Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-12Bitstream added on 2014-06-13T20:54:02Z : No. of bitstreams: 1 rangel_vf_me_sjrp.pdf: 852839 bytes, checksum: 3d2eb971002011ec16f7a22fafd009c2 (MD5) / A Hidroxiuréia (HU) é utilizada como medicamento em várias doenças humanas, incluindo anemia falciforme e câncer. Basicamente, na primeira, atua aumentando a porcentagem de hemoglobina fetal, o que diminui a gravidade do quadro clínico por inibir a polimerização da hemoglobina S e, no segundo, atua impedindo a síntese de DNA na fase S, desta forma bloqueando a divisão celular. Há preocupação com o tratamento longo com essa substância por causar efeitos colaterais, um dos quais se relaciona a problemas na fertilidade masculina. A Drosophila, devido a várias características, como ser o organismo com maior homologia com o homem quanto às doenças genéticas, por utilizar esses genes homólogos nos mesmos processos, mas de forma simplificada e por expressar os genes humanos em construções transgênicas, é hoje muito utilizada em estudos de doenças humanas e de respostas a fármacos, buscando esclarecer mecanismos de ação e conseqüências do uso. Neste trabalho, foram estudados, sob o efeito da HU em duas concentrações (0,1 e 0,25mg/ml de meio de cultura), no modelo biológico mencionado, as características taxa de oviposição, produtividade (número de descendentes), tempo de desenvolvimento, mortalidade, longevidade, tempo de pré-cópula , duração da cópula, presença de alterações morfológicas externas, morfologia do aparelho reprodutor masculino, modificações do peso das moscas, morfologia dos cromossomos politênicos e padrão de expressão das enzimas esterasicas. A produtividade foi analisada em seis gerações consecutivas, visando obter maior compreensão dos efeitos da HU. Cada característica analisada envolveu uma metodologia diferente, descrita no corpo do trabalho. A análise estatística foi baseada na aplicação da ANOVA, e nas comparações de Kruskal-Wallis e Mann-Whitney. Em algumas características o efeito do tratamento mostrou-se... / Hydroxyurea (HU) is used as a medicine in several human diseases, including sickle cell disease (SCD) and cancer. Basically, in the first, HU acts by increasing the percentage of fetal hemoglobin, thus decreasing the severity of the disease symptoms due to inhibition of the hemoglobin S polymerization. In the second mentioned disease, it acts by preventing DNA synthesis in S phase, thereby blocking cell division. There is some concern about side effects caused by the long-term treatment with HU. One of them relates to problems in male fertility. Drosophila, because of its characteristics, such as being the organism with the highest homologies with man as to genetic diseases, since it uses homologous genes in these processes in a simplified form, and also by expressing human genes in transgenic constructs, is now being widely used in studies of human diseases and responses to drugs, seeking for clarifying action mechanisms and consequences of their use. In this work, we studied, in Drosophila biological characteristics under the effect of HU in two concentrations (0.1 and 0.25 mg / ml of culture medium), including oviposition rate, productivity (number of offspring), time development, mortality, longevity, pre-mating and mating duration, presence of morphological changes in external morphology and in the male reproductive system, fly weight changes, polytene chromosome morphology and patterns of esterase enzymes. Productivity was analyzed in six consecutive generations. Each feature analyzed involved a different methodology, described in the body of the work. Statistical analysis was based on the application of ANOVA, and comparisons using the Kruskal-Wallis and Mann-Whitney tests. In some characteristics, the effect of treatment was dosisdependent. In the light of numbers, productivity was higher in control experiments (C) of six generations, and among the treated ones with... (Complete abstract click electronic access below)

La ghréline est un peptide de 28 acides aminés, produit principalement par l’estomac et caractérisé par la présence d’un groupement octanoyl sur la sérine en position 3 (Kojima et al. 1999). La ghréline stimule la libération de l’hormone de croissance (GH) et régule la prise alimentaire et le métabolisme énergétique (Gualillo et al. 2003). Ces activités biologiques sont principalement médiées par le « growth hormone secretagogue receptor » (GHS-R). Deux sous-types de GHS-R, produits par épissage alternatif d’un même gène, ont été clonés :le GHS-R 1a, dont l’activation entraîne une libération de calcium via la formation d’inositol 1,4,5-trisphosphate (IP3), et le GHS-R 1b, qui ne semble pas lié à une activité biologique (Howard et al. 1996). <p>La première partie de mon travail de thèse consistait en l’étude de la dégradation de la ghréline. La ghréline circule dans le sang principalement sous forme de des-acyl ghréline, une forme de ghréline dépourvue du groupement octanoyl qui ne se lie pas au GHS-R 1a. Peu d’études ont été réalisées sur le catabolisme de la ghréline. Les enzymes impliquées dans la dégradation de la ghréline étant des régulateurs importants de son activité biologique, le but de cette étude était d’identifier les sites de clivage et les enzymes impliquées dans la dégradation de la ghréline par du sérum, des sous-fractions plasmatiques et des homogénats de tissus. Nous avons montré qu’au contact de sérum humain et de rat, la ghréline est désoctanoylée, sans protéolyse. Dans le sérum humain, nous avons montré que la butyrylcholinestérase et la « platelet-activating factor acetylhydrolase » (PAF-AH), une phospholipase associée aux lipoprotéines de basse densité (LDL), sont impliquées dans ce phénomène (articles n°1 et n°2). En parallèle, nous avons montré que la ghréline peut être transportée dans la circulation sanguine par les lipoprotéines riches en triglycérides (TRL), les LDL, et les lipoprotéines de haute et de très haute densité (HDL et VHDL) (article n°2). Dans le sérum de rat, la désoctanoylation de la ghréline implique une carboxylestérase (article n°1). Au contact d’homogénats de tissus, la ghréline est dégradée à la fois par désoctanoylation et protéolyse N-terminale, suggérant la participation d’estérases et d’aminopeptidases. Nous avons identifié cinq sites de clivage dans la ghréline :entre les résidus Ser2-(acyl)Ser3 (dans l’estomac et le foie), (acyl ?)Ser3-Phe4 (dans l’estomac, le foie et le rein), Phe4-Leu5 (dans l’estomac et le rein), Leu5-Ser6 et Pro7-Glu8 (dans le rein) (article n°1). <p>La deuxième partie de mon travail de thèse consistait à étudier l’expression et la production de ghréline par différentes lignées leucémiques (HEL, HL-60, THP-1, SupT1), par des leucocytes poly- et mononucléés et par des plaquettes sanguines, et à étudier l’effet de la ghréline sur la prolifération cellulaire. Pour cela, nous avons mis au point des dosages radioimmunologiques (RIA) permettant de quantifier et de distinguer les formes octanoylées et non octanoylées de la ghréline, et nous avons caractérisé en détail les anticorps SB801 et SB969 obtenus. Par HPLC en phase inverse suivie des RIAs, nous avons mis en évidence la présence de ghrélines octanoylée et non octanoylée dans chaque population de cellules. Plus de 80 % de la ghréline produite est octanoylée dans les cellules HEL, les leucocytes et les plaquettes. Nous avons montré que la ghréline endogène stimule la prolifération des cellules HEL de façon autocrine impliquant un récepteur encore non identifié, distinct du GHS-R 1a (article n°3). La ghréline et la des-acyl ghréline inhibent la prolifération des leucocytes mononucléés mais sont dépourvues d’effet sur les cellules HL-60, THP-1 et SupT1. Malgré la présence du GHS-R 1a dans les leucocytes mononucléés, cet effet pourrait être médié par un récepteur différent puisque la des-acyl ghréline exerce le même effet que la ghréline sur la prolifération (article n°4). <p><p><p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

A metagenômica trouxe novas perspectivas ao estudo de comunidades microbianas no ambiente, permitindo explorar tanto a diversidade taxonômica de microrganismos ainda não-cultivados, como o acesso direto a genes e vias metabólicas. Neste trabalho, foram construídas bibliotecas metagenômicas a partir de amostras de sedimentos de mangue da Baía de Guanabara (RJ), impactadas com hidrocarbonetos de petróleo e metais pesados. Proteobacteria (33,3%), bactérias afiliadas a redutoras-de-sulfato (29,7%) e Firmicutes (20%) representaram os grupos principais nas amostras ambientais, baseado em análises filogenéticas de rDNA 16S, ao passo que isolamentos seletivos utilizando diesel e naftaleno permitiram a recuperação preferencial de delta-Proteobacteria e actinomicetos. Bibliotecas metagenômicas dos sedimentos enriquecidos com óleo diesel, com insertos entre 25 e 35 Kb clonados em fosmídeos, foram triadas para detecção de genes catabólicos de monoxigenases (alkB1) e expressão de epóxido-hidrolases, esterases, lipases e monoxigenases em ensaios de alto desempenho (HTS, high throughput screening). Clones reativos a alkB1 foram detectados, porém não foram funcionais nas condições de HTS testadas. Nas bibliotecas de fosmídeos triadas, vários clones apresentaram atividade enzimática, sendo que dois apresentaram atividade de lipase-esterase com alta seletividade, elevada taxa de conversão de substratos e excesso enantiomérico (ee >99%). Os resultados de HTS comprovaram a eficiência do uso da clonagem direta de DNA ambiental na expressão de vias metabólicas de interesse com potencial de aplicação biotecnológica. / Metagenomics brought a new perspective to the study of microbial communities in the environment, enabling access to the taxonomic diversity of uncultured microorganisms, as well as direct access to genes and metabolic pathways. In the current study, metagenomic libraries were constructed from mangrove sediment samples of the Guanabara Bay (RJ, Brazil), impacted with oil hydrocarbons and heavy metals. Proteobacteria (33.3%), sulfate-reducing affiliated bacteria (29.7%) and Firmicutes (20%) represented the main groups in the environmental samples based upon 16S rDNA phylogenetic analysis, whereas selective isolation using diesel and naphtalene yielded delta-Proteobacteria and actinomycetes. Metagenomic libraries of diesel-enriched sediment samples, with 25 to 35 Kb fosmid inserts, were screened for detecting monooxigenase genes (alkB1) and expression of epoxide hydrolases, esterases, lipases and monooxigenases in high throughput screening (HTS) assays. Clones reactive to the alkB1 probe were detected, but were not functional under the HTS conditions used. Several functional clones were detected in the clone library, and two showed lipase-esterase activity with high rates of substrate conversion and enantiomeric ratio (ee >99%). The results obtained on HTS showed the efficiency of the direct cloning of environmental DNA for the expression of metabolic pathways with potential biotechnological application.

Fluorescence-based imaging techniques provide a simple, highly sensitive method of studying live cells and whole organisms in real time. Without question, fluorophores such as GFP, fluorescein, and rhodamines have contributed vastly to our understanding of both cell biology and biochemistry. However, most of the fluorescent molecules currently utilized suffer from one major drawback, the use of visible light. Due to cellular autofluorescence and the absorbance of incident light by cellular components, fluorescence imaging with visible wavelength fluorophores often results in high background noise and thus a low signal-to-noise ratio. Fortunately, this situation can be ameliorated by altering the wavelength of light used during imaging. Near-infrared (NIR) light (650-900 nm) is poorly absorbed by cells; therefore, fluorophores excited by this light provide a high signal-to-noise ratio and low background in cellular systems. While these properties make NIR fluorophores ideal for cellular imaging, most currently available NIR molecules cannot be used in live cells. The first half of this thesis addresses the synthetic difficulties associated with preparing NIR fluorophores that can be used within living systems. Small molecule NIR fluorophores are inherently hydrophobic which makes them unsuitable for use in the aqueous environment of the cell. Water-solubility is imparted to these dyes through highly polar sulfonates, which subsequently prevents the dyes from entering the cell. The novel work presented here details vii synthetic routes to aid in the development of sulfonated NIR fluorophores, which can be delivered into live cells through the inclusion of an esterase-labile sulfonate protecting group. Application of these synthetic techniques should allow for the development of novel NIR fluorophores with intracellular applications. The second half of this thesis addresses the need for novel NIR imaging reagents. Although several classes of NIR scaffolds do exist, most NIR probes are derivatives of a single class, heptamethine indocyanines. The work described here increases this palette by displaying the ability of NIR oxazines to function as an imaging reagent in live cells and in vivo and as a molecular sensor of biologically-relevant environmental conditions. Combined, the work contained herein has the capacity to not only advance the current NIR toolkit, but to expand it so that fluorescence imaging can move out of the dark and into the NIR light.

No description available.

O. oeni est responsable de la fermentation malolactique des vins. Elle doit en permanence s’adapter aux fluctuations physico-chimiques de son environnement. La production de protéines Hsp constitue un mécanisme majeur d’adaptation de la bactérie à son environnement. Chez O. oeni, la protéine CtsR est l’unique régulateur identifié à ce jour des gènes hsp. Ce manuscrit aborde la caractérisation des mécanismes de régulation de la réponse au stress chez O. oeni. Une partie de ce travail a consisté à développer un nouvel outil d’expression de gènes chez O. oeni. Cet outil a permis l’étude de la fonction in vivo du gène hsp18 par une technique de modulation de l’expression de gènes par synthèse d’ARN antisens (ARNas). La production d’ARNas ciblant l’ARN messager du gène hsp18 entraîne une diminution du taux protéique de Lo18 et induit une perte de cultivabilité en conditions de stress. Ces résultats montrent, pour la 1ère fois in vivo, l’implication de Lo18 dans la thermotolérance et l’acidotolérance de O. oeni. Cette même approche appliquée au gène ctsR a induit une perte de cultivabilité en conditions de stress confirmant le rôle clef du locus ctsR dans la réponse au stress de O. oeni. Les mécanismes de régulation de l’activité de CtsR ont été appréhendés par complémentation d’un mutant ctsR déficient de B. subtilis via l’expression de ctsR de O. oeni. Des tests de thermoinduction mettent en évidence la thermosensibilité du CtsR de O. oeni dont l’activité est levée à une température inférieure à 33°C. Le pSIPSYN est un outil prometteur valorisé au cours de ce travail par une étude évaluant l’impact de deux estérases de O. oeni, EstA2 et EstA7 sur le profil aromatique du vin. / O. oeni is responsible for wine malolactic fermentation. As any organism, O. oeni tries to adapt its physiology to environmental fluctuations by producing Hsp proteins encoded by the hsp genes. In O. oeni, CtsR is currently the only regulator of hsp genes. As an alternative to the lack of genetic tool, with the goal of understanding the mechanisms of O. oeni stress response, we developed a new expression vector, the pSIPSYN, to produce antisense RNA targeting of hsp18 mRNA. The synthesis of hsp18 asRNA leads to the decrease in the protein level of Lo18 and induced a loss of cultivability after heat or acid shock showing for the first time in vivo involvement of Lo18 in thermotolerance and acidotolerance in O. oeni. The O. oeni ability of the membrane fluidity restoration of after ethanol stress was strongly affected in the presence of asRNAof hsp18 gene. Then, the ctsR function in O. oeni was investigated with this new genetic tool. Inhibition of the ctsR expression by asRNA approach induced a loss of cultivability after heat or acid shock confirming the key role of ctsR locus in the O. oeni stress response. B. subtilis was used to characterize the regulation of CtsR activity. The ctsR gene of O. oeni was expressed to complement a B. subtilis ctsR-deficient strain and restore the wild-type phenotype. Thermoinduction tests performed to understand the thermosensibility of CtsR showed that O. oeni CtsR is a specific thermosensor inactivated at a temperature threshold below 33°C. The pSIPSYN is a promising tool valorized in this work through an oenological study by evaluating of the impact of O. oeni two esterases, and EstA2 EstA7 on wine ester profile.

RNA interference (RNAi) is a conserved mechanism in a wide range of eukaryotes. Introduction of synthetic dsRNA could specifically target suppression of a gene or could result in off-target silencing of another gene due to sequence similarity. To verify if the observed phenotype in an RNAi transgenic line is due to silencing of a specific gene or if it is due to another nontarget gene, a synthetic gene complementation approach could be used. Synthetic gene complementation described in this method uses the technology of synthesizing a variant of a native gene (used in RNAi silencing) to maximize the difference in DNA sequences while coding for the exact same amino acids as the original native gene. This is achieved through the use of alternate codons. The new variant gene is expressed in the original RNAi transgenic lines and analyzed for complementation of the RNAi phenotype. Complementation of the RNAi-induced phenotype will indicate gene-specific silencing and not off-target silencing.

No description available.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-09-21T12:34:57Z No. of bitstreams: 1 priscila_f_v_medeiros_ioc_bp_0058_2011.pdf: 6129115 bytes, checksum: a9a22a0f84ef8206e0afcbc77535046a (MD5) / Made available in DSpace on 2012-09-21T12:34:57Z (GMT). No. of bitstreams: 1 priscila_f_v_medeiros_ioc_bp_0058_2011.pdf: 6129115 bytes, checksum: a9a22a0f84ef8206e0afcbc77535046a (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O uso de inseticidas neurotóxicos contra o mosquito Aedes aegypti ainda é um componente importante nos programas de controle de dengue. No Brasil, organofosforados (OP) e piretróides (PI) são usados para o controle de larvas e adultos de A. aegypti, respectivamente, desde 1967 e 2000. O uso frequente destes produtos selecionou populações resistentes do vetor, e motivou o Ministério da Saúde (MS) a coordenar, desde 1999, uma rede nacional de monitoramento da resistência de A. aegypti (MoReNAa), da qual nosso laboratório participa desde o início. A resistência é principalmente derivada de fatores metabólicos (enzimas que detoxificam o inseticida) ou de mutações nos sítios-alvo dos inseticidas, no Sistema Nervoso Central. Bioensaios com larvas revelaram a resistência de várias populações ao OP temephos, larvicida empregado há mais de 40 anos no país. A Rede MoReNAa conta também com bioensaios para adultos, com testes moleculares para avaliação de alteração no alvo de PI, o canal de sódio regulado por voltagem (AaNav) e com ensaios bioquímicos para quantificação, em mosquitos adultos, da atividade enzimática de Glutationa S-Transferases (GST), Esterases e Oxidases de Função Mista (MFO) (relacionadas à resistência metabólica), e de Acetilcolinesterase (Ace) (alvo de OP e carbamatos - CB). Além de serem usadas classes distintas de inseticidas contra larvas e adultos, as aplicações são feitas de maneira diferenciada: em 4-6 ciclos anuais sobre as larvas, e apenas em situações de emergência sobre os mosquitos adultos - procedimento que tem o potencial de elicitar mecanismos (e intensidades) de resistência diferentes. Além disso, bioensaios para quantificação da resistência a OP são feitos com larvas, enquanto os ensaios bioquímicos estavam disponíveis apenas para mosquitos adultos. Estes foram os principais motivos para adaptar, no âmbito desta dissertação, ensaios bioquímicos para o estágio larvar do vetor. Em relação ao ensaio da Ace, que conta com duas reações, uma na presença e a outra na ausência do CB propoxur verificamos, por meio de curvas do tipo dose- resposta, diferenças entre larvas e adultos. Além disto identificamos, em algumas populações do vetor resistentes a OP, alterações na atividade total desta enzima. Uma vez que algumas destas enzimas participam também de processos endógenos, que ocorrem naturalmente nos insetos, quantificamos sua atividade ao longo do desenvolvimento da cepa referência de suscetibilidade, Rockefeller, e de duas populações de campo. Nestes ensaios foram observadas quatro grandes “categorias” de perfis de atividade enzimática: 1) maiores atividades no estágio adulto (AChE); 2) maiores atividades no estágio larval (Esterases “α-EST” e “β-EST”); 3) atividades que aumentam no decorrer de cada estágio avaliado (MFO) e 4) atividades que tendem a aumentar no estágio larvar e a diminuir nos primeiros dias de vida adulta (DVA) (Esterase “ρNPA” e GST). Posteriormente, ensaios bioquímicos com larvas e adultos de populações de campo revelaram alterações de Ace e Esterases preferencialmente no estágio larvar, alterações de GST mais restritas ao estágio adulto, e alteração de MFO nos dois estágios do vetor. Estes ensaios possibilitam conhecer com detalhe os mecanismos de resistência em diferentes populações do vetor e podem contribuir com a definição de estratégias racionais para o controle de A. aegypti. / The use of neurotoxic insecticides against the mosquito Aedes aegypti is still an important component in dengue control programs. In Brazil, organophosphates (OP) and pyrethroids (PI) are used for the control of A. aegypti larvae and adults since, respectively, 1967 and 2000. The frequent use of these products has selected resistant vector populations, and prompted the Ministry of Health (MS) to start the coordination, in 1999, of an Aedes aegypti insecticide resistance monitoring network (MoReNAa); our laboratory participates in the network since its beginning. Resistance is mainly derived from metabolic factors (enzymes detoxifying the insecticides) or from mutations at the target sites of insecticides (in the Central Nervous System). Bioassays with larvae disclosed resistance of various populations to the OP temephos, larvicide employed for over 40 years in the country. MoReNAa network also performs bioassays with adults, molecular tests to assess substitution at the PI target site, the voltage regulated sodium channel (AaNAv), and biochemical assays that quantify, in adult mosquitoes, the activity of Glutathione S-Transferases (GST), Esterases and Mixed Function Oxidases (MFO) (related to metabolic resistance), and of Acetylcholinesterase (Ace) (target of OP and carbamates - CB). Besides using different insecticide classes against larvae and adults, the treatment are performed differently: in 4-6 times per year for larvae and only in emergency situations in the case of adult mosquitoes – a procedure that has the potential to elicit different mechanisms (and intensities) of resistance. Furthermore, bioassays for quantification of resistance to OP are made with larvae, while biochemical assays were available only for adult mosquitoes. These were the main reasons to adapt, in the context of this dissertation, biochemical assays for the larval stage of the vector. Considering the Ace test, consisting of two reactions, in the presence or in the absence of the CB propoxur, we identified - through the use of inhibition curves - differences between larvae and adults. We also detected, in some OP resistant vector populations, changes in the total activity of this enzyme. Since some of the enzymes of metabolic resistance are also involved in endogenous processes, that occur naturally in insects, we quantified their activity during the development of the reference strain of susceptibility, Rockefeller, and of two field populations. In these experiments we observed four major "categories " of enzyme activity profiles: 1) higher activity in the adult stage (AChE); 2) higher activity in the larval stage (“α-EST” and “β- EST” Esterases); 3) activities that increase during each stage evaluated (MFO) and 4) activities tending to increase in the end of the larval stage and to decrease in the first days of adult life (DVA) (Esterase “ρNPA” and GST). Subsequently, biochemical assays with larvae and adults of field populations revealed main changes in Ace and Esterases in the larval stage, GST changes preferably in the adult stage, and MFO alterations on both vector stages. These assays enable the detailed knowledge of resistance mechanisms of different vector populations and can contribute to define rational strategies for A. aegypti control.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid synthase (FASN) is over-expressed in many cancers, and novel inhibitors that target FASN may find use in the treatment of cancers. It has been shown that orlistat, an FDA approved drug for weight loss, inhibits the thioesterase (TE) of FASN, but can be hydrolyzed by TE. To understand the mechanisms of TE action and for designing better FASN inhibitors, I examined the mechanism of orlistat hydrolysis by TE using molecular dynamics simulations. I found that the hexyl tail of orlistat undergoes a conformational transition, destabilizing a hydrogen bond that forms between orlistat and the active site histidine. A water molecule can then hydrogen bond with histidine and become activated to hydrolyze orlistat. These findings suggest that rational design of inhibitors that block hexyl tail transition may lead to a more potent TE inhibitor. To search for novel inhibitors of TE, I performed virtual DOCK screening of FDA approved drugs followed by a fluorogenic assay using recombinant TE protein and found that proton pump inhibitors (PPIs) can competitively inhibit TE. PPIs, which are used for the treatment of gastroesophageal reflux and peptic ulcers, work to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase in the stomach. Recently, PPIs have been reported to reduce drug resistance in cancer cells when used in combination with chemotherapeutics, although the mechanism of resistance reduction is unknown. Further investigation showed that PPIs are able to decrease FASN activity and cancer cell proliferation in a dose-dependent manner. These findings provide new evidence that FDA approved PPIs may synergistically suppress cancer cells by inhibiting TE of FASN and suggests that the use of PPIs in combinational therapies for the treatment of many types of cancer, including pancreatic cancer, warrants further investigation.