Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-24T12:13:14Z No. of bitstreams: 2 Tese - Viníciu Fagundes Bárbara - 2017.pdf: 2916938 bytes, checksum: e3841362774c4c7ee6cde936621d4747 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-24T12:13:45Z (GMT) No. of bitstreams: 2 Tese - Viníciu Fagundes Bárbara - 2017.pdf: 2916938 bytes, checksum: e3841362774c4c7ee6cde936621d4747 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-24T12:13:45Z (GMT). No. of bitstreams: 2 Tese - Viníciu Fagundes Bárbara - 2017.pdf: 2916938 bytes, checksum: e3841362774c4c7ee6cde936621d4747 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-27 / The mining industry is known to trigger intense environmental impacts. When the exploitation is done in the open, pit lakes are formed, units still little known in environmental terms, and increasingly common in several countries. Demonstrating complex behavior, open pit lakes differ greatly from natural lakes, what makes their management a challenge. Although there is a need to develop research issues that contribute significantly to a deeper understanding of the degree of environmental commitment of open pit mining lakes, the studies developed so far are few and have a predominantly Analytical Chemistry approach, an important approach, but that only offers momentary and limited answers. The main objective of this research was to analyze the environmental liabilities formed by waters and sediments of open pit mining lakes by application a combined method of environmental assessment based on Chemical, Ecotoxicological and Genotoxicological aspects. With this purpose, water samples were collected in profile and sediments from three lakes of deactivated open - pit gold mines existing in Mara Rosa, Goiás, Brazil, in different climatic seasons and subjected to metal and anion analysis, to ecotoxicological acute tests and genotoxicological tests- Comet assay - with the Danio rerio fish. The results showed different degrees of environmental commitment of the lakes, which present chemically altered waters and sediments, and offer toxicological risks, mainly the Lago Azul. This, being the most used by the local population for recreational purposes and water sports, proved to be subject to intense geological control from acid drainage processes. It is concluded that the environmental assessment methodology used in this thesis, in order to obtain broader and deeper responses to pit lakes, emerging environmental problems, was efficient, being able to constitute an analytical tool that will significantly contribute to the expansion of knowledge to the Environmental Sciences. / A indústria da mineração é conhecida por desencadear impactos ambientais intensos. Quando a exploração se dá a céu aberto, são formadas cavas, unidades ainda pouco conhecidas em termos ambientais e cada vez mais comuns em diversos países. De comportamento complexo, lagos de mineração se diferem muito dos naturais, o que torna sua gestão desafiadora. Embora exista a necessidade do desenvolvimento de questões de pesquisa que contribuam significativamente para um entendimento mais profundo a respeito do grau de comprometimento ambiental de cavas, os estudos desenvolvidos até então foram poucos e tiveram enfoque predominante na Química Analítica, abordagem importante, mas que obtém respostas momentâneas e limitadas. O objetivo principal desta pesquisa foi analisar os passivos ambientais formados por águas e sedimentos de cavas de mineração mediante a aplicação de uma metodologia combinada de avaliação ambiental embasada em aspectos da Química, Ecotoxicologia e Genotoxicologia. Para tanto, amostras de águas coletadas em perfil e de sedimentos de três cavas de ouro desativadas localizadas em Mara Rosa, Goiás, Brasil, foram obtidas em diferentes estações climáticas e submetidas à análise de metais e ânions e a testes ecotoxicológicos agudos e genotoxicológicos – Ensaio Cometa – com peixes da espécie Danio rerio. Os resultados indicam diferentes graus de comprometimento ambiental dos lagos, que apresentam águas e sedimentos alterados quimicamente e oferecem riscos toxicológicos, principalmente o Lago Azul. Este, apesar de ser o mais utilizado pela população local para fins de lazer e prática de esportes aquáticos, demonstrou estar submetido a intenso controle geológico advindo de processos de drenagem ácida. Concluiu-se que a metodologia de avaliação ambiental empregada nesta tese com a finalidade de obter respostas mais amplas e profundas a respeito de cavas, problemas ambientais emergentes, se mostrou eficiente, podendo se constituir em uma ferramenta analítica que contribuirá de forma significartiva para a ampliação do conhecimento na área das Ciências Ambientais.

As fosfolipases A2 (PLA2s, ou fosfatidil-acil hidrolases EC 3.1.1.4) catalisam especificamente a hidrólise das ligações ácido-éster na posição sn-2 de glicerofosfolipídios liberando, como produto da catálise, ácidos graxos e lisofosfolipídio. São encontradas em plantas, mamíferos e em veneno de animais vertebrados e invertebrados e estão envolvidas em uma ampla variedade de processos fisiológicos. A fosfolipase A2 secretada humana do grupo IIA (hsPLA2 gIIA) é uma proteína de fase aguda da resposta imunológica, pois sua expressão é induzida por endotoxinas e citocinas via processos autócrinos e/ou parácrinos durante processos inflamatórios de relevância clínica. A hsPLA2 gIIA mostra efeito bactericida contra infecção por Staphylococcus aureus, e tem marcada preferência por fosfolipídios aniônicos tais como fosfatidilglicerol (PG) encontrados em membranas bacterianas. Uma grande variedade de inibidores de PLA2 do grupo IIA foi descrita na literatura, incluindo substâncias polianiônicas que atuam contra os efeitos inflamatórios destas enzimas. Suramina é um derivado de naftiluréia polissulfonado que recentemente mostrou ligação com os resíduos catiônicos no sítio de reconhecimento interfacial de Bothropstoxina-I (BthTX-I), uma PLA2-Lys49 isolada do veneno de Bothrops jararacussu, inibindo a atividade miotóxica da proteína. Devido ao tipo de interação diferenciada da suramina com BthTX-I em relação aos inibidores competitivos de PLA2, nós avaliamos a especificidade de ligação da suramina na hsPLA2 gIIA como um modelo para estudar este novo tipo de inibidor de PLA2s. O efeito da suramina nas atividades biológicas e de membranas artificiais da hsPLA2 gIIA foi avaliado. A suramina aboliu tanto a atividade hidrolítica da hsPLA2 gIIA quanto a atividade de danificação de membranas artificiais Ca2+ independente. Embora a suramina não tenha inibido a atividade bactericida da hsPLA2 gIIA contra a linhagem Micrococcus luteus, a ativação de macrófagos foi abolida pela mesma de maneira dependente de hidrólise. Além disso, técnicas de simulação de dinâmica molecular, calorimetria de titulação isotérmica e mutagênese sítio dirigida foram utilizadas para mapear os sítios de ligação da suramina na proteína. A interação da suramina com a hsPLA2 gIIA resultou de interações eletrostáticas entre grupos sulfonados com cadeias laterais de aminoácidos da região do sítio ativo e dos resíduos em torno das posições 15 e 116 localizados, respectivamente, na N- e Cterminal. Portanto, estes resultados permitem sugerir que a suramina pode atuar como inibidor de sPLA2s / Suramin is a polysulphonated napthylurea used as an antiprotozoal drug that presents inhibitory activity against a broad range of enzymes. We have evaluated the effect of suramin against the artificial and biological activities of the secreted human group IIA phospholipase A2 (hsPLA2 gIIA), a protein involved in inflammatory processes. To map the suramin binding sites on the hsPLA2 gIIA, proteins with mutations in the active site region and in the protein surface that makes contact with the phospholipids membrane were expressed in E. coli and refolded from inclusion bodies. The activation of macrophage cell line RAW 264.7 by hsPLA2 gIIA was monitored by nitric oxide release, and bactericidal activity of the protein against Micrococcus luteus was evaluated by colony counting and by flow cytometry. The hydrolytic activity of the hsPLA2 gIIA against lipossomes composed of a mixture of dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) was inhibited by a concentration of 100 nM suramin. The activation of macrophages by hsPLA2 gIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA2 gIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 M. The affinity of interaction of the suramin with hsPLA2 gIIA was evaluated by suramine fluorescence and the mutants K15A, K38A, R54A and K123A presented a reduced affinity. The binding of the suramin/hsPLA2 gIIA complex was investigated by molecular dynamics simulations, which indicated two conformations of the bound inhibitor, which involve cationic amino-acid side chains in the active-site region and residues around positions 15 and 116 located in the N- and C-termini respectively in the substrate recognition surface. These results were correlated with isothermal titration calorimetry data, which demonstrated 2.7 suramin-binding sites on the hsPLA2 gIIA. These results suggested that suramin represents a novel class of phospholipase A2 inhibitor

O sistema SOS controla a expressão de diversos genes, muitos envolvidos com o reparo de DNA. Caulobacter crescentus vem emergindo como um modelo alternativo interessante para o estudo de mecanismos de reparo de DNA. Temos como objetivos realizar uma análise funcional de genes de função desconhecida regulados por SOS, e investigar a indução de SOS por antibióticos beta-lactâmicos em C. crescentus. Análises funcionais dos genes CC_3424 e CC_3467 mostraram que deleções nestes genes resultam em fenótipo de sensibilidade à mitomicina C (MMC). CC_3424 possui similaridade com glioxalases e CC_3467 com endonucleases. Acreditamos que CC_3467 atue no reparo de ligações intercadeia no DNA, e que CC_3424 atue detoxificando a MMC das células. Estudos dos efeitos biológicos da indução do sistema SOS mostram que a cefalexina (CFE) induz este regulon em concentrações subinibitórias. Células tratadas com CFE apresentam mais danos oxidativos do tipo 8-oxoguanina. Estes resultados mostram que concentrações subinibitórias de CFE resultam em estresse oxidativo em C. crescentus. / The SOS response controls the expression of several genes, many of which are involved in DNA repair mechanisms. Caulobacter crescentus has emerged as an alternative bacterial model for DNA repair. As aims, we will undertake a functional analysis of some of the genes regulated by the SOS response, and will investigate the SOS induction by beta-lactam antibiotics in C. crescentus. Functional analysis of the genes CC_3424 and CC_3467 showed that deletions in these genes result in a phenotype of sensitivity to mitomycin C (MMC). CC_3424 has similarity to glyoxalase and CC_3467 to endonucleases. We believe that the CC_3467 gene plays a role in the repair of interstrand crosslinks in the DNA, while CC_3424 acts in MMC cellular detoxification. Studies of biological effects of SOS induction showed that subinibitory concentrations of cephalexin (CFE) induce the SOS regulon. Cells treated with CFE have higher concentrations of 8-oxoG oxidative damage. These results show that subinibitory concentrations of cephalexin leads to cellular oxidative stress in C. crescentus.

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_simone_simionatto.pdf: 333806 bytes, checksum: 6d2a7ca25216004656318115c514c721 (MD5) Previous issue date: 2008-10-10 / Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), a respiratory disease responsible for significant economic losses. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. Besides, their preparation is expensive because of fastidiously growth of M. hyopneumoniae in vitro. Therefore, the development of alternatives for EP prophylaxis is important for improving health conditions of pigs. Recombinant DNA technology can be used to overcome problems with conventional vaccines. Nevertheless, because of the use of TGA and not TGG to code for tryptophan, translation of mycoplasmal genes terminates prematurely when cloned in other bacteria, such as Escherichia coli. Because of this, the number of antigenic proteins characterized to date in vaccine formulations or immunodiagnostic tests is still restricted. In this work, 63 genetic fragments were selected, which correspond to 48 sequences coding (CDS) of M. hyopneumoniae. First, an overlap extension-PCR method for site-directed mutagenesis of M. hyopneumoniae genes was standardized, aiming at substituting TGA codon by TGG. With this improved method, site-directed mutagenesis was successfully achieved in 14 M. hyopneumoniae genes. Other selected genes were amplified by PCR and cloned into Champion pET200D/TOPO®. A total of 59 genetic fragments were efficiently cloned. From these, 49 had their proteins expressed in E. coli and 35 were purified by affinity chromatography using Ni- Sepharose columns (HisTrap ). Immunogenic and antigenic properties of these proteins were analyzed. For this, the proteins were tested against sera from hyperimmune and from convalescent pigs through ELISA (enzyme-linked immunosorbent assay) and Western blot. Nineteen recombinant proteins were specifically recognized by convalescent pig sera indicates they are expressed during infection. Immune humoral response against recombinant proteins was evaluated in BALB/c mice by ELISA. The results showed that antigens induce variable levels of antibodies, which allows inferring the immunogenicity of each antigen. Sera from inoculated mice with twenty recombinant proteins were able to recognize the native protein by ELISA. This study allowed identifying and characterizing new immunogenic proteins according to their potential for use in diagnostic tests and/or vaccine. These data represent an important contribution for the development of more efficient serological tests and a subunit vaccine for controlling M. hyopneumoniae infections in pigs. / Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína (PES), uma doença respiratória responsável por significativas perdas econômicas. Vacinas comerciais são mundialmente utilizadas no controle desta doença, porém proporcionam apenas proteção parcial. Além disso, são caras devido ao crescimento fastidioso do M. hyopneumoniae in vitro. Desta forma, o desenvolvimento de alternativas para a profilaxia da PES é fundamental para a melhoria da sanidade dos suínos. A tecnologia do DNA recombinante pode ser usada para superar problemas com as vacinas convencionais. No entanto, pela utilização de códons TGA e não TGG para codificar o amino ácido triptofano, a tradução de genes de micoplasmas termina prematuramente quando clonados e expressos em outras bactérias, como Escherichia coli. Devido a isso, o número de proteínas antigênicas de M. hyopneumoniae caracterizadas e testadas como vacinas ou em testes de imunodiagnóstico ainda é restrito. Neste trabalho, 63 fragmentos gênicos foram selecionados, os quais correspondem a 48 seqüências codificadoras (CDS) de M. hyopneumoniae. Num primeiro momento, foi padronizado um método de PCR de sobreposição para mutagênese sítio-dirigida de genes de M. hyopneumoniae, objetivando a substituição do códon TGA por TGG, na seqüência do DNA. Com esse método, a mutagênese sítio-dirigida foi realizada com sucesso em 14 genes de M. hyopneumoniae. Os demais genes selecionados foram amplificados por PCR e clonados no vetor Champion pET200D/TOPO®. No total, 59 fragmentos gênicos foram clonados eficientemente. Destes, 49 tiveram suas proteínas expressas em E. coli e 35 foram purificadas por cromatografia de afinidade em coluna de Ni- Sepharose (HisTrap ). As propriedades antigênicas e imunogênicas destas proteínas foram analisadas. Para isso, as proteínas foram testadas contra soro de suínos convalescentes e soro hiperimune através de ELISA (enzyme-linked immunosorbent assay) e Western blot. Dezenove proteínas recombinantes foram reconhecidas especificamente por soro de suínos convalescentes, indicando que elas são expressas durante a infecção. Resposta imune humoral contra as proteínas recombinantes foi avaliada em camundongos BALB/c através de ELISA. Os resultados demonstraram que os antígenos induzem um nível variável de anticorpos, o que nos permite inferir quanto à capacidade imunogênica de cada antígeno. O soro dos camundongos inoculados com 20 proteínas foi capaz de reconhecer as proteínas nativas através de ELISA. Este estudo permitiu identificar e caracterizar novas proteínas imunogênicas de acordo com o seu potencial para uso em testes de diagnóstico e/ou vacina. Estes dados representam uma importante contribuição para o desenvolvimento de testes sorológicos mais efecientes e vacinas de subunidade para o controle da infecção causada por M. hyopneumoniae em suínos.

Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

No description available.

Chez les eucaryotes, le complexe kinase TORC1 (Target Of Rapamycin Complex 1) joue un rôle central dans le contrôle de la croissance cellulaire. Il intègre de nombreux signaux et agit en modulant l’état de phosphorylation de différents effecteurs, principalement des protéines impliquées dans des processus anaboliques ou cataboliques. Parmi ces signaux, on distingue notamment les acides aminés. Ces derniers agissent sur TORC1 via l’action de protéines de la famille des GTPases Rag, elles-mêmes régulées par des facteurs GEF et GAP. Des études récentes sur des cellules mammifères ont mis en évidence l’existence de senseurs d'acides aminés, capables de moduler l'activité des facteurs GEF et GAP. Chez la levure, ces senseurs restent toutefois inconnus. Chez la levure, TORC1 contrôle la fonction de plusieurs protéines y compris des transporteurs d'acides aminés de la membrane plasmique, comme la perméase générale des acides aminés, Gap1. C’est via sa branche de signalisation Tap2-PP2A/Npr1 que TORC1 contrôle l'ubiquitylation et le trafic intracellulaire de ces transporteurs, et ceci, en modulant l’activité d’adaptateurs de type α-arrestine de l'ubiquitine-ligase Rsp5.Dans ce travail, nous avons combiné des approches de génétique et de biochimie chez la levure afin d’étudier la régulation de TORC1 et son rôle dans l'endocytose en réponse au substrat de Gap1, la perméase générale des acide aminés, et de Can1, la perméase spécifique de l'arginine.Dans la première et la deuxième partie de ce travail, je décris ma contribution à l'étude qui visait à élucider le mécanisme d'ubiquitylation de Gap1 et de Can1 induite par le transport de leurs substrats. Le modèle déduit de ce travail propose que cette régulation négative n'est pas due à l'accumulation intracellulaire des acides aminés transportés, mais à un changement conformationnel des perméases, couplé à la réaction de transport et qui fait apparaitre un état ouvert vers l'intérieur, entraînant ainsi le remodelage de la queue cytoplasmique N-terminale et en conséquence l’exposition d'un site de liaison caché pour des adaptateurs de Rsp5 de type α-arrestine. Dans le cas de Can1, l'α-arrestine principale impliquée est Art1 et doit être stimulée via TORC1. Cependant, les α-arrestines Bul1/2, impliquées dans la régulation négative de Gap1, sont capables de promouvoir son ubiquitylation induite par le transport de substrat, qu'elles aient ou non été stimulées via TORC1. Nous fournissons également des preuves que d'autres perméases d'acides aminés spécifiques (Mup1, Lyp1) sont régulées par leurs propres substrats d'une manière similaire à Can1.Dans la dernière partie de ce travail, nous avons étudié le mécanisme par lequel le transport des acides aminés chez la levure stimule l'activité de TORC1 via les GTPases Rag, Gtr1 et Gtr2. En analysant en Western Blot l’état de phosphorylation de Npr1 et Sch9, deux kinases effectrices de TORC1, nous avons révélé que le signal général qui déclenche l'activation Gtr-dépendante de TORC1 est le flux de H+ couplé au transport des acides aminés généré par des symporteurs H+/acide-aminés. Dans ce contexte, nous avons identifié la pompe à H+ de la membrane plasmique, Pma1, comme étant un régulateur essentiel de l'activité de TORC1. L'activité de transport de Pma1 étant elle-même stimulée par une augmentation des protons cytosoliques, nous suggérons que Pma1 module TORC1 par un effet de signalisation.Collectivement, nos résultats fournissent de nouvelles perspectives sur le rôle central de TORC1 dans le contrôle des transporteurs de nutriments chez la levure. / The Target of Rapamycin Complex 1 (TORC1) plays a pivotal role in controlling cell growth in probably all eukaryotic organisms. It operates by integrating upstream signals like growth factors and nutrients to modulate by phosphorylation multiple downstream effectors, mostly proteins involved in anabolic or catabolic processes. Among the various signals that impinge on TORC1, nitrogen sources, in particular amino acids are primordial input signals modulating TORC1 activity through the conserved Rag family of GTPases. Recent studies in mammals have shed the light on the existence of various sensor systems of internal amino acids that modulate the activity of the GEF and GAP factors acting on the Rag GTPases. Yet, in yeast the amino acid sensing events acting upstream of the Rag GTPase (named Gtr1 and Gtr2) regulators remain poorly known. Yeast TORC1 controls the function of many proteins including several plasma membrane amino acid transporters, e.g. Gap1, the general amino acid permease. It does so via the Tap2-PP2A/Npr1-signaling branch that controls the ubiquitylation and intracellular trafficking of these proteins through regulation of α-arrestin-type adaptors of the ubiquitin-ligase Rsp5. In this work, we combined yeast genetics and biochemical assays to study TORC1 regulation by amino acids and to illustrate the role of TORC1 in substrate-transport mediated endocytosis of Gap1 and of the arginine specific permease, Can1. In the first and second part of this work, I describe my contribution to the study that aimed at elucidating the mechanism of substrate-transport-mediated ubiquitylation and endocytosis of Gap1 and Can1. The model deduced from this work states that this down-regulation is not due to intracellular accumulation of the transported amino acids, but to substrate-transport-induced conformational transition of the transporters to an inward-facing state, resulting in remodeling of their N-terminal cytoplasmic tail and subsequent exposure of a hidden binding site for α-arrestin-like adaptors of Rsp5. In the case of Can1, the main α-arrestin involved is Art1 and needs be stimulated via TORC1. The Bul1/2 α-arrestins involved in Gap1 down-regulation, however, are able to promote its substrate-transport-elicited ubiquitylation regardless of whether they have been stimulated via TORC1 or not. We also provide evidence that other specific amino acid permeases (Mup1, Lyp1) are regulated by their own substrates in a manner similar to Can1. In the last part of this work, we investigated how amino acid uptake by yeast cells triggers Rag/Gtr-dependent activation of TORC1. By assaying the phosphorylation on western blot of two TORC1-downstream effectors, the Npr1 and Sch9 kinases, we showed that uptake by Gap1 of ß-alanine, which cannot be used as a nitrogen source, unexpectedly stimulates TORC1 activity. Further analysis of this response allowed us to show that the general signal triggering Gtr-dependent activation of TORC1 in response to amino acid uptake is the influx of H+ coupled to transport via H+/amino-acid symporters. Furthermore, we identified Pma1, the H+-ATPase establishing the H+ gradient at the plasma membrane, as a central player of this control of TORC1 activity. As the transport activity of Pma1 itself is known to be stimulated by an increase of cytosolic protons, we suggest that Pma1 modulates TORC1 via signaling. Altogether, our results provide new insights on the central role of TORC1 in control of nutrient permeases in yeast. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Abstract Osteoporosis is a disease characterized by abnormalities in the amount and architectural arrangement of bone tissue, which leads to impaired skeletal strength and increased susceptibility to fractures. Type 5 tartrate-resistant acid phosphatase (TRACP, AcP5) has been suggested to participate directly in bone resorption. In this study, baculovirus expression vector system in insect cells was used to gain large amounts of recombinant type 5 acid phosphatase for structure determination, structure-based functional studies and production of monoclonal antibodies. Active and inactive forms of the enzyme were separated from each other by cation-exchange chromatography, and characterized. The enzyme was crystallized and the three-dimensional structure was determined. Based on the three-dimensional structure of the active site five different enzyme variants were constructed, produced in insect cells, and purified. The wild type enzyme and the mutated forms were characterized, and their kinetic parameters were determined. The importance of amino acids that were expected to be essential for the acid phosphatase activity was confirmed. The acid phosphatase activity and reactive oxygen species generating activity of this dual enzyme proved to exploit different amino acids in their reaction mechanisms. Further studies are needed to clarify the physiological substrates of TRACP in vivo. The findings of this study could form a base for construction of inhibitors for TRACP that could be useful therapeutic agents for osteoporosis and related bone disorders.

La détection de molécules odorantes représente un enjeu important dans divers domaines tels que l’industrie alimentaire, le diagnostic médical et la sécurité du territoire, par exemple. En effet, les odorants, présents par milliers dans notre environnement, véhiculent de nombreuses informations, via leur nature chimique ou leur concentration. Notre système olfactif est capable de discriminer des milliers de molécules différentes via des mécanismes biochimiques impliquant l’association de nombreux partenaires protéiques et un codage combinatoire de l’information. Ces biomolécules, qui englobent notamment les récepteurs olfactifs et les protéines de liaison aux odorants (OBP), constituent une source intéressante d’éléments de détection pour la conception de biocapteurs. Les OBP sont de petites protéines solubles présentent dans le mucus nasal à des concentrations de l’ordre du millimolaire. Leur poche de liaison hydrophobe leur confère la capacité de lier de façon réversible les molécules odorantes. Leur robustesse et leur manipulation aisée en font de bonnes candidates pour l’élaboration de biocapteurs. Au cours de ce travail, nous nous sommes intéressés à la détection de molécules odorantes en associant des OBP comme biorécepteur et l’électrochimie comme méthode de transduction. Par une méthode de mutagenèse dirigée, nous avons montré qu’en modifiant un seul des acides aminés dans la poche de liaison de deux OBP de rat (rOBP2 et rOBP3), il était possible de moduler leurs affinités envers les odorants. En parallèle, nous avons décrit la détection qualitative et quantitative de molécules volatiles à partir d’OBP. Nous avons montré que rOBP3 lie la 2-méthyl-1,4-naphtoquinone (MNQ), une sonde électrochimique. La quantité de MNQ déplacée de la poche de liaison de rOBP3 par la 3-isobutyl-2-méthoxypyrazine (IBMP), un odorant modèle, a été mesurée par voltammétrie cyclique à vagues carrées. Nous avons déterminé les constantes de dissociation des complexes rOBP3 / MNQ et rOBP3 / IBMP. Les valeurs mesurées par électrochimie ont été confirmées par compétition avec une sonde fluorescente et par titration calorimétrique isotherme. En combinant cette nouvelle méthode analytique à des variants de rOBP3 qui présentent des profils de liaison différents et complémentaires, nous avons détecté sélectivement chacun des constituants d’un mélange ternaire d’odorants. Ces travaux, qui allient ingénierie des OBP et électrochimie, offrent des perspectives intéressantes dans le domaine des nez électroniques. / The detection of odorant molecules has become an important challenge in different research area, such as the food industry, medical diagnostics and homeland security. Indeed, the thousands of odorants in our environment provide information on their chemical nature or their concentration. Human olfactory system is capable of discriminating thousands of different molecules thanks to biochemical mechanisms involving multiple protein receptor partners and a combinatorial coding. These biomolecules that include olfactory receptors and odorant-binding proteins (OBP) represent an interesting source of detectors for the design of biosensors. OBPs are small soluble proteins present in nasal mucus at millimolar concentrations. Their hydrophobic binding pocket gives them the ability to reversibly bind odorant molecules. OBPs are robust and easy to produce and are thus good candidates for the design of biosensors. In this work, we focused on the detection of odorant molecules associating OBPs as a bioreceptor and electrochemistry as a transduction method. Using site-directed mutagenesis, we have shown that by substituting a single amino acid in the binding pocket of two rat OBPs (rOBP2 and rOBP3), it is possible to modulate their binding affinities towards odorants. In parallel, we described a qualitative and quantitative method for the detection of volatile molecules using OBPs. We have shown that rOBP3 binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe. The amount of MNQ displaced from the binding pocket of rOBP3 by the model odorant 3-isobutyl-2-methoxypyrazine (IBMP), was measured using square-wave voltammetry. We determined the dissociation constants of the rOBP3 / MNQ and rOBP3 / IBMP complexes. These values measured by electrochemistry were confirmed by a competitive fluorescent assay and isothermal titration calorimetry. By combining this new analytical method to rOBP3 variants with different and complementary binding profiles, we were able to selectively detect each of the components of a ternary mixture of odorants. This work, that combines the engineering of OBPs and electrochemistry, offers us interesting perspectives in the field of electronic noses.

A major factor in the success of Staphylococcus aureus as a pathogen is its vast arsenal of virulence determinants and, more importantly, the tight and precisely- timed regulation of these factors. Here we investigate the product of the S. aureus gene, SAUSA300_1984, encoding a putative transmembrane protein. This as yet uncharacterized protein belongs to the Abi (abortive infection) family, which are commonly annotated as CAAX-proteases, and are significantly understudied in prokaryotes. In S. aureus the disruption of SAUSA300_1984 results in a drastic reduction of proteolytic and hemolytic activity, as well as diminished pigmentation. This phenotype appears to be mediated through reduced agr expression, as determined by qPCR analysis. Importantly, known regulators of agr, such as CodY, MgrA, and ArlR, demonstrate no significant changes in transcription upon 1984 disruption, whilst major alterations were observed for downstream effectors of agr, such as sarS, RNAIII, rot and hla. Complementation and site-directed mutagenesis of 1984 demonstrated that proteolytic activity (via conserved EE residues) was not required for this phenotype, suggesting a potential protein-protein interaction mechanism of interaction. Proteome analysis of the 1984 mutant confirmed a number of our transcriptional observations, such as an increased abundance of Rot and surface associated proteins, as well as a marked decrease in Agr-system proteins levels, with the most striking being AgrB. Virulence profiling revealed a decreased ability of the 1984 mutant to evade constituents of the innate immune response, and impaired survival during murine models of infection. Given that SAUSA300_1984 is encoded 3 genes downstream of RNAIII, our current working hypothesis is that this Abi protein functions to control agr activity through communication with membrane components of this system, potentially via interaction with AgrB. Confirming this, and determining the upstream effectors of this regulatory system are studies currently ongoing in our laboratory.