Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-04-02T15:02:43Z No. of bitstreams: 1 GustavoHenriqueOliveiraCavalcante_DISSERT.pdf: 14332647 bytes, checksum: 07f54cbd966599c411e90b07dbb86ba8 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-04-05T13:57:34Z (GMT) No. of bitstreams: 1 GustavoHenriqueOliveiraCavalcante_DISSERT.pdf: 14332647 bytes, checksum: 07f54cbd966599c411e90b07dbb86ba8 (MD5) / Made available in DSpace on 2018-04-05T13:57:34Z (GMT). No. of bitstreams: 1 GustavoHenriqueOliveiraCavalcante_DISSERT.pdf: 14332647 bytes, checksum: 07f54cbd966599c411e90b07dbb86ba8 (MD5) Previous issue date: 2018-02-23 / O papilomav?rus humano (HPV) ? um pequeno v?rus de DNA de dupla fita circular, caracterizado como um dos mais comuns agentes sexualmente transmiss?veis no mundo, cuja detec??o e genotipagem acuradas s? s?o poss?veis por meio de t?cnicas de biologia molecular. Diferentes propriedades biol?gicas t?m sido reportadas dentre os mais de 170 tipos j? caracterizados, de maneira que um grupo particular de HPVs est? fortemente relacionado a infec??es persistentes, les?es intraepiteliais de diferentes graus e progress?o para c?nceres tais como cervical, anal, vulvar, vaginal, orofar?ngeo e de p?nis. No presente trabalho foram realizadas an?lises de variabilidade gen?tica e evolu??o molecular nos genomas dos principais HPVs de import?ncia cl?nica. Reconstru??es filogen?ticas e an?lises dos perfis de assinatura gen?tica nos genomas de cada gen?tipo sugeriram a presen?a de subgrupos de HPVs definidos por diferen?as nas sequ?ncias dos genes E1, E6, L1 e L2. Testes de evolu??o em n?vel de DNA revelaram uma atua??o mais forte da sele??o natural em c?dons espec?ficos, mais frequentemente nos genes E1, E2, L1 e L2. A partir dos dados obtidos nas an?lises de variabilidade, foi desenhado um novo conjunto de primers para a detec??o e genotipagem dos HPVs de import?ncia cl?nica por meio da t?cnica de rea??o em cadeia da polimerase (PCR). O gene E1 foi escolhido como alvo molecular devido a presen?a de uma regi?o conservada com tamanho vari?vel entre gen?tipos. O sistema proposto teve sua efici?ncia avaliada in vitro e foi comparado ao protocolo de PCR mais utilizado para detec??o do HPV em amostras cl?nicas. Utilizando a amplifica??o de ?cido nucleico de forma semianinhada (seminested), o sistema proposto foi capaz de detectar com boa sensibilidade alguns dos principais HPVs de alto risco oncog?nico e mostrou melhor especificidade em rela??o aos primers gen?ricos GP5+/6+, mesmo aplicando uma temperatura de anelamento consideravelmente maior. A an?lise do tamanho dos fragmentos amplificados usando a separa??o por eletroforese em gel de agarose pode favorecer a identifica??o do tipo de HPV presente nas amostras, permitindo a discrimina??o entre aqueles mais prevalentes na popula??o e a redu??o do tempo e do custo necess?rios para a identifica??o do agente. Opcionalmente, a separa??o dos produtos em matrizes de alta resolu??o e o sequenciamento direto podem ser usados para a tipagem, possibilitando a identifica??o de uma ampla variedade de gen?tipos de HPV descritos. / Human papillomavirus (HPV) is a small circular double-stranded DNA virus, characterized as one of the most common sexually transmitted agents in the world, whose accurate detection and genotyping is only possible through molecular biology techniques. Different biological properties have been reported among the more than 170 types already characterized, so that a particular group of HPVs is strongly related to persistent infections, intraepithelial lesions of different degrees and progression to cancers such as cervical, anal, vulvar, vaginal, oropharyngeal and penis. In the present work, analyzes of genetic variability and molecular evolution were performed in the genomes of the main clinically important HPVs. Phylogenetic reconstructions and analyzes of genetic signature profiles in the genomes of each genotype suggested the presence of subgroups of HPVs defined by differences in the E1, E6, L1 and L2 gene sequences. Evolution tests at DNA level have shown a stronger acting of natural selection at specific codons, more often in the E1, E2, L1 and L2 genes. From the data obtained in the analyzes of variability, a new set of primers was designed for the detection and genotyping of HPVs of clinical importance by polymerase chain reaction (PCR) technique. E1 gene was chosen as the molecular target due to the presence of a conserved region of variable size among genotypes. The proposed system had its efficiency evaluated in vitro and was compared to the most used PCR protocol for HPV detection in clinical samples. Using the seminested nucleic acid amplification, the proposed system was able to detect some of the major oncogenic HPVs with good sensitivity and showed a better specificity than the generic primers GP5+/6+, even applying a considerably higher annealing temperature. The analysis of the size of the amplified fragments using agarose gel electrophoresis may favor the identification of the HPV type present in the samples, allowing the discrimination between those more prevalent in the population and the reduction of the time and cost necessary for the identification of the agent. Optionally, the separation of products into high resolution matrices and direct sequencing can be used for typing, enabling the identification of a wide variety of HPV genotypes described.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-03T21:43:19Z No. of bitstreams: 1 KleytonThiagoCostaDeCarvalho_DISSERT.pdf: 2993890 bytes, checksum: 64c5c1e9194dcf2ddec049dc4f793967 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-10T19:42:28Z (GMT) No. of bitstreams: 1 KleytonThiagoCostaDeCarvalho_DISSERT.pdf: 2993890 bytes, checksum: 64c5c1e9194dcf2ddec049dc4f793967 (MD5) / Made available in DSpace on 2017-04-10T19:42:28Z (GMT). No. of bitstreams: 1 KleytonThiagoCostaDeCarvalho_DISSERT.pdf: 2993890 bytes, checksum: 64c5c1e9194dcf2ddec049dc4f793967 (MD5) Previous issue date: 2016-07-27 / C?ncer cervical (CC) ? o terceiro tipo de c?ncer mais comum em mulheres no mundo todo e a quarta principal causa de morte em mulheres nos pa?ses em desenvolvimento. Os Papilomav?rus humano (HPV) de alto risco tais como HPV 16, 18, 31 e 33 s?o o principal fator de risco para esse tipo de c?ncer. Entre estes, o HPV-16 e -18 s?o respons?veis por quase 70% dos casos de CC. Quimioterapia com compostos ? base de platina em combina??o com a radioterapia ou a cirurgia ? o tratamento de escolha para CC, mas sua efic?cia ? limitada, especialmente em est?gios avan?ados da doen?a. Al?m disso, estes tratamentos podem facilmente levar a rea??es adversas e resist?ncia ?s drogas. Assim, a busca por novos agentes antitumorais seletivos e de alta efic?cia para o tratamento deste tipo de tumor ? necess?ria. Croton blanchetianus (CB), popularmente conhecida como ?marmeleiro preto?, ? um arbusto pertencente ? fam?lia Euphorbiaceae e amplamente disseminado no nordeste brasileiro. Alguns estudos t?m demonstrado a atividade citot?xica de plantas do g?nero Croton em linhagens tumorais humanas. Contudo, at? o momento, n?o h? nada descrito na literatura quanto ao efeito citot?xico da esp?cie Croton blanchetianus. Assim, o presente trabalho teve como objetivo investigar, in vitro, os efeitos de fra??es obtidas das folhas e raiz de CB nas linhagens de c?ncer cervical humano HeLa e SiHa. As fra??es foram obtidas pelo m?todo de varia??o do pH, a partir do qual foram obtidas duas fra??es ?cidas, uma das folhas (CBaF) e outra da raiz (CBaR), e duas b?sicas, tamb?m das folhas (CBbF) e raiz (CBbR). O perfil fitoqu?mico das fra??es foi avaliado por Cromatografia em camada delgada (CCD). A atividade citot?xica e a avalia??o do tipo de morte celular foram determinados pelos ensaios de MTT e Anexina V/PI, respectivamente, enquanto que para avalia??o de altera??es morfol?gicas nucleares e ensaio do ciclo celular foram utilizados, respectivamente, microscopia de fluoresc?ncia com o corante DAPI e citometria de fluxo. De acordo com os resultados, a maioria das fra??es apresentou terpenos, alcaloides e flavonoides. Al?m disso, todas as fra??es testadas foram capazes de diminuir significativamente a viabilidade celular de HeLa e SiHa de maneira concentra??o e tempo dependentes, promoveram modifica??es morfol?gicas celulares e nucleares, al?m de induzirem apoptose e parada do ciclo celular. Este ? o primeiro estudo que demonstrou os efeitos citot?xicos e pr?-apopt?ticos de CB em linhagens de c?ncer cervical humano. Portanto, CB parece ser uma fonte natural promissora para o desenvolvimento de agentes para o tratamento do CC. No entanto, mais estudos s?o necess?rios para isolar, purificar e elucidar os poss?veis mecanismos de a??o dos compostos ativos. / Cervical cancer (CC) is the third most common cancers in women worldwide and the fourth major cause of cancer death in the woman in developing countries. High-risk human papilloma viruses (HPVs) such as HPV 16, 18, 31 and 33 have been attributed to be the major risk factors for cervical cancer, out of which HPV-16 and -18 account for almost 70% of the cancers. Platinum-based chemotherapy in combination with radiotherapy or surgery is now mainly used to treat CC, but the efficacy is limited especially in advanced-stage disease. Furthermore, these treatments could easily lead to adverse reactions and drug resistance. Thus, it is necessary to seek antitumor drugs of high efficacy for the treatment of this kind of tumor. Croton blanchetianus (CB), known as ?black marmeleiro?, belongs to the family Euphorbiaceae and it is a widely disseminated shrub found in northeast Brazil. Some studies have demonstrated cytotoxic activity of plants of this genus against human tumor cell lines. However, to date, there is nothing described in the literature as to the cytotoxic effect of the Croton blanchetianus. Therefore, the present study aimed to investigate, in vitro, the effects of leaves and root fractions from Croton blanchetianus (CB) against human cervical cancer HeLa and SiHa cells. Fractions were obtained by pH variation method, from which were obtained two acidic fractions, one of the leaves (CBaF) and root (CBaR) and two basic also obtained from leaves (CBbF) and root (CBbR). Phytochemical screening was evaluated by thin layer chromatography. Cytotoxic activity and cell death evaluation were determined with MTT and annexin V/PI assays by flow cytometry, respectively. Nuclear morphological changes were evaluated by fluorescence with DAPI stanning and flow cytometry was used to cycle assay. According to results, most of the fractions exhibited terpenoids, alkaloids and flavonoids. All fractions decreased significantly cell viability of HeLa and SiHa cells in a concentration- and time-dependent manner, promoted cellular and nuclear morphological changes and induced apoptosis and cell cycle arrest. This is the first study that demonstrated cytotoxic and pro apoptotic effects of CB on HeLa and SiHa cells. Therefore, CB appears to be a valuable natural source for the development of agents for the treatment of CC. However, more studies are needed to isolate, purify and elucidate the possible action mechanisms of the active compounds.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-01-12T15:47:41Z No. of bitstreams: 1 EmanuellyBernardesDeOliveiraDaSilva_DISSERT.pdf: 1659174 bytes, checksum: 5200a1d5bddcb328c2b32c3aac297d85 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-01-20T14:20:37Z (GMT) No. of bitstreams: 1 EmanuellyBernardesDeOliveiraDaSilva_DISSERT.pdf: 1659174 bytes, checksum: 5200a1d5bddcb328c2b32c3aac297d85 (MD5) / Made available in DSpace on 2017-01-20T14:20:37Z (GMT). No. of bitstreams: 1 EmanuellyBernardesDeOliveiraDaSilva_DISSERT.pdf: 1659174 bytes, checksum: 5200a1d5bddcb328c2b32c3aac297d85 (MD5) Previous issue date: 2015-05-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Entre os tipos mais incidentes de c?ncer no Brasil, o c?ncer cervical ? o quarto tipo mais comum entre as mulheres, com mais de 99,7% dos casos associados ? infec??o pelo Papilomasv?rus Humano (HPV), tipos 16 e 18 principalmente. A resposta limitada dos tumores malignos ? quimioterapia convencional levou ao desenvolvimento de novas estrat?gias farmacol?gicas baseadas no conhecimento de novos alvos terap?uticos. Considerando a diversidade dos escorpi?es e serpentes da fauna brasileira, esses animais pe?onhentos possuem um potencial farmacol?gico em seu veneno, sendo de grande interesse na pesquisa e desenvolvimento de novos f?rmacos a partir do veneno bruto ou fra??es. Desta forma, frente a esses venenos abre-se uma expectativa na busca de novos compostos com a??o terap?utica no tratamento do c?ncer. O objetivo deste trabalho foi avaliar o efeito citot?xico e apopt?tico do veneno bruto do escorpi?o Tityus serrulatus (TSR) e das serpentes Bothrops jararaca (BJ) e Bothrops erythromelas (BE) em linhagens de carcinoma cervical SiHa e HeLa, tranformadas pelos HPV 16 e 18, respectivamente. Em rela??o ? viabilidade celular pelo m?todo de MTT, obteve-se citotoxicidade nas linhagens tumorais desafiadas com os venenos das serpentes BJ e BE quando comparada ao veneno TSR. De forma similar, o processo de morte celular, avaliada por citometria de fluxo, foi superior quando desafiados com BJ e BE. Portanto, os venenos Bothrops demonstraram uma a??o citot?xica e apopt?tica nas linhagens estudadas, de maneira dose tempo-resposta dependentes. O presente trabalho sugere, pela primeira vez, um papel citot?xico e apopt?tico dos venenos das serpentes B. jararaca e B. erythromelas em linhagens de carcinoma cervical. / Among the types of cancer incidents in Brazil, cervical cancer is the fourth most common type among women, with more than 99.7% of cases associated with infection by human papillomaviruses (HPV) types 16 and 18 mainly. The limited of malignant tumors to conventional chemotherapy response led to the development of new pharmacological strategies based on knowledge of new therapeutic targets. Considering the diversity of scorpions and snakes of Brazilian fauna, these poisonous animals have a pharmacological potential in their venom, being of great interest in the new drugs research and development from raw or fractions poison. Thus, faced with these poisons opens an expectation in the search for new compounds with therapeutic action in the treatment of cancer. The objective of this study was to evaluate the cytotoxic effect and apoptosis of the poisons of the scorpion Tityus serrulatus (TSR), Bothrops jararaca (BJ) and Bothrops erythromelas (BE) in cervical carcinoma cell lines SiHa and HeLa, tranform by the respective HPV 16 and 18. In relation to cell viability by MTT method was obtained challenged cytotoxicity in tumor cell lines with the poisons of snakes BJ and BE compared to the TSR poisons. Similarly, the process of cell death was greater when challenged with BJ and BE. Therefore, Bothrops poisons showed a cytotoxic and apoptotic action on the lines studied, dose-dependent and time-response manner. This study suggests for the first time, a cytotoxic and apoptotic role of poisons of snakes B. jararaca and B. erythromelas in cervical carcinoma cell lines.