Orientador: Iscia Teresinha Lopes-Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T21:43:20Z (GMT). No. of bitstreams: 1 Pascoal_ViniciusD'AvilaBitencourt_D.pdf: 6752850 bytes, checksum: 4a1854e85492962b7451dd4fceef2360 (MD5) Previous issue date: 2010 / Resumo: As epilepsias afetam aproximadamente 2% da população mundial. Dentre as diferentes síndromes epilépticas descritas, a epilepsia de lobo temporal (ELT) é a mais freqüente, representando 40% dos casos, os quais são frequentemente refratários ao tratamento clínico. Uma das ferramentas mais utilizadas para tentar compreender a fisiopatologia da ELT são modelos experimentais, os quais apresentam epileptogenicidade similar àquela observada em tecidos "epilépticos" humanos quando estudados ex vivo. Dentre os vários modelos disponíveis, aquele induzido pela pilocarpina, além de muito bem estabelecido em nosso meio, tem ampla caracterização molecular, histológica, fisiológica e fenomenológica. Estudos moleculares prévios nesse modelo experimental já demonstraram o aumento da expressão de vários genes durante a fase aguda (fase de estado de mal epiléptico), porém, permanece ainda obscuro, quais desses genes e/ou vias de sinalização poderiam ser críticos para a determinação das lesões (morte celular em regiões hipocampais específicas determinando a chamada esclerose mesial temporal) e responsáveis pela epileptogênese na fase crônica do modelo (crises parciais complexas espontâneas). Baseando-nos nesses achados prévios propomos investigar diretamente o papel do gene interleucina 1- b (Il1b), o qual tem a putativa função de aumentar a liberação de glutamato durante a fase aguda do modelo da pilocarpina. Nossa estratégia experimental foi baseada no silenciamento pós-transcricional pela técnica de interferência por RNA (RNAi) aplicada "in vivo". Nossos resultados indicam que o gene Il1b tem um papel relevante no controle da homeostase no SNC, pela alteração da atividade e expressão de fatores de transcrição, com 22 impacto na expressão de recaptadores de glutamato e na taxa de mortalidade dos animais pós status epilepticus (SE). Além disso, o silenciamento do gene Il1rn, foi capaz de diminuir a morte neuronal nas regiões CA1, CA3 e giro denteado do hipocampo, como observado no material obtido dos animais na fase crônica. No entanto, apesar das ações da Il1b que observamos na fase aguda, o aumento da sua ação pelo silenciamento do Il1rn, não foi capaz de alterar a ocorrência de crises crônicas recorrentes ou a reorganização sináptica (sprouting) observados na fase crônica. Portanto, nossos resultados demonstram que a alteração na expressão do gene Il1b foi capaz de mudar a resposta fenotípica dos animais na fase aguda e teve um impacto na redução da morte neuronal em regiões críticas do hipocampo na fase crônica, porém não parece ter um papel crítico na determinação da epileptogênese na fase crônica. Além disso, nosso trabalho demonstrou a viabilidade do uso da RNAi na investigação funcional de genes envolvidos na patogênese das epilepsias in vivo / Abstract: The epilepsies affect approximately 2% of the world population; among the different epilepsy syndromes temporal lobe epilepsy (TLE) is one of the most frequent, representing 40% of all patients, who are often refractory to medical treatment. One of the strategies used to understand the pathophysiology of TLE are experimental animal models, which have a similar epileptogenicity as compared to human 'epileptic' tissues studied ex vivo. Among the several animal models available, the pilocarpine induced model is very well established and it has extensive molecular characterization, as well as histological, physiological and phenomenological studies. Previous molecular studies in this experimental model hás demonstrated an increased expression of several different genes during the acute phase (status epilepticus); however, it remains unclear, which one of these genes and / or signaling pathways could be critical in determining cell death leading to mesial temporal sclerosis, which is responsible for the epileptogenesis in the chronic phase of the model (spontaneous complex partial seizures). Based on these previous findings we propose to directly investigate the role of Il1b, which could be involved in the increased release of glutamate during the acute phase of the pilocarpine model. Our experimental strategy was based on the post-transcriptional gene silencing by the technique of RNA interference (iRNA) applied in vivo. Our results indicate that the Il1b gene has an important role in controlling homeostasis in the CNS by modulating the activity and expression of transcription factors which appear to interfere with glutamate reuptake. In addition, we observed a phenotypic impact of Il1b gene silencing in the mortality rate of animals after status epilepticus (SE). 24 However, despite the effect of Il1b observed in the acute phase, the modulation of its expression by RNAi was not sufficient to change the frequency of recurrent seizure or synaptic reorganization observed in chronic phase. In addition, silencing Il1rn was able to decreased cell death in CA1, CA3 and dentate gyrus as observed in material obtained form animals in the chronic phase. However, in spite of the effect of Il1b in the acute phase, its increased expression by means of silencing of Il1rn was not able to change the occurrence of habitual seizures or synaptic reorganization, sprouting in the chronic phase. Therefore, our results demonstrate that changes in Il1b gene expression were able to impact the phenotype of the animals in the acute phase, as well as to reduce cell death in critical hippocampus regions in the chronic phase. However, it seems not to have a critical role in the epileptogenic process in the chronic phase. Furthermore, our study demonstrated the feasibility of using RNAi as a tool for functional studies of epileptogenesis in vivo / Doutorado / Neurociencias / Doutor em Fisiopatologia Medica

A raiva é uma zoonose que afeta todos os mamíferos e causa cerca de 55.000 mortes humanas por ano, causada pelo vírus da raiva. O vírus da raiva pertence à Ordem Mononegavirales, Família Rhabdoviridae e o Gênero Lyssavirus. Ultimamente, o Protocolo de Milwaukee é a base do tratamento humano, com indução do paciente ao coma e uso de massiva terapia antiviral. O protocolo, embora tenha sido utilizado duas vezes com sucesso, inclusive em um caso brasileiro, ainda requer aperfeiçoamentos. Neste sentido, a interferência por RNA (RNAi) é uma nova abordagem para terapia de doenças virais. O objetivo deste trabalho foi avaliar a inibição da replicação do vírus da raiva in vitro e in vivo utilizando RNAi. Para este fim, foram utilizados três siRNAs (siRNA 360, siRNA 652, siRNA 649) com a fita antisenso complementar ao mRNA da fosfoproteína (P) e três siRNAs (Le 1, Le 2, Le 3) contra o RNA líder do vírus da raiva. Para o ensaio in vitro foram utilizadas as amostras PV e 4005 (AgV3) do vírus da raiva e as células de BHK-21 (Baby hamster kidney). As monocamadas celulares foram infectadas com as amostras PV ou 4005 e depois de 2 horas de incubação transfectadas com cada um dos siRNAs em combinação com Lipofectamine 2000TM. Depois de 24 e 48 horas as placas teste e controle foram submetidas à imunofluorescência direta (IFD) com conjugado globulina de coelho anti-ribonucleocapsídeo do vírus da raiva/isotiocianato de fluoresceína (Instituto Pasteur de São Paulo). Os resultados revelaram que os siRNAs contra o RNA líder do vírus não foram capazes de inibir a replicação do vírus. A utilização dos siRNAs contra mRNA P resultaram em títulos de 3,625logTCID50/ml, 3,875logTCID50/ml e 4,125logTCID50/ml para os siRNAs 360, 649 e 652, respectivamente, enquanto que, para a placa controle, o título foi 4,0logTCID50/ml nas placas infectadas com PV e período de incubação de 24h. Nas placas infectadas com a amostra 4005 e tratadas com siRNAs, a maior queda de título viral foi na placa tratada com siRNA 360, de 1,0 log, comparando-se com a placa controle de incubação de 24 para a amostra 4005. Nas placas tratadas com siRNA 649 e siRNA 652, também houve a diminuição de título viral, mas em uma escala menor (0,25log e 0,125log, respectivamente) comparando-se com o controle. Nas placas infectadas com PV e incubadas durante 48h, os títulos apresentados foram de 5,625logTCID50/ml, 4,625logTCID50/ml e 4,75logTCID50%/ml para os siRNAs 360, 649 e 652, respectivamente, enquanto que na placa controle o título foi 6,0logTCID50C%/ml. A placa com período de incubação de 48h com a amostra 4005 e tratada com siRNA 360 apresentou a maior queda de título viral entre os três siRNAs, o que resultou em 1,125log de diferença. Nas monocamadas onde foram administrados siRNA 649 e siRNA 652, observou-se também uma pequena queda de título viral igual a 0,875log e 0,295log, respectivamente, comparando-se com a placa não tratada. Para o ensaio in vivo, foram usados camundongos albino suíços de 21 dias com peso entre 11 e 14g, infectados com a cepa PV e AgV3 em 10DL50% via intracerebral. Duas horas depois da infecção, foi inoculada por via intracerebral uma solução do siRNA 360 com Lipofectamine 2000TM. Os animais com paralisia foram eutanasiados e aqueles sobreviventes foram observados até completar 30 dias de observação quando foram, então, eutanasiados. O sistema nervoso central de todos os animas foi recolhido e submetido a IFD. A utilização do siRNA 360 em camundongos resultou em 30% de animais sobreviventes frente amostra 4005, enquanto que a mortalidade nos animais não tratados foi de 90%. Nos animais inoculados com a amostra PV e tratados com este siRNA, a sobrevivência foi de 40%, enquanto que no grupo controle a mortalidade foi de 100%. O resultado do ensaio in vitro demonstra que os siRNAs utilizados são capazes de inibir a replicação do vírus da raiva, com eficiência mais pronunciada para o siRNA 360. In vivo, este siRNA foi capaz de induzir a proteção parcial dos animais inoculados com as duas variantes virais. Estes resultados, ainda que indiquem a necessidade de mais estudos, permitem concluir que a RNAi é uma tecnologia promissora como antiviral contra a raiva / Rabies is a zoonotic disease that affects all mammals and causes more than 55.000 human deaths every year, caused by rabies virus (RABV) a virus of the Mononegavirales order, Family Rhabdoviridae and the Lyssavirus genus. After the onset of the symptoms, the illness has a fast progression and the patients feel intense physical suffering. Currently, human rabies treatment has been based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. Despite this protocol has been successful in two cases, including a Brazilian one, more studies on antivirals for human rabies treatment are required. RNA interference is a new antiviral approach, which gives hope to the possibility of rabies antiviral treatment. The aim of this study was to assess the decrease in titres of rabies virus in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs (siRNA 360, siRNA 652, and siRNA 649) were used with antisense strands complementary to rabies virus phosphoprotein (P) mRNA and three other (Le 1, Le 2, Le 3) to the leader RNA. Pasteur virus strain (PV) and strain 4005 (AgV3) of rabies virus and BHK-21 cells were used, and the monolayers were transfected with each of the RNAs with Lipofectamine-2000 TM. After 22 hours, the siRNA-treated and the control plates were tested by direct fluorescent antibody test (DFAT) with anti-rabies virus nucleocapsid antibody conjugate with fluorescein isothiocianate (Pasteur Insitutte, Brazil). The plates transfected with siRNA against phosphoprotein mRNA were also incubated for 48 hours and subjected to IFD assay. Virus titres were calculated by the Spearman-Karber method. The results showed that siRNAs against virus leader RNA were not able to inhibit the replication of the virus. The use of siRNAs against P mRNA resulting titres of 3.625logTCID50/ml 3.875logTCID50/ml and 4.125logTCID50/ml for siRNAs 360, 649 and 652, respectively, while, for the control plate, the titre was 4.0logTCID50/ml in plates with PV and 24h incubation period. In plates with strain 4005 and treated with siRNAs, the highest viral titre decrease was obtained with siRNA 360, with a 1.0 log difference compared to the control plate of strain 4005 incubated for 24h. The plates treated with siRNA 649 and siRNA 652 have was also shown a decrease in viral titres, but on a smaller scale (0.25log and 0.125log, respectively) compared to the control. The plates infected with PV and incubated for 48 hours showed titre of 5.625logTCID50/ml, 4.625logTCID50/ml and 4.75logTCID50%/ml for siRNAs 360, 649 and 652, respectively, while for the control plate the titre was 6.0logTCID50C%/ml. The plate with strain 4005 and then treated with siRNA360 and incubated for a total of 48h had the highest viral titre decrease among the three siRNAs, which resulted in a 1.125log difference compared to the control plate. In monolayers treated with siRNA649 and siRNA652 there was also a discrete drop in viral titres (0.875log and 0.295log, respectively) compared to the control plate. For the in vivo assay, 21-day old Swiss albino mice weighing between 11 and 14g were intracerebrally inoculated with PV or 4005 strains (10DL50%). Two hours after inoculation, a solution of siRNA360 with Lipofectamine 2000 TM was also intracerebrally injected. Mice presenting paralysis and those that survived the 30 days of observation were euthanized. The central nervous system of all animals was collected and submitted to IFD. The use of siRNA360 in mice resulted in survival of 30% of animals in the group inoculated with strain 4005, whereas 90% mortality was observed in the control group. In animals inoculated with the PV strain, and treated with siRNA360, the survival rate was 40% and in the control group the mortality was 100%. The results of the in vitro assay demonstrate that the siRNAs used are effective in inhibiting the replication of rabies virus with a more intense inhibition regarding siRNA 360. In vivo, this siRNA was able to induce partial protection of animals infected with both viral variants. These results also indicate that, despite the need for further studies, RNAi is a promising technology as antiviral against rabies

Orientador: Iscia Teresinha Lopes Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Médicas / Made available in DSpace on 2018-08-19T12:08:05Z (GMT). No. of bitstreams: 1 Marchesini_RafaelBreglio_D.pdf: 9525871 bytes, checksum: f90452b399007d69d5b96e4070e34d9a (MD5) Previous issue date: 2011 / Resumo: As epilepsias afetam aproximadamente 1% da população mundial, sendo que a dentre as diferentes síndromes epilépticas a epilepsia de lobo temporal (ELT) é a mais freqüente, e aquela com a maior proporção de pacientes adultos que apresentam crises refratárias ao tratamento clínico. Molelos animais induzidos têm sido já largamente utilizados para estudar a ELT, já que se considera que os mesmos apresentam epileptogenicidade similar àquela observada em tecidos "epilépticos" humanos quando estudados ex vivo. Dentre os vários modelos disponíveis, aquele induzido pela pilocarpina, além de muito bem estabelecido em nosso meio, tem já ampla caracterização, sendo dividido em três fases de acordo com os aspectos fenomenológicos, histopatológicos e moleculares: fase aguda, fase silenciosa e fase crônica. Baseado em padrões de expressão diferencial de genes na fase silenciosa do modelo, tem-se que a modificação de circuitos neuronais é um importante pré-requisito para a plasticidade funcional no sistema nervoso central sob condições patológicas. Levando-se em conta que a remodelação hipocampal ocorre durante o período livre de crises (fase silenciosa), danos estruturais causados pelo status epilepticus (SE) induzido pela pilocarpina na fase aguda acabam resultando em crises espontâneas recorrentes na fase crônica do modelo. Dessa maneira, esse trabalho teve como objetivo principal investigar a ação de genes que têm suas expressões induzidas durante a fase silenciosa do modelo da pilocarpina, e que estariam possivelmente relacionados com danos neuronais e epileptogenêse através de mecanismos associados à plasticidade e reorganização neuronal. Além disso, o trabalho investigou mais profundamente o papel do gene interleucina1-beta (Il1b), o qual foi demonstrado previamente estar envolvido no aumento da liberação de glutamato durante a fase aguda do modelo da pilocarpina. A principal estratégia experimental utilizada foi baseada no silenciamento póstranscricional pela técnica de interferência por RNA (RNAi) aplicada in vivo. Foi definido um padrão de expressão gênica para os genes TrkB e Plat, que sabidamente estão envolvidos com a fase silenciosa, e também para os genes da família dos Toll like receptors, durante o início da fase silenciosa, demonstrando suas expressões alteradas. Tais alterações parecem participar dos processos críticos que ocorrem na fase silenciosa, processos inflamatórios que associadas à remodelação sináptica geradas pelos genes estruturais (TrkB e Plat), culminam na fase crônica do modelo. A expressão dos genes Il1b e Il1ra também foram definidas, além do silenciamento dos mesmos. O silenciamento na fase aguda do modelo da pilocarpina levou a alterações fenotípicas detectáveis nos animais silenciados. Sendo assim, acreditamos que nossos resultados fornecem dados relevantes referentes ao papel crítico dos genes investigados na determinação da epileptogênese durante as fases aguda e silenciosa do modelo da pilocarpina para ELT, além de ter demonstrado a viabilidade do uso da RNAi na investigação funcional de genes envolvidos na patogênese das epilepsias in vivo / Abstract: The epilepsies affect approximately 1% of the population worldwide. Temporal lobe epilepsy (TLE) is the most frequent and commonly resistant to clinical treatment. Animal models have been widely used in order to study the mechanisms underlying TLE, especially those induced by chemical agents or electrical stimulation. It is believed that these models present similar epileptogenesis to that of human epileptic tissues studied ex vivo. Among the various animals models available we decided to use the one induced by pilocarpine injections. This model is characterized by three different phases accordingly to phenomenological, histopathological and molecular aspects: acute phase, silent phase and chronic phase. Based on the presence of genes with differential expression patterns in the silent phase, it is believed that it is in this phase that occur the modifications in neuronal circuits that are essential to functional plasticity of the central nervous system under pathological conditions. The changes in hippocampal circuits occuring during the silent phase are the result of the damage caused by the status epilepticus (SE) induced by pilocarpine in the acute phase. These events will ultimately result in spontaneous recurrent seizures in the chronic phase of the model. The main objective of this work was to explore the action of genes that are induced during the silent phase of the pilocarpine model. These would be possibly related with the neuronal damage and the development of epileptogenesis through mechanisms associated with plasticity and neuronal reorganization. In addition, we aimed to explore futher the role of Interleukin1-beta (Il1b), which had been previously demonstrated to be involved in the increase of glutamate release during the acute phase of the pilocarpine model. To achieve these objectives we used a pos-transcriptional gene silencing strategy named RNA interference (RNAi) applied in vivo. It was defined a pattern of gene expression for Trkb and Plat, that are known to be involved with the silent phase. Besides that, the Toll like receptors genes family had the pattern of expression defined during the beginning of the silent phase, demonstrating the expression altered. These alterations seem to participate in the critical processes that occur in the silent phase, inflammatory processes that associated to the synaptic reorganization generated by the structural genes (TrkB and Plat), culminate in the chronic phase of the model. The expression of the genes Il1b and Il1ra were defined too, besides the silencing of them. The silencing in the acute phase of the pilocarpine model led to phenotypic alterations detectable in the animals that had the genes silenced. We believe that our results can provide relevant new information regarding the role of genes in the determination of epileptogenesis during the acute and silent phases of the pilocarpine model. In addition, we have demonstrated the fisibility of the using RNAi to study epileptogenesis in vivo / Doutorado / Neurociencias / Doutor em Ciências

Delivery vehicles are necessary for many therapeutics to overcome the various challenges in their path. It is clear, however, that the relationship between delivery vehicles and the immune system is a complex one. One such delivery vehicle is the lipidoid nanoparticle, which has been shown to be potent in several cell types. This thesis details the first time lipidoids have been used for wound delivery, and demonstrates the successful silencing of an inflammatory protein, TNFα, in the context of diabetic ulcers. Knockdown is seen in an in vitro macrophage-fibroblast coculture model, as well as in nondiabetic and diabetic mice wound models. Lipidoids silence roughly half of the TNFα gene expression in the diabetic wound and have been shown to help the wound close faster than untreated controls. Of course, immune activation can decrease therapeutic efficacy or trigger dangerous reactions in the patient. Learning more about what chemical moieties cause an immune response would allow for the design of a particle that could better resist immune clearance and avoid the creation of a secondary response. This thesis investigated the effect of a lipidoid library on the immune system using a two pronged approach. The lipidoids were first tested against human peripheral blood mononuclear cells and then were injected into mice to probe the in situ immune response. Several types of B cells were examined in this latter case, namely germinal center B cells, plasma cells, and memory B cells. A T cell dependent response occurred, favoring memory B cells for most of the lipidoids tested. There was an increase in free antibody in the blood that reflected this increase in antibody producing cells. Nitrogen rings and carbon tail lengths of eleven and twelve carbons were particularly reactive, though it appears that the amine head group determines immune response more than the tail. Further work will analyze whether these increases in immune cells reflect a loss of therapeutic efficacy, as current ramifications are unclear. An in-depth T cell subset analysis with flow cytometry would also help complete the picture.

It is well known that tight coordination of cell division timing is essential for proper cell fate specification and tissue growth during metazoan development. However, how cell divisions are coordinated in vivo is largely unknown. In this thesis, a high- content screening was conducted to identify genes responsible for temporal coordination of cell division during Caenorhabditis elegans embryogenesis. A total of 822 genes were depleted using RNA interference (RNAi). The genes were prioritized based on their degree of conservation in human, as well as their roles in development. In addition to RNAi, an experimental pipeline was established, including 3D time-lapse imaging of an RNAi perturbed C. elegans embryos followed by automated lineaging, which allows systematic quantification of division timing of each individual cells up to approximately 350-cell stage. To identify genes with a significant reduction in the asynchrony of division between sister cells (ADS) upon perturbation, average division timings of each cell between at least two replicate perturbed embryos was compared against the average division timings of 92 wild type embryos. It was found that cell fate determinants were not only important for maintaining fate asymmetries, but are also imperative for establishing ADS regardless of cellular context. Hence, the results demonstrate that fate and temporal asymmetries share a common genetic architecture. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Given the observation that perturbations of signalling pathways, especially Wnt and Notch pathways, are frequently associated with the ADS, it would be essential to map the exact signalling event that takes place at cellular level which is responsible for a given ADS or fate asymmetry. To this end, a miniMos transposon-meditated transgenic technique was adopted to insert a fusion between GFP and a promoter derived from individual components of the two signalling pathways into the C. elegans genome. The resulting insertion was crossed into a strain expressing lineaging markers, which allows automated lineaging and gene expression profiling to map the expression of each component with single cell resolution. A combination of cellular expression and cell-cell contact data would lead to a comprehensive map of each signalling event during C. elegans embryogenesis. Our preliminary results on cell-cell contacts and cellular expression validated the existing signalling events and identified potential novel ones that are associated with either ADS and/or fate asymmetry.

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Huntington Disease (HD) is a dominantly inherited neurological disease attributed to a CAG expansion within the HD gene. The HD mutation gives rise to a polyglutamine expansion in exon 1 of the protein huntingtin (Htt). Since the discovery of the HD mutation in 1993, various HD gene mouse models have been developed to contain either fragments or full-length copies of the mutant HD gene. The existence of these HD mouse models enables focused therapeutic testing to develop potential treatments for HD. RNA interference (RNAi) therapy is a targeted gene silencing approach whereby synthetic RNA constructs are shuttled into the cell by viral vectors and used by the cell’s endogenous RNAi machinery to silence a gene of interest. RNAi therapy holds promise for mutant huntingtin (muHtt) allele-specific silencing as a treatment for HD. The purpose of this thesis was to develop the tools for pre-clinical testing of RNAi-mediated gene silencing of human muHtt in the YAC128 mouse model of HD. First, AAV serotypes were compared for delivery to striatal neurons, the neurons most affected in HD. From this work AAV serotype 1 was selected as the most effective serotype for construct delivery. Second, synthetic RNAi constructs including short-hairpin RNA (shRNA) and microRNA-based constructs (miR-shRNAs) were compared for silencing of human muHtt expression in vivo. Here, miR-shRNAs were found to have increased gene silencing and improved tolerance in avoiding immune activation compared to shRNAs. Alternatively, the shRNAs induced dramatic immune activation and morbidity in some cases. Ultimately these findings will contribute to a pre-clinical trial in YAC128 mice investigating Htt RNAi-mediated gene silencing in the treatment of HD, which is also discussed in this thesis. This future work provides proof-of-principle for muHtt allele-specific silencing as a treatment of HD. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Since their first discovery in the 19th century, microsporidian species have been found to be successful obligate intracellular parasites capable of infecting a wide variety of hosts including economically and ecologically important organisms as well as model organisms for biomedical research. Recently, cases of infection of the widely used animal model Danio rerio, commonly known as the zebrafish, by the newly described microsporidium Pseudoloma neurophilia have been reported in an increasing number of research facilities. Current knowledge of the biology of this parasite found in 75% of the Zebrafish Resource Center facilities is limited to microscopic analyses on its lifecycle as well as its physical, behavioral and psychic impact on its hosts. Despite the growing relevance of this parasite in biomedical research no current data is available on its genome. In this dissertation, I provide additional knowledge on the basic biology of P. neurophilia by acquiring and exploring the content and structure of the first genome draft of the zebrafish parasite. My findings reveal that the 5.25 Mb genome of P. neurophilia harbors an unusually high amount of transposable elements as well as numerous inserts found in coding regions typically conserved in microsporidia and other organisms. This peculiar obligate parasite demonstrates strong phylogenetic and genetic relationships with other fish-mosquito microsporidia. Similar to what is observed in closely related species, intra-genomic analyses of P. neurophilia’s genome suggest that it is diploid and possesses a large repertoire of over a thousand putative genes unique to this specie. Overall, my findings provide new insights into the basic biology of this parasite and represent a milestone in the understanding of P. neurophilia and D. rerio host-parasite interaction and ultimately in the development of treatments against this parasite that has been infecting the zebrafish research industry for the past decades.