Esta tese se fundamenta na necessidade de novos tratamentos para o câncer do colo de útero visando o aumento da adesão dos pacientes aos tratamentos, assim como à qualidade de vida dos mesmos. Nesse sentido, formulações nanotecnológicas foram desenvolvidas com o objetivo de carrear o fármaco imiquimode para um local específico – a mucosa vaginal – esperando gerar melhores desempenhos nesse tratamento quando comparados com a formulação comercial. Três nanoestruturas com morfologias distintas foram propostas visando potencializar o efeito do fármaco em células de câncer cervical (SiHa). As formulações desenvolvidas compreenderam: nanoemulsões (NEimiq), nanocápsulas poliméricas (NCimiq) e nanocápsulas poliméricas revestidas com quitosana (NCimiq-chit). Observou-se que nanocápsulas poliméricas produzidas com poli(ε-caprolactona) apresentaram efeito mais pronunciado frente às células SiHa. Para tanto, essas formulações (NCimiq e NCimiq-chit) foram incorporadas em hidrogéis de quitosana e de hidroxietilcelulose a fim de possibilitar uma melhor futura aplicação para o paciente. Estudos envolvendo mucosa vaginal suína demonstraram que ambas as formulações são mucoadesivas e permeiam a mucosa vaginal. Porém, a formulação produzida com hidrogel de quitosana (NCimiq) apresentou maior desempenho. Esta foi a formulação escolhida para dar continuidade aos estudos deste trabalho, sendo objeto de estudo posterior em cultura de células SiHa a fim de elucidar o mecanismo de ação da mesma. Esses estudos demonstraram que há uma ocorrência de processos combinados de diminuição da viabilidade celular de maneira tempo-dependente e que mecanismos como apoptose, autofagia e parada de ciclo celular estão presentes. Essa formulação (NCimiq) apresentou porcentagens de morte celular significativas, mesmo utilizando baixas concentrações do fármaco. Portanto, os achados desta tese constataram que nanoestruturas modulam efetivamente a interação do fármaco com as células. / This thesis deals with the need of new treatments for cervical cancer in order to increase the adherence of patients to the treatment as well as to improve their quality of life. In this sense, nanotechnological formulations were developed to carry imiquimod to a specific site – the vaginal mucosa – expecting to obtain better performance than the commercial drug in the cervical cancer treatment. Three nanostructures with different morphologies were proposed to potentilize the drug effect on cervical cancer cells (SiHa). The developed formulations are: nanoemulsions (NEimiq), polymeric nanocapsules (NCimiq) and polymeric nanocapsules coated with chitosan (NCimiq-chit). It was observed that polymeric nanocapsules produced with poly(ε-caprolactone) presented a stronger effect against SiHa cells. Therefore, formulations NCimiq and NCimiq-chit were incorporated into hydrogels of chitosan and hydroxyethylcellulose to enable a better future application on patients. The studies of this thesis involving porcine vaginal mucosa demonstrated that both formulations are mucoadhesive and that they provided a good drug permeation. However, the formulation produced with chitosan hydrogel (NCimiq) showed a better performance. This formulation was therefore chosen to follow the next steps of this work, conducted in SiHa cell culture to elucidate its action mechanism. This study demonstrated that there is an occurrence of combined processes of decreasing cell viability in a time-dependent type. The study also showed that mechanisms such as apoptosis, autophagy and cell cycle arrest are simultaneously present. The formulation NCimiq presented a significantly percentage of cellular death, even when low concentrations of the drug were used. Consequently, the findings of this thesis indicate that nanostructures effectively modulate the interaction of the drug with the cancer cells.

The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. £]-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, £]-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/£]-catenin signaling. In our previous study, we have established that Cby over-expression attenuated £]-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of £]-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

Esta tese se fundamenta na necessidade de novos tratamentos para o câncer do colo de útero visando o aumento da adesão dos pacientes aos tratamentos, assim como à qualidade de vida dos mesmos. Nesse sentido, formulações nanotecnológicas foram desenvolvidas com o objetivo de carrear o fármaco imiquimode para um local específico – a mucosa vaginal – esperando gerar melhores desempenhos nesse tratamento quando comparados com a formulação comercial. Três nanoestruturas com morfologias distintas foram propostas visando potencializar o efeito do fármaco em células de câncer cervical (SiHa). As formulações desenvolvidas compreenderam: nanoemulsões (NEimiq), nanocápsulas poliméricas (NCimiq) e nanocápsulas poliméricas revestidas com quitosana (NCimiq-chit). Observou-se que nanocápsulas poliméricas produzidas com poli(ε-caprolactona) apresentaram efeito mais pronunciado frente às células SiHa. Para tanto, essas formulações (NCimiq e NCimiq-chit) foram incorporadas em hidrogéis de quitosana e de hidroxietilcelulose a fim de possibilitar uma melhor futura aplicação para o paciente. Estudos envolvendo mucosa vaginal suína demonstraram que ambas as formulações são mucoadesivas e permeiam a mucosa vaginal. Porém, a formulação produzida com hidrogel de quitosana (NCimiq) apresentou maior desempenho. Esta foi a formulação escolhida para dar continuidade aos estudos deste trabalho, sendo objeto de estudo posterior em cultura de células SiHa a fim de elucidar o mecanismo de ação da mesma. Esses estudos demonstraram que há uma ocorrência de processos combinados de diminuição da viabilidade celular de maneira tempo-dependente e que mecanismos como apoptose, autofagia e parada de ciclo celular estão presentes. Essa formulação (NCimiq) apresentou porcentagens de morte celular significativas, mesmo utilizando baixas concentrações do fármaco. Portanto, os achados desta tese constataram que nanoestruturas modulam efetivamente a interação do fármaco com as células. / This thesis deals with the need of new treatments for cervical cancer in order to increase the adherence of patients to the treatment as well as to improve their quality of life. In this sense, nanotechnological formulations were developed to carry imiquimod to a specific site – the vaginal mucosa – expecting to obtain better performance than the commercial drug in the cervical cancer treatment. Three nanostructures with different morphologies were proposed to potentilize the drug effect on cervical cancer cells (SiHa). The developed formulations are: nanoemulsions (NEimiq), polymeric nanocapsules (NCimiq) and polymeric nanocapsules coated with chitosan (NCimiq-chit). It was observed that polymeric nanocapsules produced with poly(ε-caprolactone) presented a stronger effect against SiHa cells. Therefore, formulations NCimiq and NCimiq-chit were incorporated into hydrogels of chitosan and hydroxyethylcellulose to enable a better future application on patients. The studies of this thesis involving porcine vaginal mucosa demonstrated that both formulations are mucoadhesive and that they provided a good drug permeation. However, the formulation produced with chitosan hydrogel (NCimiq) showed a better performance. This formulation was therefore chosen to follow the next steps of this work, conducted in SiHa cell culture to elucidate its action mechanism. This study demonstrated that there is an occurrence of combined processes of decreasing cell viability in a time-dependent type. The study also showed that mechanisms such as apoptosis, autophagy and cell cycle arrest are simultaneously present. The formulation NCimiq presented a significantly percentage of cellular death, even when low concentrations of the drug were used. Consequently, the findings of this thesis indicate that nanostructures effectively modulate the interaction of the drug with the cancer cells.

Esta tese se fundamenta na necessidade de novos tratamentos para o câncer do colo de útero visando o aumento da adesão dos pacientes aos tratamentos, assim como à qualidade de vida dos mesmos. Nesse sentido, formulações nanotecnológicas foram desenvolvidas com o objetivo de carrear o fármaco imiquimode para um local específico – a mucosa vaginal – esperando gerar melhores desempenhos nesse tratamento quando comparados com a formulação comercial. Três nanoestruturas com morfologias distintas foram propostas visando potencializar o efeito do fármaco em células de câncer cervical (SiHa). As formulações desenvolvidas compreenderam: nanoemulsões (NEimiq), nanocápsulas poliméricas (NCimiq) e nanocápsulas poliméricas revestidas com quitosana (NCimiq-chit). Observou-se que nanocápsulas poliméricas produzidas com poli(ε-caprolactona) apresentaram efeito mais pronunciado frente às células SiHa. Para tanto, essas formulações (NCimiq e NCimiq-chit) foram incorporadas em hidrogéis de quitosana e de hidroxietilcelulose a fim de possibilitar uma melhor futura aplicação para o paciente. Estudos envolvendo mucosa vaginal suína demonstraram que ambas as formulações são mucoadesivas e permeiam a mucosa vaginal. Porém, a formulação produzida com hidrogel de quitosana (NCimiq) apresentou maior desempenho. Esta foi a formulação escolhida para dar continuidade aos estudos deste trabalho, sendo objeto de estudo posterior em cultura de células SiHa a fim de elucidar o mecanismo de ação da mesma. Esses estudos demonstraram que há uma ocorrência de processos combinados de diminuição da viabilidade celular de maneira tempo-dependente e que mecanismos como apoptose, autofagia e parada de ciclo celular estão presentes. Essa formulação (NCimiq) apresentou porcentagens de morte celular significativas, mesmo utilizando baixas concentrações do fármaco. Portanto, os achados desta tese constataram que nanoestruturas modulam efetivamente a interação do fármaco com as células. / This thesis deals with the need of new treatments for cervical cancer in order to increase the adherence of patients to the treatment as well as to improve their quality of life. In this sense, nanotechnological formulations were developed to carry imiquimod to a specific site – the vaginal mucosa – expecting to obtain better performance than the commercial drug in the cervical cancer treatment. Three nanostructures with different morphologies were proposed to potentilize the drug effect on cervical cancer cells (SiHa). The developed formulations are: nanoemulsions (NEimiq), polymeric nanocapsules (NCimiq) and polymeric nanocapsules coated with chitosan (NCimiq-chit). It was observed that polymeric nanocapsules produced with poly(ε-caprolactone) presented a stronger effect against SiHa cells. Therefore, formulations NCimiq and NCimiq-chit were incorporated into hydrogels of chitosan and hydroxyethylcellulose to enable a better future application on patients. The studies of this thesis involving porcine vaginal mucosa demonstrated that both formulations are mucoadhesive and that they provided a good drug permeation. However, the formulation produced with chitosan hydrogel (NCimiq) showed a better performance. This formulation was therefore chosen to follow the next steps of this work, conducted in SiHa cell culture to elucidate its action mechanism. This study demonstrated that there is an occurrence of combined processes of decreasing cell viability in a time-dependent type. The study also showed that mechanisms such as apoptosis, autophagy and cell cycle arrest are simultaneously present. The formulation NCimiq presented a significantly percentage of cellular death, even when low concentrations of the drug were used. Consequently, the findings of this thesis indicate that nanostructures effectively modulate the interaction of the drug with the cancer cells.

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.