cochrane, and terms related to chronic diuretic heart failure of the loop and sildenafil were used, respectively. After full-text evaluation, only human studies were included in the meta-analysis. The drug sildenafil was evaluated in 9 randomized placebo-controlled studies and demonstrated a reduction in hospitalization (RR 0.29, 95% CI 0.11 to 0.78) and progressive improvement in functional and hemodynamic parameters. The use of a loop diuretic was tested in 7 clinical trials and did not show significant deterioration in renal function, electrolyte disturbance and weight change. III. METHODS AND RESULTS: In a double-blind randomized, non-inferiority, multicenter study, the safety and tolerability of furosemide withdrawal from patients with chronic and stable HF with ventricular dysfunction were compared. Randomization started at September 2015, to the moment 96 patients were randomized. CONCLUSION: Regarding sildenafil, we already have evidence of a beneficial and time-related effect on the improvement of functional capacity, hemodynamic profile and reduction of hospitalization in patients with HF with ventricular dysfunction and elevated pulmonary artery pressure. The recommendation for the use of a loop diuretic in stable patients with HF remains an unknown and the ongoing clinical trial will provide us with an important clinical impact response in the decision making to maintain the use of diuretics.

Submitted by DIOGO SOUSA ZANONI (zanoni_19@hotmail.com) on 2019-01-31T11:46:22Z No. of bitstreams: 1 Defesa Doutorado - entrega Pós.pdf: 2160080 bytes, checksum: a016ec7f20ec85dc0433f4452821780e (MD5) / Approved for entry into archive by Ana Lucia de Grava Kempinas (algkempinas@fca.unesp.br) on 2019-01-31T19:07:54Z (GMT) No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2160080 bytes, checksum: a016ec7f20ec85dc0433f4452821780e (MD5) / Made available in DSpace on 2019-01-31T19:07:54Z (GMT). No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2160080 bytes, checksum: a016ec7f20ec85dc0433f4452821780e (MD5) Previous issue date: 2018-12-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O glaucoma é a principal causa global de cegueira irreversível e o número de pessoas com glaucoma em todo o mundo aumentará para 111,8 milhões em 2040, mesmo com os tratamentos vigentes. Deste modo, enseja uma necessidade de desenvolver terapias neuroprotetoras que possam ser usadas para reduzir os efeitos perniciosos do glaucoma, como a morte de células ganglionares da retina (CGR). Avanços na compreensão da fisiopatologia do glaucoma é um fator chave na compreensão da patogênese da neuropatia glaucomatosa. Neste contexto a isquemia retiniana desempenha um papel central em várias doenças da retina. A patogênese da isquemia retiniana envolve alterações temporais da morfologia e morfometria da retina assim como mudanças na expressão gênica e protéica. O Citrato de Sildenafila (SC) mostrou efeito protetor nos modelos de isquemia/reperfusão (I/R) com efeitos neuroprotetores. Contudo, a administração oral de CS, em humanos, encontra inúmeros efeitos colaterais, tais como redução da pressão arterial, dores de cabeça, rubor e congestão nasal são concomitantes, assim como pacientes com moderada a grave doença cardiovascular ou aqueles submetidos a terapia a base de nitrato apresentam riscos secundários aumentados para efeitos cardiovasculares adversos. Deste modo, propomos que a administração tópica de CS pode ser uma alternativa à via oral e também ser igualmente neuroprotetor no glaucoma e podem minimizar os riscos indesejados no uso sistêmico desse fármaco, além de oferecer uma nova abordagem para a intervenção terapêutica na patogênese da neuropatia glaucomatosa. Caso nossos resultados sejam positivos, em um estudo tão abrangente, pode levar a um ensaio clínico deste fármaco seguro e promissor em pacientes com glaucoma. / Glaucoma is the leading global cause of irreversible blindness and the number of people with glaucoma worldwide will increase to 111.8 million by 2040, even with current treatments. Thus, there is a need to develop neuroprotective therapies that can be used to reduce the deleterious effects of glaucoma, such as retinal ganglion cell death (RBC). Advances in understanding the pathophysiology of glaucoma are a key factor in understanding the pathogenesis of glaucomatous neuropathy. In this context, retinal ischemia plays a central role in various diseases of the retina. The pathogenesis of retinal ischemia involves temporal changes in retinal morphology and morphometry as well as changes in gene and protein expression. Sildenafil Citrate (SC) showed protective effect in the ischemia / reperfusion (I / R) models with neuroprotective effects. However, oral administration of CS in humans finds numerous side effects such as reduced blood pressure, headaches, flushing and nasal congestion are concomitant, as well as patients with moderate to severe cardiovascular disease or those undergoing nitrate therapy have increased secondary risks for adverse cardiovascular effects. Thus, we propose that the eye drops administration of CS may be an alternative to the oral route and also be equally neuroprotective in glaucoma and may minimize the undesirable risks in the systemic use of this drug, as well as offer a new approach for the therapeutic intervention in the pathogenesis of neuropathy glaucomatous. If our results are positive, in such a comprehensive study, it may lead to a clinical trial of this safe and promising drug in patients with glaucoma. / 142471\2015-1

Submitted by DIOGO SOUSA ZANONI (zanoni_19@hotmail.com) on 2019-02-01T17:03:15Z No. of bitstreams: 1 Defesa Doutorado - entrega Pós.pdf: 2159159 bytes, checksum: c32db30aea42543516cc0f930e17164d (MD5) / Approved for entry into archive by Ana Lucia de Grava Kempinas (algkempinas@fca.unesp.br) on 2019-02-01T18:10:48Z (GMT) No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2159159 bytes, checksum: c32db30aea42543516cc0f930e17164d (MD5) / Made available in DSpace on 2019-02-01T18:10:48Z (GMT). No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2159159 bytes, checksum: c32db30aea42543516cc0f930e17164d (MD5) Previous issue date: 2018-12-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O glaucoma é a principal causa global de cegueira irreversível e o número de pessoas com glaucoma em todo o mundo aumentará para 111,8 milhões em 2040, mesmo com os tratamentos vigentes. Deste modo, enseja uma necessidade de desenvolver terapias neuroprotetoras que possam ser usadas para reduzir os efeitos perniciosos do glaucoma, como a morte de células ganglionares da retina (CGR). Avanços na compreensão da fisiopatologia do glaucoma é um fator chave na compreensão da patogênese da neuropatia glaucomatosa. Neste contexto a isquemia retiniana desempenha um papel central em várias doenças da retina. A patogênese da isquemia retiniana envolve alterações temporais da morfologia e morfometria da retina assim como mudanças na expressão gênica e protéica. O Citrato de Sildenafila (SC) mostrou efeito protetor nos modelos de isquemia/reperfusão (I/R) com efeitos neuroprotetores. Contudo, a administração oral de CS, em humanos, encontra inúmeros efeitos colaterais, tais como redução da pressão arterial, dores de cabeça, rubor e congestão nasal são concomitantes, / Glaucoma is the leading global cause of irreversible blindness and the number of people with glaucoma worldwide will increase to 111.8 million by 2040, even with current treatments. Thus, there is a need to develop neuroprotective therapies that can be used to reduce the deleterious effects of glaucoma, such as retinal ganglion cell death (RBC). Advances in understanding the pathophysiology of glaucoma are a key factor in understanding the pathogenesis of glaucomatous neuropathy. In this context, retinal ischemia plays a central role in various diseases of the retina. The pathogenesis of retinal ischemia involves temporal changes in retinal morphology and morphometry as well as changes in gene and protein expression. Sildenafil Citrate (SC) showed protective effect in the ischemia / reperfusion (I / R) models with neuroprotective effects. However, oral administration of CS in humans finds numerous side effects such as reduced blood pressure, headaches, flushing and nasal congestion are concomitant, as well as patients with moderate to severe cardiovascular disease or those undergoing nitrate therapy have increased secondary risks for adverse cardiovascular effects. Thus, we propose that the eye drops administration of CS may be an alternative to the oral route and also be equally neuroprotective in glaucoma and may minimize the undesirable risks in the systemic use of this drug, as well as offer a new approach for the therapeutic intervention in the pathogenesis of neuropathy glaucomatous. If our results are positive, in such a comprehensive study, it may lead to a clinical trial of this safe and promising drug in patients with glaucoma. / 142471\2015-1