Thesis (Ph.D.) - University of Glasgow, 2007. / Ph.D. thesis submitted to the Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references.

Made available in DSpace on 2015-12-06T23:47:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2007 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O controle traducional mediado pela fosforilacao da subunidade alfa do fator de inicio de traducao 2 (eIF2a) e um ponto central para programas de expressao genica induzidos por estresse. Tripanossomatideos, importantes patogenos humanos, apresentam processos de diferenciacao desencadeados pelo contato com os distintos ambientes encontrados em seus insetos vetores e hospedeiros mamiferos, provavelmente representando situacoes de estresse. Trypanosoma brucei, o agente causador da tripanossomiase africana, codifica tres potenciais quinases de eIF2α (TbeIF2Kl-K3). Neste trabalho, nos mostramos que TbeIF2K2 e uma glicoproteina associada a membrana, expressa tanto na forma prociclica quanta na sanguinea. 0 dominio catalitico de TbeIF2K2 fosforila eIF2α de levedura e de mamiferos na Ser51. A quinase tambem fosforila a incomum forma de eIF2α encontrada em tripanossomatideos, especificamente no residuo Thr169, que corresponde a Ser51 em outros eucariotos. 0 eIF2α de T brucei, no entanto, nao e um substrato para GCN2 ou PKR in vitro. 0 dominio regulatorio putativo de TbeIF2K2 nao apresenta nenhuma similaridade de sequencia com as quinases de eIF2α conhecidas. Tanto na forma sanguinea quanto na prociclica, TbeIF2K2 esta localizada principalmente na bolsa flagelar, organela que e o local exclusivo de exo e endocitose nesses parasitas. Ela tambem pode ser detectada em compartimentos endociticos, mas nao em lisossomos, sugerindo que a quinase e reciclada entre os endossomos e a bolsa flagelar. A localizacao de TbeIF2K2 sugere que ela possa funcionar como um sensor do transporte de nutrientes ou proteinas em T brucei, um organismo que depende de mecanismos regulatorios pos-transcricionais para controlar a expressao genica em diferentes situacoes. Essa e a primeira quinase de eIF2α associada a membrana descrita em eucariotos unicelulares / Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1-K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169, that corresponds to Ser51 in other eukaryotes. T. brucei eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes. / FAPESP: 02/13783-9 / BV UNIFESP: Teses e dissertações

Plasmodium spp. and Trypanosoma brucei spp. are protozoan parasites with complex lifecycles, each having to adapt to the diverse environments of their insect vector and mammalian host. Each has multiple developmental forms that differ in their morphology and metabolism. Differentiation between these forms is a tightly regulated and often synchronised process in response to changes in the parasite's environment. The molecular mechanisms by which they perceive and respond to such environmental changes are largely unknown. The Plasmodium and T. brucei genomes encode a homologue of the phylogenetically widespread GPR89 family of putative receptors or channels. The mammalian GPR89 homologue has been implicated in the regulation of Golgo acidification, whilst in plants it has been shown to be involved in G protein signally pathways. This study set out to characterise the Plasmodium and T. brucei GPR89 proteins in order to assess their potential role as environmental sensors. Bioinformatic analyses demonstrated that the GPR89 proteins are a highly divergent family of multi-transmembrane domain proteins that may perform a channel or transporter function. Several expression strategies were employed to evaluate the role of the GPR89 proteins. Functional insight was gained from the ectopic expression of both the T. brucei and Arabidopsis thaliana GPR89 proteins in T. Brucei cells. Over-expression of TbGPR98 causes premature stumpy formation in pleomorphic T. brucei cells. This phenotype was replicated by over-expression of a A. thaliana homologue in T. brucei despite signigicatnt sequence divergence. Furthermore, both were demonstrated to act on the same pathway as the putatuve RNA binding protein, RBP7 that was receontly identified in a genome-wide screen for components of the stumpy differentiation pathway. Hence, TbGPR89 is likely a compnent of the slender to stumpy differentiation pathway in bloodstream form trypanosomes and there appears to be functional complementarity between T. brucei and A. thaliana GPR89 proteins.

Two aspects of trypanosomiasis have been investigated in this study. First, molecular methods were applied to the diagnosis of T.evansi in camels in South Libya. The aim of the study was to determine if FTA card blood sampling and PCR amplification could detect parasites and this be used as tool for diagnosis and epidemiology. Targeted samples of 70 camels were identified on the basis of symptoms of infection and blood was collected on FTA cards. PCR primers and conditions for the amplification of T.evansi DNA were developed on the basis of the literature and a positive control clone grown in the laboratory. The assay found 84.3% of camel samples positive using TBR primers (177bp amplicon) and ITS nested primers (611-1513bp amplicons). This result demonstrated that Surra is endemic in this area, and that T.evansi was the species that was involved. The ITS and TBR loci in the parasites identified in Libya were almost identical to those previously reported in the genbank database, though with some polymorphisms. Dullness and emaciation were the clinical signs of camels infected by trypanosomes, and these two symptoms were significantly related to the 1200bp ITS nested PCR amplicon. These two symptoms can be thus used as a sign an initial diagnosis of T.evansi infection in camels. The second aspect of trypanosomiasis studied was the occurrence of endotoxaemia in infection. The first part of this research investigated endotoxin levels in clinical human African trypasnosomiasis using the Limulus Amoebocyte lysate assay. Endotoxin levels were significantly increased over control individuals in the plasma of T.b.rhodesiense patients. This endotoxaemia was unrelated to infection duration, parasitaemia or clinical stage but resolved after clearance of parasites by drug treatment. In the cerebrospinal fluid there was no significant difference in endotoxin level between early and late stage cases and no relationship to parasite loads. It is argued on the basis of the data that endotoxaemia in trypanosomiasis most likely results from increases in permeability of the gut to endotoxins from gram negative enter bacteria. This conclusion was further supported from a study using cell culture adapted T.brucei and secreted products which gave no evidence of any endotoxin activity. Also samples of an acute experimental mouse infection with T.brucei gave no endotoxin activity, suggesting that this phenomenon requires a more chronic infection in mice. No relationships were found between plasma or CSF endotoxin levels to neurological signs of infection. However the presence of a gross inflammatory clinical symptom, splenomegaly, was associated with endotoxaemia and the concentrations of 3 plasma cytokines associated with the immune response in trypanosome infection were associated with correlated to plasma endotoxin levels. In order to determine the nature of the endotoxin activity, a biosensor cell assay for LPS was used, based on human embryonic kidney cells transfected with TLR4/MD3 and a NF-κB induced alkaline phosphatase reporter gene. This assay revealed low or undetectable levels of LPS in clinical samples from T.b.rhodesiense patients, in mouse samples from T.b.brucei infections and in vitro cultured trypanosomes. This suggests that either the endotoxin activity detected using the LAL assay is an unconventional endotoxin signalling via a TLR4 independent pathway or that the human plasma was in some way toxic to the reporter cell and this requires further investigation. In conclusion, this study has provided the first clear evidence of an association of endotoxaemia and inflammatory responses in clinical African trypansomiasis and helps resolve the question of whether endotoxaemia is a parasite or host-microbiota related phenomenon.

The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to persist within its mammalian host. Trypanosomes evade the hosts' immune system by antigenic variation of their surface coat, consisting of variant surface glycoproteins (VSGs). Out of a repertoire of thousands of VSG genes, only one is expressed at any given time from one of the 15 telomeric expression sites (ES). The VSG is stochastically exchanged either by a transcriptional switch of the active ES (in situ switch) or by a recombinational exchange of the VSG within the active ES. However, for infections to persist, the parasite burden has to be limited. The slender (sl) bloodstream form secretes the stumpy induction factor (SIF), which accumulates with rising parasitemia. SIF induces the irreversible developmental transition from the proliferative sl to the cell cycle-arrested but fly-infective stumpy (st) stage once a concentration threshold is reached. Thus, antigenic variation and st development ensure persistent infections and transmissibility. A previous study in monomorphic cells indicated that the attenuation of the active ES could be relevant for the development of trypanosomes. The present thesis investigated this hypothesis using the inducible overexpression of an ectopic VSG in pleomorphic trypanosomes, which possess full developmental competence. These studies revealed a surprising phenotypic plasticity: while the endogenous VSG was always down-regulated upon induction, the ESactivity determined whether the VSG overexpressors arrested in growth or kept proliferating. Full ES-attenuation induced the differentiation of bona fide st parasites independent of the cell density and thus represents the sole natural SIF-independent differentiation trigger to date. A milder decrease of the ES-activity did not induce phenotypic changes, but appeared to prime the parasites for SIF-induced differentiation. These results demonstrate that antigenic variation and development are linked and indicated that the ES and the VSG are independently regulated. Therefore, I investigated in the second part of my thesis how ES-attenuation and VSG-silencing can be mediated. Integration of reporters with a functional or defective VSG 3'UTR into different genomic loci showed that the maintenance of the active state of the ES depends on a conserved motif within the VSG 3'UTR. In situ switching was only triggered when the telomere-proximal motif was partially deleted, suggesting that it serves as a DNA-binding motif for a telomere-associated protein. The VSG levels seem to be additionally regulated in trans based on the VSG 3'UTR independent of the genomic context, which was reinforced by the regulation of a constitutively expressed reporter with VSG 3' UTR upon ectopic VSG overexpression. / Der eukaryotische Parasit Trypanosoma brucei hat komplexe Strategien entwickelt, um in seinem Säugetierwirt zu überleben. Die Grundlage der Immunevasion ist die antigene Variation des Oberflächenmantels, der aus dem variablen Oberflächenglykoprotein (VSG) besteht. Von mehreren tausend VSG-Genen wird zu jedem Zeitpunkt nur ein einziges aus einer der 15 telomerischen Expressionsstellen (ES) exprimiert. Das VSG kann entweder durch einen transkriptionellen Wechsel der aktiven ES (in situ Wechsel) oder durch einen rekombinatorischen Wechsel des VSG-Gens innerhalb der aktiven ES stochastisch ausgetauscht werden. Damit jedoch eine langanhaltende Infektion des Wirts möglich wird, muss gleichzeitig der Parasitenbefall begrenzt werden. Mit ansteigender Parasitämie akkumuliert der 'stumpy induction factor' (SIF), welcher von der 'slender' (sl) Blutstromform sekretiert wird. Sobald ein Schwellenwert in der SIF-Konzentration erreicht ist, wird die irreversible Differenzierung der proliferativen sl in die zellzyklusarretierte 'stumpy'(st) Form eingeleitet, welche infektiös für den Fliegenvektor ist. Somit stellen antigene Variation und st- Differenzierung das Persistieren der Infektion und die Übertragung des Parasiten sicher. Eine frühere Arbeit mit monomorphen Zellen deutete darauf hin, dass die Attenuierung der aktiven ES eine Rolle für die Differenzierung der Trypanosomen spielen könnte. Diese Hypothese wurde in der vorliegenden Dissertation untersucht, indem in pleomorphen Zellen mit vollständiger Entwicklungskompetenz ein ektopisches VSG induzierbar überexprimiert wurde. Diese Studien offenbarten eine erstaunliche phänotypische Plastizität: während das endogene VSG nach Induktion runter reguliert wurde, arretierten die VSG-Überexpressoren in Abhängigkeit von der ES-Aktivität entweder im Wachstum oder teilten sich weiter. Die vollständige ES-Attenuierung löste die Differenzierung zu echten st Zellen unabhängig von der Zelldichte aus und ist somit der bisher einzige natürliche SIF-unabhängige Differenzierungsauslöser. Eine mildere Abnahme der ES-Aktivität verursachte keinen Phänotyp, scheint aber die Zellen auf die SIF-induzierte Differenzierung vorzubereiten. Diese Ergebnisse zeigen, dass antigene Variation und Differenzierung verbunden sind und deuteten an, dass die ES und das VSG unabhängig voneinander reguliert werden. Daher habe ich im zweiten Teil meiner Dissertation untersucht, wie ES-Attenuierung und VSG-Stilllegung vermittelt werden können. Die Integration eines Reporters mit funktioneller oder defekter VSG 3'UTR an verschiedenen Orten im Genom zeigte, dass die Aufrechterhaltung der ES-Aktivität von einem konservierten Motiv in der VSG 3'UTR abhängig ist. Ein in situ Wechsel wurde nur ausgelöst, wenn Teile des Telomer-proximalen Motiv deletiert wurden, was nahelegt, dass das Motiv auf DNA-Ebene von einem Telomerbindeprotein erkannt wird. Die VSG-Level scheinen unabhängig vom genomischen Kontext zusätzlich in trans basierend auf der VSG 3'UTR reguliert zu werden, was durch die Regulation eines konstitutiv exprimierten Reporters mit VSG 3'UTR nach VSG-Überexpression bekräftigt wurde.

The nuclear envelope serves as important mRNA surveillance system. In yeast and humans, several control mechanisms act in parallel to prevent nuclear export of unprocessed mRNAs. However, trypanosomes lack homologues to most of the proteins involved. In addition, gene expression in trypanosomes relies almost completely on post-transcriptional regulation as they transcribe mRNAs as long polycistrons, which are subsequently processed into individual mRNA molecules by trans-splicing. As trans-splicing is not error-free, unspliced mRNAs may be recognized and prevented from reaching the cytoplasm by a yet unknown mechanism. When trans-splicing is inhibited in trypanosomes, the formation of a novel RNA granule type at the cytoplasmic periphery of the nucleus, so called nuclear periphery granules (NPGs) was previously observed. To identify potential regulators of nuclear export control, changes in protein localization which occur when trans-splicing is inhibited, were globally analyzed during this work. For this, trypanosome nuclei were purified under conditions maintaining NPG attachment to the nucleus, in the absence and presence of trans-splicing. Mass spectrometry analyses identified 128 proteins which are specifically enriched in nuclear preparations of cells inhibited for trans-splicing. Amongst them are proteins, which change their localization to the nucleus or to the nuclear pores as well as many proteins that move into NPGs. Some of these proteins are promising candidates for nuclear export control proteins, as the changes in localization (to the nucleus or nuclear pores) were specific to the accumulation of unspliced mRNAs. The NPG proteome almost exclusively contains proteins involved in mRNA metabolism, mostly unique to trypanosomes, notably major translation initiation factors were absent. These data indicate that NPGs are RNP complexes which have started or completed nuclear export, but not yet entered translation. As a byproduct of these proteomic studies, a high-quality dataset of the yet unknown T. brucei nuclear proteome is provided, closing an important gap in knowledge to study trypanosome biology, in particular nuclear related processes. NPGs were characterized in more detail by microscopy. The granules are cytoplasmic and present in at least two different trypanosome life cycle stages. There are at least two distinct granule subsets, with differences in protein composition. A closer analysis of NPGs by electron microscopy revealed that the granules are electron dense structures, which are connected to nuclear pores by string-like structures. In order to approach the function of NPGs, on the one hand, the hypothesis that NPGs might be related to perinuclear germ granules of adult gonads of C. elegans was tested: we found no relation between the two granule types. On the other hand, initial single molecule mRNA FISH experiments performed in trypanosomes showed no accumulation of unspliced transcripts in NPGs, arguing against an involvement of the granules in mRNA quality control. / Die Kernhülle um den Zellkern dient als wichtiges mRNA-Überwachungssystem in Eukaryoten. Bei Hefen und Menschen wirken dabei beispielsweise mehrere Kontrollmechanismen parallel, um den Export von unverarbeiteten mRNAs aus dem Kern heraus zu verhindern. Trypanosomen fehlen jedoch Homologe zu den Meisten hierbei beteiligten Proteinen. Außerdem basiert Genexpression in Trypanosomen fast ausschließlich auf posttranskriptioneller Kontrolle, da die Parasiten mRNAs als lange Polycistrons transkribieren, die anschließend durch Transspleißen zu einzelnen mRNA-Molekülen verarbeitet werden. Da der Prozess des Transspleißen nicht fehlerfrei zu sein scheint, gibt es möglicherweise einen noch unbekannten Mechanismus, der nicht gespleißte mRNAs erkennt und diese daran hindert, das Zytoplasma zu erreichen. Unter Bedingungen, in denen Transspleißen in Trypanosomen blockiert ist, konnte eine neue Art von RNA-Granula an der zytoplasmatischen Peripherie des Zellkerns beobachtet werden, sogenannte Nuclear Periphery Granules (NPGs). Um potentielle Regulatoren einer Kontrolle des mRNA-Exports zu identifizieren, wurde während dieser Arbeit umfassend analysiert, inwieweit sich die Lokalisation von Proteinen ändert, wenn Transspleißen gehemmt wird. Zu diesem Zweck wurden Zellkerne von Trypanosomen unter Bedingungen aufgereinigt, bei denen die Bindung der NPGs an den Zellkern in Abwesenheit und Gegenwart von Transspleißen erhalten blieb. Mit Hilfe von massen-spektrometrischen Analysen konnten 128 Proteine identifiziert werden, die spezifisch in den Kernpräparaten von Zellen angereichert sind, in denen Transspleißen blockiert ist. Darunter befinden sich Proteine, die ihre Lokalisation in den Kern hinein oder zu den Kernporen hin verändern, sowie viele Proteine, die sich in NPGs bewegen. Einige dieser Proteine stellen vielversprechende Kandidaten für eine potenzielle Rolle in der Kernexportkontrolle dar, da die Veränderungen in der Lokalisation (zum Kern oder zu den Kernporen) spezifisch für die Akkumulation von nicht gespleißten mRNAs waren. Das hier erarbeitete NPG-Proteom enthält fast ausschließlich Proteine, die am mRNA-Metabolismus beteiligt sind und nur in Trypanosomen vorkommen. Insbesondere fehlen im NPG-Proteom wichtige Translationsinitiationsfaktoren. Die Daten zeigen, dass NPGs RNP-Komplexe sind, die den Export aus dem Zellkern bereits begonnen oder abgeschlossen haben, aber noch nicht mit dem Translationsprozess begonnen haben. Als Nebenprodukt dieser proteomischen Analyse, kann ein qualitativ hochwertiger Datensatz des noch unbekannten Kernproteoms von T. brucei bereitgestellt werden. Damit wird eine wichtige Wissenslücke bei der Forschung zur Trypanosomenbiologie, insbesondere zu nuklearen Prozessen geschlossen. Im Rahmen dieser Arbeit wurden außerdem NPGs anhand mikroskopischer Untersuchungen detaillierter charakterisiert. Die Granula sind zytoplasmatisch und liegen in mindestens zwei verschiedenen Lebenszyklusstadien von Trypanosomen vor. Es gibt mindestens zwei Untergruppen der Granula mit Unterschieden in der Proteinzusammensetzung. Eine genauere elektronenmikroskopische Analyse ergab, dass es sich bei NPGs um elektronendichte Strukturen handelt, die durch fadenartige Strukturen mit den Kernporen verbunden sind. Um mehr über die potenzielle Funktion von NPGs herauszufinden, wurde zum einen die Hypothese untersucht, dass NPGs mit perinuklearen Keimgranula adulter Gonaden in C. elegans verwandt sind: Es konnte keine Beziehung zwischen den beiden Granulatypen gefunden werden. Zum anderen zeigten erste Experimente mittels Einzelmolekül-mRNA-FISH in Trypanosomen keine Akkumulation von ungespleißten Transkripten in NPGs, was gegen eine Beteiligung an mRNA-Qualitätskontrollmechanismen spricht.

No description available.

As doenças tropicais negligenciadas, que atingem as populações mais carentes do mundo, representam em termos humanitários e socioeconômicos uma grande preocupação global. As tripanossomíases estão entre as doenças parasitárias mais importantes, e, particularmente, a tripanossomíase africana, ou doença do sono, destaca-se como uma grave condição de saúde, causada pelo parasita unicelular Trypanosoma brucei. Dentre os principais alvos metabólicos considerados para o desenvolvimento de novos fármacos para o tratamento das tripanossomíases, a glicólise recebe especial atenção em função de seu papel vital no processo de produção de ATP para o parasita que vive na corrente sanguínea. Esta tese de doutorado tem como objetivo identificar novos candidatos a inibidores da enzima aldolase (EC 4.1.2.13) da via glicolítica de T. brucei. Considerando-se que o alvo macromolecular em questão é validado para o planejamento de fármacos, inibidores desta enzima são candidatos a novos agentes quimioterápicos. Este trabalho explora a integração de métodos experimentais e computacionais através de estratégias de planejamento de fármacos baseado na estrutura do receptor (SBDD, na sigla inglesa para structure-based drug design) e na estrutura do ligante (LBDD, na sigla inglesa para ligand-based drug design) para a identificação de inibidores da enzima alvo. Foram produzidos resultados significativos, tais como a identificação através de triagens virtuais em larga escala de novas moléculas capazes de inibir a atividade da aldolase. Adicionalmente, destaca-se a obtenção de protocolos de expressão, purificação e cristalização para a enzima alvo. Como parte da estratégia de identificação de novos inibidores da aldolase, foram desenvolvidos modelos de QSAR 2D e 3D e estudos de dinâmica molecular. / Neglected tropical diseases, which affect the poorest populations across the developing world, are a major global concern. The trypanosomiases are amongst the most serious neglected tropical diseases, and particularly, African trypanosomiasis (sleeping sickness), caused by the unicellular parasite Trypanosoma brucei, appears as a fatal condition. The glycolytic pathway emerges as a promising target among the metabolic pathways for the development of new drugs, due to its essential role in the ATP generating process in the bloodstream form of the parasite. The goal of this work is to identify new inhibitors for the glycolytic enzyme aldolase (EC 4.1.2.13) from Trypanosoma brucei. Inhibitors of this enzyme are drug candidates with high potential for clinical development, as the respective target enzyme was validated as a molecular target for the therapy of trypanosomiasis. The strategy employed in this study includes the integration of SBDD (structure-based drug design) and LBDD, (ligand-based drug design) for the identification of inhibitors of the target enzyme, through the combination of computational and experimental methodologies. Significant results were obtained, such as the identification of new small molecule inhibitors of the aldolase enzyme through high-throughput virtual screening. Additionally, it is highlighted the standardization of expression, purification and crystallization protocols for the target enzyme. As a component of the strategy for the identification of novel aldolase inhibitors, 2D and 3D QSAR models were developed, as well as molecular dynamics studies.

A tripanossomose africana, ou doença do sono, como é popularmente conhecida, atingiu 25 mil pessoas só em 1995. Estima-se que a doença seja responsável pela morte de 10.000 pessoas a cada ano, segundo os dados da Organização Mundial da Saúde. Sua distribuição é exclusivamente africana, devido ao fato de sua transmissão depender da Tsé-Tsé, mosca do gênero Glossina que encontra lá condições favoráveis para sua proliferação. Atualmente os medicamentos existentes não são eficientes e devem ser ministrados em doses altas, provocando graves efeitos colaterais. O parasita Tripanosoma brucei, na forma encontrada no sangue dos mamíferos (tripomastigota), é o responsável pela doença e, assim como toda a família Trypanosomatidea, é altamente dependente de glicose. Esses parasitas consomem a quantidade de glicose equivalente à sua massa em aproximadamente sete horas. A via glicolítica torna-se, portanto, chave para o desenvolvimento de inibidores que possam ser utilizados no combate a estes parasitas. Nesse sentido, temos realizado estudos com a enzima glicose-6-fosfato isomerase (glucose-6-fosfato isomerase; Phosphoglucose Isomerase PGI; EC 5.3.1.9), responsável pela isomerização reversível da D-glicose-6-fosfato e D-frutose-6-fosfato e participa na glicólise como a segunda enzima da via. Os trabalhos realizados até aqui culminaram na sua expressão em forma recombinante, purificação através de coluna de afinidade, e caracterizações enzimáticas. Sua atividade específica foi determinada através de métodos já estabelecidos encontrados na literatura. O IC50 da enzima frente a quatro inibidores da reação foi determinado. O trabalho finaliza com a construção de um modelo estrutural da enzima determinado através de métodos de modelagem molecular por homologia. / The African trypanosomiasis, or sleeping sickness as is popularly known, affected 25 thousand people only in 1995. It is estimated that the disease is responsible for 10 thousand deaths per year, according to data provided from the World Health Organization (WHO). The distribution of the disease is exclusively African due to the transmission being dependent on the tsé-tsé vector. A fly, belonging to the Glossinia genus, finds in the African continent favorable conditions for its proliferation. Presently the existing drugs are not efficient and have to be applied in high dosage, resulting in severe side effects. The bloodstream form (tripomastigote) of the parasite Trypanosoma brucei is responsible for the disease and such as the whole Trypanosomatidae family is dependent on glucose. Those parasites consume a quantity of glucose equivalent to its mass in approximately seven hours. This characteristic results in the glycolitic pathway been a key target for drug development against those parasites. In this direction we are developing research with the enzyme glucose-6-phosphate isomerase (Phosphoglucose lsomerase PGI; EC 5.3.1.9) responsible for the reversible isomnerisation of D-glucose6-phosphate and D-fuctcose-6-phosphate. PGI participates as the second enzyme in the glycolytic pathway. The work developed so far resulted in the expression of the recombinant form of the parasite PGI, its affinity purification and enzimatic characterization. The specific activity was determined with established methods. The IC50 of four inhibitors was determined and a structural model of T brucei PGI was built by molecular modeling techniques.

A tripanossomose africana, ou doença do sono, como é popularmente conhecida, atingiu 25 mil pessoas só em 1995. Estima-se que a doença seja responsável pela morte de 10.000 pessoas a cada ano, segundo os dados da Organização Mundial da Saúde. Sua distribuição é exclusivamente africana, devido ao fato de sua transmissão depender da Tsé-Tsé, mosca do gênero Glossina que encontra lá condições favoráveis para sua proliferação. Atualmente os medicamentos existentes não são eficientes e devem ser ministrados em doses altas, provocando graves efeitos colaterais. O parasita Tripanosoma brucei, na forma encontrada no sangue dos mamíferos (tripomastigota), é o responsável pela doença e, assim como toda a família Trypanosomatidea, é altamente dependente de glicose. Esses parasitas consomem a quantidade de glicose equivalente à sua massa em aproximadamente sete horas. A via glicolítica torna-se, portanto, chave para o desenvolvimento de inibidores que possam ser utilizados no combate a estes parasitas. Nesse sentido, temos realizado estudos com a enzima glicose-6-fosfato isomerase (glucose-6-fosfato isomerase; Phosphoglucose Isomerase PGI; EC 5.3.1.9), responsável pela isomerização reversível da D-glicose-6-fosfato e D-frutose-6-fosfato e participa na glicólise como a segunda enzima da via. Os trabalhos realizados até aqui culminaram na sua expressão em forma recombinante, purificação através de coluna de afinidade, e caracterizações enzimáticas. Sua atividade específica foi determinada através de métodos já estabelecidos encontrados na literatura. O IC50 da enzima frente a quatro inibidores da reação foi determinado. O trabalho finaliza com a construção de um modelo estrutural da enzima determinado através de métodos de modelagem molecular por homologia. / The African trypanosomiasis, or sleeping sickness as is popularly known, affected 25 thousand people only in 1995. It is estimated that the disease is responsible for 10 thousand deaths per year, according to data provided from the World Health Organization (WHO). The distribution of the disease is exclusively African due to the transmission being dependent on the tsé-tsé vector. A fly, belonging to the Glossinia genus, finds in the African continent favorable conditions for its proliferation. Presently the existing drugs are not efficient and have to be applied in high dosage, resulting in severe side effects. The bloodstream form (tripomastigote) of the parasite Trypanosoma brucei is responsible for the disease and such as the whole Trypanosomatidae family is dependent on glucose. Those parasites consume a quantity of glucose equivalent to its mass in approximately seven hours. This characteristic results in the glycolitic pathway been a key target for drug development against those parasites. In this direction we are developing research with the enzyme glucose-6-phosphate isomerase (Phosphoglucose lsomerase PGI; EC 5.3.1.9) responsible for the reversible isomnerisation of D-glucose6-phosphate and D-fuctcose-6-phosphate. PGI participates as the second enzyme in the glycolytic pathway. The work developed so far resulted in the expression of the recombinant form of the parasite PGI, its affinity purification and enzimatic characterization. The specific activity was determined with established methods. The IC50 of four inhibitors was determined and a structural model of T brucei PGI was built by molecular modeling techniques.