Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-23Bitstream added on 2014-06-13T18:09:48Z : No. of bitstreams: 1 moraes_jdd_me_arafcf.pdf: 2965279 bytes, checksum: e4b88e85e5a2c07402cdd99a87bdd614 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Diversos antioxidantes demonstram considerável efeito na prevenção do câncer por reduzir o estresse oxidativo causado por uma ampla variedade de ataques: biológicos, químicos e físicos, dentre os quais a radiação solar ultravioleta (UV) é o estresse físico ambiental descrito entre os mais freqüentes ataques que ocorrem sobre a pele, o qual está associado no desenvolvimento na maioria das doenças, incluindo o câncer. Vários componentes da dieta podem ser utilizados sozinhos ou em combinação com fármacos convencionais para prevenir doenças. Ainda, a busca por metodologias alternativas à experimentação com animais para o teste de eficácia de formas cosméticas de uso tópico é devido à pressão pública existente neste sentido, introduzida principalmente pela Comunidade Européia (93/95/EC). O presente estudo teve por objetivo avaliar a atividade do ácido alfa– lipóico em cultura de células, além do estudo de estabilidade de emulsões O/A e seu comportamento reológico. Para isto, foram utilizadas culturas de células como modelo experimental, na tentativa de desenvolver um método alternativo à experimentação com animais que poderá ser utilizado pela indústria cosmética. Foram utilizadas culturas de células HepG2 (metabolizadoras de xenobióticos) e cultura de células HaCaT (queratinócitos) / Several antioxidants show significant effect in preventing cancer by reducing oxidative stress caused by a wide variety of attacks: biological, chemical and physical agents, amongst which the sun's ultraviolet (UV) is the most physical stress environment described among the most frequent attacks that occur on the skin, which is associated with development in most diseases, including cancer. Several dietary components can be used alone or in combination with conventional drugs to prevent disease. Still, the search for alternative methods to animal testing to test the efficacy of topical cosmetic forms is due to public pressure that exists in this sense, introduced mainly by the European Union (93/95/EC). This study aims to assess the activity of alpha-lipoic acid in cultured cells, in addition to studying the stability of O / W emulsions and their rheological behavior. For this, cell cultures are used as experimental models in an attempt to develop an alternative method to animal testing that may be used by the cosmetics industry. Will be used HepG2 cell cultures (xenobiotic metabolizing) and cell culture of HaCaT (keratinocytes)

Ce travail a pour but de développer des nouveaux substrats d’étude en culture cellulaire. Nous avons d'abord fabriqué des réseaux de micro-piliers en élastomère et en polymères thermoplastiques. En particulier, nous avons réalisé des réseaux de micro-piliers adjacents et de différentes hauteurs, qui dépend de la rigidité de la surface de culture. Nos résultats ont montré que les cellules étaient sensibles à la hauteur des piliers lorsque la rigidité effective du substrat était similaire à celle de la cellule et que les cellules se déplacent préférentiellement vers la partie plus rigide. Nous avons également développé une méthode pour fabriquer des nanofibres sur les piliers élastomère pour créer un substrat qui reproduit la matrice extracellulaire in vivo. Nos résultats ont montré que les neurones primaires de l'hippocampe sur un tel substrat étaient plus actifs que sur des substrats plats. En outre, nous avons analysé le confinement et la déformation des noyaux cellulaires dans les espaces inter-piliers pour les études de cellules tumorales et de cellules souches. Enfin, nous avons intégré les réseaux de micro-piliers dans un dispositif microfluidique afin de montrer que la migration cellulaire soumise à un gradient de concentration était influencée par la rigidité du substrat. En conclusion, les micropiliers ainsi fabriqués peuvent être utilisés pour réguler la rigidité d’un substrat afin d’étudier divers mécanismes en culture cellulaire. / This work aimed to provide new substrates for cell culture studies. We first developed a method to fabricate micropillars in both elastomer and thermoplastic polymer. In particular, we produced adjacent micropillar arrays with different heights to evaluate the surface stiffness dependent migration of cells. Our results showed that cells were sensitive to the height of the pillars when the effective stiffness of the substrate is compatible to that of the cell and that the cells were preferentially localized on the stiffer surface area. We also developed a method to fabricate nanofibers on the elastomer pillars to create in-vivo like extracellular matrix. Our results showed that primary hippocampal neurons on such a substrate were more active than on flat substrates. Furthermore, we analyzed the confinement and deformation of cell nuclei in the inter-pillar areas for both cancer and stem cell studies. Finally, we integrated the micro-pillar arrays into a microfluidic device and showed that the cell migration under concentration gradient was influenced by the substrate stiffness. Altogether, the fabricated pillar arrays can be used to regulate the stiffness of the substrate for cell culture studies.

Orientador: Vera Lúcia Borges Isaac / Banca: Marcos Antonio Corrêa / Banca: Pedro Alves da Rocha Filho / Resumo: Diversos antioxidantes demonstram considerável efeito na prevenção do câncer por reduzir o estresse oxidativo causado por uma ampla variedade de ataques: biológicos, químicos e físicos, dentre os quais a radiação solar ultravioleta (UV) é o estresse físico ambiental descrito entre os mais freqüentes ataques que ocorrem sobre a pele, o qual está associado no desenvolvimento na maioria das doenças, incluindo o câncer. Vários componentes da dieta podem ser utilizados sozinhos ou em combinação com fármacos convencionais para prevenir doenças. Ainda, a busca por metodologias alternativas à experimentação com animais para o teste de eficácia de formas cosméticas de uso tópico é devido à pressão pública existente neste sentido, introduzida principalmente pela Comunidade Européia (93/95/EC). O presente estudo teve por objetivo avaliar a atividade do ácido alfa- lipóico em cultura de células, além do estudo de estabilidade de emulsões O/A e seu comportamento reológico. Para isto, foram utilizadas culturas de células como modelo experimental, na tentativa de desenvolver um método alternativo à experimentação com animais que poderá ser utilizado pela indústria cosmética. Foram utilizadas culturas de células HepG2 (metabolizadoras de xenobióticos) e cultura de células HaCaT (queratinócitos) / Abstract: Several antioxidants show significant effect in preventing cancer by reducing oxidative stress caused by a wide variety of attacks: biological, chemical and physical agents, amongst which the sun's ultraviolet (UV) is the most physical stress environment described among the most frequent attacks that occur on the skin, which is associated with development in most diseases, including cancer. Several dietary components can be used alone or in combination with conventional drugs to prevent disease. Still, the search for alternative methods to animal testing to test the efficacy of topical cosmetic forms is due to public pressure that exists in this sense, introduced mainly by the European Union (93/95/EC). This study aims to assess the activity of alpha-lipoic acid in cultured cells, in addition to studying the stability of O / W emulsions and their rheological behavior. For this, cell cultures are used as experimental models in an attempt to develop an alternative method to animal testing that may be used by the cosmetics industry. Will be used HepG2 cell cultures (xenobiotic metabolizing) and cell culture of HaCaT (keratinocytes) / Mestre

Orientador: Débora Aparecida Pires de Campos Zuccari / Banca: Dorotéia Rossi Silva / Banca: Maria Tercília Vilela de Azeredo Oliveira / Resumo: O estudo de marcadores prognósticos e preditivos no câncer tem se mostrado efetivo na pesquisa e rotina diagnóstica. A glutationa (GSH) e as enzimas glutationa peroxidase (GPX) e glutationa S transferase pi (GSTpi) exercem papel fundamental na defesa antioxidante das células e na detoxificação de quimioterápicos. Nesse contexto, o objetivo deste estudo foi avaliar a expressão das proteínas GSH, GPX e GSTpi em pacientes com câncer de mama, além de avaliar a expressão gênica dessas proteínas em amostras tumorais in vitro após o tratamento com quimioterápico. As proteínas foram detectadas no tecido tumoral de 63 pacientes por imuno-histoquímica e quantificadas pela técnica de densitometria óptica. A expressão dos genes que sintetizam GSH, glutamato cisteina ligase (GCLC) e glutationa sintetase (GSS) e dos genes codificadores da GPX e GSTpi foi analisada por PCR em tempo real em células cultivadas provenientes de 12 amostras tumorais de mama. As células foram submetidas in vitro ao quimioterápico doxorrubicina, e a expressão gênica foi analisada antes e após o tratamento. A expressão da GSH relacionou-se com tumor receptor de estrógeno (RE) negativo (p<0,05). A expressão da GPX foi maior em tumor receptor de progesterona (RP) negativo e em pacientes que vieram a óbito (p<0,05). Alta expressão da GSTpi relacionou-se com características tumorais de prognóstico desfavorável como positividade para p53, grau histológico III, maior tamanho tumoral e óbito (p<0,05). Além disso, as pacientes foram divididas em subgrupos de acordo com o tratamento recebido. Assim, a alta expressão da GSH relacionou-se com a ocorrência de metástase no grupo de pacientes tratadas apenas com quimioterapia adjuvante (p<0,05). Nas pacientes que receberam quimioterapia e radioterapia adjuvantes, a alta expressão da GPX foi relacionada com óbito e a alta expressão da GSTpi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The study of prognostic and predictive markers in cancer has been proven effective in research and diagnostic routine. Glutathione (GSH) and glutathione peroxidase (GPX) and glutathione S transferase pi (GSTpi) play a crucial role in antioxidant defense of cells and detoxification of chemotherapeutic agents. In this context, the objective of this study was to evaluate the protein expression of GSH, GPX and GSTpi in patients with invasive ductal carcinoma, and to evaluate the expression of genes encoding these proteins in tumor samples in vitro before and after treatment with chemotherapy. The proteins were detected in tumor tissue of 63 patients by immunohistochemistry and quantified by optical densitometry technique. The expression of genes that synthesize GSH, glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) and the genes encoding GPX and GSTpi were analyzed by real time PCR in cultured cells from 12 tumor samples from patients with breast cancer. The cells were treated in vitro to doxorubicin chemotherapy, and gene expression was analyzed before and after treatment. The expression of GSH was related to tumor estrogen receptor (ER) negative (p <0.05). The expression of GPX was higher in tumor progesterone receptor (PR) negative and patients who died (p <0.05). High expression of GSTpi was related to tumor characteristics of poor prognosis such as p53 positivity, histologic grade III, larger tumor size and death (p <0.05). In addition, patients were divided into subgroups according to treatment received. Thus, high expression of GSH was related to the occurrence of metastasis in patients treated only with adjuvant chemotherapy (p <0.05). In patients who received adjuvant chemotherapy and radiotherapy, high expression of GPX was associated with death and high expression of GSTpi was correlated to local tumor recurrence, metastasis and death (p <0.05)... (Complete abstract click electronic access below) / Mestre

INTRODUCTION: One important characteristic of solid tumors is heterogeneity at multiple levels of genetic and non-genetic organization. This can include gene mutations, epigenetic alterations, copy number changes, and chromosomal aberrations. Collectively, these alterations contribute as parts of a genome-defined system. Thus, when genetic information is passed from mother to daughter cell in the context of cancer evolution, in contrast to normal cellular processes, an altered system inheritance is often transmitted. When the genome of a somatic cell is highly unstable, such as during certain phases of cancer initiation and progression, many novel alterations to the genome can be introduced in a short timeframe, effectively resulting in the macro-evolution of the somatic cell population (i.e., through the transition stages of cancer, including transformation, metastasis, and drug resistance). Unfortunately, these continually introduced, non-clonal alterations to the cell’s genetic information have often been described as background “noise” that does not function significantly in cancer. Rather, the driving force of cancer has largely been attributed to the accumulation of gene mutations in several key, driver genes. Despite the presumed significance of these driver genes by the gene mutation and clonal evolutionary theories of cancer, recent sequencing efforts have failed to identify common driver genes in the majority of cancer types. Based on this fact, and on the overwhelming presence of non-clonal alterations at multiple levels of organization in the cells comprising tumors, the paradigm of cancer research requires re-examination. A better understanding of genome-level heterogeneity is necessary, as the genome, rather than individual genes, defines system boundaries and unifies the diverse individual molecular mechanisms of cancer through their contribution to major evolutionary transitions. Because inheritance is traditionally defined as a precise process of relaying bio-information with extreme low frequencies of errors, it is challenging to explain how genetics work in cancer evolution. It is thus timely to consider that potentially novel processes of inheritance occur in many types of cancer. The maintenance of a massive extent of multi-level heterogeneity in the cells of solid tumors over generations suggests that a less precise process is taking place. We have described this with a new term, “fuzzy inheritance,” wherein a range of variants, rather than specific variants (such as specific gene mutations or chromosomal aberrations), is recapitulated in the cell division process. This study aimed to elucidate the mechanism of fuzzy inheritance by examining the relationship between genome instability-linked karyotypic heterogeneity and growth heterogeneity, based on single-cell analysis of an in vitro cell culture model. By demonstrating that increased genome-level heterogeneity is reflected by increased and more variable levels of growth heterogeneity, it was hoped to establish that fuzzy inheritance correctly explains the maintenance of high levels of heterogeneity in these somatic cell populations. An example of this phenomenon was also studied in giant cancer cells, as they undergo division processes which appear to contribute to and facilitate genome instability. METHODS: To examine these concepts, various cellular profiling methods were used, including in-situ cell growth, cellular morphological comparison, and karyotype analysis. We first quantified the extent of variation in the growth rates of single cells; by selecting the fastest- and slowest-growing colonies from the parent population, and examining the extent to which growth heterogeneity was passed in subsequent generations of cells, the correlation between genome-level heterogeneity (as reflected by the karyotype) and growth heterogeneity was determined. We then examined an extreme example of fuzzy inheritance, wherein giant cancer cells containing massive amounts of DNA undergo extremely abnormal cell division events, yielding many normal-sized daughter cells with genomes significantly different from those of both the parent cell and other daughter cells. By studying the frequency and other aspects of these cells in two unequally stable cell lines, we sought to gain insight on one specific mechanism of fuzzy inheritance. RESULTS: The data suggested that fuzzy inheritance can be demonstrated in multiple cell culture models. The extent and variability of karyotypic heterogeneity was reflected by those of growth heterogeneity, indicating the karyotype’s importance in facilitating cancer evolutionary processes. Moreover, the cells with giant nuclei can generate diverse genome-level heterogeneity. DISCUSSION: Because fuzzy inheritance allows for the less precise passage of bio-information over generations in cancer cell populations, and for the effective introduction of numerous alterations to the genome in often brief spans of time, the cell population can constantly increase its evolutionary potential, which is essential for the major transition steps of cancer evolution. The mechanism of fuzzy inheritance should be explored further, due to its clear importance in the processes underlying cancer initiation, progression, and drug resistance.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in women and fourth in men globally. While expansions in preventative measures have increased the detection of CRC at the early stages of disease, only 40% of CRC patients are diagnosed when the disease is at a local stage. Moreover, many anti-cancer drugs fail to significantly improve the life expectancy of patients due to innate and acquired resistance, underscoring a need for better diagnostic and therapeutic strategies for CRC. Immunoglobulin-containing and proline-rich receptor-1 (IGPR-1) is a novel cell adhesion molecule (CAM) that was recently identified in our laboratory. IGPR-1 is expressed in epithelial and endothelial cells and promotes cell-cell adhesion. Expression of IGPR-1 in endothelial cells regulates angiogenesis; however, its role in epithelial cells, particularly cancer cells with an epithelial origin, remains unknown. The overall goal of this study was to investigate the possible function of IGPR-1 in CRC tumor cell growth and response to chemotherapeutic agents. Specifically, we aimed to test the hypothesis that increased expression of IGPR-1 in CRC tumor cells promotes cell survival and contributes to the resistance of tumor cells to doxorubicin. Human CRC tumor cell lines, HCT116 and HT29, were transduced via a retroviral system to express IGPR-1 or empty retroviral vector pQCXIP. The effect of overexpression of IGPR-1 in HCT116 and HT29 cells was measured by MTT assay in non-adherent 24-well plates. In addition, cells were viewed under a light microscope, and images were taken to assess multicellular aggregation. Results demonstrated that expression of IGPR-1 in HCT116 and HT29 tumor cells promoted CRC tumor cell growth, increased multicellular aggregation, and stimulated resistance to the conventional chemotherapeutic agent doxorubicin in non-adherent cell culture conditions in vitro. Intriguingly, treatment of cells with doxorubicin promoted phosphorylation of IGPR-1 at serine 220 (Ser220), suggesting a critical role for phosphorylation of IGPR-1 in the development of resistance to chemotherapeutics. In addition, non-adherent cell culture conditions promoted activation of the key pro-apoptotic kinase, p38 MAPK in CRC tumor cells. Ectopic expression of IGPR-1 reversed this activation. This data suggests that IGPR-1, by suppressing p38 activity, in part, promotes tumor cell survival and increases the resistance of tumor cells to the killing effects of doxorubicin. Our findings are the first to demonstrate that IGPR-1 promotes CRC tumor cell growth and increases the resistance of CRC tumor cells to the cytotoxic effects of chemotherapeutic agents. The data suggests that IGPR-1 plays an important role in CRC by inhibiting the cellular apoptotic response and promoting chemotherapeutic resistance. Finally, IGPR-1 phosphorylation at Ser220 in response to doxorubicin may account for the IGPR-1-mediated development of resistance to doxorubicin in CRC.

Orientador: Márcia Carneiro Valera / Banca: Idomeo Bonetti Filho / Banca: Carlos Henrique Ribeiro Camargo / Resumo: A proposta deste trabalho foi avaliar a citotoxicidade do peróxido de hidrogênio liberado por agentes clareadores, utilizados para clareamento de dentes tratados endodonticamente, sobre cultura de fibroblastos provenientes do tecido gengival humano (FMM1). As células foram cultivadas em DMEM e quando apresentaram-se em quantidade suficiente e entre a quinta e décima passagens foram plaqueadas em placas de 96 poços onde receberam os meios de cultura condicionados de acordo com os grupos experimentais (n=12): G1- Perborato de Sódio + água; G2- Perborato de sódio + Peróxido de Carbamida 20%; G3- Peróxido de Carbamida 20%; G4- Perborato de Sódio + Peróxido de Hidrogênio 35%; G5- Peróxido de Hidrogênio 35%. O grupo controle (n=12) correspondeu à curva de crescimento e viabilidade celular, onde as células não receberam tratamento. O ensaio com MTT foi realizado nos períodos de 24 e 48 horas para avaliar a viabilidade celular. Paralelamente, mediu-se em espectrofotômetro a quantidade de peróxido de hidrogênio liberado nas condições experimentais. Os dados foram analisados através dos testes de ANOVA e Tukey. Todos os grupos experimentais apresentaram diferença significativa em relação ao controle. O tempo de avaliação mostrou diferença estatística, exceto para o G1 (PS + H2O). Concluiu-se que: todos os agentes clareadores testados foram citotóxicos, diminuindo significantemente o metabolismo e viabilidade celular; a associação do perborato de sódio com água destilada foi o agente clareador mais tóxico e o peróxido de carbamida 20% o menos tóxico. / Abstract: The propose of this study was to evaluate the cytotoxicity from five bleaching agents, used for the technique of intracanal bleaching, on human gingival fibroblasts (FMM1). The cells were cultivated in DMEM and when they were presented in enough amount and between the fifth and tenth passages they were placed in plates of 96 wells; where they received the conditional culture according to the experimental groups (n=12): G1- SP + H2O; G2- SP + CP20%; G3- CP20%; G4- SP + HP35%; G5- HP35%. The control group (n=12) corresponded to the curve of cell growth and viability, where the cells didn't receive any treatment. The MTT assay was carried through in the periods of 24 and 48 hours to evaluate the cellular viability. The amount of set free hydrogen peroxide in the experimental conditions was also measured in a spectrophotometer. The data were submitted to statistical analysis of variance and Turkey's test. All the experimental groups presented significant difference in comparison to the control. The evaluation time showed statistical difference, except for the G1 (SP + H2O). Conclusion: all the bleaching agents had showed cytotoxicity effects, reducing significantly the cell metabolism and viability; the association of sodium perborate with distilled water was the most toxic bleaching agent and carbamide peroxide 20% the least. / Mestre

Mesenchymal stem cells are the stem cells that replace the bone, fat and cartilage tissues of the human body. In addition, these cells can form muscles, ligaments and neurons. This wide multipotency has made mesenchymal stem cells of particular interest in the fields of tissue engineering and regenerative medicine. Furthermore, mesenchymal stem cells can modulate the immune system by reducing factors that increase inflammation and immune recognition. This immune recognition suppression has resulted in their application as part of bone marrow transplantation in the prevention of 'graft versus host‘ disease. There are hundreds of on-going clinical trials using these cells for the treatment of autoimmune diseases such as type I diabetes, arthritis and multiple sclerosis. The increasing importance of these cells has brought in to focus the culture methods used to for their expansion and manipulation. Currently, animal derived components are used as surfaces for their growth and as components in the culture media. This exposes these cells to animal pathogens and antigens that can be passed to the recipients of these cells. In the first part of this thesis, polymer microarrays were employed to identify alternatives to the biological surfaces currently used for mesenchymal stem cell culture. This platform allowed hundreds of polyacrylates/acrylamides and polyurethanes to be simultaneously scrutinised to identify surfaces that could support their growth and maintain their stem cell characteristics. Identified polymer surfaces were monitored in long-term culture (10 passages) and were shown to retain the cell phenotype and capacity to differentiate, thus providing chemically defined substrates for long-term mesenchymal stem cell culture. In the second part of this thesis, a 'smart‘ polymer microarray of hydrophilic cross-linked polymers (hydrogels) were used to remove another key biological component of culture, trypsin. These 'smart‘ hydrogels modulated their properties depending on the temperature. Hydrogels that could trigger mesenchymal stem cell release after a reduction in temperature were identified. A unique passaging system using a modest temperature reduction for 1h was developed as a passaging method. Cells were maintained and monitored for 10 passages using this novel enzyme free passaging method. Analysis of the mesenchymal stem cell phenotype and differentiation capacity revealed this method superior than conventional culturing methods. In the final part of this thesis, a 'knowledge-based‘ small molecule library was designed, which could potentially yield small molecules to manipulate/enhance the mesenchymal stem cell state without the use of biological components. The key protein pathways that control the stem cell state were examine with the bioinformatics tool GeneGo was used to identify compounds that affected these pathways, resulting in selection of 200 small molecules. The effect of the small molecules on the mesenchymal phenotype was examined and 5 small molecules were identified that enhanced the phenotype of these cells. The anti-inflammatory properties associated with the hit compounds led to the investigation of their effects on key surface proteins associated with the immune-modulatory state of the cells. In this preliminary study, two of the small molecules, estriol and spermine, increased the expression of a key mesenchymal stem cell marker STRO-1 and down regulated ICAM-1, a critical component of the immune modulation capacity of this cell type.

Submitted by Raniere Barreto (raniere.barros@ufvjm.edu.br) on 2018-05-10T17:58:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) alcides_alves_freitas.pdf: 1707403 bytes, checksum: f9233629066db73fd877b2885aa875bd (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-05-14T14:47:32Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) alcides_alves_freitas.pdf: 1707403 bytes, checksum: f9233629066db73fd877b2885aa875bd (MD5) / Made available in DSpace on 2018-05-14T14:47:32Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) alcides_alves_freitas.pdf: 1707403 bytes, checksum: f9233629066db73fd877b2885aa875bd (MD5) Previous issue date: 2017 / O boldo da terra (Plectranthus barbatus) ? popularmente utilizado para o tratamento de dist?rbios gastrintestinais e para doen?as hep?ticas. Devido ? exist?ncia de um grande n?mero de esp?cies dispon?veis para pesquisa e estudos farmacol?gicos, o estudo dessa planta torna-se importante para o conhecimento t?cnico-cient?fico, especialmente com a finalidade do desenvolvimento de novos f?rmacos. Com isto, o objetivo desse trabalho foi detectar prote?nas ativas de Plectranthus barbatus (boldo da terra) e avaliar a atividade biol?gica em c?lulas A549 e RAW264.7. As amostras dos procedimentos de extra??o das folhas e caule do P. barbatus foram submetidas ? quantifica??o de prote?na. Foi detectado em gel de poliacrilamida SDS-PAGE 12% prote?nas com peso molecular em torno de 30kDa e 94kDa o que ? descrito na literatura como lectinas e lipoxigenases. Os extratos foram caracterizados por cromatografia l?quida de alta efici?ncia com picos aparentes em 16 e 27 minutos. N?o foi detectada atividade de inibi??o da tripsina. Os resultados dos testes biol?gicos em cultura de c?lulas demonstraram que o extrato purificado de inibidores de protease n?o alterou a viabilidade celular de ambas as linhagens, no entanto, foi capaz de inibir a produ??o de ?xido n?trico na concentra??o de 10 ?g/ml para folha e caule e 100 ?g/ml para folha. Este trabalho demonstra pela primeira vez a extra??o de prote?nas em folhas e caule de Plectranthus barbatus e a atividade dessa mol?cula em cultura celular. Esse extrato n?o alterou a viabilidade celular de ambas as linhagens celulares, podendo ser caracterizados como n?o citot?xico nas concentra??es testadas. Conclui-se, portanto, que embora as folhas, caules e flores do Plectranthus barbatus seja utilizado amplamente pela popula??o esse trabalho demonstrou a detec??o de lectina e lipoxigenase at? agora desconhecidos nessa esp?cie em estudo. / Disserta??o (Mestrado Profissional) ? Programa de P?s-Gradua??o em Tecnologia, Sa?de e Sociedade, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The boldo da terra (Plectranthus barbatus) is popularly used for the treatment of gastrointestinal disorders and for liver diseases. Due to the existence of a large number of species available for research and pharmacological studies, the study of this plant becomes important for technical-scientific knowledge, especially for the purpose of developing new drugs. With this, the objective of this work was to detect active proteins of Plectranthus barbatus and to evaluate the biological activity in cells A549 and RAW264.7. Samples of P. barbatus leaf and stem extraction procedures were submitted to protein quantification. SDS-PAGE was detected in 12% proteins with molecular weight around 30kDa and 94kDa which is described in the literature as lectins and lipoxygenases. The extracts were characterized by high performance liquid chromatography with apparent peaks at 16 and 27 minutes. No trypsin inhibition activity was detected. The results of the biological tests in cell culture demonstrated that the purified protease inhibitor extract did not alter the cell viability of both strains, however, it was able to inhibit the production of 10 ?g / ml nitric oxide to leaf and And 100 ?g / ml for leaf. This work demonstrates for the first time the extraction of proteins in leaves and stem of Plectranthus barbatus and the activity of this molecule in cell culture. This extract did not alter the cellular viability of both cell lines and could be characterized as non-cytotoxic at the concentrations tested. It was concluded, therefore, that although the leaves, stems and flowers of Plectranthus barbatus were used extensively by the population, this work demonstrated the detection of lectin and lipoxygenase hitherto unknown in this species under study.

Two groups of growth regulators were described in Drosophila imaginal disc cell culture Cl.8+. Imaginal disc growth factors (IDGFs) belonging to chitinase-like protein family of carbohydrate binding proteins and Adenosine deaminase-related growth factors (ADGFs), which are active adenosine deaminases influencing homeostasis of key cellular metabolite adenosine. The functions of two of the IDGFs, as well as the effects of extracellular adenosine and its receptor were studied primarily in in vitro cell culture. Our results supported their roles in the regulation of cell survival and energy homeostasis especially in imaginal disc cells. Both the IDGFs and adenosine also play important roles in organismal responses to stress and infection and may interact in vivo.