The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

Although F1-ATPase is extremely conserved among organisms, a putative subunit p18 was identified in Trypanosoma brucei F1-ATPase complex. To explore its function in the procylic, bloodstream and dyskinetoplastic trypanosomes, three different RNAi cell lines were created. Upon p18 silencing the F1-moiety structural integrity was impaired suggesting that p18 is indeed a bona fide subunit of this complex. Since F1-ATPase is crucial for the bloodstream form survival, its potential inhibitor from the 4-oxopiperidine-3,5-dicarboxylates class (JK-11) was examined. JK-11 inhibited growth of the bloodstream trypanosomes, decreased mitochondrial membrane potential and reduced ATPase and ATP synthase activity in mitochondrial lysates. Our results suggest that JK-11 may act on FoF1-ATP synthase/ATPase and its inhibition may contribute to the cytotoxicity of this drug.

xii, 86 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / My dissertation has focused on identifying functions for non-essential genes in essential process, using the early C. elegans embryo as a model system. The fully sequenced C. elegans genome contains ∼19,800 protein coding genes of which about half have identifiable homologs in humans. Classical forward genetic mutagenesis screens, and more recently, genome-wide RNA interference (RNAi) screens has led to the identification of most essential genes in the genome. Analysis of the phenotypic data from mutants and RNAi screens shows that roughly 15% of the genes are essential and an additional 15% produce some other easily identifiable knockdown phenotype. This leaves about 70% of genes that have no functional information. Genetic modifier screening allows for the identification of roles for genes that do not produce a loss of function phenotype on their own but are able to modify the phenotype of a specific mutant. In my first chapter, I introduce approaches to identifying new gene functions and the usefulness V of C. elegans as a model system in this pursuit. In Chapter II, I describe a type of high-throughput genetic modifier screen that combines the sensitized genetic background of temperature-sensitive (ts) embryonic lethal mutants, and RNAi, to identify genes that either enhance or suppress embryonic lethality seen in the mutant background. I also summarize results from screening four ts mutants using this method. The following two chapters describe the identification and characterization of genetic modifier genes for two different ts embryonic-lethal mutants. Chapter III describes modifiers of rfl-1 , a conserved gene required for proper cytoskeletal regulation in the early C. elegans embryo. Chapter IV describes modifiers of lit-1 , also a conserved gene, that is required for regulation of Wnt signaling and cell fate specification in C. elegans . These findings reveal novel genetic interactions and provide functional information about many conserved but non-essential genes that have had no previous characterization. Conclusions are also made about the effectiveness of ts mutant/RNAi screening in the pursuit of identifying new gene functions. This dissertation contains co-authored material that has been previously published, and material that is currently in review, or is being prepared for publication. / Adviser: Bruce Bowerman

The function of chitin binding protein (CBP) and two isoforms of cathepsin B (cathB1, cathB2) were tested by using RNA interference in the tick I. ricinus. Two different methods have been used to deliver dsRNA for RNAi in ticks {--} injection and capillary feeding. The synthesized dsRNA was used to find out the impact of RNAi in the tick tissues, which were tested by RT-PCR and Western blot. The expression of CBP was successfully silenced by RNAi in the salivary glands. The silencing of cathB1 and cathB2 in the gut was less effective, but still limited tick`s ability to feed.

In this study the importance of prohibitin1 and prohibitin2 genes for Trypanosoma brucei was examined. RNA interference showed both of them essential for parasites to survive. Knocking down of these genes resulted in altered morphology of the mitochondrion, changes in membrane potential and shut down of mitochondrial translation. No changes were observed in levels of Reactive Oxygen Species and respiration. Both prohibitines are part of big complex present in the mitochondrion.

Made available in DSpace on 2014-06-12T15:07:01Z (GMT). No. of bitstreams: 2 arquivo3001_1.pdf: 1265385 bytes, checksum: 195917f7eadaa711ca792e1630574a84 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco / Um dos determinantes envolvidos no complexo ciclo de transmissão da dengue é o nível de susceptibilidade do Aedes aegypti ao vírus dengue (DENV), ou seja, a competência vetorial, que varia entre populações de mosquitos. Identificar moléculas envolvidas na interação mosquito-vírus pode auxiliar no conhecimento dos mecanismos envolvidos na competência vetorial, até então pouco elucidados. Estudos recentes mostraram a participação de certos mecanismos na interação mosquito-DENV, porém, pouco se sabe do real papel destes na modulação da competência vetorial em mosquitos de campo ou até da relação entre eles. Mediante isso, objetivamos analisar a expressão de três moléculas representantes de diferentes mecanismos de defesa antiviral no Ae. aegypti, em resposta à infecção com vírus dengue sorotipo 2 (DENV-2), sendo elas REL1, HOP e Dicer-2, em populações de campo e de laboratório do mosquito. Para isso, as diferentes linhagens foram artificialmente infectadas com DENV-2, e tecidos variados foram coletados em diversos momentos após infecção. Tanto a quantificação viral quanto a expressão das moléculas selecionadas nas amostras foram realizadas por PCR em tempo real quantitativo (qRT-PCR). Os resultados mostraram que tanto o padrão de infecção viral quanto a expressão das moléculas variaram entre as populações de A. aegypti nos diferentes momentos após infecção com DENV-2. Os resultados aqui obtidos poderão ser bastante relevantes na pesquisa da interação vetor-vírus e poderão auxiliar no desenvolvimento de novas estratégias de controle da dengue, como na pesquisa com mosquitos transgênicos

Orientador: Iscia Teresinha Lopes-Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T21:23:01Z (GMT). No. of bitstreams: 1 Araujo_PatriciaAlineOliveiraRibeirodeAguiar_D.pdf: 7351643 bytes, checksum: 6725a32e7f347be0b903efe1b6034c62 (MD5) Previous issue date: 2008 / Resumo: Introdução/Objetivo: Mutações no gene LGI1 foram descritas como causa da Epilepsia Parcial Autossômica Dominante com Sintomas Auditivos em algumas famílias. Alguns estudos apontam para um possível envolvimento do gene LGI1 com migração e/ou proliferação neuronal, porém a função exata desse gene permanece desconhecida. O objetivo deste trabalho foi determinar o perfil de expressão do gene Lgi1 em cérebro de camundongos durante o desenvolvimento do sistema nervoso central (SNC) e na fase adulta e, ainda, silenciar este gene em cérebros de camundongos utilizando a técnica de interferência por RNA (RNAi). Métodos: Acasalamentos programados foram realizados, utilizando camundongos Balb/c, para a obtenção de fetos de diferentes idades. Os cérebros de três animais, nas seguintes idades, foram retirados e os hemisférios direito e esquerdo separados: E15, E17, E18 dias (E:dias embrionários), P1, P7, P14 dias (P: dias pós-natal), 4, 6, 8 e 24 semanas. Também utilizamos cabeças inteiras de animais E13. Além disso, foram utilizados três animais adultos para a análise do gene Lgi1 em neocórtex, hipocampo e cerebelo. O perfil de expressão gênico foi determinado pela PCR em tempo real utilizando o sistema TaqMan® e por western blot. A técnica de RNAi foi realizada utilizando diferentes métodos de introdução de pequenas moléculas interferentes no cérebro de animais neonatos e adultos. Utilizamos também vários parâmetros diferentes no que se refere ao desenho das moléculas interferentes, suas concentrações, o local e o número de injeções. Além disso, experimentos de RNAi in vitro foram realizados, utilizando uma linhagem celular de glioblastoma humano, a U138MG, e uma linhagem de neuroblastoma murino, a Neuro2a. A confirmação do silenciamento gênico foi feita por PCR em tempo real e, em alguns experimentos, também por western blot. Resultados: A expressão do gene Lgi1 se apresentou baixa durante as idades intra-uterinas, aumentando progressivamente. Os animais em idade adulta apresentaram um aumento de expressão de 35 vezes quando comparadas às amostras E13, utilizando Gapdh como normalizador e de aproximadamente 28 vezes, utilizando ?-actina. Embora o teste estatístico não tenha encontrado diferença na expressão do gene Lgi1 entre os hemisférios cerebrais, ele revelou uma diferença significativa entre as idades estudadas. Os experimentos de western blot confirmaram o perfil de expressão determinado pelos estudos de PCR em tempo real, encontrando-se, a proteína Lgi1, em maior quantidade nas idades mais avançadas analisadas. O estudo de expressão das três regiões do cérebro não resultou em diferença estatisticamente significativa. O silenciamento do gene Lgi1 foi realizado com sucesso pela técnica de RNAi, em cérebro de animais adultos, sendo que os resultados mais consistentes, redução da expressão de aproximadamente 50%, foram observados com o método de eletroporação local e confirmação do silenciamento por PCR em tempo real. Além disso, nós conseguimos demonstrar silenciamento de até 99% do gene Lgi1 em cultura de células. Conclusões/Discussão: O padrão de expressão baixo do gene Lgi1 durante o desenvolvimento, com aumento progressivo e alta expressão na idade adulta aponta para uma potencial função inibitória da proliferação celular. Tal suposição encontra apoio em achados de neuroimagem de alguns pacientes com mutação em LGI1. O silenciamento do gene Lgi1 em cérebro de camundongos, utilizando a técnica de RNAi, foi alcançado, porém com grande dificuldade técnica. Esses obstáculos encontrados apontam para a existência de possíveis características moleculares próprias do gene LGI1 que poderiam dificultar seu silenciamento pela técnica da RNAi, tais como: um RNA mensageiro rico em proteínas associadas, impedindo o acesso da maquinaria de RNAi ou, ainda, LGI1 poderia ser um gene essencial, onde a diminuição de sua expressão ativaria processos celulares compensatórios / Abstract: Introduction/Objectives: Mutations in the LGI1 gene were described in some patients with autosomal dominant partial epilepsy with auditory features and preliminary functional studies point to a possible involvement of LGI1 with migration and/or neuronal proliferation. However, the precise function of LGI1 remains unknown. The objective of the present study was to determine the expression pattern of the Lgi1 gene in mice brain during central nervous system (CNS) development and in adult animals. In addition, we aimed to silence Lgi1 in mouse brain using RNA interference (RNAi). Methods: Programmed mating was carried with Balb/c mice in order to obtain embryos of different ages. The brains of three animals at the following ages were removed and the right and left hemispheres were separated: E15, E17, E18 days (E: embryo days), P1, P7, P14 (P: post-natal days), 4, 6, 8 and 24 weeks old. We also studied E13 whole head animals. In addition we studied three different regions from 5 weeks-old animal brains: neocortex, hippocampus and cerebellum. Gene expression assays were carried out using real time PCR with the TaqMan® system and western blot experiments. RNAi was performed using different methods for injection of interfering molecules into the neonate and adult brains. We also used different molecule designs and concentrations, as well as the number and the local of injections was varied. Furthermore, we performed in vitro RNAi experiments using a glioblastoma cell line, U138MG, and a murine cell line, Neuro2a. Gene silencing confirmation was carried out by real time PCR and western blot assays. Results: Lgi1 gene expression was significantly low during the intrauterine ages increasing progressively until the adult stages. Samples from adult animals presented a 35 fold increase in expression as compared to E13 samples, using Gapdh as endogenous control, and when we use ?-actin, adult samples presented approximately 28 fold increase in Lgi1 expression. There were no statistical differences between Lgi1 gene expression test between right and left hemispheres. However, a significant difference in expression was found among the different ages studied. The western blot showed higher expression of the Lgi1 protein in the most advanced ages analyzed, confirming the expression profile observed in the real time PCR studies. However, we did not find any statistic difference between the three regions of the brain studied. In addition, we achieved significant gene silencing of Lgi1, reduction of expression of approximately 50%, in brain of adult animals using RNAi and the local electroporation method. In addition, we demonstrated up to 99% silencing of LGI1 in cell culture. Conclusions/Discussion: The Lgi1 expression profile, which is characterized by low expression in the initial stages of development with progressive increase as the animal developed, could be explained by a possible inhibitory functional role in neuronal proliferation during CNS development. Lgi1 gene silencing in adult brain using RNAi technique was achieved after several attempts. These difficulties in gene silencing, point to the presence of intrinsic molecular characteristics of LGI1 which could be preventing silencing by RNAi, such as a message RNA too rich of associated proteins that may impairing the action of RNAi machinery; or LGI1 could be an essential gene, with very strong and stringent compensatory mechanisms; therefore, when attempting to decreased its expression one would activate the compensatory processes / Doutorado / Neurociencias / Doutor em Fisiopatologia Medica

Mitochondria exist in a dynamic network whereby fusion and fission events are critical to the health of the mitochondria, the cell, and the organism. Dysfunctional mitochondrial dynamics underlie a plethora of diseases including various cancers, heart diseases, diabetes, neurodegenerative diseases, and a number of mitochondrial disorders. Despite a strong molecular knowledge of a handful of functional mediators of mitochondrial dynamics, much less is known about how this process is regulated at a cellular level, and what genes are involved in signaling pathways. A previously completed mitochondrial morphology genome screen was repeated with an automated confocal microscope resulting in the identification and validation of MTX2 as a novel regulator of mitochondrial dynamics. Functional characterization of the role of MTX2 in mitochondrial dynamics will further our understanding of mitochondrial biology, and has the future potential to inform therapies for some of the many diseases underscored by dysfunctional mitochondrial dynamics.

Spinocerebellar ataxia type 1 (SCA1) is an adult onset, autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in ataxin-1, which encodes the ataxin-1 protein. SCA1 is one of nine polyQ-expansion gain-of-function diseases which includes Huntington's disease, spinal-bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and other ataxias. Clinical symptoms of SCA1 include ataxia, dysarthria, ophthalmoparesis, muscle wasting, and extrapyramidal and bulbar dysfunction. Cerebellar Purkinje cells (PCs), neurons in the inferior olive and nuclei of the brainstem are affected. No disease-modifying therapy exists for SCA1. The goals of my thesis were to assess the safety and efficacy of AAV-delivered artificial miRNAs targeting ataxin-1 to alleviate neuropathological and behavioral phenotypes in the knock-in and transgenic SCA1 mouse models. In the knock-in SCA1 mouse model AAVs expressing an artificial miRNA (miSCA1) targeting sequences conserved in mouse and human ataxin-1 were injected directly to the deep cerebellar nuclei. This achieved long term silencing of ataxin-1 mRNA and significantly improved rotarod performance, gait deficiencies, and neuropathology of the cerebellum. In the transgenic SCA1 mouse model the same method of delivery was executed with an artificial microRNA (miR) (miS1) designed to optimize potency, efficacy and safety to suppress Atxn1 expression. Additionally the therapeutic potential of continuous overexpression of ataxin-1-like was examined. Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mouse cerebellum resulted in widespread cerebellar Purkinje cell transduction. There was significant improvement to rotarod performance, gait deficiencies, coordination and balance, as well as the neuropathology of cerebellar Purkinje cells. In summary, these data indicate the utility of these approaches as possible therapies for SCA1 patients.

For more than a decade, studies focused on RNA interference (RNAi) pathway as a pivotal gene regulatory mechanism. RNAi components are attracting considerable interest due to the recent evidence demonstrating that they play a role not only in post-transcriptional regulation but also in transcriptional level. The involvement of RNAi components in heterochromatin formation and RNA Pol II processivity and alternative splicing in different organisms has been shown. Dicer protein, a highly conserved protein among kingdoms, is one of the main effectors in this pathway. There is a considerable amount of literature on Dicer’s role in the cytoplasm; however, there is still vast ambiguity concerning nuclear Dicer. More recent evidence reveals the existence of Dicer1 variants that are differentially expressed in some cancer cells. Our experiments set out to investigate one of these variants that we hypothesise is responsible for the nuclear function. We undertook genomic and biochemical approaches applied to HAP 1 cells as a model system to characterise Dicer1-s, taking advantage of a custom-made antibody in our research group. Here, as anticipated, our experiments proved that Dicer1-s is enriched in the nuclear compartment compared to full-length Dicer1, indicating that it might be a putative contributor to nuclear gene regulation activity. Unfortunately, it was not possible to establish a mutant cell line to investigate the significant nuclear function of Dicer1-s, due to the need for further optimisation of the methods used. Exploitation of previously optimised gene knock-out tools might accelerate shedding light on protein, DNA, and RNA partners, disclosing the exact nuclear mechanisms that might exhibit similar activity.