Orientador: Paulo Eduardo Martins Ribolla / Resumo: Introdução: O desenvolvimento de estratégias para redução no custo do sequenciamento de genoma completo (WGS) é importante para projetos que demandam por grandes quantidades de amostras. Uma estratégia de baixo custo é o sequenciamento de baixa cobertura aliado a técnicas de imputação para genotipagem eficiente e de confiabilidade adequada. A malária é uma das principais doenças transmitidas por artrópodes no mundo e o Brasil é considerado um país com alta incidência de malária, principalmente na região Amazônica, sendo principal vetor o mosquito Anopheles darlingi. Objetivo: O objetivo do presente estudo foi desenvolver estratégia para analisar dados de WGS de baixa cobertura de mosquitos Anopheles darlingi coletados no município de Mâncio Lima no Acre e verificar associação entre dados genéticos e dados de importância epidemiológica, tais como comportamento de picada, horário de atividade e distanciamento em escala microgeográfica. Materiais e métodos: Amostras de mosquitos Anopheles darlingi foram coletadas no município de Mâncio Lima - AC, entre 2016 e 2017. As bibliotecas foram preparadas com Nextera™ XT e sequenciadas no NextSeq500 da Illumina. Foi realizado genotipagem por sequenciamento e aplicado imputação. Estudos de associação ampla do genoma foram realizados com comportamento de picada e horário de atividade. Sinais de estratificação na população foram investigados por FST amplo no genoma e teste de permutação para significância. Resultados: Sinais fracos porém si... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Strategy development to reduce the cost of whole genome sequencing (WGS) is important for projects that demand large quantities of samples. A low-cost strategy is low-coverage sequencing combined with imputation techniques for efficient genotyping and sufficient confiability. Malaria is one of the main diseases transmitted by arthropods in the world and Brazil is considered a country with a high incidence of malaria, especially in the Amazon region with the main vector being the Anopheles darlingi mosquito. Objective: The objective of the present study was to develop a strategy to analyze low-coverage WGS data from Anopheles darlingi mosquitoes collected in the municipality of Mâncio Lima in Acre State and verify associations between genetic data and data of epidemiological importance, such as biting behavior, time of activity and distance on a microgeographic scale. Materials and methods: Samples of Anopheles darlingi mosquitoes were collected in the municipality of Mâncio Lima - AC, between 2016 and 2017. The libraries were prepared with Nextera ™ XT and sequenced on Illumina's NextSeq500. Genotyping by sequencing was performed and imputation was applied. Genome wide association studies were performed with biting behavior and time of activity. Population stratification signals were investigated by genome-wide FST and permutation test applied for significance. Results: Weak but significant stratification signals were identified considering distances of 2 to 3 k... (Complete abstract click electronic access below) / Mestre

The system of mixed function oxygenase (MFO system) participates in significant roles in the metabolism of endogenous compounds and xenobiotics. This system contains cytochrome P450, NADPH:cytochrome P450 reductase, and also there are assigned NADH: cytochrome b5 reductase and cytochrome b5. It was proved that cytochrome b5 can stimulate or inhibit cytochrome P450 (CYP)-dependent reactions and even change the ratio of resulting metabolites. The mechanism of cytochrome b5 action has not been fully elucidated yet. Elucidation of protein-protein interactions in MFO system and determination of topology of this system could explain the mechanism of cyt b5 action. The covalent cross- linking technique is suitable method for identifying protein-protein interactions within the membrane. Cytochrome b5 contains 3 methionines and in 2 cases the methionines are localized in a short hydrophobic C-terminal membrane anchor. Interactions with cytochrome P450 in the membrane environment can be identified by substitution of two methionine for photoactivatable analogue of methionine (photo-methionine) and subsequent photoactivation. This work is focused on expression and isolation of photo-cytochrome b5 (photo- cyt b5), cytochrome b5 analogue with incorporated photo-methionine. Conditions for photo-methionine...

Tumour-related diseases are the second most common cause of death in the Czech Republic, right after cardiovascular diseases. Nanomedicine - a novel scientific discipline - shows captivating potential in anticancer treatment with help of so called nanotranporters - nanoparticles capable of transporting other molecules. Encapsulation of a cytostatic drug into a nanoparticle improves its pharmacokinetical and pharmacodynamical properties which helps to reduce adverse side effects on non-tumour healthy tissue. In the scope of this diploma thesis apoferritin - apo-form of ferritin - was studied, since this nanotransporter shows promise for clinical use in anticancer treatment. Effect of hepatic microsomes from premedicated and control rats on biotransformation of doxorubicin cytostatic (Dox) in free and apoferritin nanoparticle-bound forms was investigated at pH 7,4. Over the course of biotransformation two types of metabolites - M1 and M2 - were observed. Regardless of the employed inductor all studied microsomes have exhibited similar metabolism of free doxorubicin and its apoferritin encapsulated form (ApoDox). Our results also imply that doxorubicin can be metabolically processed by rat hepatic microsomes in both free and ApoDox form with similar efficiency. We have also studied biotransformation...

Heme containing proteins and their reactivity play a central role in biological systems; they have a vast range of functions including electron transfer, catalysis, and respiration. Cytochromes c and heme c containing proteins have been used widely as model systems to understand how structure and dynamics lead toward function. In this thesis, a variety of biophysical methods are used to investigate two heme c containing model systems to gain insight into how redox potential and reactivity are modulated through changes in the local environment. Mitochondrial cytochrome c undergoes several pH dependent conformational rearrangements that involve different heme ligation and have associated changes in redox potential. Under basic conditions (pH greater than 8), the axial methionine (Met) residue is replaced by one of several nitrogen based ligands, usually a nearby lysine residue, and is coined the "alkaline transition". It is accompanied by a large downward shift in redox potential. The functional utility of this conformational change is not fully understood however it is strongly implicated in the signaling cascade for apoptosis. Bacterial monoheme cytochromes c exhibit similar phenomenological Met-loss behavior as a function of electrode material. In Chapter 2 we utilize Hydrogenobacter thermophilus cytochrome c552 as a model system for the assessment of redox thermodynamics and changes in redox potential associated with the Met-loss form. In Chapter 3 we extend our investigation to homologous cytochromes c. Bacterial cytochrome c peroxidases catalyze the two-electron reduction of hydrogen peroxide to water utilizing cytochrome c as an endogenous electron donor. Chapter 4 describes the first recombinant construct of the diheme Nitrosomonas europaea cytochrome c peroxidase (Ne CCP); a defining family member of constitutively active cytochrome c peroxidases. A variety of biophysical techniques were used to confirm similarity between the recombinant Ne CCP and native enzyme. Chapter 5 extends our investigation to the role of constitutively conserved glutamine and glutamic acid residues within the active site, and two conserved tryptophan residues; the first situated between hemes and the second distal to the active site. In Chapter 6, stopped flow spectroscopy is used to investigate the first intermediates of the Ne CCP catalytic mechanism.

Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...

Malaria, a disease caused by parasites of the Plasmodium genus, infects over 220 million people annually, resulting in over 400,000 deaths. Most of these deaths occur in Africa in children < 5 years of age. Artemisia annua L., an ancient Chinese medicinal herb, is known for its foremost phytochemical constituent, artemisinin (AN). Semisynthetic derivatives of AN form the primary component of artemisinin combination therapies (ACTs), the frontline treatment for malaria worldwide. However, ACTs have several drawbacks including cost and availability. Thus, cheaper, more readily available antimalarials are needed. Recent clinical data suggested dried leaves of A. annua (DLA) administered orally as a tea infusion may be as efficacious as ACTs despite a significantly lower AN dose delivered. In mice, AN plasma concentration was improved when administered as DLA compared to pure AN. I therefore hypothesized that phytochemicals within DLA enhanced the oral bioavailability of AN. To investigate this hypothesis, here I examined the effects of DLA on the underlying mechanisms that govern oral bioavailability. Using an in vitro human digestion model, I showed that AN solubility was greater when delivered as DLA, largely due to essential oil in the plant. Furthermore, AN intestinal permeability was enhanced in a Caco-2 cell model of the intestinal epithelium. Extracts, teas, and phytochemicals produced by Artemisia also inhibited the activity of CYP2B6 and CYP3A4, the enzymes responsible for first-pass AN metabolism in the liver. Additionally, AN tissue distribution was improved when delivered as DLA and AN accumulation in tissues was higher in female vs. male rats. Finally, I showed that DLA was a more efficacious anti-inflammatory than pure AN in rats, potentially due to enhanced AN bioavailability. Taken together, these results shed light on the mechanisms behind enhanced oral bioavailability afforded by DLA and demonstrate the potential for DLA to be used as a therapeutic for malaria and other diseases.

We have examined phylogenetic relationships of suckers of tribe Moxostomatini (Cypriniformes, Catostomidae) using cytochrome-b and Growth Hormone gene intron sequences. Phylogenies were significantly different from recent estimates of relationships based primarily on morphology (Smith, 1992) and cytochrome-b sequences (Harris et al., 2002). Overall, there was little support for many basal nodes in the phylogeny, however it was clear that Scartomyzon and Moxostoma were not monophyletic, despite morphological evidence provided Robins and Raney (1956, 1957), Jenkins (1970), and Smith (1992). Growth Hormone sequences provided good support for a monophyletic Western Scartomyzon lineage and thus suggested a single ancestral invasion of Scartomyzon-like fishes into drainages of Texas and Mexico. Phylogenetic relationships of Western Scartomyzon are structured geographically and do not conform well to current taxonomy.

While the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein-glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury.

The greenside darter, Etheostoma blennioides (Teleostomi: Percidae), is a wide-ranging polytypic taxon that occurs throughout eastern North America. A previous morphological study recognized four subspecies (blennioides, newmanii, gutselli, and pholidotum), several morphological races, and three zones of morphological intergradation. We generated complete cytochrome b (1140 bp) sequence data for 51 individuals from across the range of the greenside darter inclusive of all of the currently recognized taxa to assess genetic variation and taxonomic boundaries. Both maximum parsimony and mixed model Bayesian analyses resulted in two strongly supported deeply divergent clades including (1) a Tennessee River drainage clade, and (2) an Ohio River and Great Lakes basins, Interior Highlands, and Atlantic slope clade. Etheostoma blennius, a closely related congener, nested within the Tennessee River clade of E. blennioides, rendering the complex paraphyletic. Test of alternative topologies failed to support the current taxonomic designations. The inclusion of nuclear sequence data from intron 1 of the S7 ribosomal protein (523 bp) from a subset of the populations was included to independently test whether the currently recognized taxa conform to distinct evolutionary lineages and also to clarify potential issues associated with ancestral hybridization. Although the nuclear data was less variable than the mitochondrial data, the monophyly of several of the subspecies could not be rejected.

The reduction of ferricytochrome c ( cyt c^III), as well as some iron ligand complexes and chemically modified derivatives of cyt c^III, by two molybdenum(V)-cysteine complexes has been investigated as a model for electron transfer in molybdenum enzymes. The reduction of cyt c^III by di-μ-oxo-bis[oxo(L-cysteinato)molybdate(V)] (I) is first order in cyt c^III and zero order in I over a wide range in concentration of I at neutral pH. The reduction of cyt c^III by μ-oxo-bisfoxodihydroxo(L-cysteinato) molybdate(V)] (II) is first order in both reactants and has a rate several orders of magnitude greater than the rate of reduction by I. Activation parameters have been determined for both reactions. The reduction of two pyridoxal phosphate derivatives of cyt c^III by I proceeds at the same rate as the reduction of native cyt c^III, while the mononitromonotyrosyl derivative is reduced somewhat faster. The reduction of the N-formyltryptophyl derivative of cyt c^III by I is biphasic, with the fast phase proceeding with a rate similar to the native cyt c^III and the slow phase being somewhat slower. The reduction by I of all derivatives studied is first order in cyt c^III and zero order in I, suggesting the mechanism does not change with these chemically modified forms of cyt c^III. The pyridoxal phosphate and mononitromonotyrosyl derivatives of cyt c^III are reduced by II with rates similar to the native cyt c^III and by the same mechanism. The reduction of the N-formyltryptophyl derivative by II has complicated kinetics which suggest the presence of at least two formyl-cyt c^III species. The reduction of the cyanide, azide and imidazole complexes of cyt c^III by II occurs at a greatly reduced rate and by a different mechanism than the reduction of native cyt c^III The rate of reduction of the cyanide and azide complexes is dependent on the rate of dissociation of the ligand cyt c^III complex and independent of the concentration of II. The reduction of the imidazole complex by II is much more complicated, with the rate independent of II at very low concentrations but shows a dependence on the concentration of II at higher concentrations. Rate constants have been determined for the reactions and possible mechanisms have been developed. The results have been discussed with regard to electron transfer in molybdenum enzymes and electron transfer to cyt c^III This study is especially valuable as a model for the hepatic sulfite oxidase enzyme.