Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-05-02T11:53:47Z No. of bitstreams: 1 julianaviciniflorentino.pdf: 13579179 bytes, checksum: bacc80aab9aabc49f5ee8f5edbdcc799 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-05-09T12:00:54Z (GMT) No. of bitstreams: 1 julianaviciniflorentino.pdf: 13579179 bytes, checksum: bacc80aab9aabc49f5ee8f5edbdcc799 (MD5) / Made available in DSpace on 2018-05-09T12:00:54Z (GMT). No. of bitstreams: 1 julianaviciniflorentino.pdf: 13579179 bytes, checksum: bacc80aab9aabc49f5ee8f5edbdcc799 (MD5) Previous issue date: 2018-02-22 / A esquistossomose é uma doença parasitária mais comum em áreas tropicais e subtropicais e é uma das doenças mais negligenciadas em todo o mundo. Devido ao fato de até o momento apenas um fármaco (PZQ) ser utilizado no tratamento da esquistossomose e como consequências, diversas dificuldades já estão aparecendo como resistência a sua utilização, não prevenção contra reinfecção e nem contra formas imaturas do parasito, a busca por alternativas tem despertado bastante interesse. Desta forma, neste trabalho foram preparados vinte e oito compostos sendo quatorze chalconas e quatorze flavonas sendo apenas duas flavonas inéditas. As chalconas foram obtidas através da reação de condensação de Claisen-Schimdt, pela reação de cinco aldeídos alquilados e nove aldeídos comerciais com a 2-hidroxiacetofenona. As quatorze flavonas correspondentes foram obtidas através da reação de ciclização oxidativa das chalconas na presença de I2 e DMSO. Todas as chalconas foram obtidas em sua configuração E e todos os compostos sintetizados foram caracterizados por RMN de ¹H e ¹³C. Quatro flavonas foram testadas com relação a sua atividade esquistossomicida in vitro e mostraram potencial como antiparasitários. Além disso, as flavonas foram testadas com relação a sua atividade enzimática frente a enzima ATP-difosfohidrolase e destas, quatro mostraram um potencial interessante frente a inibição desta enzima. / Schistosomiasis is a common parasitic disease in tropical and subtropical areas and it is one of the most neglected diseases in the world. The search for alternatives has aroused a lot of interest since, so far, only one drug (PZQ) is used in the treatment of schistosomiasis and because of this, several consequences are appearing as resistance to its use and non-prevention against reinfection or against immature forms of the parasite. Therefore, in this work twenty-eight compounds were prepared, fourteen chalcones and fourteen flavones which only two flavones are unpublished. Chalcones were obtained by reaction of five alkyl aldehydes and nine commercial aldehydes with 2-hydroxyacetophenone via the Claisen-Schimdt condensation reaction. The corresponding fourteen flavones were obtained through the oxidative cyclization reaction of chalcones in the presence of I2 and DMSO. All chalcones were obtained in their E configuration and all compounds synthesized were characterized by ¹H and ¹³C NMR. Also, the infrared spectroscopy was obtained for the flavones. Four flavones were tested for their schistosomicidal in vitro activity and showed great potential as antiparasitics. In addition, these flavones were tested for their enzymatic activity against the ATP-diphosphohydrolase enzyme, which four of them showed an interesting potential against the inhibition of this enzyme.

Les liposomes d’ATP incluant des ligands hépatiques pourraient contribuer à améliorer le statut énergétique du greffon hépatique. Une première phase de développement a mis en évidence la nécessité de stabiliser la forme (i) et de valider un modèle cellulaire à statut énergétique altéré (ii) afin de pouvoir tester différentes formulations liposomales d’intérêt. Afin de résoudre la première problématique, différentes stratégies ont été mises en place lors de la lyophilisation de liposomes blancs ou chargés en ATP. Le saccharose et le tréhalose ont conduit à une stabilisation de la forme avant et après lyophilisation. Néanmoins, quel que soit le procédé, la lyophilisation s’est accompagnée d’une fuite en ATP. L’ajout d’un agent inhibant la cartinine palmitoyl transferase, l’Etomoxir, s’est révélé un modèle intéressant pour moduler le niveau énergétique des HepG2 en fonction de la température et de la concentration utilisée. En conclusion, ce travail contribue à mieux comprendre les facteurs critiques liés à la lyophilisation des liposomes (i) et décrit des modèles cellulaires hépatiques à statut énergétique altéré qui pourraient être mis à profit pour tester différents principes actifs ou formes galéniques innovantes. / ATP liposome incorporating hepatic ligands may contribute to improve the energetic status of the liver graft. In a first phase of development, it has been emphasized the great need of stabilizing the liposome (i) and of validating a cellular model with an altered energetic status in order to test the formulations of interest. To provide a stable liposomal preparation, different strategies have been carried out to freeze-dry liposome with or without ATP. Sucrose and trehalose better stabilize the liposome preparation during the freeze-drying process. Nevertheless, in all conditions, a significant ATP leakage has been observed. An inhibitor of the cartinine palmitoyl transferase (i.e. Etomoxir) has shown great interest to modulate the energetic level in HepG2 cells by varying Etomoxir concentration and culture temperature. In conclusion, this study contributes to a better understanding of the critical factors related to liposome freeze-drying and describes hepatic cell models with altered energetic status that may have great interest to test specific agent or innovating formulations.

The NTPDase1 enzymatic activity (E.C.3.6.1.5, Apyrase, ecto-ATP-diphosphohydrolase, CD39) which hydrolyses ATP and ADP, was analyzed in peripheral lymphocytes from patients with B chronic lymphocytic leukemia (B-CLL) which the main characteristic is the accumulation of monoclonal mature B-lymphocyte. The sample was composed by 23 patients with B-CLL whose were diagnosed according to the clinical and laboratorial criteria and also by the immunophenotypic characteristics. The patients were divided, according to the Binet staging system, in 4 groups: stage A (early), stage B (intermediate), stage C (advanced) and control group. The results demonstrated a significant increase in ATP hydrolysis (F(29,3)=26.79 p<0.001) for all the stages of the disease and related to the control as a function of length of disease advance with higher activity in stage C. ADP hydrolysis (F(29,3)=23.76 p<0.001) was also enhanced according to ATP results. Besides this it was found a great correlation between the activity of NTPDase-1 and the total absolute white cells count in peripheral blood. The alteration in NTPDase-1activity in lymphocytes from patients with B-CLL at the different stages, contributes to confirm the ectonucleotidases role in regulate the ATP and ADP levels, which actuates like signalling molecules in many blood cells like the lymphocytes. Therefore more studies need to be done so that we can elucidate such enzymatic system in the nucleotides and its role in the B-CLL. / A atividade da enzima NTPDase1 (E.C.3.6.1.5, Apyrase, ecto-ATP-difosfoidrolase,CD39) que hidrolisa ATP e ADP, foi analisada em linfócitos periféricos de pacientes com leucemia linfocítica crônica (LLC-B), doença que possui como característica principal o acúmulo de linfócitos B maduros. A amostra foi composta por 23 pacientes com LLC-B, diagnosticados de acordo com critérios clínicos, laboratoriais e resultados de imunofenotipagem. Os pacientes foram divididos, conforme o sistema de classificação de Binet, em 4 grupos: estágio A (inicial), estágio B (intermediário), estágio C (avançado) e um grupo controle. Os resultados demonstraram que a hidrólise do ATP foi significativamente aumentada (F(29,3)=26.79 p<0.001) em todos os estágios da doença e em relação ao controle e esse aumento é compatível com o avanço da doença mostrando a maior hidrólise no estágio C. A hidrólise do ADP acompanhou os resultados do ATP e também estava aumentada (F(29,3)= 23.76 p<0.001). Além disso, verificou-se uma forte correlação entre a atividade da enzima NTPDase1 e a contagem absoluta de glóbulos brancos do sangue periférico. A alteração na atividade da enzima NTPDase1 em linfócitos de pacientes com LLC-B em seus diferentes estágios, vem confirmar o importante papel das ecto-nucleotidases na regulação dos níveis de ATP e ADP, os quais atuam como moléculas sinalizadores em várias células sangüíneas como os linfócitos. Entretanto, mais estudos são necessários para melhor elucidar as funções desse sistema enzimático no controle de nucleotídeos e o seu papel na LLC-B.

La F1 Fo ATP synthase est un complexe enzymatique localisé au sein de la membrane interne mitochondriale qui utilise le gradient électrochimique en protons formé par la chaîne respiratoire pour synthétiser de l'ATP à partir d'ADP et de Pi. Cette enzyme conservée de la levure S. cerevisiae aux cellules de mammifères s'organise dans les membranes internes mitochondriales sous forme de structures supramoléculaires d'ATP synthases. Chez la levure, il est aujourd'hui parfaitement identifiée que cette organisation nécessite la présence de deux sous-unités accessoires de l'enzyme : les sous-unités e et g.Les travaux présentés dans ce manuscrit visaient à étudier l'implication des sous-unités e et g dans les mécanismes de dimérisation et d'oligomérisation des ATP synthases ainsi que dans la morphogénèse des crêtes mitochondriales chez la levure S. cerevisiae et dans les cellules humaines en culture.Chez la levure, l'étude réalisée nous a permis de déterminer la stœchiométrie des sous-unités e et g, élément indispensable à la modélisation de l'agencement des sous-unités membranaires de l'enzyme dans la membrane interne mitochondriale.Dans les cellules humaines en culture, nous avons pu établir que les sous-unités e et g participent à la stabilité des dimères d'ATP synthases. Cependant l'implication de ces sous-unités dans la stabilité de l'enzyme semble différente des observations effectuées dans les cellules de levure / The F1Fo ATP synthase is an enzymatic complex embedded in the inner mitochondrial membrane which use the electrochemical proton gradient generated by the phosphorylation oxydative pathway to synthesize ATP from ADP and inorganic phosphate. This enzyme is conserved from yeast to mammalian cells and displays supramolecular organization in the inner mitochondrial membrane. In yeast, it is actually well-known that the supramolecular assembly required two accessory subunits : e and g subunits.The present work was realized to understand the involvement of subunits e and g in dimerization and oligomerization of mitochondrial ATP synthases as well as their effect on mitochondrial inner membrane morphogenesis in yeast S. cerevisiae and human cultured cells.In yeast, this study led us to determine subunits e and g stoechiometry, which was cruelly missing to establish a model of the ATP synthases membranous subunits layout in the inner mitochondrial membrane.In human cells, we have demonstrated that subunits e and g are implicated in ATP synthase dimer stabilization. However, their involvement in this stabilization seems to be quietly different of what have been observed in yeast cells.

O gênero Hypostomus (Siluriformes: Loricariidae) com cerca de 130 espécies nominais, se destaca como um dos mais diversos e amplamente distribuídos gêneros de peixes de água doce neotropical. Devido a sua ampla distribuição e alta diversidade, os conhecimentos taxonômicos, filogenéticos e biogeográficos para as espécies do gênero são ainda consideravelmente incompletos. Consequentemente, pouco se sabe sobre processos naturais envolvidos em diversificação e variação morfológica para o gênero. Hypostomus ancistroides é uma espécie descrita para a bacia do Alto Paraná, uma eco-região hidrográfica tradicionalmente reconhecida por seu endemismo ictiofaunístico, ocorrendo também na bacia costeira do rio Ribeira de Iguape. Esta espécie apresenta considerável variação morfológica, cariotípica e isoenzimática em suas diferentes populações, sugerindo a existência de um complexo de espécies. Sua ampla área de distribuição, somada aos novos conhecimentos sobre padrões biogeográficos para diversas espécies de peixes do Alto Paraná, reforça essa possibilidade. Entretanto, a variação encontrada na morfologia das populações de H. ancistroides é ampla e contínua, impedindo que se defina diferentes espécies através das abordagens taxonômicas clássicas em ictiologia. Assim, este trabalho se propõe a utilizar ferramentas da sistemática molecular, genética de populações e filogeografia para responder questões fundamentais sobre a evolução desse potencial complexo de espécies. Sequências nucleotídicas completas do marcador mitocondrial ATP sintase (subunidades 6 e 8; 842 pb) foram obtidas para diversas espécies de Hypostomus, incluindo 162 exemplares de H. ancistroides provenientes de doze localidades abrangendo toda a sua área de ocorrência, além de outros gêneros da família Loricariidae, utilizados como grupos externos. Análises filogenéticas de Máxima Verossimilhança, Máxima Parcimônia e Neighbor Joining resultaram em topologias essencialmente semelhantes, sustentando a monofiletismo da espécie, e apontando como seus parentes mais próximos espécies de bacias hidrográficas adjacentes ao Alto Paraná. Esses resultados mostram ainda a existência de quatro filogrupos distintos para a espécie, com áreas de distribuição parcialmente sobrepostas. Análises populacionais e filogeográficas incluiram comparação de distância genética P, estruturação populacional baseada em distribuição de haplótipos e índices de diversidade, testes de neutralidade, índice de fixação FST, análise de variância molecular (AMOVA), análise espacial de variância molecular (SAMOVA), construção de rede haplotípica de parcimônia, e análise de clados hierarquizados (NCPA). Os resultados mostram 48 haplótipos repartidos em doze populações bem estruturadas, com baixo ou nenhum fluxo gênico entre si. Eventos de expansão geográfica podem ser identificados ao longo da história demográfica, sugerindo que a estruturação encontrada atualmente reflete não só as características ecológicas da espécie, como também uma história de mudanças nas condições ambientais, eventualmente favoráveis a migração e dispersão. Contatos entre populações de diferentes bacias podem ser mais frequentes através de capturas de cabeceiras do que ao longo do corpo dos rios principais. A hipótese mais plausível para a presença da espécie na bacia do Ribeira é a de uma captura de cabeceira do alto rio Tietê. Apesar de ser formado por quatro filogrupos distintos, algumas linhagens derivadas de H. ancistroides apresentam sobreposição de suas áreas de distribuição. Esse contato secundário revelado apenas por um marcador de herança matrilineal impossibilita a delimitação de diferentes espécies correspondentes aos filogrupos, sob os paradigmas clássicos de especiação em peixes neotropicais. / The genus Hypostomus (Siluriformes: Loricariidae), comprising ca. 130 nominal species, is one of the most species-rich and widely distributed genera of neotropical freshwater fish. Because of its wide distribution and vast diversity, knowledge on the taxonomy, phylogenetic relationships and biogeography of Hypostomus is still severely incomplete. Consequently, little is known about the processes involved in the diversification and morphological variation for the genus. Hypostomus ancistroides is a species described from the upper Rio Paraná drainage, a freshwater ecoregion known for its ichthyological endemism, also occurring in the coastal basin of Rio Ribeira de Iguape. This species shows considerable morphological, karyotypic and isoenzimatic variation among different populations, suggesting the existence of a species complex. Its wide distribution area, coupled with recent understanding on biogeographic patterns of several fish species from the Upper Parana, reinforces that possibility. However, morphological variation in populations of H. ancistroides is wide and continuous, and does not allow recognition of potential different species by means of traditional taxonomic approaches. Thus, this paper uses tools from molecular systematics, population genetics, and phylogeography in order to answer major questions about the evolution of this potential species complex. Complete sequences of the mitochondrial marker ATP synthase (subunits 6 and 8; 842 bp) were obtained for several species of Hypostomus, including 162 specimens of H. ancistroides from twelve localities covering its entire area of distribution, plus other loricariid genera as outgroups. Phylogenetic analysis using methods of Maximum Likelihood, Maximum Parsimony, Neighbor Joining and Bayesian Inference resulted in mostly similar topologies, supporting the monophyly of the species, and showing as its closest relatives other species from river basins bordering the Upper Parana. Results also reveal four distinct phylogroups for the species, with partially overlapping distribution areas. Population and phylogeographic analysis included comparisons of genetic distance P, population structure based on the haplotype distribution and diversity indices, neutrality tests, fixation index FST, analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA), construction of a parsimony haplotype network, and nested clade phylogeographical analysis (NCPA). Results show 48 haplotypes distributed into twelve well-structured populations with little or no gene flow. Geographic range expansion events can be identified along the demographic history of H. ancistroides, suggesting that the structure found today reflects not only the ecology of the species, but also a history of changing environmental conditions that on occasion weree favorable for migration and dispersal. Contact between populations from differente basins may be more intense through headwater stream capture than through the river channel. The most supported hypothesis for the presence of the species in the Rio Ribeira basin is a headwater capture from the upper Rio Tiete. Although H. ancistroides is split into four distinct phylogroups, some derived lineages of the species have overlapping distribution ranges. Such secondary contact is revealed only by a matrilineal inheritance marker and does not allow the recognition of separate species for the different phylogroups under the current paradigm of speciation and species limits in Neotropical fishes.

La mitochondrie forme un réseau dynamique de tubules, dont la morphologie et la distribution sont étroitement régulées. Les mitochondries sont des organelles à double membrane dont l’architecture interne est complexe. Les crêtes mitochondriales forment des invaginations de la membrane interne. Elles sont le lieu des phosphorylations oxydatives, réactions par lesquelles l’ATP synthase produit l’ATP. L’ATP synthase est également connue pour son rôle clé dans la morphogenèse des crêtes. Dans cette étude j’ai mis en évidence in vivo la localisation en cluster de l’ATP synthase au sein du réseau mitochondrial de S. cerevisiae se développant sur substrat fermentescible. Mes résultats suggèrent que ces clusters correspondent aux crêtes mitochondriales, ce qui ouvre de nouvelles perspectives pour l’étude du remaniement de la membrane interne.La morphologie du réseau mitochondrial est maintenue par un équilibre entre les processus de fusion et de fission des tubules mitochondriaux. Dans la deuxième partie de ma thèse, j’ai mis en évidence une fragmentation progressive du réseau mitochondrial lors de l’entrée des cellules en quiescence, un état cellulaire non prolifératif réversible. En quiescence, le réseau mitochondrial est constitué de petites vésicules sous corticales immobiles au contenu enzymatique variable. Lors d’un retour à l’état prolifératif ces vésicules fusionnent rapidement pour reformer un réseau tubulaire, et ce, avant l’émergence de la cellule fille. De façon surprenante j’ai mis en évidence que ni les machineries canoniques de fusion ou de fission, ni le cytosquelette d’actine ne sont requis lors du remaniement du réseau mitochondrial dans les transitions entre prolifération et quiescence. / Mitochondria form a dynamic tubular network which organization and distribution is highly regulated.Mitochondria are double membrane organites with a complex internal architecture. Cristae, which areinner membrane invaginations, are the site of oxidative phosphorylation, reactions by which ATP synthaseproduces ATP. ATP synthase also play a key role in cristae morphogenesis. In this study, I have shown thatATP synthase localized as discrete clusters along the mitochondrial network in living S. cerevisiae cellsgrown on a fermentable carbon source. Overall our data suggest that ATP synthase clusers correspond tomitochondrial cristae, opening new avenues to explore the mechanisms involved in inner membraneremodelling.Mitochondrial network morphology is regulated by a dynamic equilibrium between the fusion and fissionof mitochondrial tubules. In the second part of my thesis, I highlight a progressive mitochondrialfragmentation during quiescence establishment, a state defined as a reversible absence of proliferation.Quiescent cells mitochondrial network is composed of immobile small cortical mitochondrial vesicles witha variable enzymatic content. Upon quiescence exit, cortical mitochondrial vesicles rapidly fuse and atubular network is reconstituted prior to bud emergence. Astonishingly, neither the canonical fusion orfission machineries nor the actin cytoskeleton are required for the mitochondrial network modificationduring quiescence / proliferation transition.

The rhythm of ATP accumulation is an one of examples of circadian rhythmicity which is demonstrated across the animal kingdom. It is located in the suprachiasmatic nucleus of hypothalamus, which is a centre of circadian clock and imposes rhytmicity on a whole organism. The question concering the ATP rhythm in an organotypic culture and the impact of melatonin on it has been discussing in this thesis. The synthesis of melatonin is regulated by the activity of the SCN and the hormon itself is known for its feedback on the SCN and a capability of regulating it. It has been shown that ATP rhythm is significantly inhibited under the constant control of melatonin and this inhibition is dose-dependent on a scale of 0,1-10 nM. In case of one-time applied melatonin in 4 p.m. there is no reduction in the accumulation of extracellular ATP but there is a phase shift in ATP rhythm. It has also been shown that ATP rhythm is inhibited and desynchronized under the control of tetrodotoxin which blocks an electric activity of neurons. These results show that melatonin is capable of inducing phase delay of ATP rhythm in SCN and reducing an amount of extracellular ATP, that the effect of melatonin is probably mediated by specific receptors and lastly that ATP rhythm is a result of cell-cell interaction between neurons...

Postnatal adaptation of neonate to extrauterine life is among others dependent on maturation of mitochondrial oxidative phosphorylation system (OXPHOS). It depends on effective mitochondrial biogenesis during fetal developement. The inadequate capacity of mitochondrial OXPHOS system plays an important role in the neonatal mortality and morbidity. Therefore the study of mitochondrial biogenesis on molecular and biochemical level is important to improve the care of very premature neonates, especially critically ill premature neonates. This thesis has been worked out in The laboratory for study of mitochondrial disorders (Department of Pediatrics, 1st Faculty of Medicine, Charles University in Prague). The thesis is based on molecular genetic analyses, which are focused on characterisation of ATP synthase gene expression and on changes in mitochondrial DNA content during human and rat fetal development. The results provide the better insight into mitochondrial respectively ATP synthase biogenesis during human and rat fetal development.

In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimicrobial/ antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented.

In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimicrobial/ antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented.