Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 91-100).

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.

Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l’agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n’est retrouvé qu’en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l’aspect versatile de l’obtention de transcrits infectieux de ces différents clones, j’ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d’un promoteur constitutive végétal pour initier l’infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l’infection virale et étudier l’interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j’ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV. / The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

O glioblastoma (GBM) é o tumor cerebral maligno mais comum e agressivo em adultos. Uma combinação de terapia padrão com outras terapias baseadas no conhecimento de sua biologia é necessária para melhorar a sobrevida de pacientes com GBM. Muitos estudos foram desenvolvidos em busca de proteínas de membrana expressas em GBM, pois são potenciais alvos para imunoterapia. A proteína transmembrânica CD99 foi descrita como altamente expressa em astrocitomas de diferentes graus de malignidade. Embora seu mecanismo de ação ainda não seja totalmente compreendido, CD99 está envolvido na adesão e migração celular em diferentes tipos de tumores. O gene CD99 codifica duas proteínas distintas, denominadas isoforma 1, maior, de 32 kDa, e isoforma 2, gerada por splicing alternativo e menor, de 28 kDa. No presente estudo, foi demonstrada a expressão predominante da isoforma 1 em astrocitomas de diferentes graus de malignidade em comparação com o cérebro normal, bem como na linhagem celular de GBM humano U87MG. O transcriptoma das células U87MG transfectadas com siRNA para CD99 foi analisado em relação ao controle. Um total de 2.670 genes diferencialmente expressos foi identificado. Uma análise de enriquecimento no banco de dados DAVID revelou os seguintes processos como os mais significativos: junções aderentes célula-célula; adesão célula-célula envolvendo ligação de caderina e adesão celular. Ensaios funcionais baseados nestes achados (migração, invasão e adesão) foram realizados com células U87MG após o silenciamento de CD99 com dois shRNAs diferentes. A eficiência de silenciamento foi de 80 e 97%, para o shCD991 e 2, respectivamente, confirmada a nível de expressão do gene e da proteína. O silenciamento de CD99 reduziu a migração e invasão para ambos os shRNAs, com diminuição mais acentuada da migração para o shCD99 2, com maior nível de silenciamento de CD99. No ensaio de adesão, a linhagem U87MG shCD99 1 apresentou propriedades adesivas mais baixas que o controle, enquanto o shCD99 2 apresentou resultado oposto, com maior adesão celular do que seu controle. Provavelmente o silenciamento de CD99 afetou a redução da adesão celular em um padrão distinto, sugerindo que o resultado pode ser dependente do nível de expressão remanescente de CD99. Além disso, o CD99 e a faloidina colocalizaram nos lamelipódios e filopódios, sugerindo um papel importante no rearranjo do citoesqueleto. Foi demostrado, ainda, que o silenciamento de CD99 levou à redução da proliferação celular in vitro e diminuição do tumor in vivo. Camundongos imunodeficientes nos quais foram implantadas células silenciadas no cérebro apresentaram uma maior sobrevida que os animais que receberam células controle. A via de sinalização pela qual CD99 modula a proliferação no GBM ainda precisa ser elucidada. Migração, invasão e proliferação são as principais características do GBM que limitam uma ressecção cirúrgica completa e, consequentemente, levam frequentemente à recorrência. Portanto, análises posteriores das vias ativadoras do CD99 no contexto da migração, invasão, proliferação celular e apoptose são válidas para revelar novas estratégias terapêuticas para limitar a progressão do GBM / Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. A combination of standard therapy with other biologically based therapies is necessary to improve the survival of patients with GBM. Many studies have been developed in pursuit of expressed membrane proteins in GBM, which are potential targets for immunotherapy. The transmembrane protein CD99 is highly expressed in different malignant grades of astrocytomas. Although its mechanism of action is not still fully understood, CD99 is involved in cell adhesion and migration in different type of tumors. The CD99 gene encodes two distinct transmembrane proteins, named isoform 1, longer with 32 kDa, and isoform 2, generated by alternative splicing, shorter with 28 kDa. In the present study, we demonstrated predominant expression of isoform 1 in astrocytomas of different malignant grades compared to normal brain, and in the human GBM cell line U87MG. The transcriptome of U87MG cell line transfected with siRNA for CD99 was analyzed in relation to control. A total of 2.670 differentially expressed genes were identified. An enrichment analysis by DAVID Bioinformatics Database revealed the following processes as the most significant: cell-cell adherens junction; cadherin binding involved in cell-cell adhesion and cell-cell adhesion. Functional assays based on these findings (migration, invasion and adhesion) were performed with U87MG cells after knocking down CD99 with two different shRNAs. The CD99 silencing efficiency was 80 and 97%, for shCD99 1 and 2, respectively, confirmed at gene and protein level. The CD99 knockdown reduced migration and invasion for both shRNA, with the highest decrease of migration observed in the higher CD99 knocked down cells. In adhesion assay, shCD99 1 U87MG showed lower adhesive properties than the control, whereas shCD99 2 cells presented opposite results, with higher cell adhesion than control. Probably CD99 knockdown affected in the reduction of cell adhesion in a distinct pattern, suggesting that the result is dependent on CD99 remaining expression level. Additionally, CD99 and phalloidin colocalized at lamellipodia and filopodia, sugesting that CD99 plays an important role to cytoskeleton rearrangement. It has also been demonstrated that CD99 silencing caused reduction of cell proliferation in vitro and decreased tumor in vivo. Immunodeficient mice in which knocked down cells were implanted in the brain had a longer survival than animals that received control cells. The signaling pathway by which CD99 modulates proliferation in GBM still needs to be elucidated. Migration, invasion and proliferation are major characteristics of GBM, which limits the complete surgical tumor resection, and consequently leads to tumor recurrence. Therefore, further analysis of CD99 activating pathways in the context of cell migration, invasion, proliferation and apoptosis is worthwhile to unveil new therapeutic strategies to halt GBM progression

Orientadores: Fernando Ferreira Costa, Anderson Ferreira da Cunha / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T17:06:48Z (GMT). No. of bitstreams: 1 Branco_DianaSantos_D.pdf: 12860329 bytes, checksum: 5c5da209d2a41c9596907283c3c9e5e8 (MD5) Previous issue date: 2013 / Resumo: Os mecanismos moleculares envolvidos no perfil de expressão durante a eritropoese tem sido objeto de numerosas investigações como, por exemplo, o estudo da regulação gênica em células de linhagem eritroide. Esses estudos permitem a identificação de novos genes com potencial participação nesse processo e, adicionalmente, possibilitam um melhor entendimento dos genes já identificados na maturação das células eritroides e que possam estar envolvidos na produção de hemoglobinas. Nosso grupo de pesquisa identificou os diversos genes diferencialmente expressos durante a eritropoese. Dentre eles, os fatores de transcrição, EYA3 e HES6 e a latexina, LX, apresentaram maior expressão na fase final da eritropoese in vitro. Nossos dados sugerem participação dos genes EYA3 e LX, nas fases intermediaria e final da diferenciação eritroide, na expressão dos genes das globinas alfa e gama e na produção de HbF in vitro. Adicionalmente, no modelo in vivo zebrafish, os genes eya1, eya2, eya3, eya4, hes6 e hes13 apresentaram padrão de expressão ubíquo, enquanto que o gene lxn apresentou expressão especifica na ICM, tornando-o o candidato mais promissor para ser silenciado. O silenciamento desse gene em zebrafish apresentou fenótipo anêmico em embriões 72hpf, mas não em 48hpf, sugerindo que a anemia e decorrente de um processo no final da diferenciação eritroide, corroborando os dados encontrados em cultura in vitro. Estudos adicionais são necessários para compreensão dos mecanismos e vias envolvidos na participação do gene LX, durante o processo de diferenciação eritroide. Outros genes com potencial participação no processo de eritropoese são CLPX, TRAK2 E GFI. O gene CLPX codifica a caseinolytic peptidase X, uma proteína xvii altamente conservada durante a evolução e que apresenta função de chaperona dependente de ATP. Os dados deste trabalho mostram que o silenciamento do gene clpx1 reduziu significativamente os níveis de hemoglobinizacao e produção de eritrócitos em zebrafish. Contudo, estudos adicionais para o gene clpx2 precisam ser realizados para melhor compreender a possível função desses genes na produção de Heme. O gene TRAK2, por sua vez, e uma Trafficking Protein, Kinesin-Binding 2, envolvida no movimento da mitocôndria ao longo dos microtubulos. Os resultados obtidos em colaboração com o pesquisador Jeffrey Miller, M.D. (NIH/NIDDK) mostraram envolvimento desse gene na eritropoese em modelo in vitro de cultura primaria. No presente estudo, dentre os ortologos para o gene TRAK2 humano avaliados, apenas o trak1.1 parece ter sua função conservada nos teleósteos. O silenciamento desse gene gerou fenótipo anêmico nos embriões avaliados, corroborando os dados obtidos originalmente em cultura de células primaria. Finalmente, os fatores de transcrição de zebrafish gfi1aa, gfi1ab e gfi1b, ortologos aos fatores de transcrição da família Grow Factor Independence (GFI) em humanos também tiveram seu papel avaliado na hematopoese. Nossos dados mostram participação de gf1aa fase inicial de hematopoese e de gf1b na hematopoese definitiva. Também foi determinada a relação epistática entre os fatores gfi e os fatores de transcrição chave hematopoiéticos, mostrando que gfi1aa e gfi1b, juntamente com lmo2, scl, runx-1 e c-myb atuam como reguladores de HSPC em teleósteos / Abstract: Molecular mechanisms involved in expression profile during erythropoiesis have been the subject of numerous investigations such study of gene regulation in erythroid cell culture. These studies allow us to identify new genes potentially involved in erythroid differentiation and additionally to investigate genes already known as regulators of red blood cells and hemoglobin production. Our research group identified several genes differentially expressed during erythropoiesis. Among them, the transcription factors, EYA3 and HES6 and the latexin, LX, were found to have higher expression in the final phase of the in vitro erythropoiesis. Our data suggest that EYA3 and LX, are involved in the intermediate and final stages of erythropoiesis, expression pattern of alfa and gama globin and HbF production in vitro. Additionally in zebrafish model, eya1, eya2, eya3, eya4, hes6 and hes13 showed a ubiquitous expression pattern, while lxn showed specific expression in the ICM, making it the most promising candidate to be knockdowned. lxn knockdown in zebrafish showed anemic phenotype at 72hpf embryos, but not at 48hpf, suggesting that the anemia results is due to a process in the end of the erythroid differentiation, corroborating the results found for in vitro cultures. Additional studies are necessary to understand the mechanisms and pathways involved in the participation of the LX, gene during the process of erythroid differentiation. CLPX, TRAK2 and GFI transcription factors are also potentially candidates to be involved in erythropoiesis. CLPX gene codes for caseinolytic peptidase X, a protein highly conserved during evolution, which presents an ATP-dependent chaperone function. Data from this study showed that clpx1 knockdown reduced significantly hemoglobinization levels and erythroid production in zebrafish. However, future studies xv for the clpx2 gene is needed to better understand the function of these genes in the heme production. TRAK2 gene, in turn, is a Trafficking Protein, Kinesin-Binding 2, involved in mitochondrial movement along microtubules. Results obtained in collaboration with the researcher Jeffrey Miller, M.D. (NIH/NIDDK), showed the involvement of this gene in erythropoiesis in primary culture in vitro models. In this study, from the orthologs for the human TRAK2 gene analyzed, only trak1.1 appears to have its function conserved in teleosts. The silencing of this gene generated anemic phenotype in the embryos tested, corroborating the original results obtained in primary cell culture. Finally, gfi1aa, gfi1ab and gfi1b zebrafish transcription factors, orthologous to the Grow Factor Independence (GFI) family transcription factors in humans, also had their function evaluated in hematopoiesis. Our data suggest is involved in the initial phase of hematopoiesis while gf1b has a role in the definite hematopoiesis. The epistatic relation between the gfi and the hematopoietic key transcription factors was also determined, showing that gfi1aa and gfi1b, together with lmo2, scl, runx-1 and c-myb also act as regulators of HSPC in teleosts / Doutorado / Genetica Vegetal e Melhoramento / Doutora em Genética e Biologia Molecular

La tristeza es la enfermedad viral más grave del cultivo de los cítricos y su agente causal es el virus de la tristeza de los cí­tricos (CTV). CTV induce uno o más de los siguientes sí­ndromes: I) decaimiento y muerte de los naranjos dulces (ND), pomelos y mandarinos injertados sobre el patrón naranjo amargo (NA), sí­ndrome conocido como "tristeza", II) enanismo, acanaladuras en la madera y fruta de pequeño calibre (stem pitting, SP), y III) enanismo y amarillamiento de plantas de semilla de limonero, pomelo y NA (seedling yellows, SY). La gama de huéspedes de CTV es muy restringida y hasta hace poco no se conocí­a ningún huésped herbáceo experimental. Actualmente, se sabe que la agroinfiltración de Nicotiana benthamiana, con clones de DNA complemantario (cDNA) del aislado T36 de CTV produce la infección sistémica de la planta, acompañada de sí­ntomas similares a los inducidos en cí­tricos, si bien la infección no queda limitada al floema. El aislado T36 induce SY y SP de lima Mejicana (LM), pero no en otros huéspedes como pomelo o ND. El estudio de los determinantes genéticos responsables de la inducción del sí­ndrome de SP requerí­a desarrollar un sistema genético basado en clones agroinfecciosos de un aislado inductor de estos sí­ntomas, como el aislado español T318A. Para ello, se partió de clones de cDNA de longitud completa de T318A previamente desarrollados en el laboratorio, capaces de replicarse en hojas de N. benthamiana pero incapaces de inducir infección sistémica y que presentaban varias mutaciones en su proteína de cápsida minoritaria p27. La corrección de dichas mutaciones y la construcción de nuevos clones de longitud completa de T318A marcados con el gen gfp, mostraron una correcta replicación en hojas agroinfiltradas de N. benthamiana, pero resultaron incapaces de inducir infección sistémica en este huésped experimental. La respuesta diferencial de N. benthamiana frente a distintas cepas de CTV permite estudiar los factores implicados en la interacción virus-huésped. Se analizó la interacción de las proteí­nas virales p20 y p25 de los aislados T36 y T318A con proteí­nas de N. benthamiana utilizando un abordaje consistente en: i) la expresión transitoria de p20/p25 marcadas con una etiqueta Strep-Tag en hojas de N. benthamiana, ii) purificación de los complejos proteí­na CTV-proteína huésped y análisis interactómico de los datos, y iii) estudio de la interacción directa entre p20/p25 y proteínas seleccionadas del huésped mediante análisis del doble hibrido en levadura y complementación bimolecular de fluorescencia (BIFC). Este abordaje proteómico mostró claras diferencias entre aislados que pueden explicar, en parte, el comportamiento diferencial de los aislados T36 y T318A en dicho huésped experimental. La inducción el síndrome de decaimiento por parte de CTV ha obligado a utilizar patrones tolerantes al decaimiento. Dichos patrones son menos adecuados. Las plantas de cí­tricos propagadas sobre NA e infectadas por CTV muestran necrosis en los tubos cribosos y disminución del floema funcional. Éstos desórdenes podrí­an ser consecuencia de la activación de los mecanismos de defensa como la reacción de hipersensibilidad desencadenada por la ruta del ácido salicí­lico o el silenciamiento génico mediado por RNA (post-transcriptional gene silencing, PTGS). Con el objetivo de avanzar en el mecanismo molecular de la resistencia del NA a la infección por CTV, se estudió el papel de diferentes genes de la planta implicados en las rutas mediante el uso de un vector viral basado en el genoma del virus del manchado foliar de los cítricos (citrus leaf blotch virus, CLBV). El silenciamiento génico de las rutas del AS o del PTGS en plantas NA y la inoculación de tres aislados de CTV patogénicamente diferentes mostró la implicación de ambas rutas en la defensa del NA frente a CTV. / Tristeza is the most important viral disease affecting citrus plants and Citrus tristeza virus (CTV) is the causal agent of this disease. CTV induces at least one of this syndromes: I) decline and death of sweet orange (SwO), grapefruits and mandarin trees grafted on sour orange (SO) rootstock, this syndromes is known as "tristeza", II) stunting, stem pitting (SP) and small fruits, and III) stunting and leaf chlorosis of lemon, grapefruit and SO seedlings (seedling yellows, SY). The host range of CTV is restricted and until recently no experimental herbaceous host was known. The agroinoculation Nicotiana benthamiana with clones of complementary DNA (cDNA) from the CTV isolate T36 cause the systemic infection of the plant and similar symptoms to those observed in citrus, although the infection is not limited to the phloem. T36 isolate induces SY and SP of Mexican lime (ML), but not in other hosts such as grapefruit and SwO. Therefore, to study the genetic determinants responsible of the SP syndrome induction was necessary to develop a genetic system based on agroinoculated clones from an isolate able to induce these symptoms, such as the Spanish isolate T318A. To do this, full length cDNA clones from T318A were obtained. They are able to replicate in N. benthamiana leaves but unable to induce systemic infection and showed several mutations in their protein of the minor coat, p27. The correction of these mutations and the construction of new clones of complete length from T318A labeled with the gfp gene, showed a proper replication in agroinoculated leaves of N. benthamiana, but they were still unable to induce systemic infection in this experimental host. The differential response of N. benthamiana to different CTV strains allows the study of the potential factors involved in the virus-host interaction. The aim of this work was study the interaction between the viral proteins p20 and p25 from the isolates T36 and T318A with N. benthamiana proteins with an analysis consisted in: I) the transitory expression of p20/p25 fused to Strep-Tag in N. benthamiana leaves, II) purification of the CTV protein-host protein complex and interatomic analysis of the data, and III) the study of the direct interaction between p20/p25 and selected plant proteins by the analysis of the double hybrid in yeast and bimolecular complementation of fluorescence (BIFC). The proteomic analysis showed strong differences between isolates that may partially explain the differential behavior of the T36 and T318A isolates in this experimental host. The induction of decline syndrome by CTV in citrus has leaded the use of tolerant rootstocks to decline. However, the use of such rootstocks is less suitable. Citrus plants propagated on SO rootstock and infected by CTV show phloem necrosis below the bud union that reduces the flow of carbohydrates to the roots. These symptoms may be a consequence of the activation of defense pathways in the plant, such as the hypersensitive reaction, hormone salicylic acid (SA) pathways or the RNA mediated post-transcriptional gene silencing (PTGS). Their relation is essential to know their implication in the decline. Therefore, the role of different genes involved in SA and PTGS has been studied by the silencing of plant genes using a viral vector (VIGS) based in the genome of the citrus leaf blotch virus (CLBV). The gene silencing of the SA and PTGS in SO and the inoculation of three different pathogenicity CTV isolates showed that both pathways are involved in the SO defense against CTV. The analysis of the proteins p20, p23 and p25 as possible suppressors of the AS indicating that the more virulent CTV isolates possess the more powerful suppressors. / La Tristesa és la malaltia viral més greu del cultiu dels cítrics. CTV induïx un o més de les sí­ndromes següents: I) decaïment i mort de taronger dolç§ (ND), pomelo i mandariner empeltats sobre el patró taronger amarg (NA), sí­ndrome conegut com "Tristesa", II) nanisme, estries en la fusta i fruita de xicotet calibre (SP) i III) nanisme i tonalitat groguenta de plantes de llavor de llimera, pomelo i taronger amarg (SY). El rang d'hostes de CTV és molt restringit i fins fa poc no es coneixia cap hoste herbaci experimental. Actualment es sap que la infecciò sistèmica en Nicotiana benthamiana amb clons de DNA complementari (cDNA) de l`aïllat de T36 provoca la infecció sistemàtica de la planta, acompanyada de síntomes similars als induïts en cí­trics, si be la infecció no queda llimitada al floema. L' aïllat T36 induïx SY i estries en la fusta de Llima Mexicana (LM), però no en altres hostes com a pomelo, ND o NA, l'estudi dels determinants genètics responsables de la inducció de la síndrome de SP requeria desenvolupar un sistema genètic basat en clons agroinfecciosos d'un aïllat inductor d'estos sí­mptomes, com l'aïllat espanyol T318A. Per a això, es va partir de clons de cDNA longitud completa de T318A prèviament desenvolupats al laboratori, capaços de replicar-se en fulls de N. benthamiana però incapaços d'induir infecció sistèmica i que presentaven varies mutacions en la seua proteïna de càpsida minoritatia p27. La correcció d`aquestes mutacions i la construcció de nous clons T318A de longitud completa marcats amb el gen gfp, van mostrar una correcta replicació en fulls agroinfiltradas de N. benthamiana però van resultar incapaços d'induir infecció sistèmica en aquest hoste experimental. La resposta diferencial dependent d'aïllat en N. benthamiana front CTV permet estudiar els possibles factors de la interacció virus- hoste. Es va dur a terme l'estudi de la funció de les proteínes virals p20 i p25 dels aïllats T36 i T318A amb proteïnes de N. benthamiana utilitzant un abordatge consistent en: i) l' expressió transitòria de les dues proteïnes p20/p25 marcades amb una etiqueta Strep-Tag en fulls de N. benthamiana, ii) purificació dels complexos proteïna CTV-proteïna hoste i anàlisi interactómic de les dades, i iii) estudi de la interacció directa per mitjà  de doble híbrid en llevat i complementació bimolecular de fluorescència (BIFC) de les proteïnes virals i determinades proteïnes de N. benthamiana. Aquest abordatge proteòmic va mostrar clares diferències entre aïllats que poden explicar el comportament diferencial dels aïllats T36 i T318A en aquest hoste experimental. La inducció de la sí­ndrome de decaïment per part de CTV en cí­trics ha obligat la utilització de patrons tolerants al decaïment. No obstant, aquestos patrons són agronòmicament menys adequats. Les plantes de cítrics propagades sobre NA i infectades por CTV mostren necrosi als tubs cribosos i disminució del floema funcional. Aquestos sí­mptomes poden ser conseqüència de l'activació de les rutes de defensa de la planta com la reacció d'hipersensibilitat, desencadenada per la ruta de l'àcid salicí­lic o el silenciamient gènic mediat per RNA (PTGS). Amb l'objectiu d'analitzar la implicació d¿aquestes rutes en la defensa, es va estudiar el paper de diferents gens implicats en la ruta de l'AS i del PTGS per mitjà  del silenciamient gènic induït per virus basat en el genoma del tacat foliar dels cítrics (CLBV). El silenciamient gènic de les rutes AS o PTGS en plantes NA i la inoculació de tres aïllats de CTV patogènicament diferents va mostrar la implicació de les dues rutes en la defensa del NA front CTV. L'analisis de les proteïnes p20, p23 i p25 com a possibles supressors de la ruta de l'AS va indicar que els aïllats més virulents de CTV posseïxen supressors més potents. / Gómez Muñoz, N. (2017). ESTUDIO DE LA INTERACCIÓN DIFERENCIAL ENTRE AISLADOS DEL VIRUS DE LA TRISTEZA DE LOS CÍTRICOS (CTV) Y SUS HUÉSPEDES [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/94624 / TESIS

Small non-coding RNAs regulate gene expression at posttranscriptional level in eukaryotic cells. Two classes of such small (~21-25 nt) RNAs that have been extensively studied in gene silencing are short interfering RNAs (siRNAs) and microRNAs (miRNAs). RNA interference (RNAi) is process whereby double-stranded RNA induces the sequence-specific degradation of homologous mRNA. The RNAi machinery can also be programmed in human cells by introducing 21-nt siRNA duplexes that are assembled into RNA-induced silencing complexes (RISC). In this dissertation, systematic analysis of siRNAs with deletions at the passenger and/or guide strand reveals that a short RNAi trigger, 16-nt siRNA, induces potent RNAi in human cells. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene. In vitro kinetic analysis of human RISC indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that 16-nt duplexes can be designed as potent triggers for RNAi. RISC can be programmed by small interfering RNAs (siRISC) to cleave a perfectly complementary target mRNA, or endogenous microRNAs (miRISC) to inhibit translation by binding imperfectly matched sequences in the 3’-untranslated region (3’-UTR) of target mRNA. Both RISCs contain Argonaute2 (Ago2), which localizes to cytoplasmic mRNA processing P-bodies. This dissertation shows that RCK/p54, a DEAD box helicase, interacts with Ago2, in affinity-purified active siRISC or miRISC, facilitates formation of P-bodies. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm, but did not significantly affect siRNA-mediated RNAi. Depleting RCK/p54 releases general and miRNA-induced translational repression. These findings imply that miRISC-mediated translation repression requires RCK/p54, also suggest that location of miRISC to P-bodies is not required for miRNA function, but is the consequence of translation repression. To elucidate the function of RCK/p54 in miRNA-mediated gene silencing, analysis of a series of YFP-tagged RCK/p54 mutants reveals the motif required for P-body localization, interaction with Ago2, and/or facilitating the miRNA-mediated translation repression. Additionally, rabbit reticulocyte lysate system was used to recapitulate the miRISC function in a cell-free system and confirmed the requirement of RCK/p54 for miRNA function in vitro. Analysis of Ago2 distribution in the polysome profiling in RCK/p54-depleted cells, compared to that in normal cells, revealed that RCK/p54 facilitates miRISC by trapping it at translation initiation complex. These data suggest that interaction of RCK/p54 with Ago2 is involved in the repression of translation initiation of miRNA function.

Au cours des cinq dernières années l’existence d’un contrôle de la transcription par les ARN non-codants longs (lncRNAs) a été décrite dans une large variété d’eucaryotes. Cependant, les mécanismes par lesquels les lncRNAs régulent la transcription restent en grande partie méconnus. Les premiers travaux effectués dans le cadre de cette thèse ont participé à la caractérisation du mécanisme mis en jeu par un lncRNA, nommé nam1, dans le contrôle de l’entrée en différenciation sexuelle chez la levure Schizosaccharomyces pombe. Il a ainsi été montré qu’au cours de sa synthèse le lncRNA nam1 est ciblé par la protéine de liaison à l’ARN Mmi1 et une machinerie de surveillance des ARN qui comprend l’exosome, un complexe de dégradation des ARN conservé au cours de l’évolution. La fixation de Mmi1 au lncRNA nam1 contrôle la terminaison de la transcription de nam1 et empêche ainsi la transcription de se poursuivre et d’interférer alors avec la transcription du gène situé en aval (codant pour une MAP kinase essentielle à l’entrée en différenciation). Les travaux suivant montrent l’implication dans ce mécanisme de la protéine Cti1, un des co-facteurs connus de l’exosome. Fait marquant, ces travaux rapportent aussi l’existence d’un mode de production inédit pour un lncRNA. En effet, ils révèlent que la transcription non-interrompue d’un gène codant conduirait à la production d’un ARN bi-cistronique. La maturation co-transcriptionnelle de cet ARN bi-cistronique produirait, d’un côté, un ARN messager et, de l’autre, le lncRNA nam1. Enfin, ils ont permit la caractérisation initiale d’un nouveau composant de la machinerie de surveillance des ARN recrutée sur nam1 par Mmi1. Ainsi, dans leur ensemble, ces travaux contribuent à une meilleure connaissance des mécanismes pouvant être mis en jeu par un lncRNA et agissant en cis pour réguler l’expression génique et, à travers elle, des processus cellulaires majeurs, tel que la différenciation cellulaire. De plus, ils décrivent un nouveau mécanisme de biogénèse d’un lncRNA. / Over the last five years, the control of transcription mediated by long non-coding RNAs (lncRNAs) has been reported to take place in a wide variety of eukaryotes. However, the mechanisms by which lncRNAs regulate transcription remain relatively poorly described. The first work conducted in the context of this PhD thesis has contributed to the characterization of the mechanism used by a lncRNA, named nam1, to control entry into sexual differentiation of the fission yeast Schizosaccharomyces pombe. It was shown that, while the lncRNA nam1 is being produced, it is targeted by the RNA binding protein Mmi1 and a RNA surveillance machinery that includes the exosome, a conserved complex throughout evolution. The binding of Mmi1 to nam1 lncRNA controls the termination of transcription of nam1, which prevents this non-coding transcription from interfering with the transcription of the downstream gene, coding for a MAP kinase essential to entry into differentiation. The following work shows the importance of the protein Cti1, one of the known co-factor of the exosome, in the nam1-dependent control of sexual differentiation. Remarkably, it also strongly suggests the existence of a new way of producing a lncRNA. Indeed, it reveals that read-through transcription of a protein-coding gene leads to the production of a bi-cistronic RNA, which is co-transcriptionally matured to produce on one side a messenger RNA and on the other side the lncRNA nam1. Finally, this work initiated the characterization of a new component of the RNA surveillance machinery targeting nam1. Collectively, this work brings several insights into the mechanisms used by cis-acting lncRNAs to regulate gene expression and, thereby, major cellular processes such as cell differentiation. Moreover, it also provides insights into the biogenesis of lncRNAs by reporting a new mode of production of lncRNAs.

Small extracellular vesicles (small EVs) are endogenous membrane-enclosed nanocarriers released from essentially all cells. They have been shown to carry proteins, lipids, nucleic acids to transmit biological signals throughout the body, including to the brain. Some evidence has suggested that small EVs can cross the blood-brain barrier (BBB), moving from the peripheral circulation to the central nervous system (CNS). The BBB is a dynamic barrier that regulates molecular trafficking between the peripheral circulation and the CNS. As a result, small EVs have attracted attention for their potential as a novel delivery platform for nucleic acid-based therapeutics across the BBB. Silencing RNAs (siRNAs) are a potent drug class but using “naked” siRNA is not feasible due to their short half-life, vulnerability to degradation and low penetration in cells. Despite the excitement for the development of small EV-based therapeutics, their clinical development is hampered by the lack of reliable methods for packing therapeutics into them. Reshke et al. has shown that cells can be genetically engineered to produce customizable small EVs packaged with siRNA against any protein by integrating the siRNA sequence into the pre- miR-451 structure. Mounting evidence has established that in a misfolded state, α-synuclein becomes insoluble and phosphorylated to form intracellular inclusions in neurons (known as Lewy bodies) which leads to Parkinson’s disease (PD) pathogenesis. Given that increased α-synuclein expression causes familial and idiopathic PD, decreasing its synthesis by using siRNA is an attractive therapeutic strategy. Here, we genetically engineered cells to produce small EVs packaged with siRNA against α-synuclein integrated in the pre-miR451 backbone, tested their ability to cross an in vitro BBB, and deliver its cargo to silence endogenous α-synuclein in neuron- like cells. The therapeutic potential of α-synuclein siRNA delivery by these small EVs was demonstrated by the strong mRNA (60-70%) and protein knockdown (43%) of α-synuclein in neuron-like cells. We also demonstrated that approximately at 4% and 2%, respectively of small EVs-derived from human brain endothelial cells (hCMEC/D3) and human embryonic kidney (HEK293T) were transported cross the in vitro BBB model. Interestingly, we observed that small EVs-derived from HEK293T deliver their cargo to induced brain endothelial cells (iBECs) (~74% α-synuclein mRNA reduction) but their rate of transport across BBB was lower and did not reduce α-synuclein mRNA expression in neuron-like cells, seeded on the far side of the BBB. Small EVs- derived from hCMEC/D3 reduced α-synuclein mRNA (40%) in neuron-like cells across the BBB model. This finding suggests that small EVs derived from different cell sources can undergo different intracellular trafficking routes, providing various opportunities to influence the efficiency of delivery and fate of intracellular cargo. Using small EVs-derived from hCMEC/D3, two different routes of administration, a single bolus intravenous (IV) or intra-carotid (ICD) injection, showed small EVs largely accumulated in the liver, spleen, small intestines and kidneys; and only a small amount of small EVs were detected in the brain. These results indicate that human brain endothelial cells may serve as a promising cell source for CNS treatments based on small EVs.

The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...