O nosso grupo está explorando como alternativa para a identificação de novas estratégias de tratamento o reposicionamento de drogas. Nesse contexto, nosso laboratório mostrou que memantina, um antagonista de receptores de glutamato NMDA, apresenta uma atividade tripanocida no ciclo de vida do T. cruzi, in vitro. Com base nessas informações, avaliamos o efeito da memantina em animais infectados com a cepa Y. O tratamento apresentou uma redução da parasitemia (40%) no 8º dpi e redução da carga parasitária no tecido cardíaco no 15º dpi. Uma vez que a cepa Y invade preferencialmente macrófagos, avaliamos o efeito de memantina em células RAW 264.7. Memantina diminuiu a produção de NO por células estimuladas com LPS, diminuiu o Ca2+i, e não houve uma redução de EROs. Memantina também interferiu no ciclo intracelular do parasita, reduzindo o número de células infectadas. Nossos resultados sugerem que o tratamento com memantina pode direcionar o sistema imune de modo a modular os receptores de tipo NMDA, através de um efeito direto ou indireto produzido pelo tratamento. / The search for new therapeutic targets has been of extreme importance for the specific treatment of Chagas disease. Our group is exploring as an alternative, a strategy for drug repurposing. In this context, our laboratory showed that memantine, an antagonistic of NMDA glutamate receptors, has a trypanocidal activity along the life cycle of T. cruzi, in vitro. Based on this information, we evaluated the effect of memantine in animals infected with Y strain. The treatment showed a reduction of parasitemia (40%) in 8th dpi and reduced parasitic load in cardiac tissues at 15th dpi. As the Y strain preferably invades macrophages, we evaluated the effect of memantine in RAW 264.7 cells. Memantine decreased NO production by cells stimulated with LPS, decreased Ca2+i, and did not cause a reduction in ROS. Memantine also interfere with the intracellular parasite cycle, reducing the number of infected cells. Our results suggest that treatment with memantine can target the immune system to modulate the NMDA receptor, through direct or indirect effect caused by the treatment.

Foi demonstrado que a L-prolina é fundamental para o metabolismo das formas intracelulares de T.cruzi e está envolvida em processos de resistência a diferentes condições de estresse. O ácido L-tiazolidina-4-carboxílico (T4C) age como inibidor competitivo do transporte de prolina para o interior do parasita, desta forma, pode-se assumir que, quando aplicado como agente terapêutico simula uma situação de hipoprolinemia no hospedeiro mamífero. Sobre esta base, propomos que a prolinemia poderia estar relacionada com a severidade da infecção pelo T. cruzi, portanto, decidimos inverter o racional e obter nesse trabalho um modelo de hiperprolinemia. Inicialmente foi estabelecido um modelo murino de hiperprolinemia transiente. Camundongos da linhagem BALB/c que foram tratados com prolina, por via intraperitonial (i.p), em 30 minutos já apresentaram concentrações plasmáticas de 1,359 ± 0,121 mM, porém, após 3 horas seus níveis plasmáticos retornaram ao normal, 0,4361 ± 0,03496. Utilizando este modelo, foi avaliado o efeito da hiperprolinemia transiente em camundongos infectados pelo T.cruzi. Em três ensaios de um total de seis foi observado um aumento significativo da parasitemia em camundongos tratados, sem nenhuma diferença na mortalidade e na carga parasitária de diversos tecidos. Essa inconsistência observada no perfil da parasitemia redirecionou os experimentos para um modelo de hiperprolinemia hereditário previamente estabelecido. Interessantemente, foram observados diminuições na parasitemia, porém, a mortalidade foi aumentada. Foi hipotetizado que, diferentemente do que acontece com a hiperprolinemia transiente, com os níveis plasmáticos aumentados de maneira estável, as formas intracelulares de T.cruzi teriam acesso ao aminoácido em quantidades e tempo maiores. No entanto, não se pode descartar como hipótese complementar, que a hiperprolinemia possa afetar a resposta imune e por sua vez, imunossuprimir ou imunoestimular o hospedeiro mamífero. Por esse motivo, avaliaram-se alguns parâmetros da resposta imune ex vivo. Ensaios ex vivo mostraram que tratamento com prolina diminui a produção de NO sob ativação por LPS, no entanto quando células peritoneais não ativadas por LPS são infectadas por T. cruzi, o tratamento com prolina não altera o perfil de NO. A expressão gênica da óxido nítrico sintase induzível (iNOS) das células peritoneais diminui quando elas são cultivadas na presença de prolina, confirmando esses resultados. Mostrou-se dessa maneira que a hiperprolinemia pode interferir com a resposta imune do hospedeiro, o que levaria a uma eventual imunossupressão. Observou-se que, tanto células peritoneais infectadas como não infectados tratadas com prolina apresentam redução de seu volume celular o que poderia ser indício de sinal apoptótico. Ensaios de infecção (ex vivo) em células peritoneais de camundongos BALB/c com tripomastigotas da cepa MJL superexpressando o transportador de prolina apresentaram aumento da taxa de infecção enquanto que as infectadas por tripomastigotas da cepa Y superexpressando o mesmo transportador de prolina apresentaram diminuição da taxa de infeçção, quando comparadas aos controles, mostrando que a reposta de redução ou aumento da taxa de infecção ao tratamento com prolina é determinada também pela cepa de T. cruzi. Análises da prolinemia em soro de pacientes com sintomas de cardiopatia chagásica severa mostraram menores níveis de prolina sérica quando comparados aos controles (pacientes não infectados). Juntos, todos esses resultados colocam a prolina como um fator de severidade na infecção pelo T. cruzi. / It was shown that L-proline is essential for the metabolism of the intracellular forms of T. cruzi and is involved in resistance to different stress conditions. L-thiazolidine-4-carboxylic acid (T4C) acts as a competitive inhibitor of the proline transport to the inside of parasite, thus it can be assumed that when T4C is applied as a therapeutic agent simulates a hipoprolinemia situation in the mammalian host. On this basis, we propose that prolinemia could be related to the severity of T. cruzi infection, then, in this work we decided to reverse the rational and obtain a hyperprolinemia model. As a starting point, it was established a mouse transient hyperprolinemia model. BALB/c mice were intraperitoneally injected with proline showed an increased proline levels in sera after 30 min (1,359 ± 0,121 mM). However, these increased levels were diminished to normal levels after 3 h (0,4361 ± 0,03496 mM). Once established this model, was initially used to evaluate the effect of transient hyperprolinemia in mice infected by T.cruzi. In three out of six experiments an increase in parasitemia but not in mortality or in tissue parasite loads was observed. In the remaining three experiments no differences were detected. These inconsistencies directed the work to the search to a previously established hereditary model of mouse hyperprolinemia model. Interestingly, in this new model, a diminished parasitemia was recorded, however, mortality was higher. From this information it was hypothesized that, differently to what happens in the transient hyperprolinemia, a permanent hyperprolinemia exposes the T. cruzi infected cells (and so, the intracellular parasites) to higher concentrations of proline as well as for longer times. In addition, it cannot be disregarded the complementary hypothesis that hyperprolinemia could be affecting the immune response and, at the same time of its action on the parasite, it could be immunosupresing or immunostimulating the mammalian host. This possibility led us to evaluate some parameters of the immune response both, in vivo and ex vivo. Ex vivo assays showed that proline-treated LPS-activated peritoneal cells had a diminished production of NO while proline-treatment of T. cruzi infected, non-LPS-activated peritoneal cells did not affect their NO production. This data showed that hyperprolinemia could interfer the immune response leading the host to an eventual immunosupresion. In addition, both, infected and non-infected macrophages had their cellular volume diminished when treated with proline, which could be attributed to the iniciation of an apoptotic process. Infection assays (ex vivo) of perioneal cells from BALB/c mice with MJL strain trypomastigotes overexpressing a proline transporter had an increased infection rate, while the same type of cells infected with Y strain trypomastigotes overexpressing the same proline transporter showed a diminution in the infection rate. These results show that changes in the infection rate as a function of intracellular proline availability depends on T. cruzi strain. The analysis of prolinemia in patients serum with symptoms of Severe Chronic Chagasic Cardiopathy showed tat proline levels were diminished in comparison to control (non-infected patients). Taken together, these results prompt proline as a factor modulating the severity of T. cruzi infection.

The aims of the research presented in this thesis were concerned with investigating the effect of inducible nitric oxide (NO) synthase expression during Trypanosoma brucei infections on both host and parasite. NO was shown to exhibit a potent cytostatic effect on parasite proliferation. Oxyhaemoglobin is a potent scavenger of NO. The cytostatic effects of NO on the trypanosomes were completely prevented through the addition of erythrocytes to the cultures. This implies that in the host blood-stream, NO is unlikely to be involved in the eradication of the parasites. Through the adoptive transfer of suppressor macrophages from T.brucei-infected donor mice to naive recipients, it was demonstrated that NO mediates a suppressive effect on host lymphocyte responses in vivo. Furthermore, suppressor macrophages were shown to have a finite life-span and undergo NO-mediated apoptosis. Evidence also suggested that elevated NO production in the bone marrow of T.brucei -infected mice is likely to play a significant role in the anaemia resulting from T.brucei infection. Experiments demonstrated that a soluble lysate prepared from freeze-thawed blood-stream form T.brucei, activated interferon (IFN)-gamma primed macrophages to express high levels of NO synthase. Experiments also demonstrated that viable blood-stream forms, but not procyclic form trypanosomes, released a soluble factor which in combination with IFN-gamma induced NO synthase. The absolute requirement of IFN-gamma priming for NO synthase activation by T.brucei was studied using macrophages from mutant mice lacking functional IFN-gamma receptors (IFN-gamma R -/- mutant mice). In comparison to macrophages from wild-type mice, cells from IFN-gamma-R-/- mutant mice were unable to produce NO following stimulation in vitro or infection in vivo. Finally, utilising mice with specific immunodeficiencies it was demonstrated that natural killer cells and a/b T-lymphocytes were important sources of IFN-gamma during murine T.brucei infections.

Memoria para optar al Título Profesional de Médico Veterinario / Se estudió la presencia de Trypanosoma cruzi en roedores silvestres y cosmopolitas (reservorios del ciclo silvestre), en sectores de las comunas de Calera de Tango y Til-Til, ubicados a 16 y 59 Km. respectivamente de la ciudad de Santiago. Se capturó un total de 138 roedores incluyendo ejemplares de Octodon degus, Rattus rattus, Phyllotis darwini, Abrothrix olivaceus y Abrocoma bennetti. Se les extrajo 0,5 ml de sangre mediante punción cardiaca, con el objetivo de determinar la frecuencia de T. cruzi por medio de la reacción de la polimerasa en cadena (PCR). Las muestras fueron sometidas a extracción del ADN contenidas en ellas, amplificación y detección de la región hipervariable de los minicírculos del ADNk de T. cruzi. Con el fin de visualizar el producto de la amplificación, se sometieron a electroforesis en gel de agarosa y se observó en transiluminador de luz ultravioleta. Se detectó la presencia de T. cruzi en 19 (14,84%) de las 128 muestras, diez de R. rattus, (7,25%), siete de O. degus, (5,07%), y dos de P. darwini, (1,45%). Los ejemplares de A. olivaceus y A. bennetti resultaron negativos. Estos resultados demostraron la circulación del parásito en la comunidad de micromamíferos, de lo que se puede concluir que los triatominos usan la sangre de roedores (silvestres y sinantrópicos), como alimento lo que asegura el mantenimiento del ciclo silvestre y peridoméstico del parásito, en las zonas rurales estudiadas en el presente trabajo

Memoria para optar al Título Profesional de Médico Veterinario / En cirugía ortopédica, el uso de factores tróficos que favorezcan la formación de tejido de granulación y eviten o retrasen la formación de nuevo hueso es deseable en resoluciones quirúrgicas de ciertas patologías en pequeños animales. Calreticulina (CRT) es una proteína chaperona que asegura el correcto plegamiento de proteínas y actúa en la regulación del metabolismo del calcio dentro del retículo endoplásmico (RE). Esta proteína ha sido localizada además fuera de la célula donde cumple variadas funciones, entre ellas, favorece la formación de tejido de granulación en piel y además, inhibe la formación de hueso. Es así como, en este estudio se evaluó y se comparó el efecto de CRT recombinante humana (rHuCRT) y parasitaria proveniente de Trypanosoma cruzi (rTcCRT) sobre la regeneración ósea, mediante liberación controlada desde una esponja de quitosano al interior de una lesión circular en hueso cortical de conejo. rHuCRT y rTcCRT demostraron inhibir la regeneración ósea in vivo, sin embrago, rTcCRT demostró ser más eficaz en esta función que su contraparte humana / Financiamiento: Proyecto Fondecyt 3100021

Memoria para optar al título de Bioquímico / El agente etiológico de la enfermedad de Chagas es el parásito hemoflagelado Trypanosoma cruzi (T. cruzi). Esta enfermedad posee un carácter endémico en América Latina y se estima un promedio de 8 millones de personas infectadas. El ciclo de vida de T. cruzi involucra insectos vectores y mamíferos hospederos, en los cuales se diferencia en tres formas celulares finales: epimastigote (extracelular y replicativa) presente en los vectores triatominos; tripomastigote (extracelular no replicativa e infectiva) que se encuentra tanto en insectos vectores como en hospederos mamíferos; y amastigote (intracelular y replicativa) presente en hospederos mamíferos. En ambos hospederos, T. cruzi se encuentra sometido a la acción de especies reactivas de oxígeno y nitrógeno (ROS/RNS) que dañan su DNA. No obstante, el parásito sobrevive a la acción del estrés oxidativo. En la mayoría de los eucariontes, el daño oxidativo al DNA es reparado principalmente por la vía de reparación por escisión de bases (BER), proceso conservado en el que participan una serie de proteínas con actividad enzimática, siendo fundamentales las endonucleasas apurínicas/apirimidínicas (endonucleasas AP). En el genoma de T. cruzi se identificó la secuencia de la endonucleasa AP TcAP1, homóloga al endonucleasa AP de humano (APE1). Se demostró que la sobrexpresión de esta enzima incrementa la viabilidad de epimastigotes y tripomastigotes expuestos a ROS/RNS y que la inhibición de la actividad de TcAP1 genera el efecto opuesto; es decir, disminuye la viabilidad de los parásitos tratados con agentes oxidantes. Actualmente, se sabe que APE1 humana se relaciona directamente con otras enzimas de la vía BER y modula la expresión de proteínas que participan de otras vías de reparación del DNA. Hasta el momento no se ha determinado si TcAP1 se asocia a otras proteínas, como ocurre con su ortóloga APE1 en humanos. En esta memoria de título se propuso generar herramientas que permitan a futuro identificar las proteínas parasitarias asociadas a la endonucleasa TcAP1 de T. cruzi, las cuales podrían modular o ser moduladas por ella. Para tales efectos, se diseñaron dos construcciones plasmidiales (pTREX-(ha)3-(flag)3-tcap1 y pTREX-cmyc-tcap1) con el objetivo de generar proteínas de fusión de TcAP1 asociadas a diferentes epítopes ((HA)3-(FLAG)3 o c-Myc). Estos vectores de expresión se transfectaron en XIepimastigotes de la cepa Y de T. cruzi, los que se seleccionaron con el antibiótico G-418. Solo se obtuvieron epimastigotes transfectantes que expresaron la proteína de fusión N-cMyc-TcAP1, evidenciado mediante ensayos de Western blot. Utilizando un kit comercial (anti-cMyc), se realizaron ensayos de inmunoprecipitación sobre homogeneizados de proteínas totales obtenidas desde los epimastigotes transfectantes que expresan N-cMyc-TcAP1 y epimastigotes control no transfectados. Las proteínas obtenidas se separaron mediante electroforesis bidimensional en condiciones desnaturantes con el objetivo de evidenciar patrones electroforéticos diferenciales entre parásitos transfectantes y controles. Las proteínas detectadas de manera exclusiva para los parásitos transfectantes se enviaron a identificar mediante espectrometría de masa, sin embargo, no se obtuvieron resultados concluyentes. A pesar de los problemas presentados, esta memoria de título presenta una gran relevancia para nuestro laboratorio, ya que permitió generar herramientas para la futura identificación de proteínas reguladoras de la actividad de TcAP1 o que son reguladas por ella / The etiologic agent of Chagas disease is the hemoflagellate parasite Trypanosoma cruzi (T. cruzi). This disease is endemic in Latin America and an estimated 8 million people are infected. The life cycle of T. cruzi involves triatomine insect vectors and mammalian hosts, in which it differs in three final cellular forms: epimastigote (extracellular and replicative) present in triatomine vectors; trypomastigote (non-replicative and infective extracellular form) found in both, insect vectors and mammalian hosts; and amastigote (intracellular and replicative) present in mammalian hosts only. Inside both hosts, T. cruzi is exposed to reactive oxygen and nitrogen species (ROS/RNS) that damage the parasite DNA, however the parasite survives. In most eukaryotes, oxidative DNA damage is repaired mainly by the base excision repair pathway (BER), a conserved mechanism, in which a series of proteins with enzymatic activity participate, being apurinic/apyrimidinic endonucleases (AP endonuclease) essential to this process. In the T. cruzi genome the nucleotide coding sequence for an AP endonuclease (TcAP1), homologous to human AP endonuclease (APE1), was identified. In previous reports it has been demonstrated that the overexpression of this enzyme in T. cruzi, increases the viability of epimastigotes and trypomastigotes exposed to ROS/RNS and that the inhibition of TcAP1 activity generates the opposite effect, decreasing the parasite viability. Currently, human APE1 is known to directly interact with other enzymes of the BER pathway and modulates the expression of proteins involved in other DNA repair pathways. To date, it has not been determined whether T. cruzi TcAP1 is associated with other proteins, similar to those reported for APE1 ortholog human protein. In this work, it was proposed to generate tools that allow the future identification of parasitic proteins that interacts with the TcAP1 endonuclease, which could modulate their activity or or proteins that modulate their activity product of its interaction with TcAP1. For this purpose, two plasmid constructs (pTREX-(ha)3-(flag)3-tcap1 and XIIIpTREX-cmyc-tcap1) were designed with the aim of generating TcAP1 fusion proteins associated with different tags ((HA)3-(FLAG)3 or c-Myc). These expression vectors were transfected into epimastigotes of the T. cruzi Y strain, which were selected with the G-418 antibiotic. Only transfectant epimastigotes expressing the N-cMyc-TcAP1 fusion protein, evidenced by Western blot assays, were obtained. Using a commercial kit (anti-cMyc), immunoprecipitation assays were performed on total protein homogenates obtained from transfectant epimastigotes expressing N-cMyc-TcAP1 and untransfected control epimastigotes. The proteins obtained were separated by two-dimensional electrophoresis under denaturing conditions with the aim of evidencing differential electrophoretic patterns between transfectant and control parasites. The proteins exclusively detected for the transfectant parasites were sent to be identified by mass spectrometry. However, these could not be identified. Despite the presented problems, this title memory presents a great relevance for our laboratory, since it allowed to generate tools for the future identification of proteins that interacts with the TcAP1 enzyme

The biosynthesis of GDP-Fucose, the activated donor for fucose, has been recently shown to be essential in the parasite Trypanosoma brucei. Fucose is a common sugar modification on eukaryotic glycan structures, but it has not been well described in trypanosomatids. To elucidate the role of fucose in T. brucei we searched for putative fucosyltransferases in this parasite. A single putative T. brucei fucosyltransferase (TbFT) was identified and recombinantly expressed in Escherichia coli. The protein was active and structural characterization of its reaction product identified it as a GDP-Fuc: ß-D-galactose a-1,2-fucosyltransferase with preference for Galß1,3GlcNAc containing structures as glycan acceptors. A procyclic form conditional null mutant for TbFT was generated and this glycosyltransferase shown to be essential for parasite growth in vitro, with the mutant cells displaying a slightly abnormal morphology and an apparent reduction in the surface high molecular weight glycoconjugate complex. Here we also describe the various experimental approaches that were used to try to identify the fucosylated glycocojugates in T. brucei. Lastly, to better understand the biosynthesis of GDP-Mannose, the starting metabolite for the biosynthesis of GDP-Fuc, we biochemically characterized T. brucei phosphomannomutase (TbPMM). Here we show this enzyme could interconvert not only mannose-phosphates, but also glucose-phosphates.

The distribution of trypanosomes infecting wild ruminants of North America has only recently been investigated . Many isolates have been mensurally studied and were determined to be conspecific with Trypanosoma cervi. Widely divergent forms exist however, between host species and seasonally within a host . To determine the validity of all inclusions in the taxon and the extent of intraspecific variability , trypanosome isolates of moose, reindeer , antelope, muledeer, Roosevelt Elk and two discrete transplant populations of Rocky Mountain elk were characterized and differentiated using recombinant DNA techniques.

Die ausschließliche Expression von nur einem Gen aus einer großen Genfamilie ist ein weit verbreitetes Phänomen, das als monoallele Expression bezeichnet wird. In dem Blutparasiten Trypanosoma brucei stellt die Expression eines einzigen variablen Oberflächenglykoproteins (VSG) aus einem Repertoire von über 1000 verschiedenen Genen die Grundlage für die Immunevasion dar. Durch einen periodischen Wechsel der VSG Expression (Antigene Variation) bleibt der Parasit vom Immunsystem des Wirtes unerkannt. Die VSG Gene werden aus telomerischen Blutstromform Expressionsstellen (BES) transkribiert, von denen nur eine zu einem bestimmten Zeitpunkt aktiv ist. Die Kontrolle der monoallelen VSG Expression ist somit einer der wichtigsten Virulenzfaktoren von T. brucei. Ziel dieser Arbeit war es, die Vorgänge eines transkriptionellen Wechsels zwischen zwei BESs zu beschreiben. Das Ausschalten des aktiven VSGs durch RNA-Interferenz hatte zuvor gezeigt, dass dies nicht zu einer erhöhten Wechselrate führt. Es wurde daher untersucht, welche Auswirkungen das Anschalten einer zweiten BES auf die monoallele Expression hat. Da es bisher keine Möglichkeit gibt, eine inaktive BES gezielt zu aktivieren, wurde ein artifizielles System gewählt, das die gezielte induzierbare Expression eines Gens ermöglicht. Die BESs unterscheiden sich in der Anzahl und Zusammensetzung der Expressionsstellen-assoziierte-Gene (ESAGs), jedoch besitzt jede BES ein telomernahes VSG. Somit wird, bei einer BES Aktivierung, in jedem Fall ein neues VSG exprimiert. Durch die induzierbare Expression eines zweiten VSGs wurde so das Anschalten einer neuen BES simuliert. Mithilfe dieses Systems konnte gezeigt werden, dass das VSG selbst für die Kontrolle der monoallelen Expression verantwortlich ist. Die ektopische Überexpression eines zweiten VSGs führte zu einer graduellen Inaktivierung der BES. Infolge dessen verlangsamte sich der Zellzyklus und die Zellen verblieben bis zu fünf Tage in einem ruhenden Zustand. Genauere Analysen dieses Zustandes zeigten, dass es sich hierbei um ein bisher unbekanntes, reversibles Zwischenstadium zwischen proliferierenden sogenannten Long Slender und arretierten sogenannten Short Stumpy Formen handelt. Die Ergebnisse dieser Arbeit führten zu einem neuen Modell, das die Kontrolle der monoallelen VSG Expression mit der Entwicklung der Trypanosomen mechanistisch verbindet. / The exclusive expression of only one gene from a gene family is a common phenomenon, known as monoallelic expression. The blood parasite Trypanosoma brucei evades the host immune system by expressing only one variant surface glycoprotein (VSG) from a repertoire of hundreds of different VSG genes. By periodically switching VSG expression (antigenic variation) the parasites evade the host antibody response. The VSG genes are transcribed from specialized telomeric bloodstream form expression sites (BESs), of which only one is active at any given time. Thus, monoallelic VSG expression is one of T. brucei's most important virulence factors. The aim of this work was to describe the processes occuring while transcription switches from one BES to another. The depletion of the active VSG by RNA interference (RNAi) was shown previously to have no effect on switching frequency. It was therefore investigated here, which influence the activation of a new BES would have on monoallelic expression. So far, it has not been possible to specifically activate a silent BES. Therefore, an artificial system was chosen which allows for inducible expression of a particular gene. The BESs differ in number and composition of expression site associated genes (ESAGs), but all contain a telomeric VSG gene. Thus, activation of a new BES will inevitably lead to expression of a second VSG. To simulate - in the most straightforward manner - the activation of a new BES, a second VSG was inducibly expressed. Using this system, it was shown that the VSG itself controls its own monoallelic expression. The ectopic overexpression of a second VSG led to a gradual inactivation of the BES. This, in turn, led to a prolonged cell division cycle and the cells remained in a dormant stage for up to 5 days. Further analyzes of this stage revealed a new, reversible intermediate stage between proliferating long slender and arrested short stumpy forms. The results of this work led to a new model that mechanistically links the control of monoallelic VSG expression and development in trypanosomes.

No description available.