[pt] A aparição do HIV na década de 80 e de outros vírus presentes no sangue dos doadores, assim como a expansão de doenças como a hepatite C, tem estimulado as pesquisas para desenvolver substitutos do sangue. Pesquisas recentes têm sido realizadas em derivados artificiais da albumina de soro humano (human serum albumin - HSA) associados com heme, representando uma linha promissora nesse sentido. No presente trabalho, fazemos um estudo computacional da ligação do heme e de seu precursor, a protoporfirina IX, com a HSA, com o objetivo de conhecer os aminoácidos que contribuem à estabilidade da ligação, a natureza do sítio de ligação, a formação de possíveis sítios secundários de ligação e a influência das porfirinas na estrutura global da proteína. Para conseguir este objetivo ut ilizamos técnicas de cálculo quântico usando a Teoria do Funcional de Densidade, Docking Molecular e Dinâmica Molecular. Finalmente fazemos um estudo da correlação existente entre as diferentes regiões da HSA e de como este padrão muda na presença das porfirinas. Para este propósito usamos os coeficientes de correlação generalizada baseados na formulação de Kraskov. Com base nestas informações, discutimos a contribuição que poderiam fornecer nossos cálculos para a reengenharia do HSA, no sentido de fornecer- lhe características de hemoproteínas. / [en] The uprising of HIV and other viruses in the beginning of the 80s, as well as the expansion of other diseases like hepatitis C has stimulated research in order to develop blood substitutes. Research based on modified Human Serum Albumin associated with heme has shown a promissory line in this direction. In the present work we make a computational study about the heme and its precursor, the Protoporphyrin IX complexed with HSA, in order to know the contribution of the main amino acids to the stability of the binding, the character of the binding site, secondary binding sites formation, and the influence of the porphyrins in the global protein structure. To find this objective we use quantum calculations based on Functional Density Theory, Molecular Docking and Molecular Dynamics. Finally, a correlation study between different regions of HSA and the pattern modifications caused by the porphyrins were performed, using the generalized correlation coefficients based on the Kraskov formulation. We also discuss the contribution that our calculations could give to the reengineering of HSA, to provide them with hemeprotein characteristics.

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Dans cette thèse, j'ai essayé d'identifier le mode d'action de composés biscationiques récemment synthétisés et leurs interactions avec les parasites résistants à la chloroquine. Les activités de divers représentants des composés ammonium bisquaternaire, des amidines alkyl (provenant du groupe de professeur Vial, Montpellier, France), des amidines bisbenzyl (provenant du groupe Chimie de l'Université de Liverpool, Royaume-Uni) ont été comparés à la chloroquine et la pentamidine. Leurs potentiels de résistance croisée avec la chloroquine ont également été étudiés. Dans ce but, deux lignées cellulaires modifiées génétiquement par échange allélique, C3Dd2 et C2GCO3 furent utilisées.Parmi les amidines bisbenzyl, une série de composés appartenant aux guanidines, thiazoles et triazoles ont été criblés pour leur activité contre des souches résistantes et sensibles à la chloroquine chez Plasmodium falciparum. Une hypersensibilité significative est observée pour les amidines bisbenzyl parmi les isolats affichant un PfCRT mutant. Aucune différence n'est observée pour les composés provenant du groupe Vial. Pour comprendre le mode d'action et le rôle de PfCRT, j'ai réalisé des expériences de fixation compétitives (competitive binding') et de cristallisation d'hème. Tous les composés ont montré à différents degrés des interactions avec l'hème, cependant il fut observé que leur activité ne corrélait pas avec l'inhibition de la cristallisation d'hème. Une des raisons possibles à cela est que les différences structurales peuvent jouer un rôle important dans le transport du composé. De plus, j'ai étudié l'effet du pH sur l'activité des composés en utilisant les lignées cellulaires modifiées génétiquement par échange allélique afin d'observer l'effet du gradient de proton sur le transport de la chloroquine et de la pentamidine. Des différences significatives de l'activité de la chloroquine furent observées chez les deux souches. Malgré les valeurs de pKa élevées pour la pentamidine, il y avait une différence significative dans la sensibilité pour ce composé chez les souches quand le pH a changé.Car les diamidines requièrent des transporteurs pour traverser les barrières membranaires et qu'un possible transporteur de choline a été caractérisé chez Plasmodium falciparum, j'ai également réalisé des études initiales sur la caractérisation moléculaire de ce transporteur. Un gène qui encode une protéine chez P. falciparum avec une similarité significative aux eucaryotes supérieurs fut identifié en utilisant des analyses bioinformatiques et fut employé dans une transformation et des études analyses fonctionnelles.En conclusion, ce travail suggère qu'il est possible d'utiliser de nouveaux amidines bisbenzyl pour cibler spécifiquement les souches résistantes à la quinoléine chez Plasmodium falciparum, arborant des allèles de PfCRT mutantes. En adhérant à cette hypothèse et sachant que les deux classes de composés fixent la même cible non parasitaire (soit l'hème), il serait possible de créer rationnellement une combinaison de composés quinoléine / diamidine. Ainsi, les souches résistantes à un des deux composés seraient plus sensibles à l'autre partenaire, retardant ainsi l'apparition de résistance. / In this thesis I have attempted to subject the issues of mode of action of recently synthesized bis cationic compounds and their possible interactions with chloroquine resistance. Antimalarial activities of representatives of various bis quarternary ammonium compounds, alkyl amidines (received from Dr.Vial group, Montpellier) and of bisbenzyl amidines (received from Chemistry group, Liverpool) activity have been investigated with chloroquine and pentamidine and looked for cross resistance with chloroquine. For this purpose two genetically modified allelically exchanged cell lines C3Dd2 and C2GCO3 modified on the chloroquine resistance-related PfCRT (P.falciparum chloroquine Resistance Transporter) gene were used. Among the benzyl amidines, a significant hypersensitivity tobis benzyl amidines was observed among the isolates bearing the mutant PfCRT. No such difference is observed for the bisalkyl amidines. To understand the mode of action and role of PfCRT, competitive binding assay to heme (which may mediate the well-known cellular accumulation of the compounds) and effect on heme crystallization assays (which is involved in the toxic effect against the intracellular parasite) were performed. All these compounds were shown to interact with heme in various degrees. Their activity was observed not to be correlating with heme crystallization inhibition. This is likely due to the structural differences between the compound which discriminate the compounds in the transport of the compound to the parasite and their mechanism of antimalarial activities. In addition I have studied the effect of pH on the pharmacological activity of the drugs using allelically exchanged genetically modified cell lines (for PfCRT) to characterize the importance of proton gradient on the transport of chloroquine and pentamidine to the intracellular parasite. Significant difference (reduced antimalarial activity with increased pH) in the activity of chloroquine was observed for both the strains. Despite of the high pKa values for pentamidine, there was significant difference in the sensitivity of the strains to this compound, when the pH is changed. As both the diamidines and choline analogs require transporters to cross the membrane barriers and enter the parasite where they accumulate I have also performed initial studies on the molecular characterization of a potential carrier in P.falciparum. Using basic bioinformatic tools, a gene encoding a P.falciparum protein with significant similarity to higher eukaryotes choline transporter was identified and preliminary work for its functional analysis was performed. In conclusion, this work establishes substantial differences between the various classes of bis-cationic compounds essentially (based on benzamidine and choline-analogs alkylkamidine series) concerning their interaction with the infected erythrocyte and their antimalarial activity. The series are diffentallly affected by the PfCRT mutation and the chloroquine resistance. Results suggest that it may be possible to use novel bisbenzyl amidines to specifically target quinoline resistant Plasmodium falciparum malaria, harbouring mutant pfcrt alleles. Taking this idea further and since both classes of compound target the same non-parasite target (heme), it may even be possible to rationally design a quinoline / diamidine drug combination, in which isolates resistant to one partner drug become more sensitive to the other partner, thus delaying the onset of resistance.

Heme is one of the most important and most studied cofactors that are essential for proper function of many proteins. Heme-containing proteins comprise of a large group of biologically important molecules that are involved in many physiological processes. The presented dissertation is focused on two groups of heme sensor proteins, namely prokaryotic heme-based gas sensors and eukaryotic heme-responsive sensors. Heme-based gas sensors play an important role in regulation of many bacterial processes and consist usually of two domains, a sensor domain and a functional domain. The dissertation thesis aims at the study of two model bacterial heme-based gas sensors, histidine kinase AfGcHK and diguanylate cyclase YddV, in order to elucidate their mechanism of interdomain signal transduction. Using X-ray crystallography and hydrogen-deuterium exchange coupled to mass spectrometry approaches, significant differences in the structure of the AfGcHK protein between the active and inactive forms were described. The signal detection by the AfGcHK sensor domain affects the structural properties of the protein, and these conformational changes then have indirect impact on the enzyme activity of the functional domain. Further, the dissertation pays more attention to the effect of a sensor domain dimerization...

Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.

Au cours de ma thèse, je me suis intéressée à l'interaction du complexe Nitrate Reductase A (NarGHI) avec les quinones et les lipides de la membrane chez E. coli. Nous avons identifié que les intermédiaires ménasmiquinones interagissent avec une liaison hydrogène avec l'histidine 66 du site Qd. Par ailleurs, nous avons mis en évidence par la fixation spécifique d'une molécule de cardiolipine est indispensable au fonctionnement du complexe NarGHI en permettant la fixation du quinol. Enfin, nous avons démontré l'existence d'une liaison fonctionnelle entre la voie de biosynthèse des hèmes et les complexes respiratoires via la protéine HemG, qui couple la réduction des quinones avec l'oxydation du protoporphyrinogène IX. Ces éléments prouvent qu'une voie catalytique peut contribuer à la synthèse ATP. L'ensemble de ces résultats indique une étroite interconnexion physique et fonctionnelle entre tous les éléments qui composent la membrane cytoplasmique d'E. coli / In this thesis, I study the interaction between the nitrate reductase A comlex (NarGHI) with the quinines and lipids of the E. coli cytoplasmic membrane. We demonstrate that His66 present at the Qd site is directly hydrogen bonded to both menasemiquinone and ubisemiquinone species. In addition, we show that functionning of the enzyme complex is controlled by cardiolipin binding in a specific cavity allowing quinol binding at the nearby QD site. Finally, we relealed that heme biosynthesis is a quinone-depended metabolic reaction during anaerobic growth of E. coli, in wich the HemG protein will direct electron transfer issued from oxidation of a heme biosynthetic intermediate towards quinone molecules via interaction between quinones, lipids and membrane- associated complexes that couple respiration and anabolic pathways to ATP generation in specialized domains of E. coli membrane

The obligate human pathogen Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed “nutritional immunity” by expression of TonB-dependent transporters (TdTs): outer membrane receptors that facilitate nutrient transport in an energy-dependent manner. N. gonorrhoeae encodes eight TdTs, five of which facilitate utilization of iron or iron-chelates from host derived proteins including transferrin, lactoferrin and hemoglobin, in addition to siderophores from neighboring bacteria. The transferrin utilization system was previously shown to be critical for establishing infection in human males; demonstrating the possible contributions of TdTs to gonococcal pathogenesis. As such, studies describing the biological function and contribution to pathogenesis of the remaining three uncharacterized TdTs (TdfG, TdfH and TdfJ) are needed. In this study we report that neither TdfG, TdfH nor TdfJ are heme receptors as gonococcal heme utilization occurs passively, independent of energy derived from the TonB system. We also report that TdfH and TdfJ are zinc (Zn) regulated and identify virulence associated regulators that modulate expression of these TdTs, which is in some cases strain-specific. We report that both TdfH and TdfJ contribute to Zn acquisition in N. gonorrhoeae and we characterize TdfH as a calprotectin receptor. Calprotectin, an immune effector protein highly expressed in neutrophils, has antimicrobial activity due to its ability to sequester Zn and Mn. We present evidence that TdfH confers resistance to calprotectin and that TdfH facilitates gonococcal calprotectin binding and Zn accumulation in the presence or absence of calprotectin. Finally, we demonstrate that TdfH expression enhances N. gonorrhoeae NET survival. These studies identify for the first time a novel gonococcal defense strategy to host-mediated nutritional immunity, in which N. gonorrhoeae, via the TdT TdfH, utilizes calprotectin as a Zn source neutralizing its antimicrobial activity. These studies have yielded novel insights into the function and regulation of TdfG, TdfH and TdfJ in N. gonorrhoeae and have laid the framework for future investigation of TdT-mediated Zn acquisition and its role in bacterial pathogenesis.

The smallest free living eukaryote known as Ostreococcus tauri became along with some related species great experimental models for iron uptake research in marine picoplankton. The ecological context of such topic is very interesting considering the nature of adaptation to iron limitation and its connection to the copper metabolism. Our experiments has simulated iron and copper limiting conditions of the ocean, as a control we used iron and copper repleted cultivation media. The maximum cell counts were two- to threefold higher in iron-repleted medium compared to iron- depleted one. There was the only exception showing no difference in growth - O. lucimarinus coming from open ocean with high irradiance levels and very low iron concentrations, which all together made it the best adapted species. The reinoculation of cell cultures after a week cultivation into to the same iron/copper containing media led to unmasking the copper effect on growth, which was much weaker than encountered with iron (again except of O. lucimarinus). Iron sufficiency positively affects heme b and chlorophyll a and b content with no significant copper dependency. The circadian timing of heme b and chlorophyll a and b content shows the increasing trend during the day followed by decrease at night. This might be caused by...

O monóxido de carbono (CO) é uma das moléculas mais antigas encontradas na atmosfera, sendo popularmente associado à asfixia por sua alta taxa de ligação à hemoglobina. Contudo, estudos também demonstram seu envolvimento em diversas funções fisiológicas através da produção endógena pela enzima heme-oxigenase (HO), que catalisa o metabolismo do grupo heme em quantidades equimolares de CO, biliverdina e ferro. Destes, o CO é um de seus produtos mais ativos, podendo atuar como neurotransmissor e neuromodulador do sistema nervoso, molécula de sinalização cardiovascular com propriedades vasoativas e com potencial participação em processos nociceptivos. Inúmeras pesquisas associam os mecanismos de ação da via da HO/CO com a ativação da enzima guanilato ciclase, GMPc ou modulação direta de canais de potássio, sendo este último, amplamente conhecido pelo seu envolvimento em respostas antinociceptivas, em especial sua classe sensível ao ATP (K+ATP). Assim, nosso objetivo neste estudo, foi verificar o possível envolvimento de K+ATP, na antinocicepção desencadeada pela via da HO/CO após indução de hiperalgesia pela administração de carragenina e exposição aos testes nociceptivos de Von Frey eletrônico e Randall Selitto. Os resultados obtidos em nossos experimentos demonstram que a aplicação intraplantar de hemina (substrato da HO) ocasionou ação antinociceptiva, sendo excluída através do tratamento contralateral a ocorrência de mecanismos sistêmicos, frente à manipulação periférica da via. Por sua vez, a administração do inibidor da via da HO (tin protoporphyrin IX dichloride) potencializou a hiperalgesia ocasionada pela carragenina. Efeitos similares não foram observados frente à administração dos demais subprodutos, biliverdina e ferro (sulfato ferroso), sugerindo o CO como sendo o mais ativo subproduto da via na modulação de processos nociceptivos. Já a administração conjunta de glibenclamide (bloqueador de K+ATP) e hemina, acarretou o bloqueio da ação antinociceptiva ocasionada pela hemina, sendo que a administração conjunta da menor dose efetiva de diazoxide (ativador de canais de potássio) e hemina evidenciou uma ação sinérgica entre as drogas potencializando a resposta antinociceptiva do substrato da via. Também observamos que o efeito antinociceptivo da hemina não foi alterado pela administração de naloxone (antagonista opióide), sugerindo assim, a exclusão da participação do sistema opióide na resposta antinociceptiva da via da HO/CO. Neste contexto, os resultados apresentados em nosso estudo sugerem fortemente que a ação antinociceptiva periférica da via HO/CO, pode estar relacionada com a ativação de canais de potássio sensíveis ao ATP / Carbon monoxide (CO) is one of the oldest molecules found in the atmosphere, being popularly associated to asphyxia by its high binding to hemoglobin. However, studies have also demonstrated its involvement in various physiological functions through the endogenous production by the enzyme heme-oxygenase (HO), which catalyzes the metabolism of heme in equimolar amounts of CO, biliverdin and iron. Of these, the CO is one of its most active and may act as a neurotransmitter and neuromodulator nervous system, cardiovascular signaling molecule with vasoactive properties and potential involvement in nociceptive processes. Numerous research associate of the mechanisms of action of the HO/CO pathway by activation of the enzyme guanylate cyclase, cGMP or direct modulation of potassium channels, the latter being widely described as involved in various nociceptive mechanisms in particular its class ATP-sensitive (K+ATP). Thus, our goal in this study was to investigate the possible involvement of K+ATP in an antinociceptive via triggered by the HO/CO after hyperalgesia induced by administration of carrageen and exposure to electronic Von Frey and Randall Selitto. The results obtained in our experiments suggest that i.pl. treatment with the substrate of HO (hemin) caused a dose-dependent antinociception, and the possible systemic action was excluded by treating contralateral paw via. In turn, the administration HO pathway inhibitor (Tin protoporphyrin IX dichloride) increased the hyperalgesia caused by carrageen. Similar effects were not observed by the administration of the other two products, biliverdin, iron, showing the CO as the more active pathway in the modulation of nociceptive processes. Since the joint administration of glibenclamide (blocker of K+ATP) and hemin, caused the blockage of antinociceptive action caused by hemin, and the joint administration of the lowest dose-response diazoxide (potassium channel activator) and hemin caused a synergistic action between drugs, and potentiate the antinociceptive response to the pathway substrate. We also observed that the antinociceptive effect of hemin was not altered by the administration of naloxone (opioid antagonist), suggesting the exclusion of the opioid system in the antinociceptive response of the HO/CO. Thus, the results presented in this study strongly suggest that the peripheral antinociceptive action via the HO/CO may be related to the activation of potassium channels sensitive to ATP / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

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