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

Teladorsagia circumcincta is a parasitic nematode which is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The parasite has developed resistance to the major anthelmintic drug classes and this challenges its future control. Vaccination is a potential alternative control method since sheep are able to develop protective immunity against this parasite. Although potential vaccine candidates have been revealed, the increasing gene datasets suggest that vaccinetarget selection may be aided by screening methods such as RNAi. This is a reverse genetic mechanism that causes highly specific gene silencing which was initially described and applied to defining gene function in Caenorhabditis elegans. Nevertheless, its application was more difficult than anticipated in parasitic nematodes because of the inconsistency of the silencing effect. In the unsuccessful cases, did the dsRNA penetrate the parasite and activate the RNAi pathway? Thus far, there are no internal controls that indicate the activation of the pathway. Are the RNAi pathway genes constantly transcribed or are they ‘switched on’ in response to the dsRNA exposure? The initial aim of the study was to determine potential marker genes in the RNAi pathway that could indicate the activation of the pathway in C. elegans. After the exposure to dsRNA from two target genes, the transcript levels of three candidate marker genes (Ce-dcr-1, Ce-ego-1 and Ce-rsd-3) were examined and showed that exposure to dsRNA has no effect on the transcript levels of these genes making them inappropriate markers for the activation of the RNAi pathway. The two target-genes were Ce-cpr-4 and Ce-sod-4 which had been proven to be consistently susceptible and refractory to RNAi, respectively. Another aim of the project was to develop an RNAi platform in T. circumcincta for use as a screening method for potential vaccine candidates. The targets selected for the in vitro RNAi included: five members of the Activation-associated Secreted Proteins (ASPs); a Macrophage migration Inhibitory Factor-like (Tci-mif-1) and a Surface Associated Antigen gene (Tci-saa-1), all of which have been associated with vaccine-induced protective immunity. The selection of the ASPs was based on a bioinformatic and transcriptomic analysis of the ASPs in T. circumcincta. The results showed successful knock-down only for three out of five ASP targets after 1 hour of soaking in gene-specific double stranded RNA (dsRNA) which illustrates the inconsistency and the target specificity of RNAi in T. circumcincta which has been observed in the past with other parasitic nematodes. Inconsistencies were also observed within the successful ASP targets with the results not being reproducible after several successful experiments. Potential reasons for the inconsistencies were examined with the duration of larval storage being a critical factor. Larvae stored for a short or long period of time were susceptible and refractory to RNAi, respectively. Experiments were also conducted to investigate how the ASPs relate to extracellular microvesicles (EMVs). These vesicles are considered to play an important role in the intercellular communication between parasites and their hosts, and thus represent potentially useful vaccine and/or drug targets. Transmission electron microscopy (TEM) confirmed that EMVs are excreted / secreted by the parasite and the proteomic analysis revealed several types of proteins within the vesicles such as: ASPs, Actins, Metallopeptidases, and RAB proteins. A comparative analysis of EMVs, EMV-free ES (Excretory / Secretory) and total ES products showed that approximately 35% of the proteins found in the vesicles could also be identified in EMV-free ES and in total ES products, whilst the remaining 65% were present only in EMVs.

We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-FectTM (vehicle), and delivered as repeated daily bolus doses (0.5 mug to 4 mug) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-FectTM complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / RNA interference (RNAi) is a highly conserved gene regulatory mechanism triggered by the presence of double-stranded RNAs and results in post-transcriptional and transcriptional gene silencing. RNAi has been demonstrated to have therapeutic potential to treat chronic viral infections including HIV-1. Due to the side effects of and eventual drug resistance to highly active antiretroviral therapy, a novel anti-HIV-1 therapy is required. The most suitable exogenous RNAi triggers to use in anti-HIV-1 RNAi-based therapy are expressed short hairpin RNAs (shRNAs). Despite being highly developed, shRNA systems still pose safety concerns. Highly expressed shRNAs are at risk of over-saturating the endogenous RNAi pathway, inducing an innate immune response or silencing off-target mRNA. The purpose of this study was to minimise shRNA-associated off-target effects and simultaneously maximise the potency and specificity of expressed shRNAs for potential therapeutic application. ShRNAs shorter than 19 base pairs are not recognised by the endonuclease Dicer, which is an important component of the RNAi pathway, but miR-451 is Dicer-independent. Smaller shRNAs that retain their potency would be easier to deliver into a disease model. For this study, 25mers and miR-451-mimicking 19mers were generated. The shRNA pairs exhibited significant knockdown of their respective targets in dual-luciferase assays. The 19mers are more specific gene silencers compared to the 25mers. A 19mer that is more potent than its 25mer counterpart was identified. None of the hairpins induced an innate immune response, caused cytotoxic effects or saturated the endogenous RNAi pathway. This study concludes that the 19mers were processed in a manner similar to miR-451 resulting in a single ~30 nt mature RNA product. We dubbed these miR-451-mimicking 19mers, guide shRNAs. The single RNA strand of mature guide shRNAs abolishes the risk sense strand-associated off-targeting thus improving shRNA specificity. These revolutionary guide shRNAs can be developed into highly potent activators of the RNAi pathway in a therapeutic setting.

ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.

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

Heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) requires conserved components that mediate RNA interference (RNAi) directed methylation of histone H3 on lysine 9 (H3K9). Fission yeast heterochromatin is mainly found at centromeres, telomeres, and the mating-type locus. At centromeres, transcripts from repetitive elements are processed to siRNAs and RNAi promotes chromatin modification by recruiting the Clr4 methyltransferase. RNAi is not required to maintain silent chromatin at the mating-type locus. This RNAi-directed form of centromeric heterochromatin provides an ideal system for in vivo screening to allow the identification of compounds that inhibit the activity of proteins involved in RNA silencing, chromatin modification and heterochromatin assembly in fission yeast and may inhibit conserved proteins in other organisms. A dominant selectable marker gene system at fission yeast centromeres that reports loss of heterochromatin integrity by increased resistance to G418 in 96-well plate format liquid cultures was developed. The resulting strain was used to screen a nontargeted chemically diverse compound library in vivo to identify compounds that disrupt the integrity of RNAi-directed heterochromatin. Two compounds, Emi1 and Emi14, were identified and found to cause a significant decrease in the level of H3K9 methylation on the outer repeats at fission yeast centromeres. Growth in the presence of Emi1 or Emi14 also caused a reduction in H3K9 methylation levels at the mating-type locus, suggesting that they do not act through RNAi. Consistent with this, Emi1 and Emi14 did not cause a decrease in centromeric siRNA levels. Analyses therefore suggest that Emi1 and Emi14 do not disrupt RNAi but that they inhibit downstream events in chromatin modification and heterochromatin assembly. Cells lacking RNAi due to loss of Dicer (dcr1Δ) or cells lacking the histone deacetylase (HDAC) Sir2 (sir2Δ) retain significant but lower levels of H3K9 methylation on the centromeric outer repeats. When dcr1Δ or sir2Δ cells were grown in the presence of Emi1 or Emi14 a further reduction in H3K9 methylation levels was observed on the outer repeats. This mimics the effect of combining clr3Δ with dcr1Δ or sir2Δ and suggests that Emi1 and Emi14 may interfere with SHREC function. SHREC is a chromatin remodelling complex that includes the HDAC Clr3 and the chromatin remodeler Mit1 and is known to contribute to heterochromatin integrity. Expression profiling performed on Emi1 and Emi14 treated cells confirmed the previous results. The changes in gene expression following Emi1 and Emi14 treatment were compared to known mutants defective in heterochromatin integrity. The profile of expression changes following Emi14 treatment was found to correlate with alterations in the expression pattern observed in cells with SHREC components deleted. No correlation with mutants lacking other HDACs or RNAi components was detected. Emi1 had a weaker correlation with defective SHREC function and thus may also partially inhibit the SHREC complex. Murine erythroleukemia (MEL) cells harbouring a silenced eGFP reporter transgene were used to assess whether Emi1 and Emi14 also affect silencing in mammalian cells. Emi1 was found to disrupt silencing at the eGFP reporter and this correlated with a decrease in H3K9 methylation. Structurally related analogues of Emi1 and Emi14 were selected and tested in the fission yeast assay. Interpretation of the obtained structure-activity relationships allowed identification of the chemical moieties key to Emi1 and Emi14 activity. Overall, an approach was developed to identify two novel small molecule inhibitors of a well-characterized chromatin modification pathway. The SHREC complex was identified as the putative target of these two compounds and structurally related active analogues were identified for them. Importantly, one of the compounds was also active in mammalian cells, highlighting the usefulness of this approach in identifying compounds that affect higher organisms.

Acquired Immunodeficiency Syndromes (AIDS), a disease caused by the infection of human immunodeficiency virus (HIV), is still incurable to date. Various types of anti-viral drugs have been developed and most of these drugs are targeted on HIV reverse transcriptase and protease. Highly active antiretroviral therapy (HAART) has been used on AIDS treatment recently. However, new drugs are required to delay the resistance onset and to maximize the effectiveness of combination therapy by inhibiting a variety of targets simultaneously. / In the second part, the possibility of using the vector-based approach of RNA interference (RNAi) to reduce the expression of HIV-1 integrase and HIV replication in mammalian cells was examined. RNAi suppressed protein synthesis through the induction of sequence-specific gene silencing of 21-25 nucleotides (nt) double stranded RNA fragments, termed small interfering RNA (siRNA). pSilencer series vectors with different promoters (p Silencer 1.0-U6, pSilencer 2.0-U6, pSilencer 3.0-H1) were used on shRNA expression inside HeLa cells. Four different hairpin constructs containing the 19-nt corresponding to the nucleotide sequence of HIV integrase at positions 19-27, 79-96, 158-176 and 495-513 were generated for RNAi study. (Abstract shortened by UMI.) / Integrase is one of the important enzymes on HIV infection. It acts by integrating viral RNA to host DNA and this is one of the ideal targets for therapeutic intervention. Previous results in our laboratory demonstrated that luffin, a type-I ribosome inactivating protein (RIP), had high potency on integrase inhibition. In the first part of this thesis, alpha-luffin cDNA was cloned from the seed of Luffa cylindrica. Three different sets of expression vectors were used to produce recombinant luffin. Different deletion mutants of luffin were also generated for structural analysis on integrase inhibition. Recombinant alpha-luffin and its various deletion mutants were expressed exclusively in the form of inclusion bodies despite different expression conditions had been attempted. Various refolding strategies and conditions were carried out but the problem of insolubility was consistently found after removal of the denaturing reagents. The problem of insolubility was improved by using the maltose binding protein (MBP) luffin fusion construct. However, there is evidence that this soluble MBP-luffin formed a multimeric fusion protein complex rather than monomer and removal of MBP tag resulted in the precipitation of luffin. / Lau Tat San. / "August 2005." / Adviser: C. C. Wan. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 202-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Virus-host interactions determine virus transmissibility and virulence, and underlie coevolution that shapes interesting biological phenomena such as the genetic architecture of host resistance and host range. Characterization of the virus factors that exert selective pressure on the host, and the host genes which underlie resistance and adaptation against viruses will help to define the mechanistic pathways embroiled in host-virus coevolution. In this thesis, I describe the viral causes and host consequences of host-virus coevolution. These include genomic signatures consistent with antagonistic coevolution in antiviral RNA interference pathway genes such as high rates of positive selection and polymorphism, loci that underlie genetic variation in resistance to virus infection, and apparent conflict between NF-κB signalling and DNA virus infection. The RNA interference (RNAi) pathway is the most general innate immune pathway in insects, underlined by the observation that many viruses encode suppressors of RNAi (VSRs). The relationship between RNAi and VSRs has garnered attention as a plausible battleground for host-virus antagonistic coevolution, and genomic patterns in Drosophila support this hypothesis. However, genomic patterns in the N-terminal domain of the key RNAi effector gene, Argonaute-2, have not been described. In Chapter 2, I sequence the Argonaute-2 N-terminal domain using PacBio long-read sequencing technology to describe variation within and across Drosophila species, and test whether this variation is associated with resistance to Drosophila C Virus. The RNAi pathway evolves adaptively in Drosophila, but this has not been formally extended across invertebrate species. In Chapter 3, I quantify rates of adaptive protein evolution and describe evidence for selective sweeps in RNAi pathway genes using population genomic data from 8 insect and nematode species. These analyses indicate that RNAi genes involved in suppression of transposable elements and defence against viruses evolve rapidly across invertebrates, and I identify genes with signatures of elevated adaptation in multiple insect species. Host genes that underlie host-virus interactions have been described in RNA virus infection of Drosophila, however substantially less attention has focussed on the host response to DNA viruses, primarily because no DNA viruses have been isolated from Drosophila. In Chapter 4, I describe the isolation of Kallithea virus, a Drosophila dsDNA nudivirus, and characterise the host response to infection and genetic variation in resistance. I find that Kallithea virus infection causes early male-specific lethality, a cessation of oogenesis, and induction of undescribed virus-responsive genes. Further, I describe genetic variation in resistance and tolerance to Kallithea virus infection, and identify a potential causal variant for virus-induced mortality in Cip4. Insect viruses commonly encode viral suppressors of RNAi, however there are a multitude of antiviral immune mechanisms besides RNAi which may select for viral-encoded inhibitors. In Chapter 5, I describe the requirement for RNAi and NF-κB in immunity against Kallithea virus, and map gp83 as a virus-encoded inhibitor of NF-κB signalling. I find that gp83 inhibits Toll signalling at the level of, or downstream of NF-κB transcription factors, and that this immunosuppressive function is conserved in other nudiviruses.

DYT1 dystonia is an early onset central nervous system-based movement disorder characterized by uncontrolled sustained muscle contractions that can lead to debilitating abnormal postures. Though a genetic mutation in the gene TOR1A is responsible for most DYT1 cases, the low penetrance of the disease implicates additional genetic and environmental modifiers. Current therapeutic options for DYT1 dystonia are limited to symptomatic treatments with variable effectiveness. Currently, the underlying pathogenesis of this disease and the role of torsinA (torA), the protein product of TOR1A, in the development of this disease have yet to be established. In the first part of this thesis we aimed to further understand the effects of the TOR1A mutation at the molecular, cellular and organismal level in order to identify disease associated biomarkers that can be later used to measure the effectiveness of novel therapies. We found that expression of mutant torsinA (torA(ÄE)) in a cellular and an animal model of DYT1 had no significant effect on global transcription, despite its interaction with nuclear envelope proteins. Recent research has unearthed a role for microRNAs (miRNAs) in neuronal development and maturation. Consequently we explored whether torA(ÄE) expression in murine neural tissue was associated with changes in miRNA expression in young DYT1 knockin (KI) mice. Since the primary sight of dysfunction is still being debated, we profiled miRNA expression of the two strongest candidates, the striatum and cerebellum, both of which have well established roles in the control and coordination of muscle movements. We have identified several microRNAs that were uniquely altered in either the striatum or cerebellum and further research will be conducted to determine their usability as disease biomarkers. Finally, we were unable to identify motor phenotypes in either a DYT1 (KI) mice or a novel DYT1 transgenic model in open field, rotarod or staircase forepaw reaching tests. In the second part of this thesis we aimed to develop and evaluate the safety and efficacy of viral therapeutic RNAi constructs for in DYT1 murine models. DYT1 is an ideal candidate for this form of therapy due to its dominant inheritance, common mutation and potentially reversible phenotype. Virally delivered short-hairpin RNAs (shRNA) designed to knockdown torA(ÄE) in either an allele-specific or nonallele-specific manner were injected into the striatum of DYT1 transgenic or KI mice respectively. Unexpectedly, we found widespread lethal toxicity and behavioral abnormalities in mice injected with either therapeutic or control shRNAs that weren't observed in mice injected with no shRNAs. Further studies found that regions where toxic shRNAs were expressed corresponded with neuronal loss and glial activation. Finally, we found evidence that the severity of toxicity was influenced in part by the genetic background of the mice. In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.