Submitted by Rafael Dias do Espírito Santo null (rafael.lqof@gmail.com) on 2017-05-09T16:49:03Z No. of bitstreams: 1 Tese - Rafael Dias do Espírito Santo.pdf: 5290008 bytes, checksum: d5945a04420d68f212920428f82770e4 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-10T18:27:32Z (GMT) No. of bitstreams: 1 santo_rde_dr_bauru.pdf: 5290008 bytes, checksum: d5945a04420d68f212920428f82770e4 (MD5) / Made available in DSpace on 2017-05-10T18:27:33Z (GMT). No. of bitstreams: 1 santo_rde_dr_bauru.pdf: 5290008 bytes, checksum: d5945a04420d68f212920428f82770e4 (MD5) Previous issue date: 2017-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Leishmanioses são doenças causadas por protozoários parasitas, causadas por mais de 20 espécies do gênero Leishmania, sendo estimados 1,3 milhões de novos casos anualmente, que resultam em 20 – 30 mil mortes. Os fármacos usados para o tratamento de leishmanioses são antigos, estão relacionados a graves efeitos tóxicos, e o aparecimento de cepas de parasitas resistentes a esses fármacos tem tornado urgente a necessidade de se encontrar medicamentos novos, seguros e eficazes contra leishmanioses. Compostos do tipo guanidinas são estudados para o tratamento de doenças tropicais negligenciadas, sendo que alguns estudos testaram a efetividade de compostos guanidínicos contra leishmaniose. O principal objetivo do presente trabalho foi sintetizar, caracterizar e avaliar a atividade leishmanicidade de compostos que possuem o núcleo guanidínico. A síntese dos compostos derivados guanidínicos foi descrito e os compostos foram caracterizados por Espectrometria de Massas usando Ionização por Eletrospray e por Espectroscopia de Ressonância Magnética Nuclear de 1H e de 13C. Quatorze compostos foram avaliados quanto à atividade leishmanicida frente às cepas promastigotas de L. amazonensis e também frente a macrófagos peritoneais de camundongo infestados com leishmania amastigotas. Os valores de IS de duas moléculas contra as formas amastigotas (SI=130,25 para o composto LQOF-G7 e 36,37 para o composto LQOF-G1), sugeriram que estes podem ser candidatos promissores para estudos in vivo. As análises preliminares dos resultados de avaliação biológica mostraram que os compostos que possuem grupos eletroretiradores ligados ao átomo C4 do anel da porção anilina, foram mais ativos e seletivos contra leishmania promastigota. O teste do micronúcleo para a mutagenicidade foi realizado para cinco compostos, incluindo três dos mais ativos contra Leishmania (LQOF-G1, LQOF-G2 e LQOF-G7). Para todos os compostos avaliados não foram observados diferenças significativas entre a frequência de células micronucleadas quando comparados com o controle negativo, sugerindo que os compostos não são mutagênicos quando avaliados pelo teste de micronúcleo, mesmo nas concentrações mais altas. / Leishmaniases are diseases caused by protozoan parasites from >20 Leishmania species, being estimated 1.3 million new cases annually, which result in 20,000–30,000 deaths. The drugs used for the treatment of leishmaniases are old and have serious toxic effects. In addition, the emergence of drug-resistant parasite strains has caused an urgent requirement for novel, safe, and efficacious drugs against leishmaniasis. Several guanidine derivatives have been studied in the treatment of neglected diseases and some of these studies tested the effectiveness of guanidine compounds against leishmaniasis. The main objective of the present work was the synthesis, characterization and evaluation of leishmanicide activity of compounds having guanidine nuclei. Synthesis of the guanidine derivatives was described and the compounds were characterized by Mass Spectrometry with Eletrospray Ionization and Nuclear Magnetic Ressonance for 1H and 13C. Fourteen compounds were evaluated for their leishmanicidal activity against L. amazonensis promastigotes and mouse peritoneal macrophages infested with leishmania amastigotes. The IS values, of two molecules, against the amastigote forms (SI = 130.25 for compound LQOF-G7 and 36.37 for compound LQOF-G1), suggested that they may be promising candidates for in vivo studies. The preliminary analysis of the results of biological evaluation showed that compounds, which contain electro-withdrawing groups bound to atom C4 of aniline ring, were more active and selectives against Leishmania promastigotes. The micronucleus test for mutagenicity was performed for five compounds, including three of the most active against Leishmania (LQOF-G1, LQOF-G2 and LQOF-G7). For all compounds evaluated, no significant differences were observed between the frequencies of micronucleated cells when compared to the negative control, suggesting that the compounds are not mutagenic when evaluated by the micronucleus test, even at the highest concentrations.

Le stress oxydatif définit un phénomène cellulaire particulier caractérisé par un niveau élevé de molécules hautement réactives, essentiellement lié à l’utilisation de l’oxygène par les systèmes biologiques via la respiration. La dérégulation de l’état oxydatif de la cellule est à l’origine soit de processus d’adaptations efficaces (adaptation à l’altitude) soit de pathologies (AVC, infarctus). Ce travail de thèse s’est porté sur l’étude de deux nouvelles cibles pouvant induire une résistance/tolérance à la variation du stress oxydatif : la première est la protéine canal CFTR («Cystic Fibrosis Transmembrane conductance Regulator») et la seconde la voie d’activation d’eIF5A («eukaryotic Initiation translation Factor 5A»). Nous avons pu mettre en évidence que la protéine CFTR grâce à sa perméabilité au glutathion (l’antioxydant majoritaire cellulaire) est un modulateur de l’état oxydatif de la cellule, que ce soit lors de l’exposition à des agents cytotoxiques (cisplatine) ou lors de l’adaptation à des conditions hypoxiques chroniques. La deuxième cible identifiée est le facteur eIF5A qui est la seule protéine activée par la fixation d’un résidu hypusine. L’inhibition de cette modification post-traductionnelle protège les cellules d’une production d’espèces réactives induite par l’anoxie. Cette résistance à l’anoxie est accompagnée d’un profond remodelage métabolique et mitochondrial. Sur des modèles animaux d’ischémie (rein et cerveau), l’inhibition de l’activation d’eIF5A conduit à une protection des organes face à un manque d’oxygène. Ces études fondamentales ont des applications cliniques potentielles dans des pathologies humaines (infarctus, AVC, transplantation). / Oxidative stress represents a particular cellular condition, characterized by an intracellular increase in thereactive species level. These species are highly reactive towards biomolecules and result of oxygenconsumption by biological systems essentially through respiration. Deregulation of the cellular oxidativestate can initiate adaptive processes (as elevation adaptation) or several human pathologies (stroke,infarct). This thesis work has been devoted to the study of two news potential targets allowing atolerance/resistance towards disequilibrium of oxidative stress; the first one is CFTR, a channel protein(«Cystic Fibrosis Transmembrane conductance Regulator»), and the second one is the activation pathwayof the translation factor eIF5A («eukaryotic Initiation translation Factor 5A»). Based on the peculiaractivity of CFTR, consisting in the transport of glutathione, the major antioxidant of the cell, weevidenced the role of CFTR in the management of cellular oxidative state during cytotoxic drugexposure (cisplatin) or during adaptation to chronical hypoxia. The second target, eIF5A is the only oneprotein described as post-translationally modified by fixation of a hypusine residue. We demonstratedthat inhibition of eiF5A activation protect cells from reactive oxygen species generated during anoxia. Atcellular level, this protection is accompanied with deep metabolic and mitochondrial changes. Usinganimal models, we showed that inhibition of this eiF5A activation allows a tolerance against ischemicaccident in different organs (kidney and brain). These fundamentals results can have extensiveapplication in human clinical use (infarct, stroke, graft).

Thrombin is a critical enzyme involved in blood coagulation and haemostasis. For this reason the study of its interactions with substrates, inhibitors and modulator is essential. It is also a unique enzyme in the serine protease family because unlike enzymes like trypsin and chymotrypsin its activity is modulated by various endogenous and exogenous ligands. This is due to the presence of “exosites” on the thrombin surface. Exosite II, unlike exosite-I, has not been characterized for its allosteric effect. In order to understand the structural basis of interaction and inhibition of inhibitor 4AS, which possibly interacts with exosite-II, native bovine thrombin crystals and human thrombin crystals grown in presence of 4-AS were prepared. X-ray diffraction data was collected on 4AS soaks of native bovine thrombin as well as human thrombin crystals. The data were phased by molecular replacement using appropriate search models. The structures were refined to R factors of 0.24 and 0.27 for native bovine thrombin-4AS soaks and human thrombin-4AS co-crystals respectively. Examination of a 2Fo-Fc electron density map revealed no density for 4-AS. Low affinity of the inhibitor may be the reason for its absence in the solved structures. In the process of solving these structures, unliganded native bovine thrombin in a new crystal form, previously unreported in literature, was solved. The structure shows an overall topology similar to that found in previously published thrombin molecules. Examination of the crystal packing shows that the exosite-II is solvent exposed. This crystal form can be used in the future to study interaction of exosite-II ligands. To characterize the interaction of sucrose octasulfate with thrombin, which may interact with thrombin exosite-II, fluorimetric equilibrium binding titrations were performed using the active site fluorescent probe para-amino benzamidine. At physiological salt concentrations, the KD was found to be ~22 μM, which is lower than heparin fragment of corresponding length. The higher affinity was attributed to the high charge density of the ligand. Measurement of KD at different salt concentrations showed a significant amount of contribution to the binding energy from ionic interactions. Based on the salt dependence experiments, the number of charged interactions per sucrose octasulfate molecule interacting with thrombin was found to be 3.5. Competitive experiments of sucrose octasulfate with FDs (a sulfated dehydro-polymer being investigated in the lab for its anticoagulant properties) for inhibition of thrombin activity showed competitive effects that did not appear to follow Dixon-Webb competitive phenomenon. It was found that sucrose octasulfate itself is a weak inhibitor of thrombin. To investigate the mode of interaction, co-crystals of sucrose-ocasulfate complexed with thrombin were prepared. High resolution data (2.2 Å) was collected. The structure solved using this data showed weak density for two sucrose octasulfate molecules. Sucrose octasulfate was modeled into this density and refined. The refined structure shows that two sucrose octasulfate molecules bind to two thrombin monomers of the asymmetric unit at exosite-II. One of the sucrose octasulfate molecules interacts with both monomers, and could be present as an artifact of crystal packing. The second molecule interacts with exosite-II of only one of the thrombin monomers. The key residues involved in the interaction are Lys236, His91, Arg93 and Arg101. The thrombin-sucrose octasulfate structure does not show any major deviation from unliganded structure. It is possible that the conformational change may have been masked due to crystal packing. Characterization of this novel interaction mode of sucrose octasulfate interaction with thrombin adds one more candidate to the list of compounds that interact with exosite-II in a manner very similar to heparin, but unlike heparin can inhibit thrombin activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (Dhps) to post- translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of Dhps in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein Eif5a is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.

Le genre Pneumocystis désigne un groupe de champignons opportunistes présentant une étroite spécificité d'hôte. Il détermine lors d'immunodépression sévère une infection pulmonaire grave, la pneumonie à Pneumocystis (PPC). La transmission de Pneumocystis par voie aérienne d'un hôte développant une PPC à un hôte susceptible a été démontrée à l'aide des modèles murins. Les travaux menés chez la souris ont montré également que des sujets immunocompétents colonisés par Pneumocystis murina peuvent transmettre le champignon à des souris immunodéprimées qui développeront une PPC ultérieurement. Les individus colonisés par Pneumocystis sp., ainsi que ceux développant une PPC, participeraient au réservoir du champignon. La survenue de cas groupés de PPC en milieu hospitalier est en faveur de la transmission interindividuelle de Pneumocystis jirovecii (P.jirovecii) chez l’homme. La détection de l'ADN de P.jirovecii dans l'air exhalé par les patients développant une PPC suggère que cette transmission se fait par voie aérienne. La caractérisation des populations infectées par P.jirovecii et la caractérisation génotypique du champignon au sein de son réservoir humain constituent la base de ce travail de recherche. Nous avons montré que la prévalence de la colonisation par P.jirovecii est faible chez les patients atteints de mucoviscidose et suivis dans notre CHU. La participation de ces patients au réservoir de P.jirovecii à Brest serait donc marginale. Cette faible prévalence pourrait être le reflet d'une faible circulation du champignon dans les communautés humaines dans notre région. Nous avons évalué le dosage du ß-1,3-D glucane sérique pour dépister les populations infectées. Ce dosage couplé à la détection de P.jirovecii dans les prélèvements respiratoires par la microscopie et la PCR, permet de différencier les patients développant une PPC et les patients présentant une colonisation pulmonaire par P.jirovecii. De plus, les premières données sur le ß-1,3-D glucane au cours de la primo-infection chez le nourrisson ont été obtenues.En termes de caractérisation de P.jirovecii dans notre région, l'analyse du locus dihydropteroate synthase (DHPS) a montré que: i) le lieu habituel de résidence plutôt que le lieu de diagnostic de l’infection à P.jirovecii serait un facteur prédictif d’infection par un mutant, ii) P.jirovecii pourrait circuler en France d’une région à une autre via des voyageurs infectés, iii) la prévalence de mutants potentiellement résistants chez les patients vivant effectivement à Brest était de 0%. L'analyse des séquences des "internal transcribed spacers" (ITS) 1 et 2 de P. jirovecii conforte l'hypothèse que les patients développant une PPC et les patients colonisés sont infectés par des populations fongiques présentant des caractéristiques identiques. Tous les patients, quelle que soit la présentation clinique de leur infection, constitueraient un réservoir unique et commun de P.jirovecii. Les travaux de génotypage ont constitué l'étape préalable nécessaire à l'analyse de cas groupés d'infections à P.jirovecii survenus chez des patients transplantés rénaux au CHU de Brest. Nous avons apporté des données originales sur le rôle des patients colonisés en tant que source potentielle de P. jirovecii dans un contexte d'acquisition et de transmission nosocomiales du champignon. Par ailleurs, la concordance partielle ou complète des génotypes ITS et DHPS dans les couples "prélèvements d'air–LBA" réalisés chez des patients développant une PPC est compatible avec l’exhalation du champignon et sa diffusion aérienne dans l’environnement hospitalier. Ces données apportent des arguments pour l'application de mesures de prévention des infections nosocomiales à P. jirovecii. Les précautions "gouttelettes" recommandées par la Société Française d'Hygiène Hospitalière devraient être appliquées a minima aux patients développant une PPC. Nous proposons leur extension aux patients colonisés par le champignon. / The genus Pneumocystis represents a group of opportunistic fungi that show strong host specificity. It is the cause of severe pneumonia (Pneumocystis Pneumonia [PCP]) in immunocompromised subjects. Pneumocystis transmission from a host with PCP to another susceptible host via the airborne route has been demonstrated in rodent models. Moreover, it has been established that Pneumocystis murina can be transmitted from immunocompetent mice, transiently colonized by the fungus, to immunocompromised susceptible mice that subsequently develop PCP. Colonized subjects and those developing PCP may be part of the fungus reservoir. Reports of PCP case cluster in hospital strongly suggest that Pneumocystis jirovecii (P.jirovecii) transmission in humans may also occur. P.jirovecii DNA detection in the air surrounding PCP patients is consistent with the transmission of P.jirovecii via the airborne route.Our goals were to characterize human populations infected with P.jirovecii and to characterize P.jirovecii within its human reservoir. We showed that P.jirovecii was rarely involved in pulmonary colonization in patients with cystic fibrosis monitored in the Brest Hospital. Thus this patient population was not part of the human reservoir of the fungus in our region (Brittany, Western France). This low prevalence of colonization may reflect a low level of P.jirovecii circulation within human communities in Brittany. In order to improve the identification of patients infected with P.jirovecii, we evaluated ß-1,3-D glucan detection in serum samples. We showed that serum ß-1,3-D glucan levels combined with P.jirovecii detection in pulmonary samples using microscopic examination and a PCR assay make it possible to distinguish between PCP and pulmonary colonization. Moreover the first data on ß-1,3-D glucan levels during primary infection were obtained.In order to characterize P.jirovecii in our region, we performed the typing of P.jirovecii isolates from infected patients monitored at Brest hospital, using the dihydropteroate synthase (DHPS) and the internal transcribed spacer (ITS) 1 and 2 locus analysis. DHPS typing showed that i) the usual city of patient residence rather that the city in which the diagnosis of P.jirovecii infection has been made is a predictor of mutants, ii) mutants can be imported from one region to another through infected visitors, iii) the prevalence of mutants potentially resistant to sulfonamides was 0% in patients who effectively lived in the Brest geographic area. Results of ITS analysis in PCP patients and colonized patients are consistent with the hypothesis that these 2 patient groups are infected with similar P.jirovecii populations. All infected patients, whatever their clinical presentation, may be part of a common and unique reservoir of the fungus. We investigated an outbreak of P.jirovecii infections in 18 renal transplant recipients using the same typing method combined with patient encounter analysis. The results provided evidence of the role of colonized patients as potential sources of P.jirovecii. The same typing method was applied to pairs of pulmonary samples and room air samples of PCP patients. Full or partial matches of P.jirovecii types in pulmonary and air sample pairs were observed. These results are consistent with P.jirovecii exhalation by PCP patients in their close environment. These data support arguments for applying droplet precautions, at least to PCP patients, to prevent P.jirovecii transmission, as recommended by the "Société française d'hygiène hospitalière". We suggest extending droplet precautions to colonized patients to achieve the prevention of P.jirovecii nosocomial infections.

<p>  </p> <p>Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (<em>Dhps</em>) to post-translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of <em>Dhps</em> in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein <em>Eif5a</em> is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.</p>

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.