Orientadores: Marco Antonio Garcia de Carvalho, Marco Aurelio Takita / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia / Made available in DSpace on 2018-08-26T15:21:12Z (GMT). No. of bitstreams: 1 Palma_EloaCarolinaNavaCardoso_M.pdf: 2004126 bytes, checksum: 618ad5da7f91e98f63bfdc0a41812387 (MD5) Previous issue date: 2014 / Resumo: O surgimento dos sequenciadores automáticos de DNA por volta dos anos 1990 fez com que a quantidade de sequências e informações a serem analisadas aumentasse exponencialmente. Sendo assim, os recursos computacionais são cada vez mais exigidos em diversas tarefas de armazenamento desses dados. Para atender essas e outras necessidades, surgiu a Bioinformática, que engloba diversas áreas como Ciência da Computação, Matemática, Estatística, e a Biologia Molecular. Neste trabalho, desenvolvemos um software denominado Biodonut, que consiste em manipular plasmídeos utilizando visualização 2D e 3D. Biodonut é um produto de software de plataforma web, open source para que os pesquisadores possam manipular plasmídeos in silico, visualizando em formato 2D e 3D e possibilitando uma análise diferenciada das áreas julgadas importantes. A base de dados utilizada no software é o GenBank, por ser mundialmente conhecida e respeitada. Uma comparação com dois importantes softwares da área foi efetuada e é apresentada neste trabalho. Os resultados apontam que usar Biodonut é vantajoso em relação os outros dois softwares / Abstract: The advent of automated DNA sequencers around 1990s led to the amount of information sequences to be analyzed and increased exponentially. Thus, computing resources are increasingly demanded in various tasks such data storage. To meet these and other needs arose Bioinformatics, encompassing diverse areas as Computer Science, Mathematics, Statistics, and Molecular Biology. In this work, we developed a software called Biodonut, which involves manipulating plasmids using 2D and 3D visualization. Biodonut is a product of web software platform, open source so that researchers can manipulate plasmids in silico, visualizing 2D and 3D format, enabling a differentiated analysis of the areas deemed important. The database software is used in GenBank, to be globally known and respected. A comparison with two important software of the area was conducted and is presented in this paper. The results show that using Biodonut is advantageous over the other two softwares / Mestrado / Tecnologia e Inovação / Mestra em Tecnologia

Au cours de son cycle de vie, le principe actif se retrouve en solution dans différentes situations : dans desformes pharmaceutiques liquides, dans l’organisme et dans les eaux usées. Or, par rapport à l’état solide, lamise en solution du principe actif l’expose davantage à des facteurs susceptibles de conduire à sa dégradation.Les transformations modifient sa structure chimique et donc potentiellement ses activités pharmacologiques ettoxicologiques.L’objectif de ce travail de thèse est de présenter une méthodologie et des études visant à prédire le devenir ensolution de principes actifs et les impacts potentiels consécutifs à leur dégradation.Trois principes actifs ont été sélectionnés pour la réalisation de ce travail. Ils ont en commun de présenter,d’une part, une activité pharmacologique élevée corrélée à une toxicité potentielle de leurs produits dedégradation et, d’autre part, l'absence de données sur leurs comportements en solution. Dans tous les cas,bien que le contexte soit singulier pour chaque molécule, l’approche méthodologique suivie intègre aussi biendes travaux expérimentaux que des études ab initio et in silico.La première étude porte sur le devenir de l’apixaban, principe actif actuellement commercialisé sous formeorale solide, en solution aqueuse. Les données expérimentales ont mis en évidence des groupementschimiques du principe actif pouvant contribuer à son instabilité. L’approche ab initio a permis d’expliquer larégio-spécificité de la réaction d’hydrolyse dépendamment du pH. À partir de la structure des produits dedégradation caractérisés, l’étude de leur potentiel toxique a été réalisée par approche in silico. Ces donnéesconcourent à la démarche d'analyse et évaluation des risques déployée lors de développements de formespharmaceutiques liquides ou des situations particulières impliquant la mise en solution de l'apixaban aumoment de l'administration.De telles approches ont également été employées pour caractériser les mécanismes de photodégradation del’argatroban et évaluer le potentiel toxique des produits de dégradation. Les processus initiant laphotodégradation ont fait l’objet d’études complémentaires reposant sur des calculs d’énergies. Cesconnaissances pourront apporter le rationnel nécessaire au choix de procédés capables de réduire laphotodégradation de l’argatroban et son impact sur les patients. Elles pourront également servir à anticiper lessituations d’écarts pouvant mettre en jeu le rapport bénéfice risque du médicament telles que le mésusage oula modification de la forme pharmaceutique administrée.Enfin, dans un contexte autre que le contexte pharmaceutique, une étude de dégradation du pémétrexed parphotocatalyse via un procédé d'oxydation avancée a été réalisée. Il s'agit d'un procédé particulièrement étudiépour sa capacité à réduire l’empreinte environnementale de composés organiques en accélérant leurdégradation. Le choix de ce principe actif utilisé comme anticancéreux a été justifié par son caractère toxiqueet rémanent dans les eaux de surface, ce qui en fait un produit à haut risque environnemental. Ce travail amontré que des produits de plus faible masse résultant de la transformation photocatalytique du pémétrexedsont malheureusement plus toxiques et encore plus rémanents que la molécule mère elle-même. Ces résultatscontribuent donc à souligner que les procédés d'oxydation avancée, bien qu'efficaces pour l'élimination despolluants médicamenteux, sont à évaluer au regard de l'existence d'un risque accru pour l'environnementavant toute perspective d'utilisation à grande échelle.Les approches et les résultats présentés dans cette thèse pourront être employés pour d’autres études visant àprédire, prévenir et réduire l’impact de la dégradation du principe actif sur le patient et l’environnement. / During its life cycle, an active substance is in solution for various reasons: in a liquid pharmaceutical form, in the body and in wastewater. However, compared to the solid state, the active substance in solution exposes it more to factors likely to cause its degradation. The transformations modify its chemical structure and thus potentially its pharmacological and toxicological activities.The objective of this thesis is to present a methodology and studies aiming to predict the fate in solution of active substances and the potential impacts following their degradation.Three active ingredients have been selected for this work. They have in common, on the one hand, a high pharmacological activity correlated to a potential toxicity of their degradation products and, on the other hand, the fact that there is little information on their behaviour in solution. In all cases, although the context is specific to each molecule, the methodological approach followed integrates both experimental work and ab initio and in silico studies.The first study concerns the fate of apixaban, an active substance currently marketed in solid oral form, in aqueous solutions. The experimental data made it possible to highlight chemical groups of the active ingredient that could contribute to its own instability. The ab initio approach explained the regio-specificity of the hydrolysis reaction as a function of pH. Based on the structure of the characterized degradation products, their toxic potential was studied using an in silico approach. These data contribute to the risk analysis and evaluation process deployed at different stages of development of liquid pharmaceutical forms or in particular situations involving the solution of apixaban at the time of administration.Such approaches have also been used to characterize the photodegradation mechanisms of argatroban and assess the toxic potential of degradation products. The processes that initiate photodegradation were also addressed by calculating the energies potentially involved. This knowledge provides a rational basis for the choice of processes and formulations to limit photodegradation of argatroban and its impact on patients. They also make it possible to anticipate situations where the benefit/risk ratio of the medicinal product may be modified, such as incorrect handling or modification of the pharmaceutical form administered.Finally, in a context other than the pharmaceutical context, a study of degradation of pemetrexed by photocatalysis via an advanced oxidation process was carried out. This process is particularly studied for its ability to reduce the environmental footprint of organic compounds by accelerating their degradation. The choice of this active substance as an anti-cancer agent was justified by its toxic and persistent nature in surface waters, making it a product with a high environmental risk. This work has shown that products of lower mass produced by photocatalytic transformation of pemetrexed are unfortunately more toxic and even more persistent than the parent molecule itself. These results underline the fact that advanced oxidation processes, although effective in removing drug pollutants, must be evaluated because of an increased risk to the environment before any prospect of large-scale use.The approaches and results presented in this thesis can be used for other studies to predict, prevent and reduce the impact of active ingredient degradation on the patient and the environment.

Modelling of interactions of proteins and peptides with metal ions Ondrej Gutten - Diploma thesis Keywords: Metalloproteins, metal ion selectivity, in silico prediction Abstract: An approach for in silico prediction and estimation of selectivity properties of metal-binding peptides is suggested. An in-depth analysis is performed to disclose the justifiability and limitations of this approach. The study is divided into three parts. First part investigates the soundness of two quantum chemical methods (MP2 and DFT) for their use in the set-up quest. The testing includes comparison with CCSD(T), effect of basis selection, performance of the two methods in geometry optimizations and effect of implicit solvent model. Second part foreshadows the approach of searching for a metal selective peptide by thoroughly investigating the ability of simple representative systems, derived from their metalloprotein templates, to retain the property of interest. Final part describes the initial step of extensive combinatorial approach towards examination of vast number of simple systems that represent metal-binding sites, and which are to be used for prediction of metal-selectivity through exploitation of the described approach and, ultimately, to the de novo design of metalloproteins with desired properties.

Liver flukes of the family Fasciolidae are parasites of mammals including human. Fascioloides magna and Fasciola hepatica are considered as a veterinary and medically important species occurring also in the Czech Republic. Fascioloides magna and F. hepatica infect wide spectrum of wild and domestic ruminants and in case of F. hepatica human can be also infected. Both flukes are responsible for damage of liver tissue and/or bile-ducts of their definitive hosts causing weight lose, anemia, reduced productivity and in specific cases the death of the host. Effective diagnosis plays the key role in control of F. hepatica and F. magna infections. Current diagnostics is predominantly based on serodiagnostic methods using specific antigens e.g. from excretory-secretory products (ESPs). Due to heterogenity of ESPs, such diagnostic markers can lack the specificity and also the reproducibility of the method is poor. Particular proteins of ESPs are often used in diagnostics of fasciolid flukes. Such approach requires biological material and laboratory procedures associated with identification, purification and antigenicity testing of selected proteins. Recent development of parallel sequencing technologies results in huge amount of genomic, transcriptomic and proteomic data, which are publicly available. Such...

Understanding the role of structural patterns within complex networks is essential to establish the governing principles of such networks. Social networks, biological networks, technological networks etc. can be considered as complex networks where information processing and transport plays a central role. Complexity in these net works can be due to abstraction, scale, functionality and structure. Depending on the abstraction each of these can be categorized further. Gene regulatory networks are one such category of biological networks. Gene regulatory networks (GRNs) are assumed to be robust under internal and external perturbations. Network motifs such as feed-forward loop motif and bifan motif are believed to play a central role functionally in retaining GRN behavior under lossy conditions. While the role of static characteristics like average shortest path, density, degree centrality among other topological features is well documented by the research community, the structural role of motifs and their dynamic characteristics are not xiii well understood. Wireless sensor networks in the last decade were intensively studied using network simulators. Can we use in-silico experiments to understand biological network topologies better? Does the structure of these motifs have any role to play in ensuring robust information transport in such networks? How do their static and dynamic roles differ? To understand these questions, we use in-silico network models to capture the dynamic characteristics of complex network topologies. Developing these models involve network mapping, sink selection strategies and identifying metrics to capture robust system behavior. Further, I studied the dynamic aspect of network characteristics using variation in network information flow under perturbations defined by lossy conditions and channel capacity. We use machine learning techniques to identify significant features that contribute to robust network performance. Our work demonstrates that although the structural role of feed-forward loop motif in signal transduction within GRNs is minimal, these motifs stand out under heavy perturbations.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Os manguezais são considerados um ecossistema único, devido sua particular combinação de condições ambientais, influenciados pela sua localização na interface entre o continente e oceano. O estudo deste ecossistema se torna urgente uma vez que os manguezais estão desaparecendo em todo mundo, e sua diversidade de grupos microbianos é ainda pouco conhecida. Dentro dessa temática, o presente projeto visa descrever a possível funcionalidade de bactérias pertencentes ao filo Planctomycetes nos manguezais. Tais organismos são ainda pouco estudados, de difícil cultivo, sendo obtidos principalmente em ambientes marinhos, tratamento de água e locais de criação de peixes. Dentro desse filo são encontrados microrganismos pertencentes a gêneros descritos como capazes de realizar a oxidação anaeróbica do íon amônio (anammox), uma importante transformação do nitrogênio em ambientes com baixa disponibilidade de oxigênio. E ainda, visamos descrever a possível funcionalidade de bactérias pertencentes ao filo Planctomycetes nos manguezais. Para isso, foi inicialmente realizada uma comparação dos genomas de Planctomycetes com as sequências obtidas por análises de metagenômica e metatranscriptômica, onde foram encontradas sequências similares às afiliadas a funções desconhecidas (putative protein) e a sulfatases. Tais enzimas são descritas como hidrolíticas, que catalizam a clivagem de ésteres de sulfato, liberando o enxofre na forma assimilável. A análise de sequências de uma biblioteca metagenômica (obtida de um manguezal contaminado com petróleo) permitiu a visualização de fragmentos genômicos de Planctomycetes. Esta análise revelou também a ocorrência de genes relacionados a produção de sulfatases, além de indicar arranjos gênicos distintos dos descritos nos genomas de organismos deste grupo, possivelmente indicando a ocorrência de endemismo de Planctomycetes em manguezais. Esta observação foi reforçada pelo cultivo de isolados afiliados a este grupo, os quais tiveram sua afiliação confirmada pelo sequenciamento parcial do gene ribossomal 16S DNAr. Alguns destes isolados formaram clusters diferenciados dentro do filo Planctomycetes, indicando que podem ser estes novas espécies. Sendo assim, a caracterização desse grupo de microrganismos numa combinação de análises in sílico e in vivo, possibilitou a confirmação da ocorrência de tais organismos nos manguezais, e gerou as primeiras informações sobre sua funcionalidade neste sistema, onde parece ocorrer de forma diferenciada aos demais ambientes onde já foram descritos. / Mangroves are considered an unique ecosystem due to its particular combination of environmental conditions, influenced by its location at the interface between land and ocean. The study of this ecosystem becomes urgent since mangroves are disappearing worldwide, and its diversity of microbial groups is still poorly understood. Within this theme, this project aims to describe the possible functionality of bacteria belonging to the phylum Planctomycetes in mangroves. Such organisms are still poorly studied, difficult to cultivate and mainly isolated from marine environments, water treatment and fish farming sites. Within this phylum are found microorganisms belonging to genera described as capable of performing the anaerobic oxidation of ammonium (anammox), a major transformation of nitrogen in environments with low oxygen availability. Here we aimed to describe the possible functionality of bacteria belonging to the phylum Planctomycetes in the mangroves. In order to achieve the target, it was initially performed a comparison of the Planctomycetes genomes with sequences obtained by metagenomics and metatranscriptomics, revealing the presence of sequences with similarity to those affiliated to unknown function and sulfatases. These are hydrolytic enzymes, which catalyze the cleavage of sulfate esters, releasing sulfur on its available form. The analysis of sequences from a metagenomic library (obtained from an oil-contaminated mangrove) allowed the visualization of genomic fragments related to Planctomycetes. It also revealed the presence of genes related with the production of sulfatases, besides the indication of specific genome arrangements, distinct from those describe in organisms allocated in this group, possibly indicating the occurrence of endemicity for Planctomycetes in mangroves. It was reinforced by the cultivation of isolates affiliated to this groups, whose have their affiliation confirmed by the partial sequencing of the 16S rDNA gene. Some isolates clustered composed new clusters within the Planctomycetes phylum, indicating the occurrence of new species. In sum, the characterization of this group by combining in sílico and in vivo analyses, allowed the confirmation of the occurrence of these organisms in mangroves, and generated the first insights about its functioning on this system, where it seems to occur in a differentiated form from those observed in other environments where it has been described.

No description available.

Rhesus macaques are a widely used animal model of human diseases and related immune responses. Fc receptors (FcRs) mediate the interaction between antibody molecules and innate killing mechanisms, consequently eliminating the pathogen. In rhesus macaques, FcRs are highly polymorphic. To evaluate the potential influence of FcgR polymorphisms on the interaction with antibody molecules, we performed in silico analysis using SIFT, Provean, nsSNPAnalyzer, I-Mutant, MuSTAB and iPTREE-STAB web servers. V20G in FcγRI, I137K in FcγRII and I233V in FcγRIII were further analyzed structurally using FOLD-X, AMMP and Chimera to calculate changes in folding and interaction energy and for structure visualization. Results from our analysis suggest that the selected variations destabilize protein structure. Additionally, Q32R increases the binding affinity of FcγRI, whereas A131T decreases the binding affinity of FcγRII towards IgG1. Together, our results indicate that these substitutions might influence effector and regulatory mechanisms resulting from antibody/FcR interactions.

To produce male Muscovy only for fatty liver and meat-type production is an important economic goal in animal husbandry, although the sex-determining mechanism in poultry remains to be elucidated. Manipulation of sex-determining gene(s) in poultry provides enormous opportunities on the development of sex pre-selection reproductive systems. DSX and MAB-3 genes in Drosophila and C. elegans are conserved across the human, mice, chickens, fish, turtles, and reptiles revealing an ancient sex-determining locus DMRT1. Thus the Z-linked, DMRT1 in chicken is an excellent candidate regulatory gene controlling similar aspects of sexual development in poultry. This dissertation is aimed to clone and characterize Muscovy DMRT1 gene for further application in sex pre-selection. Partial cDNA sequences of Muscovy DMRT1 was determined and revealed 95% identity and 83% with chicken and red-eared slider turtle DMRT1 cDNA sequences. DMRT1 orthologs among various species were analyzed by Phylip program and phylogenetic tree was constructed by MEGA2 programs. Results indicated that Muscovy, chicken and red-eared slider turtle DMRT1 revealing 95%, and 83% identity at cDNA and 61%, 54% identity at amino acid level.