Das Harnblasenkarzinom (BCa) stellt in Deutschland die vierthäufigste Tumorneuerkrankung und die zehnthäufigste krebsbedingte Todesursache bei Männern dar. Nichtmuskelinvasive BCa werden organerhaltend aus der Blasenwand entfernt und zur Rezidiv- und Progressionsprophylaxe mittels intravesikaler Chemo- oder Immuntherapien behandelt. Trotz dieser adjuvanten Therapien, die mit starken Nebenwirkungen verbunden sein können, ist nur eine bedingte Minimierung des Rezidivrisikos möglich. Besonders im fortgeschrittenen Stadium weisen Harnblasenkarzinome eine schlechte Prognose auf. Obwohl das BCa eine chemosensitive Erkrankung darstellt, wird das Ansprechen auf lokale oder systemische Chemotherapien häufig durch auftretende Resistenzmechanismen limitiert. Daher stehen sowohl die Verbesserung konventioneller Chemotherapien als auch die Suche nach neuartigen Behandlungsstrategien im Fokus der experimentellen BCa-Forschung. Die Apoptose, eine Form des programmierten Zelltodes, ist ein essenzieller, streng regulierter biologischer Prozess, welcher der Aufrechterhaltung der Gewebshomöostase und der gezielten, entzündungsfreien Eliminierung geschädigter Zellen dient. Fehlregulationen in den Apoptosesignalwegen stellen ein zentrales Ereignis in der Tumorgenese dar und tragen außerdem zur Entstehung von Chemo- und Radiotherapieresistenzen bei. Eine wichtige Rolle in der Apoptoseregulation spielen die Mitglieder der BCL2- und der Inhibitor of Apoptosis Protein (IAP)-Familien, deren wichtigste antiapoptotische Vertreter BCL2, BCL-XL, XIAP und Survivin häufig in Tumoren, einschließlich des BCa, überexprimiert sind. Unter Verwendung von small interfering RNAs (siRNAs), synthetischen Nukleinsäurekonstrukten zur selektiven Geninhibition, wurde im Rahmen der Arbeit in vitro und in vivo untersucht, ob die Hemmung der Apoptoseinhibitoren BCL2, BCL-XL, XIAP und Survivin – allein und in Kombination mit Chemotherapie – eine Therapieoption zur Behandlung des BCa darstellen könnte. Da zur Tumorentstehung und -progression eine Vielzahl von genetischen Veränderungen beitragen, erscheint der Angriff eines einzelnen Zielgens unzureichend für eine effektive Tumortherapie. Aufgrund dessen wurde untersucht, ob durch simultane Reduktion der ausgewählten Apoptoseinhibitoren in BCa-Zellen stärkere wachstumsinhibitorische Effekte erzielt werden können. In der vorliegenden Arbeit wurde gezeigt, dass insbesondere die siRNA-vermittelte Hemmung von BCL-XL und Survivin in den BCa-Zelllinien EJ28 und J82 antiproliferative Effekte hervorruft und diese Tumorzellen gegenüber einer nachgeschalteten Chemotherapie mit Mitomycin C oder Cisplatin sensitiviert. Hingegen bewirkte sowohl die transiente als auch die stabile RNAi-induzierte Hemmung von BCL2 und XIAP in den untersuchten BCa-Monolayerzellkulturen, möglicherweise infolge kontinuierlicher Versorgung der Tumorzellen mit Sauerstoff und Nährstoffen, keine Reduktion des Tumorwachstums. Eine gegenüber den Einzelbehandlungen deutliche Verstärkung der antitumoralen und insbesondere der chemosensitivierenden Effekte in den BCa-Zelllinien wurde durch simultane Hemmung von BCL-XL und Survivin erzielt. Beispielsweise stieg der Anteil apoptotischer Zellen von 64 % nach Survivin-siRNA+Cisplatin-Behandlung auf 94 % nach gleichzeitiger BCL-XL+Survivin-Inhibition in Kombination mit Cisplatin. Folglich stellt die simultane Inhibition von BCL-XL und Survivin in Kombination mit Chemotherapeutika eine äußert viel versprechende BCa-Therapieoption dar. Tierexperimentelle Studien belegen die wachstumsinhibitorische Wirkung der Survivin-Reduktion und der kombinierten BCL-XL-siRNA+Chemotherapie-Behandlung, so wurde das Tumorendvolumen im Vergleich zur Kontrollbehandlung um 43 % bzw. um 48 % reduziert.

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 68 pages. Includes Vita. Includes bibliographical references.

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of a complementary target mRNA. Previous work proved that certain structural features of siRNAs are essential for RNAi in flies, including the requirement for 5´ phosphates and 3´ hydroxyl groups. In cultured mammalian cells, the requirement for a 5´ phosphate also holds true, but we found no evidence to support the necessity for 3´ hydroxyls in either system. In addition, siRNAs can act as single strands entering the pathway downstream of double-stranded siRNAs, both of which are competent in directing the cleavage of its cognate mRNA at a single site. While these key features are a requirement for functional siRNAs, alone they do not determine the efficiency to which an siRNA can enter the RISC. In fact, both strands of an siRNA can enter RISC to a different degree as determined by the stabilities of the 5´ ends of the siRNA strand, a phenomenon termed ‘functional asymmetry’. This characteristic is also reflected in another class of small RNAs involved in gene silencing known as microRNAs (miRNAs), which are processed from long hairpin RNA structures into mature, single-stranded non-coding RNAs. The asymmetric loading of siRNAs suggests that miRNAs are initially generated from siRNA-like duplexes cleaved from the stem of the hairpins. The strand whose 5´ end is less tightly paired will be processed into the mature miRNA, while the other strand is destroyed. By applying the rules of siRNA asymmetry it is possible to predict which side of the stem will be processed into the mature miRNA, a finding verified experimentally by our lab and others. This discovery also has additional implications in designing highly effective siRNAs and in reducing siRNA off-target effects. We used these results to design siRNAs that target the single nucleotide polymorphism in superoxide dismutase that causes the familial form of amyotrophic lateral sclerosis (ALS), but leave the wild-type mRNA intact and functional. Our experiments have helped define the ‘rules’ for creating SNP-specific siRNAs. In particular, we found that only siRNAs with a purine:purine mismatch to the allele not intended for destruction show good discrimination. The placement of the mismatch in a tiled set of siRNAs shows that mismatches located in the 5´ region of the siRNA, a region shown to be responsible for siRNA binding, can not discriminate between alleles. In contrast, mismatches in the 3´ region of the siRNA, the region contributing to catalysis, discriminate between wild-type and mutant alleles. This work is an important step in creating allele-specific siRNAs as therapeutics for dominant negative genetic diseases. But how does RISC cleave its target? By isolating both the 5´ and 3´ cleavage products produced by RISC in the Drosophila in vitro system, we discovered that RISC acts as a Mg2+-dependent endonuclease that cleaves a single phosphodiester bond in the mRNA target, leaving 5´ phosphate and 3´ hydroxyl groups. These findings were a critical step in the demonstration that Argonaute, a protein known to be a component of RISC, is the RNAi endonuclease.

RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex, called the RNA-induced silencing complex (RISC), as single-strands. A member of the Argonaute family of small RNA-binding proteins lies at the core of all known RNA silencing effector complexes. Plants and animals contain multiple Argonaute paralogs. In addition to endonucleolytic cleavage, Argonaute proteins can direct translational repression/destabilization of mRNA or transcriptional silencing of DNA sequences by the siRNAdirected production of silent heterochromatin. The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. That only one Dicer, RdRP and Argonaute is expressed in S. pombe might reflect the extreme specialization of RNA silencing pathways regulating targets only at the transcriptional level in this organism. We decided to test if classical RNAi can be induced in S. pombe. We introduced a dsRNA hairpin corresponding to a GFP transgene. GFP silencing triggered by dsRNA reflected a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe required dcr1, rdp1, and ago1, but did not require chp1, tas3, or swi6, genes required for transcriptional silencing. We concluded that the RNAi machinery in S. pombecould direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that, in spite of specialization in distinct siRNA-directed silencing pathways, these three proteins fulfill a common biochemical function. In Drosophila, miRNA and RNAi pathways are both genetically and biochemically distinct. Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)/Loquacious complex produces miRNAs. Argonaute proteins can be divided by sequence similarity into two classes: in flies, the Ago subfamily includes Argonaute1 (Ago1) and Argonaute2 (Ago2), whereas the Piwi subfamily includes Aubergine, Piwi and Argonaute 3. siRNAs and miRNAs direct posttranscriptional gene silencing through effector complexes containing Ago1 or Ago2. The third class of small RNAs, called repeat-associated small interfering RNAs (rasiRNAs), is produced endogenously in the Drosophilagerm line. rasiRNAs mediate silencing of endogenous selfish genetic elements such as retrotransposons and repetitive sequences to ensure genomic stability. We examined the genetic requirements for biogenesis of rasiRNAs in both male and female germ line of Drosophilaand silencing of 8 different selfish elements, including tree LTR retrotransposons, two non-LTR retrotransposons, and three repetitive sequences. We find that biogenesis of rasiRNAs is different from that of miRNAs and siRNAs. rasiRNA production appears not to require Dicer-1 or Dicer-2. rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. While siRNAs derive from both the sense and antisense strands of their dsRNA precursors, rasiRNAs accumulate in antisense polarity to their corresponding target mRNAs. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. We find that rasiRNAs silence their target RNAs posttranscriptionally: mutations that abrogate rasiRNA function dramatically increase the steady-state mRNA level of rasiRNA targets, but do not alter their rate of transcription, measured by nuclear run-on assay. Our data suggest that rasiRNAs protect the fly germ line through a silencing mechanism distinct from both the miRNA and RNAi pathways.

85 f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-04-08T13:22:40Z No. of bitstreams: 1 NORMANDO DISSERTAÇÃO FINAL (1).doc: 1373696 bytes, checksum: 6e6f4afc01a6708ec44c276e63213713 (MD5) / Rejected by Ana Hilda Fonseca(anahilda@ufba.br), reason: transformar em pdf. on 2013-06-06T12:59:17Z (GMT) / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-06-06T13:43:50Z No. of bitstreams: 1 Dissertação NORMANDO DISSERTAÇÃO FINAL (1).pdf: 1416415 bytes, checksum: 4927e2bf2b9b19287f6adb42fc059ccf (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-06-06T13:47:50Z (GMT) No. of bitstreams: 1 Dissertação NORMANDO DISSERTAÇÃO FINAL (1).pdf: 1416415 bytes, checksum: 4927e2bf2b9b19287f6adb42fc059ccf (MD5) / Made available in DSpace on 2013-06-06T13:47:50Z (GMT). No. of bitstreams: 1 Dissertação NORMANDO DISSERTAÇÃO FINAL (1).pdf: 1416415 bytes, checksum: 4927e2bf2b9b19287f6adb42fc059ccf (MD5) Previous issue date: 2012 / CAPES / O objetivo deste trabalho foi o desenvolvimento de um procedimento analítico empregado Cromatografia Líquida Ultra Rápida acoplada a Detector de Fluorescência (UFLC-FLU) para determinação de interferentes endócrinos (IEs; bisfenol A (BPA), 4n-nonilfenol (4NP), 4-octifenol (4OP), 4-t-octifenol (4TOP), estriol (E3), estrona (E1), 17β- estradiol (E2) e 17α-etinilestradiol (EE2)) em água do mar. Foi utilizado um sistema de pré-concentração em fase solida (cartucho SPE com fase estácionária C18) para extração e pré-concentração dos IE em as amostras de água do mar. A separação foi otimizada e realizada em um tempo total de corrida de 10 min, em uma coluna cromatográfica Shim-pack XR-ODS C-18 (2,0 mm ID x 50 mm), com a fase móvel de acetonitrila e água ultra pura com gradiente de eluição. A vazão foi de 0,12 mL min-1, a temperatura da coluna foi mantida em 60°C e os comprimentos de onda de emissão e excitação foram de 306 nm e 280 nm, respectivamente. O método validado foi aplicado em amostras de água coletadas na Baia de Todos os Santos, Bahia, Brasil. As amostras foram coletadas na Baía da Ribeira, Feira do São Joaquim, Santo Amaro da Purificação, São Francisco do Conde, Cachoeira e Acupe. As recuperações para o IE variaram entre 84,9% (para o composto 4nOP) e 104% (para o composto 4nNP), e a repetibilidade foi adequada (RSD < 4,5%). Os limites de detecção e quantificação encontrados para os compostos estudados variaram de 4 a 27 µg L-1 e de 19 a 185 µg L-1, respectivamente para o método cromatográfico. Considerando o fator de concentração de 2000 vezes, o LD e LQ variaram de 2 a 23 ng L-1 e de 9 a 96 ng L-1, respectivamente quando calculados para amostra real. Foi observada a ocorrência dos seguintes IE nas amostras reais: bisfenol A (BPA), 17β-estradiol (E2), estriol (E3) e 4n-octifenol (4NOP) em concentrações que variaram de 5 ng L-1 em Santo Amaro a 18,3 ng L-1 em Cachoeira para o E2, 20 ng L-1 do 4NOP na Ribeira a 135 ng L-1 no estuario do Rio Subaé (Santo Amaro), sendo o 4NOP o contaminante onipresente nas amostras analisadas. A concentração de 38 ng L-1 de E3 foi encontrada apenas nas amostras da Ribeira. A presença de BPA foi detectada em quase todas as amostras (o BPA não foi detectado na Ribeira), em níveis entre 13 ng L-1 no estuário do rio Paraguaçu em Cachoeira e aproximadamente 77 ng L-1 no estuário do rio Subaé. Os resultados sugerem que as regiões estudadas encontram-se possivelmente impactadas em relação os IE estudados e que as concentrações encontradas poderiam indicar possíveis danos ao ecossistema marinho local. O método analítico empregando SPE e UFLC-FLU se mostrou eficiente na determinação dos oito compostos de interesse usando um volume de amostra de 4 litros. / Salvador

A incidência de melanomas tem crescido mundialmente. Apesar de representar um potencial problema de saúde pública pela sua incidência crescente, melanomas ainda se apresentam como tumores de difícil tratamento, especialmente quando diagnosticados em estadios avançados. A taxa de resposta a quimioterapia não ultrapassa 30% de resposta clínica objetiva nestes casos. As bases moleculares da quimiorresistência não são ainda completamente esclarecidas e seu conhecimento será útil para o delineamento de estratégias de quimiossensibilização. Em estudos anteriores, observamos que o tratamento de linhagem de células de melanoma metastático humano com o quimioterápico cisplatina induz o acúmulo de proibitina nas células sobreviventes. Proibitina é uma molécula expressa ubiquamente na maioria das células. Há evidências de que a forma nuclear esteja envolvida com o processo de morte celular e inibição de E2F1 enquanto a forma citoplasmática parece atuar como chaperona mitocondrial, garantindo sua homeostasia. O objetivo deste projeto foi avaliar a compartimentalização subcelular e a expressão de proibitina, após tratamento com 25 M de cisplatina por 24 horas em diferentes linhagens de melanoma metastático humano; e, o efeito de sua subexpressão, usando-se small interference (si) RNA. Nós mostramos que nas linhagens de melanoma humano LB373Mel, SKMel 37 e Mel 85, a proibitina foi encontrada predominantemente no citoplasma, associada, pelo menos em parte, com a mitocôndria. Após tratamento com cisplatina, uma porção da proibitina também foi encontrada no núcleo, como pode ser detectado utilizando-se anticorpo monoclonal (clone II-14-1). Experimentos de knockdown de proibitina por siRNA obtiveram sucesso em duas de três linhagens. Nessas duas linhagens (LB373Mel e Mel 85), o bloqueio do acúmulo de proibitina após tratamento com cisplatina levou à quimiossensibilização. A quimiossensibilização à cisplatina não foi observada na linhagem SKMel 37, que foi capaz de acumular proibitina mesmo quando tratada com siRNA específico para proibitina. A conclusão deste projeto é que a expressão de proibitina é parte da resposta celular que leva a sobrevivência de células de melanoma expostas à cisplatina / The incidence of melanomas has grown world-wide. Besides representing a potential problem of public health for its increasing incidence, melanomas are tumors of difficult treatment, especially when diagnosed in advanced phases. The clinical objective response rate does not exceed 30% in these cases. The molecular bases of chemoresistance are not completely clarified and their understanding will be useful for the delineation of chemosensitization strategies. In previous studies, we observed that the treatment of a human metastatic melanoma cell line with the chemotherapeutic agent cisplatin induced the accumulation of prohibitin in the surviving cells. Prohibitin is ubiquitously expressed molecule in most cells. There is evidence that the nuclear form is involved with the process of cell death and inhibition of E2F1, while the cytoplasmic form seems to act as mitochondrial chaperone, guaranteeing its homeostasis. The aim of this project was to evaluate the subcellular compartmentalization and protein expression profile of prohibitin, after treatment with 25M of cisplatin for 24 hours in different human metastatic melanoma cell line, and the effect of its underexpression, using siRNA. We showed that, in human melanoma cell lines, LB373Mel, SKMel 37 and Mel 85, prohibitin was found predominantly in the cytoplasm, associated at least in part with the mitochondria. Upon cisplatin treatment, a fraction of prohibitin was also found in the nucleus, as detected by using a monoclonal antibody (clone II-14-10). Knockdown experiments were successful in two out of three cell lines. In these two cell lines (LB373Mel and Mel 85) blockage of the accumulation of prohibitin upon cisplatin treatment led to chemosensitization. Chemosensitization to cisplatin was not observed for SKMel 37 cells, which accumulated prohibitin even when treated with prohibitin specific siRNA oligonucleotides. Altogether, we concluded that expression of prohibitin is part of the cellular response that leads to cell survival in melanoma cells exposed to cisplatin

Orientador: Maria Cristina Cintra Gomes Marcondes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T00:25:10Z (GMT). No. of bitstreams: 1 Tomasin_Rebeka_D.pdf: 4368331 bytes, checksum: 33a1f49902e0d1a423b1a4b7ebe757e9 (MD5) Previous issue date: 2014 / Resumo: Câncer é um nome genérico para um grupo de mais de cem doenças que compartilham algumas características. Talvez a característica mais marcante das neoplasias malignas seja a rápida proliferação de células anormais para além de suas fronteiras usuais, invasão de tecidos adjacentes e finalmente dispersão para órgãos distantes. Anualmente, cerca de oito milhões de pessoas morrem devido ao câncer e outros doze milhões de novos casos são diagnosticados. Dentre os eventos associados à progressão neoplásica, destacam-se as metástases e a caquexia. Metástases são tumores em sítios secundários, sendo responsáveis por cerca de 90% do total de mortes por câncer. Já a caquexia, uma síndrome paraneoplásica, é caracterizada por extensa espoliação de gordura e massa magra, fadiga e alterações metabólicas, prejudicando a qualidade de vida e a sobrevida de cerca de 50-85% dos pacientes, dependendo do tipo de tumor. Com relação às terapias, o grande desafio ainda é combater o tumor sem prejudicar o hospedeiro, o que acredita-se ser possível através de terapias-alvo para genes específicos e/ou tratamentos coadjuvantes, incluindo aqueles que empregam suplementação e/ou drogas derivadas de produtos naturais, que muitas vezes tem menor efeito tóxico ao hospedeiro. Desse modo, este trabalho teve dois objetivos: (1) avaliar a ação da administração oral de Aloe vera e mel sobre o tecido tumoral e o hospedeiro portador de carcinosarcoma de Walker 256; e (2) identificação de genes supressores de metástase através de screen funcional in vivo empregando-se biblioteca de shRNA em modelo de câncer de mama triplo negativo. Em relação ao primeiro objetivo, os resultados sugerem que a associação entre Aloe vera e mel pode modular a proteólise e o estresse oxidativo de maneira diferencial preservando o hospedeiro em detrimento do tecido tumoral. Ainda, o tratamento com Aloe vera e mel parece ser capaz de diminuir a propensão metastática das células tumorais in vivo, através de aumento na expressão de caderina-E e redução na expressão de caderina-N, bem como inibição da angiogênese. Outros experimentos sugerem que os efeitos antitumorais observados in vivo estão, em parte, relacionados à ação imunomodulatória de alguns componentes da Aloe vera. Com relação ao segundo objetivo, foram identificados dezenas de candidatos a genes supressores de metástase. Dentre esses genes, que estão sendo validados, Mnat1, Snd1, Cul5, Gabbr1, Rorb, Adk, Ccnd3, Gdnf, Nr1d1, Ptprs e Ltah4 são os genes-candidatos de maior confiabilidade por cumprirem um ou mais dos seguintes requisitos: (a) diminuição significativa do nível de DNA e RNA em canceres de mama humanos agressivos, sendo assim relacionados à pior prognostico, (b) papel biológico sugestivo, (c) fenótipo marcante durante o screen ou ainda (d) decréscimo significativo na expressão em linhagens de câncer de mama mais agressivas / Abstract: Cancer is a generic name for a group of over a hundred diseases which share some features. The most remarkable feature in cancer disease is the fast proliferation of abnormal cells beyond their usual boundaries, invasion of surrounding tissues and finally spread to distant organs. Every year, cancer is responsible for over eight million deaths and twelve million new cases are diagnosed. Among all the events associated with the neoplastic progression, metastasis and cachexia are major issues. Metastases, which are tumours growing in secondary sites, account for 90% of all cancer deaths. Cachexia, a paraneoplastic syndrome, is characterized by severe fat and lean mass waste, fatigue and metabolic alterations, jeopardizing the quality of life and reducing the survival of about 50-85% of the cancer patients, depending on the tumour type. Regarding to the therapies, the biggest challenge is still fighting the tumour without harming the host, which is believed to be possible by targeted therapies to specific genes and/or adjuvant treatments, including supplementation and drugs derived from natural compounds, which usually have lower side effects in the host. Knowing these points, this work had two aims: (1) evaluate the effects of Aloe vera and honey in both tumour and host tissues in Walker 256-tumour bearing rats; and, (2) identification of metastasis suppressor genes using a functional in vivo shRNA screen in a triple negative breast cancer syngeneic model. Regarding to the first aim, the results suggested that the combination of Aloe vera and honey selectively modulate proteolysis and oxidative stress, damaging the tumour tissue while protected the host. Moreover, the Aloe vera and honey treatment apparently decreases the metastatic potential in vivo, by simultaneous increase in E-cadherin and decrease in N-cadherin expression, while decreased tumour vascularization. Finally, our results suggested the antitumoral effects observed in vivo are, at least partially, related to the immunomodulatory activity of some Aloe vera¿s compounds. Regarding to the second aim, dozens of putative metastasis suppressor genes were identified. High confidence candidates, which would be further analysed are Mnat1, Snd1, Cul5, Gabbr1, Rorb, Adk, Ccnd3, Gdnf, Nr1d1, Ptprs e Ltah4. Their selection was based on meeting the following requirements: (a) significant decrease at DNA or RNA level in highly aggressive human breast cancer carcinomas and thus, worse prognosis, (b) suggestive biological role, (c) occurrence of a remarkable phenotype during the screen, and (d) significant decrease in expression in more aggressive cancer cell lines / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana. / Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime. / Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631 / 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.