Trz1 chez levure est responsable du clivage endonucléolytique à l'extrémité 3 'au cours du processus de maturation des ARNt. Trz1 appartient à la famille des RNases de type b-lactamase, caractérisé par la présence d'un motif de séquence HxHxDH qui est impliqué dans la fixation des ions de zinc catalytique. La famille des RNaseZ est partagée en deux sous-familles de longueur de séquence différente: les formes courtes (300-400 acides aminés) et les formes longues (700-900 acides aminés). Les structures cristallines des enzymes RNaseZ de forme courte ont montré qu'ils sont actifs comme des homodimères. Une sous-unité englobe les ARNt substrat en utilisant un bras en saillie et l'autre fournit le site catalytique. Nous présentons ici la structure cristalline de Trz1, la première pour une RNase Z de forme longue. Trz1 est organisé en deux domaines reliés par un long peptide charnière. Chaque domaine est composé d'un repliement de type β-lactamase. Le domaine N-terminal a perdu ses résidus catalytiques au cours de l’évolution, mais il contient le bras long qui est important pour la liaison de l'ARNt; tandis que c’est l'inverse pour le domaine C-terminal. À partir des études protéomiques, on sait que Trz1 forme un complexe ternaire avec NUC1, une nucléase mitochondriale impliquée dans l'apoptose, et avec une mutarotase (codée par YMR099C). Nous avons purifié le complexe ternaire Trz1/NUC1/mutarotase caractérisé ses propriétés biochimiques. Trz1/NUC1/mutarotase forme in vitro un heterohexamère très stable en solution. A partir de nos données SAXS et MALLS nous proposons que l'homodimère NUC1 est au centre du complexe et que chaque sous-unité interagit avec une copie de Trz1 et une copie de mutarotase. / Yeast Trz1 is responsible for the endonucleolytic cleavage at the 3’-end during the maturation process of tRNAs. Trz1 belongs to the family of β-lactamase type RNases characterized by the presence of a HxHxDH sequence motif that is involved in the ligand formation of the catalytical required Zn-ions. The family consists of two subfamilies: the short forms with sequence lengths between and the long forms. A few crystal structures of short form RNase Z enzymes showed that they are active as homodimers. One subunit embraces the substrate tRNA using a protruding arm and the other provides the catalytic site. We here present the crystal structure of Trz1, the first of a long form RNase Z. Trz1 is organized in two domains connected by a large linker. Each domain is composed of a beta-lactamase type fold. The N-terminal domain has lost its catalytic residues, but contains the long arm that is important for tRNA binding; while it is the other way around of the C-terminal domain. From proteomics studies it is known that Trz1 forms a ternary complex with NUC1, a mitochondrial nuclease involved in apoptosis, and with a mutarotase (encoded by YMR099C). We purified the ternary Trz1/Nuc1/mutarotase complex and characterized its biochemical properties. Trz1/Nuc1/mutarotase forms in vitro a very stable heterohexamer in solution. From our SAXS and MALLS data we propose that the Nuc1 homodimer is at the centre of the complex and that each subunit interacts with one copy of Trz1 and mutarotase.

Submitted by PPG Odontologia (odontologia-pg@pucrs.br) on 2017-10-03T13:27:09Z No. of bitstreams: 1 MARIA_NOEL__MARZANO_RODRIGUES_PETRUZZI_TES.pdf: 3641098 bytes, checksum: 3aa8ce331659fd0a8d765be0dc6f3d45 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-10-04T12:34:26Z (GMT) No. of bitstreams: 1 MARIA_NOEL__MARZANO_RODRIGUES_PETRUZZI_TES.pdf: 3641098 bytes, checksum: 3aa8ce331659fd0a8d765be0dc6f3d45 (MD5) / Made available in DSpace on 2017-10-04T12:36:41Z (GMT). No. of bitstreams: 1 MARIA_NOEL__MARZANO_RODRIGUES_PETRUZZI_TES.pdf: 3641098 bytes, checksum: 3aa8ce331659fd0a8d765be0dc6f3d45 (MD5) Previous issue date: 2017-06-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / A late diagnosis of oral squamous cell carcinoma is related to high morbidity and mortality rates, as well as recurrence after treatment. Hence, there is an increasing interest in the validation of biological markers, chemoprevention strategies, and adjuvant treatment alternatives for combating oral cancer. The present Thesis evaluated the anticancer effect of cannabidiol (CBD) in a validated experimental murine model of oral carcinogenesis. Fifteen Fischer 344 rats were randomly divided into three groups of five animals each and subjected to a 7,12-dimethylbenzanthracene (DMBA) topical application on the ventral mucosa of their tongues, thrice a week. From the 2nd to the 12th week, they received intraperitoneally-administered vehicle (group 1), CBD 5 mg/kg (group 2), or CBD 10 mg/kg (group 3). After euthanasia, the tongues were dissected, processed and assessed by histological and immunohistochemical analysis. Group 2 and group 3 showed inhibition of severe oral epithelial dysplasia and carcinoma, and exhibited lower cell proliferation as compared to group 1. The null hypothesis was rejected when results showed statistical significance at a 0.05 level (confidence interval = 95%). In sequence are presented three manuscripts, first one regarding the original experiment and the two subsequent ones providing overall theoretical support. / O diagn?stico tardio do carcinoma espinocelular oral est? relacionado a um alto ?ndice de morbi-mortalidade e recorr?ncia ap?s o tratamento. Portanto, h? um crescente interesse na valida??o de marcadores biol?gicos que contribuam para o diagn?stico precoce, novas estrat?gias de quimiopreven??o e recursos adjuvantes para o tratamento dessa neoplasia maligna. A presente Tese centrou-se na avalia??o do efeito anticarcinog?nico do canabidiol (CBD) em um modelo experimental consagrado para a indu??o de altera??es epiteliais, com risco de malignidade, na mucosa oral de murinos. Para tanto, 15 ratos Fischer 344 foram aleatoriamente divididos em tr?s grupos, onde todos os animais tiveram o ventre de suas l?nguas expostas ao 7,12-dimetilbenzantraceno (DMBA) tr?s vezes por semana. A partir da segunda semana os grupos receberam por via intraperitoneal, ve?culo (grupo 1), 5 mg/Kg ou 10 mg/Kg de CBD (grupos 2 e 3, respectivamente). Na d?cima segunda semana ap?s o in?cio do experimento realizou-se a eutan?sia dos animais, dissec??o das l?nguas e processamento dos esp?cimes. As an?lises foram realizadas por meio das t?cnicas histol?gicas de rotina e imunoistoqu?mica. Observou-se a inibi??o do desenvolvimento de displasia oral severa e carcinoma, bem como a modula??o dos ?ndices de prolifera??o celular nos grupos 2 e 3 em rela??o ao grupo 1. A hip?tese nula p?de ser rejeitada, uma vez que os resultados obtidos apresentaram n?vel de signific?ncia de 0,05 (intervalo de confian?a = 95%). A seguir, apresentam-se tr?s artigos cient?ficos que descrevem primeiramente o experimento original desenvolvido e, em sequ?ncia, as revis?es da literatura para subsidiar a discuss?o do tema proposto.