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Single-molecule chemistry studies with engineered alpha-hemolysin poresHammerstein, Anne Friederike January 2011 (has links)
Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes. <b>Chapter 1</b> provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level. <b>Chapter 2</b> describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution. <b>Chapter 3</b> investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn<sup>2+</sup> ion. The single channel recordings reveal a two step process, in which the Zn<sup>2+</sup> ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed. <b>Chapter 4</b> demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores. <b>Chapter 5</b> explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]<sup>2+</sup> binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.
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Kinetic and mechanistic studies of oxygen sensing Fe(II)/2-oxoglutarate dependent oxygenasesTarhonskaya, Hanna January 2014 (has links)
The Fe(II)/2-oxoglutarate (2OG) dependent oxygenases are a widespread enzyme family, which are characterised by structurally similar active sites and proposed to employ a common reaction mechanism. The work described in this thesis concerned kinetic and biophysical studies on 2OG oxygenases, with a particular focus on the hypoxia-inducible transcription factor (HIF) hydroxylases and mechanistic aspects of their reaction with oxygen. The four human HIF hydroxylases regulate cellular levels and transcriptional activity of HIF by catalysing its post-translational hydroxylation in response to changes in oxygen availability. The three prolyl hydroxylase domain enzymes (PHDs1-3) and factor inhibiting HIF (FIH) are proposed to act as cellular oxygen sensors and provide a direct link between oxygen availability and the hypoxic response. Previous transient kinetic studies have shown that PHD2 (the most important human PHD isoform) reacts slowly with oxygen, a factor proposed to be related to its oxygen-sensing role. The molecular mechanisms for the slow PHD2 reaction with oxygen were investigated using a range of kinetic and biophysical techniques to probe the effects of key active site substitutions. The studies reveal that a conservative substitution to an Fe(II)/H<sub>2</sub>O binding residue results in 5-fold faster reaction with oxygen, suggesting a role for H<sub>2</sub>O release from the active site in limiting the ability of oxygen to react with PHD2. This thesis also describes the first transient kinetic studies of FIH. The obtained results show that the rate of the FIH reaction with oxygen was significantly faster than for PHD2. Further, FIH catalyses hydroxylation not only of HIF-α, but also of proteins containing ankyrin repeat domains (ARD). The rate of the FIH reaction with oxygen was shown to be substrate dependent; faster oxygen activation of the reaction in the presence of ARD compared with HIF substrates was observed. Mechanistic studies were performed to investigate a report that PHD2 is involved in the enzymatic oxidation of an oncometabolite (R)-2-hydroxyglutarate (2HG) to give 2OG, in what would be an unprecedented reaction for a 2OG oxygenase. This work found that 2HG does not substitute for 2OG in PHD2 catalysis. Instead, the non-enzymatic transformation of 2HG to 2OG was observed, which could potentially contribute to the reported 2HG-dependent PHD activation in vivo. The biophysical and transient kinetic techniques used for studying the HIF hydroxylases were also applied to study the mechanism of deacetoxycephalosporin C synthase (DAOCS, the enzyme catalysing penicillin N ring expansion). Previously, it has been suggested that the DAOCS mechanism differs from the consensus 2OG oxygenase mechanism. The results described in this thesis provide strong evidence that DAOCS employs the consensus ordered mechanism characteristic of 2OG oxygenases, supporting the proposal that the consensus mechanism is a common feature of the 2OG oxygenase family. Overall, the work described in this thesis is supportive of the proposal that most, if not all, 2OG oxygenases employ a common mechanism. However, the differences in the kinetics of their reaction with oxygen, presented throughout the thesis, suggest that different 2OG oxygenases have different rate-limiting steps. Thus, the kinetics of specific oxygenases may be adapted to their biological function, in particular that of PHD2 as the key cellular O<sub>2</sub> sensor.
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Environmental Change in South Central Chibuto Southern Mozambique 1965-2000Ombe, Zacarias Alexandre 01 March 2007 (has links)
Student Number : 9707731W -
PhD thesis -
School of Geography, Archaeology and Environmental Studies -
Faculty of Science / Southern Mozambique has experienced changes in land-use and land-cover over the
last 45 years. South Central Chibuto is a dynamic environmental change ‘hotspot’ of
this region, and because of the socio-economic development that is taking place it is
unveiling a number of land-use changes in Mozambique. In this research,
environmental changes in the region are shown to be a function of various socioeconomic
and biophysical drivers of change. A variety of research methods including
interpretation of aerial photographs and satellite images, analysis of archival material,
Participatory Rural Appraisal (PRA) and structured interviews, have been used to
derive some of the drivers of environmental change. These drivers of change include
socio-economic factors, the colonial dual economy and the increase in cash crop
production that led to negative impacts on some lands in the steep slopes of the
ancient dunes, the post-independence policy of nucleation of settlements that has
induced extensive conversions of land use together with the war and the recent shifts
into a market economy that have led to further intensification of land use. The
biophysical drivers include, among others but not limited to, fluctuations in rainfall,
with periods of alternating dry and wet spells shaping land-use change, as well as
single shock events, such as extreme droughts and floods. New factors, including the
HIV/AIDS pandemic and globalization have been identified as having an impact on
the livelihoods in the study area. A valuable, local-scale case or place-based case
study approach is shown to enable a detailed and rich analysis of the complex
trajectory of environmental change. Finally, lessons drawn from this study are shown
to have the potential to inform policies for sustainable livelihoods in Mozambique.
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Modélisation de la croissance de tumeurs cérébrales : application à la radiothérapie / Brain tumor growth modeling : application to radiotherapyLê, Matthieu 23 June 2016 (has links)
Les glioblastomes comptent parmi les cas les plus répandus et agressifs de tumeurs cérébrales. Ils sont généralement traités avec une combinaison de résection chirurgicale, suivie de chimiothérapie et radiothérapie. Cependant, le caractère infiltrant de la tumeur rend son traitement particulièrement délicat. La personnalisation de modèles biophysiques permet d’automatiser la mise au point de thérapies spécifiques au patient, en maximisant les chances de survie. Dans cette thèse nous nous sommes attachés à élaborer des outils permettant de personnaliser la radiothérapie des glioblastomes. Nous avons tout d’abord étudié l’impact de la prise en compte de l’œdème vasogénique. Notre étude rétrospective se fonde sur une base de donnée de patients traités avec un médicament anti-angiogénique, révélant a posteriori la présence de l’œdème. Ensuite, nous avons étudié le lien entre l’incertitude due à la segmentation de la tumeur et la distribution de la dose. Pour se faire, nous avons mis au point une méthode permettant d’échantillonner efficacement de multiples segmentations réalistes, à partir d’une unique segmentation clinique. De plus, nous avons personnalisé un modèle de croissance tumorale aux images IRM de sept patients. La méthode Bayésienne adoptée permet notamment d’estimer l’incertitude sur les paramètres personnalisés. Finalement, nous avons montré comment cette personnalisation permet de définir automatiquement la dose à prescrire au patient, en combinant le modèle de croissance tumoral avec un modèle de réponse à la dose délivrée. Les résultats prometteurs présentés ouvrent de nouvelles perspectives pour la personnalisation de la radiothérapie des tumeurs cérébrales. / Glioblastomas are among the most common and aggressive primary brain tumors. It is usually treated with a combination of surgical resection, followed with concurrent chemo- and radiotherapy. However, the infiltrative nature of the tumor makes its control particularly challenging. Biophysical model personalization allows one to automatically define patient specific therapy plans which maximize survival rates. In this thesis, we focused on the elaboration of tools to personalize radiotherapy planning. First, we studied the impact of taking into account the vasogenic edema into the planning. We studied a database of patients treated with anti-angiogenic drug, revealing a posteriori the presence of the edema. Second, we studied the relationship between the uncertainty in the tumor segmentation and dose distribution. For that, we present an approach in order to efficiently sample multiple plausible segmentations from a single expert one. Third, we personalized a tumor growth model to seven patients’ MR images. We used a Bayesian approach in order to estimate the uncertainty in the personalized parameters of the model. Finally, we showed how combining a personalized model of tumor growth with a dose response model could be used to automatically define patient specific dose distribution. The promising results of our approaches offer new perspectives for personalized therapy planning.
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Caracterização e mapeamento biofísico ambiental da bacia hidrográfica do rio vermelho a partir de dados remotamente sensoriados / Characterization and mapping biophsical environment of the rio Vermelho watershed from remotely sensed dataSantos, Pablo Santana 15 May 2014 (has links)
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Previous issue date: 2014-05-15 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The agricultural expansion in the Brazilian Cerrado represents nowadays more than half of the total biome area, where conversion of the natural vegetation to pasturelands is the main dynamics imposed by the productive sector along the relatively recent historical occupation process. Such transformations tend to create distinct spatial patterns regarding the biophysical behavior of the main existent vegetation formations, which can be identified and discriminated through a variety of available remote sensing products. This study, focused on the rio Vermelho watershed, a representative unit of the Cerrado biome, and considering a variety of satellite data for the 2001 – 2011 period, pursued a detailed analysis of the biophysical responses in face of the historical processes that determined and shaped the landscape transformations. Specifically, the following datasets were utilized: MODIS (vegetation index, evapotranspiration and primary productivity) and TRMM (Tropical Rainfall Measurement Mission) precipitation. Our results indicated that climatic factors, such as seasonality, have a direct influence on biomass levels of key vegetation formations, particularly those with shallow root systems, for which reductions in radiometric values, as high as 50%, could be observed during the dry season. In fact, natural vegetation areas, comparatively to areas dominated by cultivated pastures, show very distinct and significant biophysical behavior (p < 0.05). Likewise, two major landscapes could be identified, i.e. “Region 1” and “Region 2”, where converted and remnant vegetation areas predominate, respectively. The distribution of natural and cultivated pastures seems to be related to local edafoclimatic factors, such as soil (depth/lithic content) and water availability, which explain the occurrence of most grass species. Based on field biomass, it is possible to say: 1) cultivated pastures found in “Region 1” are slightly more productive comparatily to those from “Region 2” (for both dry- and wet-seasons) and 2) there is a correspondence between “natural” and “cultivated” green biomass during the dry season, as opposed to the wet season. However, and based on the seasonal moderate spatial resolution MODIS images, it is not possible to separate between natural and cultivated pastures. In fact, such separation can be accomplished via the supervised classification (Support Vector Machine) of medium spatial resolution imagery (Landsat). Based on high spatial resolution (5m) RapidEye/BlackBridge images, it was produced a land cover / land use map for the entire basin at the 1:50.000 scale, in which it was possible to discriminate the major converted and natural physiognomies, and distinct grass species, in particular (Overall Accuracy ~ 90% and Kappa = 0.83). / A expansão da atividade agropecuária no Cerrado brasileiro representa hoje mais da metade da área total deste bioma, onde a conversão das áreas naturais em pastagens cultivadas foi a principal dinâmica imposta pelo setor produtivo ao longo de um processo histórico de ocupação relativamente recente. Tais transformações tendem a resultar em padrões espaciais distintos quanto ao comportamento biofísico das principais formações vegetais existentes, os quais podem ser identificados e discriminados através dos vários produtos de sensoriamento remoto disponíveis. O presente trabalho, ao utilizar a bacia hidrográfica do rio Vermelho como unidade experimental do bioma Cerrado, teve como objetivo principal realizar uma análise detalhada das respostas biofísicas frente aos processos históricos que condicionaram as transformações na paisagem, considerando o período entre 2001 a 2011, e uma ampla base de dados satelitária, obtida a partir dos produtos MODIS (índice de vegetação, evapotranspiração, produtividade primária) e TRMM (Tropical Rainfall Measurement Mission/precipitação). Os nossos resultados evidenciam que fatores climáticos, a exemplo da sazonalidade, tem uma influência direta na biomassa das principais formações vegetais, principalmente àquelas que apresentam sistemas radiculares superficiais, com reduções nos valores radiométricos de até 50% durante o período da seca. De forma significativa, as áreas de vegetação natural, comparadas às áreas de pastagens cultivadas, apresentaram comportamentos distintos do ponto de vista biofísico (p < 0,05). Em fato, dois macroambientes puderam ser definidos para a área de estudo, onde predominam a ocupação antrópica (“Região 1”) e a vegetação natural remanescente (Região 2), respectivamente. A distribuição das pastagens cultivadas e naturais está associada a fatores edafoclimáticos locais, como características de solo (profundidade/pedregosidade) e disponibilidade hídrica, os quais explicam a ocorrência da maioria das espécies forrageiras. Com base nos dados de biomassa coletados em campo, é possível afirmar: 1) as pastagens cultivadas encontradas na “Região 1” são ligeiramente mais produtivas do que as encontradas na “Região 2”, tanto no período seco quanto chuvoso, e 2) há uma correspondência entre a biomassa verde encontrada no período seco para as duas categorias de pastagens, ao contrário do observado no período chuvoso. Entretanto, e tendo por base as imagens biofísicas sazonais de resolução espacial moderada (MODIS), não é possível separar entre pastagens nativas e cultivadas. Por sua vez, esta separação se mostra possível através da classificação supervisionada (Support Vector Machine) de imagens de resolução espacial média (Landsat). Com base em imagens RapidEye/BlackBridge, com resolução espacial de 5m, foi produzido um mapa de cobertura e uso da terra à escala de 1:50.000, no qual foi possível separar as áreas de pastagens quanto à sua tipologia, bem como individualizálas em outras formas de uso e fito-fisionomias remanescentes (Acurácia Total ~ 90% e Kappa = 0,83).
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Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni / Biochemical, biophysical and inhibitory studies of dihydroorotate dehydrogenase from Schistosoma mansoniJuliana Serafim David Costacurta 26 September 2014 (has links)
Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.
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Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni / Biochemical, biophysical and inhibitory studies of dihydroorotate dehydrogenase from Schistosoma mansoniCostacurta, Juliana Serafim David 26 September 2014 (has links)
Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.
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Protokol pro sběr biofyzikálních parametrů vegetace v terénu / Field protocol for in-situ biophysical parameters collectionŠudová, Markéta January 2019 (has links)
Field protocol for in-situ biophysical parameters collection Abstract The main aim of diploma thesis is to design a unified protocol intended for collecting of in-situ biophysical parameters of vegetation based on an up-to-now published literature as well as on my own experimental measuring. The key for measuring of high- quality field data, that are suitable for a subsequent validation of remote sensing products, is an implementation of unified rules as well as all the phases of data collecting, a recommended preparation before the very measuring, the choice of a sampling scheme and a processing of measured data. Based on an accessible literature the current methods of LAI data measuring were evaluated and the basic protocol parameters were defined. For designing of a proper parameterization of this protocol an experimental field measurement was carried out. A determination of a suitable number of partial measurements within ESU is given by a relationship between the number of partial measurements and MSE value. The sampling schemes stated in a literature as well as my own designed sampling schemes are subsequently tested in order to determine a suitable sampling scheme. Based on achieved MSE values the most suitable sampling scheme that was later verified in field measuring was evaluated. Based on a field...
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Application of Biophysical Data to an Unsupervised Classification to Map Ecoregional Boundaries in the Desert SouthwestMcClurg, Paxton 01 May 2002 (has links)
An unsupervised classification was applied to continuous biophysical variables in an attempt to delineate ecoregional boundaries in the desert southwest. Output was then compared with ecoregions delineated by the Natural Resources Conservation Service (NRCS), the Environmental Protection Agency (EPA), and the Forest Service at the national level. An attempt was made to use the same biophysical variables for input into the unsupervised classification as was emphasized by the various agencies with their ecoregional classifications at the desert level. Major constraints included data availability at such a large study area, data resolution, and data that were continuous. This eliminated categorical data such as vegetation type, geology type, or soil texture. The aim of the study was to develop a more objective and repeatable approach to identifying self-similar geographic regions.
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Molecular aspects of biomolecule structure and functionRodger, Alison January 2002 (has links)
All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.
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