O balanço redox em mitocôndrias de mamíferos é realizado pelo transportador de aspartato-glutamato (AGC), o qual é o principal mecanismo para o movimento de equivalentes redutores na forma de NADH. A citrulinemia do tipo II (CTLN2) é uma doença autossômica recessiva de início tardio, causada por mutações no gene SLC25A13 que codifica a citrina. A citrina é uma isoforma do transportador AGC e catalisa o transporte de glutamato citosólico através da troca com o aspartato mitocondrial, o qual será utilizado no ciclo da ureia. A CTLN2 promove uma deficiência no ciclo da ureia e consequente hiperamonemia. A deficiência da citrina promove um aumento da razão NADH/NAD+ citosólica. O aumento dessa razão inibe a glicólise e a gliconeogênese. O desenvolvimento de modelo in vitro da CTLN2 é importante para estudos do mecanismo da doença e de novas terapias. A expressão heteróloga de proteínas entre diferentes reinos tem sido utilizado como uma forma de corrigir algumas doenças mitocondriais. Estudos bioquímicos e moleculares em nosso laboratório demonstraram a presença de um transportador mitocondrial de nicotinamida adenina dinucleotídeo (Ndt1) em Aspergillus fumigatus. Ndt1 realiza o transporte de NAD+ citosólico para a matriz mitocondrial, sendo dessa forma uma proteína importante para manter o balanço redox em A. fumigatus. Assim, o objetivo deste trabalho foi obter uma linhagem de células de mamífero HEK293 com knockdown para o gene SLC25A13, ou seja, um modelo in vitro de CTLN2 e a expressão heteróloga da proteína Ndt1 como uma forma de recuperação do metabolismo. As células com knockdown para o gene SLC25A13 apresentaram um aumento da razão NADH/NAD+ citosólico, redução da glicólise, redução da concentração da ureia e aumento da concentração de amônia. A expressão de Ndt1 foi capaz de reduzir a razão NADH/NAD+ citosólico e recuperou a atividade glicolítica. Entretanto, a expressão de Ndt1 não foi capaz de aumentar a concentração de ureia e reduzir a concentração de amônia causadas pela CTLN2. Dessa forma, nossos resultados sugerem que a expressão da proteína Ndt1 em células de mamíferos recupera o metabolismo mitocondrial e atividade glicolítica das células com CTLN2, mas não melhora o ciclo da ureia e o aumento da concentração de amônia. / Redox balance in mammalian mitochondria is performed by the aspartate-glutamate carrier (AGC), which is the main mechanism for the movement of reducing equivalents in the form of NADH.Type II citrullinemia (CTLN2) is an adult-onset autosomal recessive disease caused by mutations in SLC25A13 gene, and that coding citrin. Citrin is an isoform of AGC and catalyzes the transport of cytosolic glutamate through exchange with mitochondrial aspartate. It will be used in the urea cycle. CTLN2 causes urea cycle deficiency and hyperammonemia. Citrin deficiency cause an increase in the cytosolic NADH/NAD+ ratio. The increase in this ratio inhibits glycolysis and gluconeogenesis. The development an in vitro CTLN2 model is important for new studies about the disease mechanism and new therapies. Heterologous expression of proteins from different organisms has been used to recover some mitochondrial diseases. Biochemical and molecular studies in our laboratory demonstrated the presence of a mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) in Aspergillus fumigatus. Ndt1 protein performs cytosolic NAD+ transport to the mitochondria matrix, thus being an important protein to keep the redox balance in A. fumigatus. Thus, the aim of this work was to obtain a line of HEK293 mammalian cells with knockdown for the SLC25A13 gene, an in vitro CTLN2 model and the heterologous expression of the Ndt1 protein as a form of metabolism recovery. Cells with citrin knockdown showed an increase of cytosolic NADH/NAD+ ratio, reduction of glycolysis, reduction of urea concentration, and increase of ammonia concentration. Expression of Ndt1 protein was able to reduce cytosolic NADH/NAD+ ratio and recovered the glycolytic activity. However, Ndt1 protein was not able to increase the urea concentration and reduce of ammonia concentration caused by CTLN2. Thus, our results suggest that expression of Ndt1 protein in mammal cells recovers the mitochondrial metabolism and glycolytic activity in CTLN2 cells but does not improve urea cycle and reduce ammonia concentration.

Pouco se sabe sobre o mecanismo de ação da pentamidina, um composto utilizado no tratamento das leishmanioses. Para uma melhor compreensão do mecanismo de ação assim como o mecanismo de resistência à pentamidina, foi isolado um gene que codifica um membro da família de transportadores ABC, chamado PRP1 capaz de conferir resistência à pentamidina em formas promastigotas e amastigotas de Leishmania spp. O tratamento dos transfectantes que superexpressam a PRP1 com pentamidina em presença de concentrações não tóxicas de verapamil, um inibidor de transportadores ABC, foi capaz de reverter a resistência mediada por esse transportador. Foram ainda isolados nesse estudo duas linhagens de L. amazonensis resistentes à pentamidina. A análise molecular dos parasitos indicou que esses mutantes não apresentaram nenhuma amplificação de DNA, inclusive do gene PRP1 que também não se mostrou superexpresso em ambas as linhagens. As duas linhagens resistentes à pentamidina tiveram sua resistência revertida quando tratadas com verapamil, indicando que o mecanismo de resistência nesses mutantes pode estar associado a um transportador ABC. Os resultados obtidos nesse estudo fornecem dados para uma melhor compreensão do mecanismo de resistência à pentamidina e sugerem um provável potencial da associação pentamidina e verapamil no tratamento da doença. / Little it is known about the mechanism of action of pentamidine, an compound used for leishmaniases chemotherapy. To understand the mechanism of action and resistance of pentamidine, it was isolated a gene that codifies a member of ABC transporter family, named as PRP1 able to confer pentamidine resistance in promastigotes and amastigotes of Leishmania spp. Treatment of transfectants overexpressing PRP1 with pentamidine in presence of non toxic concentration of verapamil, an inhibitor of ABC transporters, was able to reverse the drug resistance mediated by this transporter. Two lines of L. amazonensis resistant to pentamidine were selected. Molecular analysis of parasites indicated that these mutants do not contain amplified DNA, including the PRP1 gene either not associated with overexpression in both lines. The two lines resistant to pentamidine had their resistance reversed when treated with verapamil, indicating that the mechanism of resistance may be associated to an ABC transporter. The results of this work lead to new insights for a better understanding of the mechanism of of resistance suggesting a probably potencial of pentamidine and verapamil association in the chemotherapy.

A digestão nos insetos ocorre no intestino médio de forma compartimentada. A digestão inicial dos polímeros ocorre no interior da membrana peritrófica. Os oligômeros resultantes difundem-se para o espaço luminal exterior à membrana peritrófica onde são atacados por outras enzimas. Na digestão final os dímeros resultantes são hidrolisados por enzimas imobilizadas na superfície do epitélio do intestino médio. Após o processo de digestão final os monômeros são absorvidos pelas células do epitélio intestinal. Os Díptera ditos superiores, incluindo a mosca doméstica, apresentam peculiaridades digestivas que aparentemente resultam de adaptações para digerir uma dieta que consiste principalmente de bactérias. No ventrículo anterior ocorre uma diminuição no conteúdo de amido do bolo alimentar. Na porção seguinte, o bolo alimentar passa para o ventrículo médio onde as bactérias são mortas pela ação combinada de baixo pH, uma lisozima digestiva e uma proteinase tipo catepsina D. O material liberado das bactérias é digerido no ventrículo posterior, como ocorre no ventrículo inteiro da maioria dos insetos de outros grupos taxonômicos. Com o objetivo de compreender a peculiar digestão em Musca domestica, foram utilizadas suas larvas para identificar funcionalmente as regiões absortivas de nutrientes, identificar as moléculas envolvidas na absorção de nutrientes, identificar as moléculas envolvidas com tamponamento e fluxos de fluidos intestinais, sequenciar as enzimas digestivas principais e identificar os seus sítios de secreção. Experimentos fisiológicos de absorção de glicose e análises de atividade enzimática permitiram acessar de maneira direta os aspectos da digestão. Contudo, experimentos de sequenciamento de bibliotecas de cDNA, análise de sequências transcritas e verificação de expressão de genes em diferentes tecidos foram abordagens fundamentais na identificação das moléculas subjacentes aos processos fisiológicos intestinas de Musca domestica. Os indícios de que absorção de glicose no intestino de Musca domestica se dê por transportadores do tipo SGLT, com a possível participação de facilitadores do tipo GLUT, permitem estabelecer um foco para futuros estudos. A descrição de sequências relacionadas ao tamponamento intestinal permitiu ampliar a discussão sobre tal processo. Ao detalhar os sítios de expressão da subunidade a da V-ATPase, do canal de cloreto e do transportador de amônia foi possível testar o modelo de tamponamento proposto anteriormente e propor a participação de outras moléculas no processo. Sequências correspondentes as atividades de carboxipeptidase, maltase e aminopeptidase descritas na literatura foram pesquisadas, gerando sequências candidatas a codificarem as referidas enzimas. Com isso, é possível descrever a digestão de oligômeros e dímeros com base nos genes transcritos e nas sequências de aminoácidos que formam as enzimas digestivas. A descoberta da sequência que transcreve uma metaloproteinase, por sua vez, abre caminhos para a descrição e caracterização de sua atividade proteolítica nos tecidos digestivos da larva de Musca domestica. Essa análise permitiu também elucidar a localização dos sítios de expressão e, portanto, as zonas de secreção de enzimas. De maneira geral, este estudo contribuiu para a compreensão de diversos aspectos da digestão de Musca domestica, elucidando questões da particular fisiologia digestiva desse inseto. / Digestion in insects occurs in the midgut in a compartmentalized way. Initial digestion takes place inside the peritrophic membrane. The resulting oligomers diffuse into the luminal space outside the peritrophic membrane where they are hydrolyzed by other enzymes. In the final digestion, the resulting dimers are hydrolyzed by enzymes immobilized on the midgut epithelium. After the final digestion, the monomers are absorbed by intestinal epithelial cells. The so-called higher Diptera, including the house fly, have digestive peculiarities apparently resulting of adaptations to digest a diet consisting mainly of bacteria. In the anterior midgut there is a decrease in the starch content of the food bolus. The bolus now passes into the middle midgut, where bacteria are killed by the combined action of low pH, a special lysozyme and a cathepsin D-like proteinase. Finally, the material released by bacteria is digested in the posterior midgut, as is observed in the whole midgut of insects of other taxa. In order to understand the peculiar digestion in Musca domestica, the larvae were used to identify (a) the functionally the nutrient absorptive regions, (b) the molecules involved in the absorption of nutrients, (c) the molecules involved in buffering and fluid flows, (d) the cDNA sequences corresponding to intestinal digestive enzymes, (e) the main sites of secretion. Physiological experiments of glucose absorption and enzyme activity analysis allowed a direct access to aspects of digestion. Otherwise, cDNA library sequencing followed by sequence annotation and tissue-specific expression analysis were fundamental approaches in the understanding of intestinal physiology of Musca domestica. Evidence that glucose absorption in the gut of Musca domestica occurs through SGLT-like transporters, with the possible participation of facilitators GLUT-like, allowed us to establish a focus for future studies. The description of cDNA sequences corresponding to proteins putatively responsible for intestinal buffering widened the discussion of this process. The finding of the expression sites of V-ATPase subunits, chloride channel, and ammonia transporter led to revising the present buffering model and the inclusion of other molecules in the process. The cDNA sequences corresponding to the activities of carboxypeptidase, aminopeptidase and maltase described in the literature were searched for as candidate sequences to encode those enzymes. This made it possible to describe the digestion of oligomers and dimers based on transcribed genes and enzyme amino acid sequences. The discovery of the metalloproteinase transcribing sequence opened a new research line: the description and characterization of its proteolytic activity in the midgut of the Musca domestica larvae. This study also allowed elucidating the location of digestive enzyme expression sites and, therefore, the putative zones of enzyme secretion. Overall, this study contributed to understanding many aspects of digestion of Musca domestica, clarifying aspects of the peculiar digestive physiology of this insect.

O parálogo 3 do trocador Na+/H+ (NHE3) é essencial para a reabsorção de HCO3- nos túbulos proximais renais e sua expressão e função adaptam-se às diferentes condições ácido-base do organismo. O objetivo desta tese foi avaliar quais as diferenças entre os efeitos da acidose metabólica (AM) e respiratória (AR) sobre a regulação do NHE3 e identificar variáveis responsáveis pelas respostas adaptativas observadas. Em células OKP, a AM foi simulada diminuindo a [HCO3-] do meio de cultura e a AR aumentando a pCO2 ambiente por 24 h. Foram observados os efeitos das acidoses sobre o RNAm-Nhe3, a presença da proteína-NHE3 na membrana celular e a atividade promotora do gene do Nhe3. Concluiu-se que o pH extracelular não é a variável físico-química responsável por estimular a expressão do NHE3, contudo é um importante candidato à variável responsável por regular o tráfego da proteína para a membrana. Além disso, a região de -471 a -153 pb em relação ao sítio de início de transcrição do promotor do gene do Nhe3 contém prováveis reguladores positivos que atuam em resposta à AM. / The Na+/H+ exchanger 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules and its expression and function must adapt to acid-base conditions. The goal of the presente study was to evaluate whether there are differences between metabolic (MA) and respiratory acidosis (RA) with regard to NHE3 modulation and to identify variables that may trigger these distinct adaptive responses. In OKP cells, MA was achieved by lowering [HCO3-] in the cell culture medium and RA by increasing pCO2 in the incubator chamber for 24 h. The effects of both acidosis on Nhe3 mRNA levels, cell-surface NHE3 expression and promoter activity were evaluated. In summary, it was concluded that low extracellular pH is not the physical-chemical variable that up-regulates NHE3 expression, however, extracellular pH is a candidate for the variable related to the NHE3 displacement to the apical membrane. Moreover, the Nhe3 gene promoter region spanning from -471 to -153 base pairs upstream from the transcriptional start site contains putative enhancers regulated in response to MA.

Stroke is one of the major causes of death and disability worldwide. The area that surrounds the infarcted core is the location of the continuing damage that takes place hours and days following an insult, and is referred to as the penumbra. By creating an oxygen deprived environment in the neuronal-like PC12 and NT2 cells and an in vivo photothrombotic model of stroke (PTS) in mice, two different strategies were created to replicate the conditions of an ischaemic brain. In differentiated PC12 and NT2 cells, following hypoxia, preferential activation of HIF-2α transcription and protein expression was detected. Increased expression of the neural progenitor stem cell-like markers, thought to be transcriptionally regulated by HIF-2α, were also observed. Furthermore, hypoxia caused loss of neuronal characteristics in differentiated cells. This is highly significant as it shows neuronal cells possess molecular mechanisms which could trigger recovery following ischaemic insult. The expression of the chloride co-transporters, NKCC1 and KCC2, mediators of the GABAergic response, was assessed following hypoxia in differentiated PC12 and NT2 cells and PTS. In PC12 and NT2 cells exposed to hypoxia, the expression of KCC2 was significantly decreased at both the transcript and protein level whereas NKCC1 expression remained unmodified. In the in vivo model, the development of the penumbra in the days following injury was assessed with specific markers allowing the identification of the penumbra up to 200 ❍m from the ischaemic core and a progressive neuronal loss was observed within. Our results show an increase in the number of neurons expressing NKCC1 in the penumbra up to 5 days following the insult when compared to the contralateral hemisphere. On the contrary, KCC2 positive cells were dramatically decreased in this area. In mice treated with bumetanide, an NKCC1 antagonist, a significant reduction in neuronal loss was observed. Our results show a reversal on the chloride co-transporters expression in vitro and in vivo and how treatments targeting these channels might represent a novel strategy to reduce the damage associated with stroke.

No description available.

Fine temporal regulation of dopamine transmission is critical to its effects on behaviour. Dopamine can be cleared from the synapse either by recycling via the dopamine transporter (DAT) or by enzymatic degradation involving catechol-O-methyltransferase (COMT). DAT recycling predominates in striatum and contributes to dopaminergic regulation of reward-guided behaviour, while COMT degradation predominates in cortex and modulates executive functions. However, human functional imaging studies demonstrate interactive effects of DAT and COMT genotype, suggesting that the traditional division between DAT and COMT is not so clear-cut. Given the interdependence of mesolimbic and mesocortical circuitry and the presence of COMT in the striatum, it is possible that DAT and COMT interact to a greater extent than previously thought. We investigated the contributions of DAT and COMT to regulation of dopamine transmission and reward-guided behaviour by combining in vivo electrochemical recording, pharmacology, and behavioural testing in mice. Using fast scan cyclic voltammetry to record evoked dopamine release in anaesthetised animals, we found that systemic DAT blockade increased the size of dopamine transients in the nucleus accumbens (NAc) but not in the medial frontal cortex (MFC), demonstrating that DAT regulates phasic striatal dopamine release and confirming that DAT makes little contribution to regulation of cortical dopamine transmission. Unexpectedly, COMT inhibition did not affect evoked dopamine transients in either the NAc or the MFC. In agreement with these findings, systemic administration of a DAT blocker, but not of a COMT inhibitor, increased motivation to work for reward in a progressive ratio paradigm. COMT inhibition also had little effect on reinforcement learning (RL) strategies during reward-guided decision making. Intriguingly, however, we found that DAT blockade both decreased the influence of model-free RL and increased the influence of model-based RL on behaviour. Our study confirms that DAT regulates dopamine transmission in striatum but not in cortex and indicates that sub-second changes in dopamine transmission in both regions are largely insensitive to COMT. However, our behavioural data reveal the importance of striatal dopamine in multiple components of reward-guided behaviour, including both motivational aspects traditionally associated with striatum as well as cognitive aspects heretofore mainly associated with cortical function. Together, these findings emphasise that reward processing occurs across corticostriatal circuits and contribute to our understanding of how striatal dopamine transmission regulates reward-guided behaviours.

A creatina (Cr) é uma reserva de fosfato de alta energia, sendo a fonte mais rápida de restauração do ATP intracelular. O hormônios tireoideano participa de forma importante na manutenção da taxa metabólica, aumentando a síntese e consumo de ATP, por meio da regulação de diferentes genes-alvo. Neste sentido, avaliamos o efeitos do HT sobre a expressão gênica do transportador de Cr nos músculos esqueléticos e cardíaco de ratos. O tratamento com o hormônio regula estes processos, porém de forma distinta nos diferentes tipos de músculos. / Creatine (Cr) is a high-energy phosphate reservoir and the fastest source for intracellular ATP regeneration. The thyroid hormone plays a key role on the maintenance of basal metabolic rate, increasing the synthesis and the degradation of ATP through regulation of target-genes. In this study, we explore the effects of thyroid hormone on Cr transporter gene expression and regulation of intracellular pool of Cr in skeletal and cardiac muscles in rats. The hormone can regulate these processes in distinct ways in different muscle types.

Lors d’une infection microbienne, la défense de l’hôte comprend deux facettes complémentaires. Premièrement, le système immunitaire cible les pathogènes dans le but de les éliminer, une attaque correspondant à la résistance. Dans un second temps, l’organisme doit réparer les dégâts causés par le pathogène ou par la réponse immunitaire de l’hôte, un mécanisme appelé résilience. J’ai étudié les effets d’une infection intestinale par la bactérie Serratia marcescens chez la drosophile. Nous avons mis en évidence un processus de purge dans l’intestin, lors duquel les enterocytes -les cellules principales de l’intestin- se vident partiellement de leur contenu. L’épithélium intestinal devient alors très fin mais se régénère rapidement, protégeant ainsi la mouche des effets délétères de l’infection. J’ai identifié un transporteur d’acides aminés, CG1139, qui est nécessaire à la régénération de l’intestin. CG1139 est requis pour la mobilisation de certaines réserves métaboliques de la drosophile et pour le transport rétrograde de ces dernières vers l’intestin. / Upon microbial infections, host defenses comprise two complementary facets. First, immune effectors target and kill the invading pathogen, an attack referred to as resistance. Second, the infected host must repair the damages inflicted by microbes or by the immune response itself, a mechanism called resilience. I have studied the effects of an intestinal infection with the bacterium Serratia marcescens in Drosophila. We have discovered a purge mechanism in the intestine, where enterocytes -the main cell type in the gut- extrude some of their internal contents. The intestinal epithelium thus becomes very thin but rapidly recovers its shape, thereby protecting the fly against the deleterious effects of infection. I have identified an amino acid transporter, CG1139, which is required for the intestinal recovery. CG1139 is necessary to mobilize the fly’s internal metabolic reserves and to transport some these metabolic stores back to the gut, in a retrograde manner.

<p>The subject of this thesis was to study two intestinal barriers to oral drug bioavailability, drug efflux proteins of the ABC-transporter family, and in particular ABCB1/P-glycoprotein (Pgp), and the drug metabolizing enzyme cytochrome P450 (CYP) 3A4. At the onset of this thesis, similarities between CYP3A4 and Pgp in terms of their tissue distribution and gene regulation, along with overlapping substrate specificities, had generated the hypothesis that CYP3A4 and Pgp may have a complementary function and thus form a coordinated intestinal barrier to drug absorption and gut wall metabolism.</p><p>In the first part of this thesis, a cell culture model of the intestinal epithelium that expressed both functional Pgp and CYP3A4 was developed. This model was then used to investigate the steroselective drug efflux and metabolism of R/S-verapamil. In summary, the results indicated that the two barriers in the cell culture model were in agreement with those in the human intestine.</p><p>Both ABC-transporters and CYPs are regulated by drugs that interact with nuclear receptors. However, while the regulation of CYPs is quite well understood, less is known about how repeated drug administration regulates the most abundantly expressed ABC-transporters. Therefore, in the second part of this thesis, the effects of repeated drug administration on the gene regulation of four ABC-transporters and CYP3A4 were studied in intestinal epithelial cell lines in vitro and in the perfused human jejunum in vivo. The in vitro studies revealed that the ABC-transporters are induced by drugs that interact with slightly different sets of nuclear receptors. The in vivo study showed that repeated oral administration of St John’s wort decreased the bioavailability of verapamil, predominantly by induction of intestinal CYP3A4. This part of the thesis provides new information about the regulation of ABC-transporters, shows that the intestinal metabolism is the most significant barrier to oral bioavailability of verapamil and provides evidence for a clinically significant interaction between verapamil and St John’s wort in vivo.</p>