Fumarato hidratases ou fumarases (FH) catalisam a reação estereoespecífica reversível de hidratação do fumarato em L-malato. Essas enzimas se apresentam em todas as classes de organismos, desde procariotos a eucariotos, e podem ser encontradas nas formas mitocondrial e citosólica. A enzima tem papel importante na produção de energia pois participa do ciclo do ácido cítrico, na resposta ao dano do DNA e como supressor tumoral. A fumarase humana (HsFH), que pertence à classe II, é codificada pelo gene 1q42.1, possui 467 aminoácidos em cada monômero com peso molecular de 50,2 kDa cada. Estudos associaram mutações no gene da FH com diversas doenças humanas como acidúria fumárica, leiomiomatoses de útero e pele (MCUL), que quando associadas com um agressivo carcinoma múltiplo de células é conhecido como leiomiomatose hereditária e câncer renal (HLRCC). Apesar da grande importância da fumarase humana no metabolismo energético, ainda há pouca informação em relação ao mecanismo catalítico adotado pela enzima e o efeito estrutural e cinético causado pelas mutações envolvidas com essas doenças. Diante disso, nosso trabalho utilizou uma abordagem híbrida que envolve a caracterização biofísica, bioquímica e estrutural da enzima HsFH, e seus mutantes: N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH e N373DHsFH, identificados em pacientes. Estudos cinéticos foram realizados em sete diferentes pHs e, pela primeira vez para fumarases, o ensaio foi realizado com os dois substratos presentes na mesma mistura reacional, confirmando a contribuição da reação reversa para a velocidade global da enzima. De acordo com os estudos de termoflúor a proteína é estabilizada em pHs alcalinos e através da ligação de compostos no sítio ativo. A estrutura da enzima HsFH nativa foi resolvida a 1,8 Å e identificou a presença de moléculas de HEPES complexadas na região C-terminal da enzima. Os estudos cinéticos demonstraram um aumento da eficiência catalítica na presença do HEPES, sugerindo um possível papel alostérico de seu sítio de ligação para a atividade catalítica. Foram determinadas as estruturas para os mutantes N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH e N373DHsFH. As mutações Q185R, E362Q, S365G e N373D foram identificadas no sítio ativo afetando diretamente a capacidade da proteína em ligar os substratos, enquanto que a mutação H180R foi localizada no sítio B, que conduz os substratos e produtos para dentro e fora do sítio ativo. Já a mutação K230R está localizada no domínio central, mas os resultados de termoflúor demonstram um efeito direto na capacidade da enzima em acomodar o substrato. A mutação N107T, localizada longe do sítio ativo foi a única que permaneceu ativa e teve seus parâmetros cinéticos residuais determinados. O presente trabalho contribui para o entendimento das bases estruturais que correlacionam mutações na HsFH, deficiência enzimática e patologia. / Fumarate hydratases or fumarases (FH) catalyze the reversible stereospecific hydration of fumarate to L-malate. They are present in all classes of organisms, from prokaryotes to eukaryotes, and can be found in the mitochondrial and cytosolic forms. The enzyme has an important role in energy production as part of the well-known Citric Acid Cycle, in DNA damage response and as tumor suppressor. Human fumarase (HsFH) belongs to class II and is encoded by 1q42.1 gene. HsFH is tetrameric and has 467 amino acids per monomer, with predicted molecular weight of 50.2 kDa. Several studies associated FH gene mutations with some human diseases such as fumaric aciduria, multiple cutaneous and uterine leiomyomatosis (MCUL), which when associated with an aggressive form of multiple cell carcinoma is known as hereditary leiomyomatosis and renal cancer (HLRCC) syndrome. Although the major role of HsFH in energetic metabolism, there are still little structural and kinetic information about the mutants involved in these diseases. Thus, this study aims, through a hybrid approach, composed by biophysics, biochemical and structural characterization of mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH and N373DHsFH identified from patients. Steady-state kinetics studies were performed in seven different pHs and, for the first time, the contribution of both substrates was analyzed simultaneously in a single kinetic assay and allowed to quantify the contribution of the reverse reaction for kinetics. According to thermofluor studies, structural stability can be achieved at alkaline pHs and suggests that ligand binding can modulate the protein stability. HsFH crystal structure was solved at 1.8 Å resolution and identified HEPES molecules complexed with the enzyme C-terminal region. Kinetics studies with HEPES showed an increase of the catalytic efficiency and suggests that HEPES binding site might have an allosteric role. Crystal structures for the mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH and N373DHsFH were determined. The mutations Q185R, E362Q, S365G and N373D were identified in the active site and affect the substrate binding capacity directly, while mutation H180R was localized in the B site, which conducts the substrates and products in and out the active site. The mutation K230R is localized in the central domain, but thermofluor results demonstrate a direct effect on the ability of the enzyme to accommodate the substrate. The N107T mutation located far from the active site was the only one that remained active and had its residual kinetic parameters determined. The present work contributes to the understanding of the structural bases that correlate mutations in HsFH, enzymatic deficiency and pathology.

O presente trabalho de Mestrado teve como objetivo produzir géis carregados de emulsão contendo nutrientes incorporados, visando a complementação da alimentação de leitões neonatos. Para alcançar tal objetivo, foram produzidas emulsões utilizando-se diferentes lipídios e tensoativos para definição preliminar sobre quais destes ingredientes seriam mais eficientes na formação de emulsões estáveis. As emulsões produzidas foram avaliadas em relação à distribuição de tamanho de gota e aspecto visual dentro de 24 h após a produção. A partir de tais resultados, algumas amostras foram escolhidas e foi estudado o aumento da concentração de lipídios, bem como a razão óleo:tensoativo e intensidade de agitação. Para cada emulsão estudada, esta era incorporada ao gel polissacarídico, e então realizava-se um estudo da estabilidade térmica a 40 e 50 °C. Após a definição da melhor concentração de lipídios, ocorreu o estudo da incorporação de nutrientes em diversas concentrações na emulsão. Novamente, as emulsões foram avaliadas por meio da distribuição de tamanho de gota, bem como pelo aspecto visual. Foi avaliada a estabilidade da emulsão ao longo de 28 dias, que indicou um sistema estável para o período indicado. Os géis carregados foram analisados quanto à quantidade de energia e à atividade de água, apresentando um baixo valor energético e atividade de água alta. Foram também caracterizados por análises reológicas, que indicaram que a presença da emulsão fortaleceu a estrutura do gel, indicando adesão das gotas à matriz biopolimérica. O gel carregado de emulsão apresentou estabilidade microbiológica, não tendo havido crescimento de fungos filamentosos e não-filamentosos, bem como a manutenção do pH. No entanto, houve alteração da cor ao longo da estocagem. Assim, foi possível produzir um gel carregado de emulsão contendo nutrientes, possibilitando a formação de um sistema estável. / The aim this work was to produce emulsion filled gels with the incorporation of nutrients for the complementation of nutrition of neonates piglets. For this, the emulsions were produced from a variety of lipids and surfactants to define, preliminarily, which of these ingredients would be efficient to form stable emulsions. The emulsions produced were evaluated by droplet size distribution and visual appearance within 24 hours after production. From these results, some samples were selected and the increase of lipid concentration was studied as well as the ratio oil:surfactant and the stirring intensity. For each emulsion studied, this was incorporated into the polysaccharide gel and then the thermal stability study at 40 and 50 °C was made. After setting the optimal concentration of lipids and a stirring intensity, there was the study of nutrientse incorporation at different concentrations in the emulsion. Again, the emulsions were evaluated by droplet size distribution, as well as by visual appearance. The emulsion stability was evaluated over 28 days, which indicated a stable system for this time. The emulsion filled gels were characterized for the amount of energy and water activity and presented as results low amount of energy and high water activity. In the rheological characterization, the results showed that the presence of emulsion strengthened the gel structure, indicating adhesion of the droplets to the biopolymer matrix. The emulsion filled gel showed microbiological stability has not been growing of filamentous and non-filamentous fungi, as well as pH stability. However, there was a change in color over the storage. Thus, it was possible to produce an emulsion-filled gel with nutrients, on which occurred the formation of a stable system.

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of the blastocyst that can give rise to cells of the ectoderm, endoderm and mesoderm lineages. Once isolated from the blastocyst, ESCs can be cultured indefinitely in vitro in an undifferentiated state or can be induced to differentiate. In the case of mouse ESCs (mESCs), the cytokine leukemia inhibitory factor (LIF) is added to culture media to maintain pluripotency and is removed to induce differentiation. Although it is known that extracellular matrix (ECM) components influence stem cell maintenance, proliferation and differentiation, the precise effects of ECM environments on embryonic stem cell behavior have not been systematically studied. The main purpose of this thesis project was to investigate the behavior of undifferentiated mESCs cultured in different 3D hydrogel matrices and to determine whether viscoelastic and biochemical variations in the matrices differentially affect the ability of stem cells to self-renew; that is, retain their pluripotency or undifferentiated phenotype. Their behavior in 3D environments was compared to mESC behavior in traditional 2D culture. In addition, a new method of casting hydrogels in polydimethylsiloxane (PDMS) molds was developed in order to efficiently cast multiple hydrogels of varying sizes and shapes. The findings of this thesis project will benefit both the scientific and engineering community as it encourages researchers to re-evaluate the quality of standard 2D embryonic stem cell culture methods versus potentially novel and advantageous 3D hydrogel culture methods.

No description available.

Systemic sclerosis (SSc) is a rare connective tissue disease affecting skin and internal organs. The pathogenesis of SSc is multifactorial and includes autoimmunity, inflammation and vasculopathy. Pulmonary arterial hypertension (PAH) is among the most serious of SSc complications and is characterized by augmented vasoconstriction, neointimal remodeling and occlusion of small arteries in lung. Elevated pulmonary arterial blood pressure and volume overload in the right heart eventually lead to death from heart failure. Pre-existing elevated pro-fibrotic signaling, systemic vasculopathy and chronic inflammation are additional factors that likely contribute to the more severe PAH manifestation in SSc patients, whose response to existing therapies is suboptimal. The aim of my thesis research was to investigate the pathological role of altered transcriptional regulation of endothelium in pulmonary vasculature, as well as testing potential novel therapies for SSc-PAH in vivo. I found that endothelial downregulation of GATA6 promotes increased production of reactive oxygen species (ROS) by suppressing enzymatic machinery responsible for ROS clearance. Increased ROS production triggered ER stress and inflammation, exacerbating endothelial dysfunction and vascular injury both in vitro and in vivo. Another discovery is that simultaneous depletion of two ETS-family factors, ERG and FLI1 synergistically activates interferon signaling in pulmonary endothelial cells and promotes inflammation in lung in vivo. Based on observed pathological contribution of oxidative stress and inflammation to vasculopathy and fibrosis I tested an anti-oxidative and anti-inflammatory agent- dimethyl fumarate (DMF) in mouse models of PAH, as well as lung and skin fibrosis. DMF efficiently ameliorated increased pulmonary artery pressure and vascular remodeling, as well as fibrotic changes in lung and skin. Mechanistically, I found that DMF promotes a proteasomal, βTRCP-dependent degradation of pro-fibrotic mediators TAZ/YAP, β-catenin and Sp1. In conclusion, I characterized new elements of pathological mechanism that promote vasculopathy and fibrosis, as well as provided an insight into anti-inflammatory and anti-fibrotic DMF therapy. Importantly, elucidating the novel mechanism of DMF action and recognizing the pathological role of Hippo and Wnt signaling in fibrosis might help to design more specific and effective pharmacological intervention in SSc-PAH patients.

Introduction: Uterine fibroids are the most common benign tumours of female genital tract with the peak incidence in the 4th and 5th decennium. The aetiology of uterine fibroids still remains poorly understood. Genetic factors play undisputed role in the onset of uterine fibroids. Up to date numerous gene mutations were identified in certain percentage of patients with uterine fibroids. One of the candidate genes is Fumarate hydratase gene (FH). Heterozygous germiline mutations of FH cause two hereditary syndromes: Multiple smooth muscle tumours of the skin and uterus (MCUL1)/ Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) characterised by leiomyomata of the skin, early onset uterine fibroids between 20-30 years of age and renal papillary carcinoma. The aim of our thesis was to identify the frequency of FH mutations in patients with early onset sporadic uterine fibroids. Methods: Patients with the diagnosis of uterine fibroids up to the age of 30 years were enrolled in the study. Control group consisted of patients with absence of uterine fibroids. Activities of Fumarate hydratase and control protein Citrate synthase were measured in lymphocytes and compared to the results obtained from the healthy controls. Mutation analysis of FH gene was performed. Activity of Fumarate...

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No. of bitstreams: 2 Dissertação - Aroldo Vieira de Moraes Filho - 2013.pdf: 777889 bytes, checksum: ff3a609fe7af52fcc55701fe96a8d450 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The antiretroviral drugs appeared to prevent the multiplying HIV virus in the body, reducing its virulence, but not eliminate it from infected cells. These drugs increase the length and the quality of life of AIDS patients. In this context, Efavirenz (EFV) is non-nucleoside reverse transcriptase inhibitors. The Tenofovir Disoproxil Fumarate (TDF), oral prodrug of tenofovir, is analogue of adenosine 5 'monophosphate, belonging to the class of nucleotide reverse transcriptase inhibitors. These drugs act on the mechanisms of HIV replication by inhibiting the action of reverse transcriptase and thus preventing viral DNA synthesis. In order to assess the toxic and toxic-genetic potential of EFV and TDF, the present study used the Test for Detection of Somatic Mutation and Recombination (SMART) in Drosophila melanogaster. 3rd stage larvae originating from standard cross (ST) between males mwh and females flr³, were treated with solution of EFV and TDF and distilled water (negative control), for approximately 48 hours (chronic treatment) until they reach the pupal stage. These strains are carriers of specific gene markers, located on the left arm of chromosome 3, which allow you to monitor events related to mutation, mitotic recombination and chromosome aberrations. The statistical diagnosis was obtained by conditional binomial test. In this work, the results demonstrated that the EFV was toxic in high concentrations, but showed no induction of toxic genetic events. Inversely, the TDF showed no toxicity at the concentrations tested, but was showed induction of toxic genetic events at all concentrations, with a prevalence of recombinogenic events. Then, it is essential to analyze constantly the effects risk/benefit of isolated drugs and identify toxic and toxic genetic activity of each drug in order to ensure the quality of life for patients who use monotherapies and offers support for investigations with therapies that use combinations of antiretroviral drugs. / Os medicamentos antirretrovirais surgiram para impedir a multiplicação do vírus HIV no organismo, reduzindo a sua virulência, porém sem eliminá-lo das células infectadas. Estes medicamentos aumentaram o tempo e a qualidade de vida dos pacientes com AIDS. Dentro deste contexto, o Efavirenz (EFV) é inibidor da transcriptase reversa não-análogo de nucleosídeo. O Fumarato de Tenofovir Desoproxila (TDF), pró-fármaco oral de tenofovir, é análogo da adenosina 5`-monofosfato, pertencente à classe de inibidores da transcriptase reversa análogos de nucleotídeos. Estes fármacos atuam nos mecanismos de replicação do HIV, inibindo a ação da transcriptase reversa e, consequentemente, impedindo a síntese de DNA viral. Com o intuito de avaliar o potencial tóxico e tóxico genético do EFV e do TDF, utilizou-se o Teste para Detecção de Mutação e Recombinação Somática (SMART) em Drosophila melanogaster. Larvas de 3º estágio oriundas do Cruzamento Padrão (ST – standard cross) entre machos mwh e fêmeas flr³, foram tratadas com soluções de EFV e TDF, assim como com água destilada (controle negativo), por aproximadamente 48 h (tratamento crônico), isto é, até atingirem o estágio de pupa. Essas linhagens são portadoras de genes marcadores específicos, localizados no braço esquerdo do cromossomo 3, que permitem monitorar eventos relacionados com mutação gênica, aberrações cromossômicas e recombinação mitótica. O diagnóstico estatístico foi obtido pelo teste binomial condicional. Os resultados demonstraram que o EFV foi tóxico em altas concentrações, mas não induziu eventos tóxico genéticos. Inversamente, o TDF não apresentou toxicidade nas concentrações testadas, porém apresentou indução de efeitos tóxico genéticos em todas as concentrações, com prevalência dos eventos recombinogênicos. Então, torna-se fundamental analisar constantemente o risco/benefício de medicamentos isolados e identificar a atividade tóxica e tóxico-genética de cada fármaco com o intuito de assegurar qualidade de vida aos pacientes que fazem uso de monoterapias e oferecer suporte para as investigações com as terapias que utilizam combinações de antirretrovirais.

Monitoring the early responses of tumours to treatment is a crucial element in guiding therapy and increasing patient survival. To achieve this, we are using magnetic resonance imaging (MRI), which can provide detailed physiological information with relatively high temporal and spatial resolution. In combination with the dynamic nuclear polarisation (DNP) technique, high signal-to-noise is obtained, resulting in a powerful tool for in vivo 13C metabolic imaging. However, detection of hyperpolarised substrates is limited to a few seconds due to the exponential decay of the polarisation with the longitudinal relaxation time constant T1. This work aimed to improve the combination of hyperpolarisation and metabolic NMR/ MRI by extending the observation timescale of the technique. Working with quantum mechanical properties of the detected substrates, long lifetimes might be accessible by using the nuclear singlet configuration of two coupled nuclei. The singlet state is immune to intramolecular dipole-dipole relaxation processes, which is one of the main sources of signal decay in MRI. In favourable situations, the singlet relaxation time constant can be much longer than T1, so transfer of the polarisation into the singlet state may allow one to extend the usable time period of the nuclear hyperpolarisation. Here we studied the relaxation of hyperpolarised metabolites, including those found in the TCA cycle, and examined the possibility of extending their observation timescale by storing the polarisation in the long-lived singlet state. The polarisation remains in this state until it is eventually required for imaging. We also investigate how one may track polarised metabolites after injection into a subject due to the transfer of polarisation to the solvent by Overhauser cross-relaxation, so that the 13C polarisation remains untouched until imaging is required. In this way we should be able to interrogate slower metabolic processes than have been examined hitherto using hyperpolarised 13C MRS, and better understand metabolic changes induced in tumours by treatment.

Rhizopus oryzae is a prominent strain for producing fumarate, where biomass growth precedes fumarate production. The natural bio film growth of R. oryzae as fungal mat was investigated using different glucose addition strategies in a novel fed-batch fermenter. Batch growth was compared through three fed-batch runs, each with a different glucose addition strategy. The fed-batch runs involved a constant glucose feed (CGF) of 0.075 g h-1 and controlled glucose feeds in order to keep the respiration quotient (RQ) at either 1.3 mol CO2 mol-1 O2 (RQ1.3) or 1.1 mol CO2 mol-1 O2 (RQ1.1). Ethanol overflow via the established Crabtree mechanism was completely negated for the CGF and RQ1.1 runs, while the batch and RQ1.3 runs exhibited significant ethanol formation. Biomass yield on glucose was found to be 0.476 g g-1 (RQ1.1), 0.194 g g-1 (RQ1.3), 0.125 g g-1 (CGF) and 0.144 g g-1 (batch). The results indicate a three-fold improvement in biomass yield when comparing the batch run with the RQ1.1 run. In addition, the RQ1.1 run resulted in zero detectable byproducts, unlike the batch scenario where pyruvate and fumarate were associated with ethanol formation. Clear evidence is provided that glucose overflow can be fully eliminated during R. oryzae growth, significantly affecting the biomass yield on glucose. / Dissertation (MEng (Chemical Engineering))--University of Pretoria, 2021. / University of Pretoria postgraduate bursary / CSIR Inter-bursary Programme / Chemical Engineering / MEng (Chemical Engineering) / Unrestricted

Introduction: Uterine fibroids are the most common benign tumours of female genital tract with the peak incidence in the 4th and 5th decennium. The aetiology of uterine fibroids still remains poorly understood. Genetic factors play undisputed role in the onset of uterine fibroids. Up to date numerous gene mutations were identified in certain percentage of patients with uterine fibroids. One of the candidate genes is Fumarate hydratase gene (FH). Heterozygous germiline mutations of FH cause two hereditary syndromes: Multiple smooth muscle tumours of the skin and uterus (MCUL1)/ Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) characterised by leiomyomata of the skin, early onset uterine fibroids between 20-30 years of age and renal papillary carcinoma. The aim of our thesis was to identify the frequency of FH mutations in patients with early onset sporadic uterine fibroids. Methods: Patients with the diagnosis of uterine fibroids up to the age of 30 years were enrolled in the study. Control group consisted of patients with absence of uterine fibroids. Activities of Fumarate hydratase and control protein Citrate synthase were measured in lymphocytes and compared to the results obtained from the healthy controls. Mutation analysis of FH gene was performed. Activity of Fumarate...