Interactions between 3´ untranslated regions (UTRs) and RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation. Metazoan genomes encode hundreds of RBPs and thousands of 3’ UTRs have been experimentally identified, yet the spectrum of interactions between 3´UTRs and RBPs remains largely unknown. Several methods are available to map these interactions, including protein-centered methods such as RBP immunoprecipitation (RIP) and cross-link immunoprecipitation (CLIP), yeast three-hybrid assays and RNAcompete. However, there is a paucity of RNA-centered approaches for assaying an RNA element of interest against multiple RBPs in a parallel, scalable manner. Here, I present a strategy for delineating protein-RNA interaction networks using a gene centered approach. This approach includes annotating RBPs and identifying physical interactions between an RNA of interest and these RBPs using the Protein-RNA Interaction Mapping Assay (PRIMA). Few RBPs have been experimentally determined in most eukaryotic organisms. Therefore I show that existing RBP annotations can be supplemented using computational predictions of RNA binding domains (RBD) from protein sequences. A single RNA of interest can be tested using PRIMA against a library of RBPs constructed from these annotations. PRIMA utilizes the green fluorescent protein (GFP) in yeast as a reporter. PRIMA is based on reconstitution of the interaction between the 5´ and 3´ ends of an mRNA, which increases mRNA stability and enhances translation. PRIMA recapitulates known and uncovers new interactions involving RBPs from human, Caenorhabditis elegans and bacteriophage with short RNA fragments and full-length 3´UTRs. The development of RBP prey libraries will enable the testing of 3´UTRs against the hundreds of RBPs, which is essential to gain broad insights into post-transcriptional gene regulation at a systems level.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that posttranscriptionally regulate gene expression primarily through binding to the 3’ untranslated region (3’UTR) of target mRNAs, and are known to play important roles in various developmental and physiological processes. The work presented in this thesis was centered on understanding how Caenorhabditis elegans miRNAs are modulated by genetic, environmental, or physiological factors and how these small RNAs function to maintain the robustness of developmental processes under stressful conditions. To identify modulators of the miRNA pathway, I developed sensitized genetic backgrounds that consist of a panel of miRNA gene mutants and miRNA biogenesis factor mutants with partially penetrant phenotypes. First, I found that upon infection of Caenorhabditis elegans with Pseudomonas aeruginosa, an opportunistic pathogen of diverse plants and animals, let-7 family miRNAs are engaged in reciprocal regulatory interactions with the p38 MAPK innate immune pathway to maintain robust developmental timing despite the stress of pathogen infection. These let-7 family miRNAs, along with other developmental timing regulators, are also integrated into innate immune regulatory networks to modulate immune responses. Next, I demonstrated that loss-of-function mutations of Staufen (stau-1), a double-stranded RNA-binding protein, increase miRNA activity for several miRNA families, and this negative modulation of Staufen on miRNA activity acts downstream of miRNA biogenesis, possibly by competing with miRNAs for binding to target mRNA 3’UTRs. In summary, these studies provide a better understanding on how miRNAs are modulated by various environmental and cellular components, and further support the role of the miRNA pathway in conferring robustness to developmental processes under these perturbations.

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3′ untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.

Multidrug resistance protein 2 (MRP2) is the second member the C subfamily in the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters. MRP2 is a critical player for generation of bile acidindependent bile flow and biliary excretion of glutathione, glucuronate and sulfate conjugates of endo- and xenobiotics. Dysfunctional expression of MRP2 is associated with Dubin-Johnson Syndrome. Pathological and physiological states or xenobiotics change the MRP2 expression level. Under some conditions, expression of the human MRP2 and rat Mrp2 proteins are regulated at the translation level. There are several transcription initiation sites in MRP2/Mrp2 gene. The 5’ untranslated regions (5’UTRs) of MRP2/Mrp2 contains multiple translation start codons. The focus of this study, therefore, was investigation of the translational regulatory mechanisms mediated by the upstream open reading frames (uORF) of MRP2/Mrp2. Using in vitro translation assays and transient cotransfection assays in HepG2 cells, we showed that the rat uORF1 starting at position -109 (relative to the ATG of Mrp2) and the human uORF2 starting at position -105 (relative to the ATG of MRP2) are two major cis-acting inhibitors of translation among the rat and human multiple uORFs, respectively. Translational regulation mediated by the uORFs in the rat Mrp2 mRNA is a combined effect of the leaky scanning model and the reinitiation model, and also results from interaction of the multiple uORFs. In addition, by Ribonuclease Protection Assays (RPA), we detected multiple transcription initiation sites of MRP2/Mrp2 gene in tissues. We also found that the relative abundance of the rat Mrp2 mRNA isoforms with different 5’UTRs differed in the rat liver, kidney, jejunum, ileum, placenta, and lung. This is the first study on the translational regulatory mechanisms of the MRP2/Mrp2 gene.

Cheung Hoi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-166). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1 --- Non-coding RNAs in transcriptional output --- p.2 / Chapter 1.2 --- Diverse functions of non-coding RNAs --- p.5 / Chapter 1.3 --- HI9: imprinted non-coding RNA --- p.6 / Chapter 1.4 --- Objective --- p.7 / Chapter CHAPTER TWO: --- The ROLE OF H19 RNA IN MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- H19-Igf2 locus as a model for genomic imprinting --- p.10 / Chapter 2.1.2 --- HI9 as a non-protein coding regulatory RNA --- p.12 / Chapter 2.1.3 --- Controversial roles of H19 RNA --- p.13 / Chapter 2.1.4 --- Novel role of H19 RNA in drug resistance --- p.15 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell culture --- p.19 / Chapter 2.2.2.2 --- Plasmid construction and stable cell transfection --- p.19 / Chapter 2.2.2.3 --- Transient gene transfection --- p.20 / Chapter 2.2.2.4 --- RNA isolation and RT-PCR --- p.21 / Chapter 2.2.2.5 --- MTT drug sensitivity assay --- p.22 / Chapter 2.2.2.6 --- Western blot analysis --- p.22 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential expression of H19 RNA in different human cancer cell lines --- p.24 / Chapter 2.3.2 --- R-HepG2 cells over-expressed P-glycoprotein and H19 RNA --- p.24 / Chapter 2.3.3 --- Development of H19-silenced cell lines in HepG2 cells by RNA interference --- p.26 / Chapter 2.3.4 --- Altered drug sensitivity in H19-silenced cells --- p.28 / Chapter 2.3.5 --- Expression of P-glycoprotein in H19-silenced cells --- p.31 / Chapter 2.3.6 --- Overexpression of H19 RNA in HepG2 cells --- p.34 / Chapter 2.3.7 --- Induction of H19 RNA and MDR1 in HepG2 cells --- p.34 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- H19 regulation of MDR1 associated drug resistance --- p.38 / Chapter 2.4.2 --- The puzzle of riboregulation in drug resistance --- p.40 / Chapter CHAPTER THREE: --- The ROLES OF PTB AND IMP1 IN H19-RELATED MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- H19 RNA binding proteins --- p.43 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.46 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.48 / Chapter 3.2.2.2 --- Plasmid construction and stable cell transfection --- p.48 / Chapter 3.2.2.3 --- RNA extraction and RT-PCR --- p.48 / Chapter 3.2.2.4 --- MTT drug sensitivity assay --- p.48 / Chapter 3.2.2.5 --- Western blot analysis --- p.48 / Chapter 3.2.2.6 --- Real-time PCR analysis of gene expression --- p.49 / Chapter 3.2.2.7 --- DOX efflux assay --- p.49 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PTB knockdown increased P-glycoprotein expression --- p.51 / Chapter 3.3.2 --- IMP1 knockdown decreased MDR1 /P-glycoprotein expression --- p.54 / Chapter 3.3.3 --- Altered drug sensitivity in IMP 1 -knockdown cells --- p.60 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Antagonistic effect of PTB and IMP1 on H19/MDR1 expressions --- p.64 / Chapter 3.4.2 --- Complexity of riboregulation --- p.65 / Chapter CHAPTER FOUR: --- IDENTIFICATION OF H19 RNA BINDING PROTEINS FROM HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Overview of RNA-protein interactions --- p.69 / Chapter 4.1.2 --- Methodology in the study of RNA-protein interactions --- p.71 / Chapter 4.1.3 --- Identification of RNA-binding proteins --- p.72 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.75 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Screening of H19 cDNA from human placenta cDNA library --- p.78 / Chapter 4.2.2.2 --- Preparation of nuclear and cytoplasmic extracts from HepG2 cells / Chapter 4.2.2.3 --- In vitro RNA transcription and RNA labeling --- p.80 / Chapter 4.2.2.4 --- RNA electrophoretic mobility shift assay --- p.81 / Chapter 4.2.2.5 --- In vitro UV-crosslinking assay --- p.82 / Chapter 4.2.2.6 --- Preparation of RNA-affinity column and isolation of RNA binding proteins --- p.83 / Chapter 4.2.2.7 --- In-gel digestion and MALDI-TOF mass spectrometry --- p.84 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Screening of H19 cDNA and preparation ofH19 RNA --- p.86 / Chapter 4.3.2 --- Electrophoretic mobility shift analysis of H19 RNA with HepG2 cytoplasmic extract --- p.87 / Chapter 4.3.3 --- UV-crosslinking of H19 RNA with HepG2 nuclear and cytoplasmic extract --- p.90 / Chapter 4.3.4 --- Isolation of H19 RNA binding proteins by RNA-affmity chromatography --- p.94 / Chapter 4.3.5 --- Confirmation of PTB and IMP1 as H19 RNA binding protein --- p.96 / Chapter 4.3.6 --- MALDI-TOF mass spectrometric analysis of isolated H19 RNA binding proteins --- p.96 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- RNA-protein interactions: an initial step for mechanistic study --- p.99 / Chapter 4.4.2 --- In vitro and in vivo methods for isolation of RNA binding proteins --- p.101 / Chapter 4.4.3 --- Novel role of hnRNP M protein in H19 RNA binding --- p.103 / Chapter CHAPTER FIVE: --- THE ROLE OF PTB IN APOPTOSIS / Chapter 5.1 --- Introduction / Chapter 5.1.1 --- Overview of polypyrimidine tract-binding protein in RNA processing and post-transcriptional gene regulation --- p.106 / Chapter 5.1.2 --- Evidences of polyrimidine-tract binding protein in the regulation of apoptosis --- p.108 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Materials --- p.111 / Chapter 5.2.2 --- Methods / Chapter 5.2.2.1 --- Cell culture --- p.114 / Chapter 5.2.2.2 --- Stable cell transfection in A431 cells --- p.114 / Chapter 5.2.2.3 --- Western Blot analysis --- p.114 / Chapter 5.2.2.4 --- MTT drug sensitivity assay --- p.114 / Chapter 5.2.2.5 --- DNA fragmentation assay --- p.115 / Chapter 5.2.2.6 --- Flow cytometry analysis of apoptosis --- p.115 / Chapter 5.2.2.7 --- Caspase activity assay --- p.116 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Taxol as an apoptosis inducer in HepG2 cells --- p.117 / Chapter 5.3.2 --- PTB was cleaved during Taxol-induced apoptosis --- p.118 / Chapter 5.3.3 --- PTB knockdown increased Taxol cytotoxicity and apoptosis --- p.118 / Chapter 5.3.4 --- Effect of PTB knockdown on drug sensitivity of cells --- p.121 / Chapter 5.3.5 --- Effect of PTB knockdown on other drug-induced apoptosis --- p.121 / Chapter 5.3.6 --- Effect of PTB knockdown on the basal expressions of genes in apoptosis pathway --- p.126 / Chapter 5.3.7 --- The role of caspase-9 activation in PTB-regulated apoptosis --- p.129 / Chapter 5.3.8 --- The effect of PTB knockdown on pro-caspase-9 expression and Taxol-induced apoptosis in A431 cells --- p.133 / Chapter 5.3.9 --- The role of PTB in the regulation of intrinsic apoptosis pathway --- p.136 / Chapter 5.4 --- Discussion / Chapter 5.4.1 --- The role of PTB in intrinsic apoptosis pathway --- p.138 / Chapter 5.4.2 --- PTB in regulation of pro-caspase-9 expression --- p.139 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION AND CONCLUSION / Chapter 6.1 --- H19 as a potential target in anti-cancer gene therapy --- p.143 / Chapter 6.2 --- Conclusion --- p.144 / Chapter 6.3 --- Unanswered questions and future work --- p.145 / Chapter 6.4 --- A proposed model for H19 pathway --- p.148 / REFERENCES --- p.151

O melanoma cutâneo é um tipo de tumor formado a partir dos melanócitos, células de origem neuroectodérmica que habitam a epiderme, sendo responsáveis por sua pigmentação. Embora este tumor seja o tipo menos frequente de câncer de pele, ele está associado com altas taxas de mortalidade, principalmente devido a seu comportamento agressivo (alta capacidade metastática) e quimiorresistência aos tratamentos (quimioterapia e radioterapia). Os processos da quimiorresistência em melanoma ainda não são totalmente conhecidos. Estudos prévios de nosso grupo evidenciaram que a expressão da proteína Proibitina (PHB) encontra-se aumentada em células de melanoma frente à exposição a certos quimioterápicos, executando sua função de molécula anti-apoptótica, quando localizada no citoplasma, e/ou supressora tumoral quando no núcleo. Adicionalmente, foi visto que a repressão de PHB sensibiliza as células de melanoma, enquanto a sua superexpressão protege da morte celular induzida por cisplatina. Além disso, estudos de associação genótipo-fenótipo revelaram que o alelo menos frequente/raro do SNP rs6917 (C1703T) na região 3\'UTR do gene da PHB foi associado com o risco aumentado de desenvolvimento do melanoma. O objetivo do nosso trabalho foi avaliar o envolvimento do SNP rs6917 da 3\'UTR do gene PHB em linhagens de melanoma humano e sua resposta celular frente ao tratamento com agentes quimioterápicos indutores de estresse celular como temozolomida, cisplatina e vemurafenib. Para avaliar a contribuição do polimorfismo rs6917 no desenvolvimento de melanoma, foi desenvolvido um estudo tipo caso-controle numa população brasileira. O estudo analisou 198 pacientes com melanoma e 200 controles. Em ensaios in vitro as linhagens celulares de melanoma humano SK-Mel 05 e UACC-62 (BRAFV600E mutadas) foram transfectadas com as variantes polimórficas UTR/C e UTR/T clonadas no plasmideo pmirGLO Dual-Luciferase nos sítios de restição NheI/XhoI. A Geneticina (G418) foi utilizada para seleção estável das células, ensaios de western Blot, qRT-PCR e luminiscencia confirmaram a expresão do transgene. As diferentes doses de cisplatina, temozolomida e vemurafenib foram definidas para o tratamento das células. Para avaliar a morte celular foi realizada a técnica de citometria de fluxo após incorporação com iodeto de propidio. Após os tratamentos, foram realizados ensaios clonogênicos e de imunofluorescencia para determinar sobrevivencia celular e localização subcelular da proibitina, respectivamente. Nossos resultados revelaram que variáveis clinicas como a presença de cabelos claros (loiros ou ruivos), mais de 20 pintas, exposição solar intermitente, queimaduras solares na infância e adolescência são fatores de risco para o aparecimento de melanoma. Nos casos, os portadores do alelo T mostraram um risco aumentado em 5,6 vezes para o desenvolvimento de melanoma de crescimento vertical em comparação com os pacientes com genótipo CC. Ensaios in vitro, mostraram que células de melanoma humano superexpressando o alelo raro 3\'UTR/T após tratamento com cisplatina, temozolomida e vemurafenib apresentavam um fenotipo mais proliferativo e clonogênico com menor morte celular quando comparadas com as células 3\'UTR/C e vetor vazio. Ensaios de Western blot e bioluminescência mostraram, respectivamente, um aumento na expressão e atividade do gene da luciferase nas células 3\'UTR/T. Estes resultados mostram que o SNP rs6917 modula a expressão de PHB e que o alelo raro T representa um polimorfismo funcional promovendo a quimiorresistência em melanoma / Melanoma is a type of skin tumor formed from melanocytes, cells of neuroectodermal origin that inhabit the epidermis, being responsible for its pigmentation. Although its low incidence, melanoma is associated with high rates of mortality due to its resistance to chemotherapy. The mechanisms involved in melanoma chemoresistance are not well fully understood yet, therefore, the comprehension of this phenomenon may be useful for the development of new treatment strategies. Previous results from our laboratory showed that Prohibitin (PHB), a protein with diverse functions including regulation of cell cycle progression and apoptosis, was overexpressed in human melanoma cells lines when exposed to high-doses of the chemotherapy drug, cisplatin. PHB knockdown sensitized melanoma cells, meanwhile PHB recombinant overexpression protected melanoma cells to cisplatin-induced cell death. Studies of genotype-phenotype association revealed that the Single Nucleotide Polymorphism rs6917 at the portion 3\'UTR of the PHB gene showed an association with some risk factors for the development of melanoma. The aim of this study was to investigate if the SNP rs6917 affects PHB protein function by modulating cell proliferation and chemoresistance in human melanoma cell lines when exposed to stress inductors agents such as cisplatin, temozolomide and vemurafenib. A hospital-based case-control study was carried out in a Brazilian sample to evaluate the contribution of rs6917 polymorphism in melanoma development. The study comprised 198 melanoma patients and 200 controls. For invitro assays SK-Mel 05 and UACC-62 human melanoma cell lines (both BRAFV600E mutated) were used. The 852bp of PHB-3\'UTR harboring wild-type (3\'UTR/C) or the rare allele (3\'UTR/T) were cloned at pmirGLO Dual-Luciferase plasmid at NheI/XhoI restriction sites and stable cell lines were generated. Geneticin (G418) was used for stable selection, qRT-PCR, Western Blot and luciferase assays confirmed the transgene expression. Different doses of cisplatin, temozolomide and vemurafenib were defined to treat the cells. Cell death was evaluated using flow cytometry after propidium iodide incorporation. Cell survival was assessed by clonogenic assay after cells undergone treatment. Our results revealed that clinical variables like presence of light hair, more than 20 moles, intermintent sun expossure and sunburns at childhood are risk factors for melanoma development. We also showed that T allele carriers have a 5,6 times increased risk to develop vertical growth melanoma in comparison with the CC genotype patients. In vitro assays with transfected melanoma cells, SK-Mel 05 and UACC-62, overexpressing the rare allele 3\'UTR/T showed a more proliferative and clonogenical phenotype and less induced cell death after cisplatin, temozolomide and vemurafenib treatment when compared to cells overexpressing the wild-type allele 3\'UTR/C and empty vector. Westernblot and bioluminiscence assays showed respectively an increase in the expresion and activity of the luciferase gene of the 3\'UTR/T cells in comparison with the 3\'UTR/C and empty vector cells. All together these results showed that the SNP rs6917 modulates the expression of PHB and that the rare allele T represents a functional polymorphism by promoting a chemoresistance phenotype in melanoma

Background. Familial Amyloidosis with Polyneuropathy (FAP) is an autosomal dominantly inherited systemic amyloid disease. The disease is caused by mutations in the transthyretin (TTR) gene, where close to 100 different amyloidogenic mutations have been identified. FAP is found worldwide, but endemic areas with a high frequency of patients are found in Portugal, Japan and northern Sweden. Cases from these endemic areas all share the same TTR c.148G>A, p.V50M ("V30M") mutation, but the phenotype of the disease varies between the areas, and also within the endemic areas. The mean onset of the disease is two decades earlier in Portugal and Japan compared to Sweden, but late as well as early age at onset cases occur within all the populations. Interestingly, the different populations all display a maternal anticipation, where an earlier onset is observed for those individuals who inherit the trait from their mother. Since substantial variation in the phenotype is observed for different populations, epigenetic/genetic and/or environmental factors must exert a significant impact on the penetrance of the disease. Amyloid formation is caused by conformational changes of proteins, which facilitates their assembly into fibrils, amyloid. Oxidative stress can mediate conformational changes of proteins and since the mitochondria regulate oxidative processes within the cell, mitochondrial function may affect amyloid formation. The mitochondrial DNA is a non-nuclear DNA, which is entirely maternally inherited, and therefore could be related to the observed maternal anticipation of the disease. In addition, differences within the surrounding regions of the TTR gene may have an impact on the transcription of the gene and thereby on the expression of the different alleles. Material and methods. DNA from early and late onset V30M cases and from non-carriers (the latter utilised as controls) from Swedish, French, Japanese and Portuguese populations were analysed. In addition, DNA from healthy Swedish V30M carriers was analysed. Conventional analytical methods were employed, such as PCR, sequencing and genotyping. Conventional statistical methods used were t-test, Chi-squared test and maximum likelihood. Results. The study of V30M carrier frequency in two counties (Lycksele and Skellefteå) within the Swedish endemic area revealed a carrier frequency of 2.14% and 2.54%, respectively. The mitochondrial haplogroup analysis showed that in populations with generally late onset (French and Swedish), the haplogroup distribution of late onset cases resembled that of the controls derived from the same area, whereas haplogroup distribution for early onset patients was significantly different. The most pronounced difference was for the rare haplogroup K, of which early onset cases had a higher frequency than the controls. Analysis of the Portuguese population, with predominantly early onset, showed that haplogroup distribution for early onset cases were similar to the Portuguese control group, which had a different distribution than the Swedish control group. By analysis of pedigrees from Swedish and Portuguese patients it could be shown that mitochondrial genetic variation entirely could explain maternal anticipation in the Portuguese patients, whereas for Swedish patients, an additional parent of origin effect is present. Our analysis of the TTR gene disclosed a polymorphism (rs62093482) in the 3'UTR region of the Swedish patients. This polymorphism was found in all V30M carriers, irrespective of symptoms. In addition, homozygous TTR V30M carriers were homozygous also for the polymorphism. Since Swedish patients share a common founder this polymorphism thus is localised on the V30M allele. This polymorphism was found in only 4% of the Swedish controls. French controls showed the same frequency, but none of the French V30M patients displayed the polymorphism. In the Japanese population the polymorphism was not present at all. Interestingly, this polymorphism generates a potential binding site for microRNA and thereby possibly could down-regulate the expression of the mutated TTR allele. Conclusions. The carrier frequency in the endemic area is remarkably high, above 2% in the Lycksele and Skellefteå areas. The prevailing haplogroup distributions in the different endemic areas are consistent between the general population and the patient group with the predominant phenotype of that area. Mitochondrial genetic differences may explain maternal anticipation in Portuguese patients, and have an influence in Swedish patients. A polymorphism in the 3'UTR regulatory region of the mutated TTR allele is found in all Swedish patients. This polymorphism may down-regulate TTR V30M expression and thereby contribute to the late onset of the disease noted in the Swedish population.

Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T18:49:26Z (GMT). No. of bitstreams: 1 Alves_BeatrizSantosCapela_D.pdf: 5512305 bytes, checksum: 707ea6299bc24ddc6fb459520d79aeee (MD5) Previous issue date: 2008 / Resumo: O combate à dengue atualmente está limitado praticamente aos esforços de eliminação do mosquito transmissor, o Aedes aegypti, porém esta estratégia não tem se mostrado eficiente. O desenvolvimento de novos instrumentos de combate à dengue requer, portanto, maior conhecimento sobre a biologia do vírus com relação à sua interação com seus hospedeiros. O genoma do vírus é constituído por um RNA simples-fita de polaridade positiva e possui duas regiões não traduzidas (5¿ e 3¿ UTR). A região 5¿UTR viral possui organização similar à dos mRNAs eucarióticos, diferentemente da região 3¿UTR que é longa e não possui cauda de poli(A). Em vez disso, na região 3¿UTR encontram-se estruturas conservadas entre os diferentes Flavivirus, dentre elas a estrutura 3¿ stem-loop (3¿SL) que é indispensável para a replicação do RNA viral. O objetivo do nosso estudo foi identificar novas proteínas humanas capazes de interagir com a estrutura 3¿SL do RNA do vírus da dengue. Dados da literatura descrevem que a proteína Mov34 de camundongo interage com 3¿SL do vírus da encefalite japonesa. Devido à alta similaridade entre as proteínas ortólogas humana e de camundongo, bem como das respectivas estruturas 3¿SL dos vírus da dengue e da encefalite japonesa, foi testada a interação entre a Mov34 humana com o 3¿SL do vírus da dengue. Porém, em nenhuma das condições testadas foi possível obter evidência de interação da Mov34 humana com 3¿SL dos vírus da dengue e da encefalite japonesa. Para a identificação de novas proteínas que são capazes de interagir com a estrutura 3¿SL do RNA do vírus da dengue foi utilizado o ensaio de triplo-híbrido de levedura. A proteína humana PACT, conhecida como proteína celular ativadora de PKR, foi isolada neste ensaio utilizando 3¿SL como isca. PKR é uma quinase ativada por PACT ou RNA dupla-fita. A ativação de PKR leva a um estado antiviral adquirido pela fosforilação do fator de iniciação da tradução eIF2a e conseqüente inibição da tradução. Além disso, PKR está envolvida em outras vias de transdução de sinal e na resposta celular à proteínas desenoveladas. A ação antiviral de PACT é evidenciada pela ação de proteínas dos vírus influenza A e herpes simplex tipo 1 que inibem a ativação de PKR por PACT e por RNA dupla-fita. A interação direta de PACT com 3¿SL do RNA do vírus da dengue foi confirmada por ensaio de UVcrosslinking PACT possui três domínios de interação com RNA dupla-fita, sendo que os dois domínios N-terminais são responsáveis pela sua interação com o 3¿SL. Foi identificada uma região específica do 3¿SL, o stem-loop superior, onde PACT interage com maior afinidade. Além disso, foi mostrado que PACT endógena de células HEK293 é capaz de interagir com o 3¿SL biotinilado. Para caracterizar a função desta interação durante a infecção viral, foi desenvolvida uma linhagem celular com inibição da expressão de PACT através da técnica de RNA de interferência. Com esta linhagem poderemos analisar a importância da interação entre PACT e o RNA do vírus da dengue quanto à ativação e/ou inibição de PKR durante a infecção viral / Abstract: The combat to the dengue virus is basically limited to the efforts in eliminating the transmitter mosquito, the Aedes aegypti. But this strategy is not very efficient. The development of new instruments of combat to dengue virus requires improved knowledge about the virus biology and its relation to hosts. The dengue virus genome is a single-stranded RNA of positive polarity flanked by a 5¿ untranslated region (UTR) of ~100 bases and a highly structured 3¿ UTR of ~450 bases. As many other viruses, dengue encodes the enzymes required for its genome replication, but relies completely on the host translational machinery to synthesize its proteins. The essential difference between host cellular mRNAs and dengue virus genome RNA involves the 3¿UTR, which instead of a polyadenylate tail contains highly conserved structural elements, including the 3' stem-loop (3¿SL), located at the 3' terminus of the 3'UTR of many flaviviruses that is essential for their replication. The aim of this study is to identify new human proteins capable of interacting with dengue virus RNA 3¿SL structure. Literature data describe that the murine Mov34 protein interacts with Japanese encephalitis virus 3¿SL. Giving the high similarity between the human and murine ortholog proteins, as well as the conservation of the Flavivivirus RNA 3¿SL structure, we tested the interaction between the human Mov34 and the dengue virus 3¿SL. However, no interaction was detected under the conditions used in this work. In addition, the yeast three-hybrid system was used to screen for novel proteins that interact with the dengue virus 3¿SL. Human PACT, known as the cellular protein activator of PKR, was identified as a putative 3¿SL-interacting protein. PKR is an interferon-inducible, PACT or double-stranded RNA activated protein kinase. Activated PKR phosphorylates the translation initiation factor eIF2a, inhibiting translation of cellular and viral RNAs, leading to a cellular antiviral state. PACT and doublestranded RNA activation of PKR is inhibited by influenza A and herpes simplex type 1 virus proteins during viral infection, indicating that PACT plays a role in the cellular antiviral state. Direct interaction between PACT and 3¿SL was confirmed by UV-crosslinking assays. PACT contains three doublestranded RNA interaction motifs, but only the two N-terminal motifs are responsible for 3¿SL interaction. A 3¿SL specific region, the top stem-loop, was identified to interact with PACT with higher affinity. Furthermore, HEK293 cells endogenous PACT interacts with biotin-labeled 3¿SL. To further characterize PACT-3¿SL interaction during dengue virus infection, a cell line with low expression of PACT was developed using the RNA interference technique. This cell line will be used to determine the propagation rate of dengue virus which is expected to reveal the importance of PACT either for the cell antiviral state or for dengue virus proliferation / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

O melanoma cutâneo é um tipo de tumor formado a partir dos melanócitos, células de origem neuroectodérmica que habitam a epiderme, sendo responsáveis por sua pigmentação. Embora este tumor seja o tipo menos frequente de câncer de pele, ele está associado com altas taxas de mortalidade, principalmente devido a seu comportamento agressivo (alta capacidade metastática) e quimiorresistência aos tratamentos (quimioterapia e radioterapia). Os processos da quimiorresistência em melanoma ainda não são totalmente conhecidos. Estudos prévios de nosso grupo evidenciaram que a expressão da proteína Proibitina (PHB) encontra-se aumentada em células de melanoma frente à exposição a certos quimioterápicos, executando sua função de molécula anti-apoptótica, quando localizada no citoplasma, e/ou supressora tumoral quando no núcleo. Adicionalmente, foi visto que a repressão de PHB sensibiliza as células de melanoma, enquanto a sua superexpressão protege da morte celular induzida por cisplatina. Além disso, estudos de associação genótipo-fenótipo revelaram que o alelo menos frequente/raro do SNP rs6917 (C1703T) na região 3\'UTR do gene da PHB foi associado com o risco aumentado de desenvolvimento do melanoma. O objetivo do nosso trabalho foi avaliar o envolvimento do SNP rs6917 da 3\'UTR do gene PHB em linhagens de melanoma humano e sua resposta celular frente ao tratamento com agentes quimioterápicos indutores de estresse celular como temozolomida, cisplatina e vemurafenib. Para avaliar a contribuição do polimorfismo rs6917 no desenvolvimento de melanoma, foi desenvolvido um estudo tipo caso-controle numa população brasileira. O estudo analisou 198 pacientes com melanoma e 200 controles. Em ensaios in vitro as linhagens celulares de melanoma humano SK-Mel 05 e UACC-62 (BRAFV600E mutadas) foram transfectadas com as variantes polimórficas UTR/C e UTR/T clonadas no plasmideo pmirGLO Dual-Luciferase nos sítios de restição NheI/XhoI. A Geneticina (G418) foi utilizada para seleção estável das células, ensaios de western Blot, qRT-PCR e luminiscencia confirmaram a expresão do transgene. As diferentes doses de cisplatina, temozolomida e vemurafenib foram definidas para o tratamento das células. Para avaliar a morte celular foi realizada a técnica de citometria de fluxo após incorporação com iodeto de propidio. Após os tratamentos, foram realizados ensaios clonogênicos e de imunofluorescencia para determinar sobrevivencia celular e localização subcelular da proibitina, respectivamente. Nossos resultados revelaram que variáveis clinicas como a presença de cabelos claros (loiros ou ruivos), mais de 20 pintas, exposição solar intermitente, queimaduras solares na infância e adolescência são fatores de risco para o aparecimento de melanoma. Nos casos, os portadores do alelo T mostraram um risco aumentado em 5,6 vezes para o desenvolvimento de melanoma de crescimento vertical em comparação com os pacientes com genótipo CC. Ensaios in vitro, mostraram que células de melanoma humano superexpressando o alelo raro 3\'UTR/T após tratamento com cisplatina, temozolomida e vemurafenib apresentavam um fenotipo mais proliferativo e clonogênico com menor morte celular quando comparadas com as células 3\'UTR/C e vetor vazio. Ensaios de Western blot e bioluminescência mostraram, respectivamente, um aumento na expressão e atividade do gene da luciferase nas células 3\'UTR/T. Estes resultados mostram que o SNP rs6917 modula a expressão de PHB e que o alelo raro T representa um polimorfismo funcional promovendo a quimiorresistência em melanoma / Melanoma is a type of skin tumor formed from melanocytes, cells of neuroectodermal origin that inhabit the epidermis, being responsible for its pigmentation. Although its low incidence, melanoma is associated with high rates of mortality due to its resistance to chemotherapy. The mechanisms involved in melanoma chemoresistance are not well fully understood yet, therefore, the comprehension of this phenomenon may be useful for the development of new treatment strategies. Previous results from our laboratory showed that Prohibitin (PHB), a protein with diverse functions including regulation of cell cycle progression and apoptosis, was overexpressed in human melanoma cells lines when exposed to high-doses of the chemotherapy drug, cisplatin. PHB knockdown sensitized melanoma cells, meanwhile PHB recombinant overexpression protected melanoma cells to cisplatin-induced cell death. Studies of genotype-phenotype association revealed that the Single Nucleotide Polymorphism rs6917 at the portion 3\'UTR of the PHB gene showed an association with some risk factors for the development of melanoma. The aim of this study was to investigate if the SNP rs6917 affects PHB protein function by modulating cell proliferation and chemoresistance in human melanoma cell lines when exposed to stress inductors agents such as cisplatin, temozolomide and vemurafenib. A hospital-based case-control study was carried out in a Brazilian sample to evaluate the contribution of rs6917 polymorphism in melanoma development. The study comprised 198 melanoma patients and 200 controls. For invitro assays SK-Mel 05 and UACC-62 human melanoma cell lines (both BRAFV600E mutated) were used. The 852bp of PHB-3\'UTR harboring wild-type (3\'UTR/C) or the rare allele (3\'UTR/T) were cloned at pmirGLO Dual-Luciferase plasmid at NheI/XhoI restriction sites and stable cell lines were generated. Geneticin (G418) was used for stable selection, qRT-PCR, Western Blot and luciferase assays confirmed the transgene expression. Different doses of cisplatin, temozolomide and vemurafenib were defined to treat the cells. Cell death was evaluated using flow cytometry after propidium iodide incorporation. Cell survival was assessed by clonogenic assay after cells undergone treatment. Our results revealed that clinical variables like presence of light hair, more than 20 moles, intermintent sun expossure and sunburns at childhood are risk factors for melanoma development. We also showed that T allele carriers have a 5,6 times increased risk to develop vertical growth melanoma in comparison with the CC genotype patients. In vitro assays with transfected melanoma cells, SK-Mel 05 and UACC-62, overexpressing the rare allele 3\'UTR/T showed a more proliferative and clonogenical phenotype and less induced cell death after cisplatin, temozolomide and vemurafenib treatment when compared to cells overexpressing the wild-type allele 3\'UTR/C and empty vector. Westernblot and bioluminiscence assays showed respectively an increase in the expresion and activity of the luciferase gene of the 3\'UTR/T cells in comparison with the 3\'UTR/C and empty vector cells. All together these results showed that the SNP rs6917 modulates the expression of PHB and that the rare allele T represents a functional polymorphism by promoting a chemoresistance phenotype in melanoma

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate. Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O-methyl group at their 3´ ends, and depleted in dicer-2 mutants. Methylation of small RNAs at the 3´ end in the soma, is a consequence of assembly into a mature Argonaute2-RNA induced silencing complex. In addition to endo-siRNAs, we observed certain miRNAs or their miRNA* partners loading into Argonaute2. We discovered, that irrespective of its biogenesis, a miRNA duplex can load into either Argonaute (Ago1 or Ago2), contingent on its structural and sequence features, followed by assignment of one of the strands in the duplex as the functional or guide strand. Usually the miRNA strand is selected as the guide in complex with Ago1 and miRNA* strand with Ago2. In our efforts towards finding 3´ modified small RNAs in the fly soma, we also discovered 24-28nt small RNAs in certain fly genotypes, particularly ago2 and dcr-2mutants. 24-28nt small RNAs share many features with piRNAs present in the germline, and a significant fraction of the 24-28nt small RNAs originate from similar transposon clusters as somatic endo-siRNAs. Therefore the same RNA can potentially act as a precursor for both endo-siRNA and piRNA-like small RNA biogenesis. We are analyzing the genomic regions that spawn somatic small RNAs in order to understand the triggers for their production. Ultimately, we want to attain insight into the underlying complexity that interconnects these small RNA pathways. Dysregulation of small RNAs leads to defects in germline development, organogenesis, cell growth and differentiation. This thesis research provides vital insight into the network of interactions that fine-tune the small RNA pathways. Understanding the flow of information between the small RNA pathways, a great deal of which has been revealed only in the recent years, will help us comprehend how the pathways compete and collaborate with each other, enabling each other’s optimum function.