This thesis combines four published research papers and a book chapter investigating aspects of central metabolism in the human malaria parasite Plasmodium falciparum. The publications are preceded by a statement which explores features of the research not fully described in the published texts, incorporates a review of the development over time and the present state of relevant scientific knowledge, and discusses the place of the individual papers and book chapter within malaria research. An assessment of the impact of each publication on its field of study is also included. A general discussion of the combination of papers as representative of the progress of research into the metabolism of malaria parasites concludes the statement section. The first publication is a chapter from a book, which describes detailed methods for the in vitro cultivation of P. falciparum. Such methodology, both robust and reliable, is a prerequisite for any investigation of parasite metabolism. The following publications are all primary research papers. The second publication describes the isolation and characterisation of the gene encoding the glycolytic pathway enzyme enolase from P. falciparum. The inferred amino acid sequence included peptide insertions found only in the enolases of higher plants and other photosynthetic organisms. This raised implications concerning the deep evolutionary history of the malaria parasite and related species. The third is concerned with the elucidation of the molecular basis of resistance to the antimalarial drug sulfadoxine. Resistance was found to result from point mutations within the dihydropteroate synthetase domain of the bifunctional protein hydroxymethylpterin pyrophosphokinase-dihydroptero¬ate synthetase, an enzyme of the parasite folate pathway. Additionally, it was discovered that the presence of exogenous folate has an antagonistic effect on sulfadoxine in some parasites of a defined genotype. This highlighted the importance of folate salvage in parasite metabolism. Fourth is a paper representing the discovery of a novel metabolism in both P. falciparum and the related apicomplexan parasite Toxoplasma gondii. The use of parasite genes in rescuing an Escherichia coli tyrosine auxotroph resulted in a proof of function of the products of these genes as pterin-4a-carbinolaminedehydratases. Pterin recycling, hitherto undetected in apicomplexans, was therefore added to the known metabolic processes of these organisms. The final paper describes an investigation into the subcellular distribution of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) within P. falciparum erythrocytic stage parasites. The use of confocal laser scanning microscopy and immunofluorescent techniques showed that SHMTc, the sole enzymatically active parasite SHMT protein, was found in the cytoplasm but also showed a stage-specific localisation to both the mitochondrion and apicoplast organelles. The otherwise enigmatic, enzymatically inert, SHMTm paralogue revealed a possible function, when in complex, in allowing targeted localisation of SHMTc to the mitochondrion. The spatial distribution of SHMTm also suggested a possible role in the morphogenesis of elongating apicoplasts during schizogony.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Introdução: A resposta inflamatória sistêmica após cirurgia cardíaca está relacionada com aumento dos marcadores de lesão cerebral e o desenvolvimento de disfunção cognitiva pós-operatória (DCPO). O objetivo deste estudo foi avaliar a potencial associação entre DCPO e os níveis séricos de proteína S100beta e enolase específica de neurônio (NSE) após cirurgia de revascularização do miocárdio (RM) com circulação extracorpórea (CEC). Métodos: Foram investigados 73 pacientes submetidos à RM com CEC. A função cognitiva foi avaliada por psicólogas no pré-operatório e nos 3º, 7º, 21º, 90º e 180º dias após a cirurgia, utilizando-se testes neurocognitivos específicos para o reconhecimento de déficits de atenção, memória, função executiva e linguagem. Os níveis séricos de proteína S100beta e NSE foram medidos no pré-operatório, após a indução da anestesia, ao final da cirurgia e seis e 24 horas após a cirurgia. Para se avaliar a associação entre DCPO e os níveis séricos de proteína S100beta e NSE, utilizou-se ANOVA e ANOVA com medidas repetidas. As diferenças encontradas foram analisadas pela comparação múltipla de Tukey. Os resultados foram significantes quando p < 0,05. Resultados: DCPO foi observada em 63,1% dos pacientes após 21 dias e em 21,7% após seis meses da cirurgia. Na primeira semana após a cirurgia, 46,6% dos pacientes apresentaram delirium pósoperatório. Os maiores níveis séricos de proteína S100beta (1,8 ng/mL) e NSE (18,2 ng/mL) foram vistos no final da cirurgia e seis horas após a cirurgia, respectivamente. No entanto, não foi observada significante associação entre DCPO e aumento dos níveis séricos dos marcadores de lesão cerebral. Conclusões: O presente estudo observou uma elevada prevalência de DCPO. Adicionalmente, detectou-se um aumento dos níveis séricos de proteína S100beta e NSE no pós-operatório. Contudo, embora RM com CEC esteja significativamente relacionada a DCPO e níveis séricos elevados de proteína S100beta e NSE, o aumento dos níveis destes marcadores de lesão cerebral não foi associado à DCPO / Background: Systemic inflammatory response after cardiac surgery is associated with increased brain injury markers and development of postoperative cognitive dysfunction (POCD). The aim of this study was to evaluate the association between POCD and serum levels S100beta protein and neuron-specific enolase (NSE) after coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB). Methods: 73 patients undergoing CABG surgery with CPB were investigated. Cognitive function was assessed by psychologists preoperatively and at 3, 7, 21, 90 and 180 days after surgery, using neurocognitive tests for specific recognition deficits in attention, memory, executive function and language. Serum levels S100beta protein and NSE were measured preoperatively, after induction of anesthesia, in the end of surgery, and 6 and 24 hours after surgery. Association between POCD and serum levels S100beta protein and NSE was assessed with ANOVA and ANOVA with repeated measurements. Differences were analyzed by Tukey\'s multiple comparison. Results were significant at p <0.05. Results: The mean age of patients was 61.8 ± 9.1 years. POCD was observed in 63.1% of patients after 21 days and 21.7% after 6 months of surgery. Postoperative delirium was seen in 46.6% of patients in the first week after surgery. The highest serum levels of S100beta protein (1.8 ng/mL) and NSE (18.2 ng/mL) were seen at the end of surgery and 6 hours after surgery, respectively. However, this study not observed a significant association between POCD and increased serum levels brain injury markers. Conclusions: The present study observed a high prevalence of POCD. Additionally, serum levels S100beta protein and NSE increased postoperatively. However, while CABG surgery with CPB is significantly related to POCD and elevated serum levels S100beta protein and NSE, the increased levels of such brain injury markers was not associated with POCD

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Introdução: A resposta inflamatória sistêmica após cirurgia cardíaca está relacionada com aumento dos marcadores de lesão cerebral e o desenvolvimento de disfunção cognitiva pós-operatória (DCPO). O objetivo deste estudo foi avaliar a potencial associação entre DCPO e os níveis séricos de proteína S100beta e enolase específica de neurônio (NSE) após cirurgia de revascularização do miocárdio (RM) com circulação extracorpórea (CEC). Métodos: Foram investigados 73 pacientes submetidos à RM com CEC. A função cognitiva foi avaliada por psicólogas no pré-operatório e nos 3º, 7º, 21º, 90º e 180º dias após a cirurgia, utilizando-se testes neurocognitivos específicos para o reconhecimento de déficits de atenção, memória, função executiva e linguagem. Os níveis séricos de proteína S100beta e NSE foram medidos no pré-operatório, após a indução da anestesia, ao final da cirurgia e seis e 24 horas após a cirurgia. Para se avaliar a associação entre DCPO e os níveis séricos de proteína S100beta e NSE, utilizou-se ANOVA e ANOVA com medidas repetidas. As diferenças encontradas foram analisadas pela comparação múltipla de Tukey. Os resultados foram significantes quando p < 0,05. Resultados: DCPO foi observada em 63,1% dos pacientes após 21 dias e em 21,7% após seis meses da cirurgia. Na primeira semana após a cirurgia, 46,6% dos pacientes apresentaram delirium pósoperatório. Os maiores níveis séricos de proteína S100beta (1,8 ng/mL) e NSE (18,2 ng/mL) foram vistos no final da cirurgia e seis horas após a cirurgia, respectivamente. No entanto, não foi observada significante associação entre DCPO e aumento dos níveis séricos dos marcadores de lesão cerebral. Conclusões: O presente estudo observou uma elevada prevalência de DCPO. Adicionalmente, detectou-se um aumento dos níveis séricos de proteína S100beta e NSE no pós-operatório. Contudo, embora RM com CEC esteja significativamente relacionada a DCPO e níveis séricos elevados de proteína S100beta e NSE, o aumento dos níveis destes marcadores de lesão cerebral não foi associado à DCPO / Background: Systemic inflammatory response after cardiac surgery is associated with increased brain injury markers and development of postoperative cognitive dysfunction (POCD). The aim of this study was to evaluate the association between POCD and serum levels S100beta protein and neuron-specific enolase (NSE) after coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB). Methods: 73 patients undergoing CABG surgery with CPB were investigated. Cognitive function was assessed by psychologists preoperatively and at 3, 7, 21, 90 and 180 days after surgery, using neurocognitive tests for specific recognition deficits in attention, memory, executive function and language. Serum levels S100beta protein and NSE were measured preoperatively, after induction of anesthesia, in the end of surgery, and 6 and 24 hours after surgery. Association between POCD and serum levels S100beta protein and NSE was assessed with ANOVA and ANOVA with repeated measurements. Differences were analyzed by Tukey\'s multiple comparison. Results were significant at p <0.05. Results: The mean age of patients was 61.8 ± 9.1 years. POCD was observed in 63.1% of patients after 21 days and 21.7% after 6 months of surgery. Postoperative delirium was seen in 46.6% of patients in the first week after surgery. The highest serum levels of S100beta protein (1.8 ng/mL) and NSE (18.2 ng/mL) were seen at the end of surgery and 6 hours after surgery, respectively. However, this study not observed a significant association between POCD and increased serum levels brain injury markers. Conclusions: The present study observed a high prevalence of POCD. Additionally, serum levels S100beta protein and NSE increased postoperatively. However, while CABG surgery with CPB is significantly related to POCD and elevated serum levels S100beta protein and NSE, the increased levels of such brain injury markers was not associated with POCD

Understanding how protein sequence, structure and function coevolve is at the core of functional genome annotation and protein engineering. The fundamental problem is to determine whether sequence variation contributes to functional differences or if it is a consequence of evolutionary divergence that is unrelated to functional specificity. To address this problem, we cannot merely analyze sequence variation between homologous proteins that have different functions. For comparison, we need to understand the factors that determine sequence variation in proteins that have the same function, such as a set of orthologous enzymes. Here, we address this problem by analyzing the evolution of functionally important residues in the o-succinylbenzoate synthase (OSBS) family. The OSBS family consists of several hundred enzymes that catalyze a step in menaquinone (Vit. K2) synthesis. Based on phylogeny, the OSBS family can be divided into eight major subfamilies. We assayed wild-type OSBS enzyme activities. The results show that the enzymes from γ-Proteobacteria subfamily 1 and Bacteroidetes have relatively low values, the enzyme from Cyanobacteria subfamily 1 is intermediate, and the values for the proteins from the Actinobacteria and Firmicutes subfamilies are relatively high. We are using computational and experimental methods to identify functionally important amino acids in each subfamily. Our data suggest that each subfamily has a different set of functionally important residues, even though the enzymes catalyze the same reaction. These differences may have accumulated because different mutations were required in each subfamily to compensate for deleterious mutations or to adapt to changing environments. We assessed the roles of these amino acids in enzyme structure and function. Our method achieved 70% successful rate to identify positions that play important roles in one family but not another. The residues P119 and A329 play important role in D. psychrophila but not in T.fusca OSBS. We also observed two class switch mutations in T.fusca, P11 and P22. The mutations at these two position have a similar kinetic parameters as wild-type D. psychrophila OSBS.

The possible existence of extraterrestrial life forms has been of interest to humans for many millennia. In the past few decades space travel has provided an opportunity to search life outside of Earth. Chiral molecules are critical molecules in Earth-based life and are among the first chemical molecules sought after as proof of potential extraterrestrial life; however, identification of these chiral molecules is difficult due the lack of sensitive instruments. The objective of this work is to develop a benchmark reaction to be used as a guide in the development of instrumentation, such as a polarimeter, to be used in the search for extraterrestrial life. To achieve this objective, to investigate the enzyme mandelate racemase (MR), which catalyzes the racemization between the enantiomers of mandelate. MR is a member of the enolase superfamily, which contains a (alpha/beta)7-b barrel domain, the fold most frequently found among all known protein structures. Activity of the enzyme was measured at low temperatures and in non-aqueous media, as these are the conditions that represent extraterrestrial terrain. We find that mandelate racemase (MR) is active in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between 30C to 70C; however, the enzyme is not active in several organic cryosolvents. The stability of the structure of MR was also explored. Using differential scanning calorimetry (DSC) we observe the unfolding of the enzyme was irreversible and therefore kinetically controlled. We also found proof for divergent evolution of the enolase superfamily, providing evidence for divergent evolution across the MR and muconate lactonizing enzyme (MLE) subfamilies has been demonstrated. However, we also conclude that reactions yielding a polarimetric signal, such as racemizations employed in this work, are suitable as a tool to find signs of life.

Resumo: Paracoccidioides brasiliensis é um importante patógeno humano que causa a paracoccidioidomicose (PCM), uma micose sistêmica com ampla distribuição na América Latina. A adesão e invasão de células são eventos cruciais envolvidos na infecção e disseminação do patógeno. Além disso, patógenos utilizam suas moléculas de superfície para se ligar aos componentes da matriz extracelular para estabelecer a infecção. Uma proteína antigênica de P. brasiliensis foi isolada de géis de eletroforese bidimensional do cell-free do fungo e caracterizada. Peptídeos foram obtidos da proteína de 54 kDa e pI 5,6 e mostraram homologia com enolase de Paracoccidioides brasiliensis e outros fungos. A proteína foi purificada através de eletroeluição e utilizada para a produção de anticorpo policlonal em coelho. Por microscopia de fluorescência não foi possível observar a localização exata desta proteína, apenas que se encontra aparentemente distribuídapor todo o fungo, foi possível verificar alterações no citoesqueleto de pneumócitos durante a infecção por P. brasiliensis. A localização foi confirmada por microscopia imunoeletrônica a presença de enolase foi detectada principalmente na parede celular de leveduras de P. brasiliensis e também no citoplasma, ela se demonstrou mais expressa quando este fungo foi cultivado em meio onde houve acréscimo de sangue de carneiro e durante a situação de infecção a pneumócitos. A enolase purificada foi capaz de se ligar a fibronectina, fibrinogênio, laminina, plasminogênio, colágenos tipo I e IV. Foi confirmado que a ligação desta proteína à pneumócitos é influenciada pela sequência de aminoácidos Arg-Gly-Asp contida provavelmente nos receptores da matriz extracelular presentes na célula do hospedeiro. Essas informações indicam que a enolase possivelmente contribui para a adesão do microrganismo aos tecidos do ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioides brasiliensis is an important human pathogen that causes paracoccidioidomycosis (PCM), a systemic mycosis with a wide distribution in Latin America. The ability of P. brasiliensis to cause not only human diseases but also mycoses with a variety of clinical manifestations from localized forms to the disseminated disease progressing to lethality, probably depends on the relationship between the virulence of the fungus, its ability to interact with and to invade the surface structures of the host and the immune response of the host. The adhesion of the pathogen leads to the recognition of carbohydrate and protein ligands on the surface of the host cell or proteins of the extracellular matrix (ECM). The large number of tissue types that fungi can colonize and infect suggests that they use a variety of surface molecules for adhesion.Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis / Orientador: Ana Marisa Fusco Almeida / Coorientador: Maria José Soares Mendes Giannini / Banca: Márcia Aparecida Silva Graminha / Banca: Eduardo Bagagli / Mestre

Interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells during Chlamydia infection. To further understand the mechanism underlying this protective property in IL-10 DCs, we combined two proteomic techniques: 2DIGE and liquid chromatography mass spectrometry. We then performed western blotting on proteins from Chlamydia infected wild type (WT) and IL-10 knock out (KO) DCs. The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the ninth step of glycolysis, was significantly upregulated in Chlamydia infected IL-10KO DCs compared to WT DCs. We further studied the role of ENO1 by silencing ENO1 gene using lentiviral siRNA technology. Flow cytometry, confocal microscopy, cytokine analysis, infectivity analysis and T-cell proliferation analysis were also used to determine DC function. We then analyzed the effect of the ENO1 knockdown on DC metabolism during Chlamydia stimulation. The results showed that DC maturation and activation were decreased in ENO1 knockdown DCs that were stimulated with Chlamydia compared to the Chlamydia stimulated WT DCs. In addition, pyruvate concentration decreased significantly in ENO1 knockdown DCs stimulated with Chlamydia. The mitochondrial structure of Chlamydia stimulated ENO1 knockdown DCs appeared damaged compared to the Chlamydia stimulated WT DCs. The function of ENO1 in the immune response to Chlamydia trachomatis infection is unknown. However, the results from this study indicates that ENO1 may contribute to DC metabolism and mitochondrial homeostasis which may play a role in the maturation and antigen presenting function of DCs.

Submitted by Andrea Hayashi (deachan@gmail.com) on 2016-07-19T18:43:58Z No. of bitstreams: 1 richard.pdf: 1735785 bytes, checksum: 2df8fed54dbb769385bd94c07d15eefc (MD5) / Approved for entry into archive by Andrea Hayashi (deachan@gmail.com) on 2016-07-19T18:44:51Z (GMT) No. of bitstreams: 1 richard.pdf: 1735785 bytes, checksum: 2df8fed54dbb769385bd94c07d15eefc (MD5) / Made available in DSpace on 2016-07-19T18:44:51Z (GMT). No. of bitstreams: 1 richard.pdf: 1735785 bytes, checksum: 2df8fed54dbb769385bd94c07d15eefc (MD5) Previous issue date: 2010 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / World Health Organization (WHO) / Howard Hughes Medical Institute (HHMI) / Os agentes antifúngicos de uso corrente na medicina apresentam alta toxicidade e efeitos colaterais, tem como alvo poucos constituintes celulares, e muitos mecanismos de resistência já foram descritos. Com o objetivo de identificar potenciais novos alvos para drogas antifúngicas, duas ORFs hipotéticas de Candida albicans, potencialmente essenciais e com perfis singulares de expressão foram caracterizados. A ORF CaYlr339c é conservada em outros fungos do gênero Candida e Saccharomyces, é transcrita nas fases de levedura e hifa e a presença de seu produto protéico de aproximadamente 18 kDa foi constatada em extratos celulares de leveduras e hifas de C. albicans a partir de experimentos de western blot. A ORF CaYdr187c é conservada em poucos representantes do gênero Saccharomyces, é transcrita apenas na fase de levedura e potencialmente codifica uma proteína de superfície. Esses dados indicam que as ORFs selecionadas são genes reais e podem ser bons candidatos para a descoberta de novas funções e para o desenvolvimento futuro de novos agentes antifúngicos. Numa tentativa de testar o possível papel da proteína putativa de superfície CaYdr187cp na adesão celular, constatamos que a incubação de discos de tecido gastrointestinal com a proteína recombinante enolase, utilizada inicialmente como controle em nossos experimentos, foi capaz de inibir a adesão de C. albicans de maneira dose dependente em até 70%. O papel da enolase de superfície na adesão celular foi confirmado a partir da inibição da adesão em até 50% observada com o bloqueio da superfície de C. albicans com anticorpos anti-enolase. A capacidade de ligação da enolase de C. albicans ao plasminogênio foi confirmada a partir de experimentos de coimunoprecipitação. A pré-incubação de C. albicans com esta proteína foi capaz de inibir parcialmente sua adesão reforçando a idéia de que a enolase realmente esteja mediando à adesão deste organismo ao tecido gastrointestinal. Em contrapartida, a enolase não foi coimunoprecipitada com a fibronectina e a pré-incubação de C. albicans com esta proteína falhou em inibir sua adesão indicando que a inibição observada com a enolase recombinante não é decorrente do bloqueio da fibronectina no tecido gastrointestinal. Este trabalho é o primeiro a demonstrar que a enolase presente na superfície de C. albicans está envolvida em mecanismos de adesão, podendo ser um importante fator de virulência neste organismo. Assim, esta proteína poderia ser utilizada no desenvolvimento de vacinas ou estratégias de controles a partir da inibição da adesão de C. albicans ao tecido gastrointestinal com o intuito de prevenir sua proliferação. / Current antifungal agents in medicine presents high toxicity and collateral effects, targets limited cell constituents and different resistance mechanisms have already been described. In order to identify novel potential antifungal drug targets, two hypothetical Candida albicans genes potentially essential and with singular expression profile have been characterized. The ORF CaYlr339c is conserved among fungal species from Candida and Saccharomyces genus, is transcribed in yeast and hyphae and an approximately 18-kDa protein was identified in hyphae and yeasts C. albicans cell extracts by western blotting. The ORF CaYdr187c is conserved in a few representatives of the Saccharomyces genus, is transcribed only in yeasts and potentially codifies a surface protein. These findings indicate that the selected ORFs are real genes and could be good candidates for the discovery of new functions and the development of new antifungal agents in the future. In an attempt to test the possible role of the putative surface protein CaYdr187cp on cell adhesion, we find out that incubation of the gastrointestinal Murine disks with the recombinant protein enolase, initially employed as a control in our experiments, was able to inhibit C. albicans adhesion in a dose-dependent manner in 70%. The role of surface enolase on cell adhesin was confirmed with the 50% of the adhesion inhibition observed after blocking C. albicans surface with antibodies anti-enolase. The binding capacity of enolase to plasminogen was confirmed by co-immunoprecipitation. Preincubation of C. albicans with this protein partially inhibited the adhesion supporting the idea that enolase is really mediating the adhesion of this organism to gastrointestinal tissues. On the other hand, enolase was not co-immunoprecipitated with fibronectin and pretreatment of C. albicans with this protein failed to inhibit the adhesion, indicating that the observed inhibition with recombinant enolase was not due to the blocking of fibronectin in the gastrointestinal disks. This was the first work to demonstrate that surface C. albicans enolase is involved in cell adhesion, being a potential virulence factor in this organism. Accordingly, this protein could be useful in vaccine development and is a promising candidate for the prevention of C. albicans proliferation by the inhibition of its adhesion. Esta tese é composta de uma Introdução geral e duas partes independentes compostas de: Introdução, objetivos, materiais e métodos, resultados, discussões e conclusões específicas. A primeira parte da tese trata da caracterização parcial de genes hipotéticos de C. albicans e a segunda parte trata do papel da enolase na adesão de C. albicans ao tecido gastrointestinal.