The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma. The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of the Bad/Bcl-XL dimer. Knockdown of PKC-é by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-é may be a Bad kinase. Since, PKC-é is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-é/Bad pathway. Treatment with PI(3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-é activity and subsequent phosphorylation of Bad suggesting that PKC-é regulates the activity of Bad in a PI (3)-kinase dependent manner. Robust expression of PKC-é is a hallmark of human glioma and benign and malignant meningiomas, however, little is understood about its role in glioma cell proliferation. The cyclin dependent kinase activating kinase complex (CAK), comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator. Cdk7 phosphorylates its downstream cyclin dependent kinases (cdks) and promotes cell proliferation. Results show that PKC-é directly associated and phosphorylated Cdk7 at T170. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at T160. Purified PKC-é was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-é knockdown with siRNA, PDK1 siRNA and (PI) 3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. In addition, PKC-é knockdown reduced cell proliferation; led to cell cycle arrest and also induced apoptosis. Thus, these findings suggest the presence of a novel PI (3)-kinase/PKC-é/BAD mediated cell survival and PI (3)-kinase/PKC-é/Cdk7 mediated cell proliferation pathway.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis--University of Oklahoma. / Bibliography: leaves 115-119.

Thesis (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references. Also available online.

RNA modifications and processing events are important modulators of global gene expression. Genomic mutations in the RNA methylase NSun2 and the alternative splicing factor Srsf2 are linked to neurological disorders and cancer in humans, respectively. NSun2 methylates cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. Srsf2 is a critical component of the spliceosome and interacts with abundant ncRNAs that are methylated by NSun2. However, how precisely these processes effect homeostasis is largely unexplored. Therefore, the main aims of my PhD were (1) to dissect the molecular mechanisms of NSun2-mediated RNA methylation pathways that regulate cell survival under normal conditions and in response to oxidative stress, and (2) to investigate the importance of Srsf2 in stem cells using skin as a model system. In the context of RNA modifications, firstly I described how NSun2-expressing cells enrich for transcripts related to enhanced cell survival. Subsequently, by metabolically profiling wildtype and patient-derived dermal fibroblasts carrying loss-of-function mutations in the NSUN2 gene, I showed that the absence of NSun2 is synonymous to an energy-saving, low-translating and stressed cellular state. I further confirmed that lack of NSun2 was sufficient to instigate a cellular stress response, by monitoring BIRC5, a member of the inhibitor of apoptosis family. To further answer whether lack of NSun2 enhanced the susceptibility of patient cells to external stress stimuli, I next exposed them to oxidative stress and measured transcriptional and translational changes. I discovered that NSun2 is required to adapt global protein synthesis to the stress response, while NSun2-depleted cells failed to do so. This was concurrent with NSun2-depleted cells enriching for transcripts related to mRNA degradation and negative regulators of protein translation in response to stress. Generally, since loss of NSun2-driven methylation in tRNAs triggers their cleavage into small ncRNA fragments by angiogenin, I asked how angiogenin or tRNA-derived ncRNAs affect translation levels. In the presence of NSun2, angiogenin alone did not reduce global protein synthesis, yet tRNA fragmentation was required to modulate translation levels. Finally, to uncover how the lack of NSun2 influenced tRNA cleavage and methylation patterns in response to stress, I exposed wildtype and patient cells to sodium arsenite and measured the abundance of tRNA-derived fragments and occurrence of methylation events. With this I discovered unique tRNA fragmentation patterns and global RNA methylation profiles for wildtype and NSun2-depleted cells, that can account for the underlying molecular and phenotypical differences in response to stress. In the context of alternative splicing, and since the cellular functions of Srsf2 are largely unknown, I explored its role in cellular survival and differentiation. By conditionally deleting SRSF2 in two different stem cell populations of the mouse epidermis, I observed significant thickening of the epidermis, altered expression of cell proliferation and stem cell differentiation markers, and distorted hair follicle structures. Moreover, I demonstrated that lack of Srsf2 promotes skin regeneration following injury, thus strongly indicating that Srsf2 is required for normal skin development and regeneration after injury. In summary, my research suggests that NSun2-mediated RNA methylation pathways orchestrate transcriptional and translational programmes in response to external stress stimuli, and my studies are the first to show that the alternative splicing factor Srsf2 is required for stem cell differentiation in skin.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Vincristine is a chemotherapeutic agent used to treat various types of cancers with effective clinical outcomes. However, its effects include a number of disorders related to balance, such as ataxia, tremors and peripheral neuropathy. These damages are likely associated with poisoning by the drug in the central and peripheral nervous systems. There is evidence that guarana (Paullinia cupana) may have neuroprotective effects on the central nervous system. The aim of this study was to evaluate the effects of vincristine and guarana on cell viability and oxidative metabolism in vitro cell cultures of mice brain and cerebellum. The cells were incubated for 24 and 72 hours in a specific medium (DMEM) and treated with vincristine at a concentration 0.009 mM for 24 hours and 0.0007 mM for 72 hours and at concentrations of 10, 30, 100 and 300μg/m Lof guarana extract. Incubation was followed by MTT and Pico Green ® tests and evaluation of catalase, superoxide dismutase and thiol activity (cell viability), as well as TBARS, protein carbonyls and dichlorodihydrofluorescein (oxidative metabolism). The results indicated that vincristine may cause cytotoxic effects in mice cerebellum and brain and that guarana may reverse this cytotoxicity in concentrations 100 e 300μg/mL. / A vincristina é um quimioterápico utilizado no tratamento de vários tipos de neoplasias com grandes resultados clínicos, porém, seus efeitos incluem uma série de alterações do equilíbrio, como ataxia, tremores e neuropatia periférica. Estes danos estão provavelmente associados à intoxicação provocada pelo fármaco no sistema nervoso central e periférico. Há indícios de que o guaraná (Paullinia cupana) possui componentes capazes de produzir neuroproteção central. O objetivo deste estudo foi avaliar os efeitos da vincristina e do guaraná sobre a viabilidade celular e o metabolismo oxidativo no cérebro e no cerebelo de camundongos em cultura celular in vitro. As células foram incubadas por 24 e 72 horas em meio específico (DMEM) e tratadas com vincristina na concentração 0,009 μM para 24 horas e 0,0007 μM para 72 horas e com as concentrações de 10, 30, 100 e 300μg/mL de extrato de guaraná. Após, realizou-se os testes MTT, Pico Green®, atividade das enzimas catalase e superóxido dismutase e tióis (viabilidade celular); TBARS, diclorodihidrofloresceína e carbonilação de proteínas (metabolismo oxidativo). Os resultados indicaram que a vincristina pode causar efeitos citotóxicos em cerebelo e cérebro de camundongos e que o guaraná pode reverter essa citotoxicidade, principalmente nas concentrações de 100 e 300 μg/mL.

Made available in DSpace on 2014-06-11T19:23:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-15Bitstream added on 2014-06-13T20:30:33Z : No. of bitstreams: 1 carvalho_acgs_me_araca.pdf: 682833 bytes, checksum: 3da34d67f34af6e497f53ac11e8e701c (MD5) / O propósito deste trabalho foi avaliar a influência da reutilização de fresas de alta resistência sobre o aquecimento ósseo, a viabilidade celular imediata e a deformação das fresas após osteotomia para implantes em tíbias de coelhos. Para isso, foram utilizados 12 coelhos machos brancos (Oryctolagus cunilicus, Nova Zelândia), que foram submetidos a 200 sequências de fresagens para implantes na cortical superior de suas tíbias. Foram estabelecidos 6 grupos (G1 a G6) que correspondiam ao número de osteotomias realizadas por cada fresa (0, 10, 20, 30, 40 e 50). Cada leito cirúrgico recebia a seguinte sequência de fresagem: fresa tipo lança e helicoidais de 2,0mm, 2,8mm, 3,0mm e 3.15mm. As áreas osteotomizadas foram coletadas para análise imunoistoquímica, as oscilações térmicas foram quantificadas e as fresas utilizadas receberam análise por microscopia eletrônica de varredura. Nos resultados, houve uma correlação alta entre a porcentagem de deformação e o número de reutilização de cada fresa (Coeficiente de Correlação de Pearson r = 0,984). Observou-se também que as fresas do tipo lança apresentaram maior deformação após o uso do que as fresas helicoidais (proporção de 2:1). Não houve correlação estatística significativa entre aumento da reutilização da fresa e aquecimento ósseo (p > 0,05). No entanto, observouse que a oscilação térmica durante a fresagem da lança é maior que as demais fresagens (proporção de 3:1). Na análise imunoistoquímica, observou-se um equilíbrio fisiológico da expressão das proteínas Osteoprotegerina e RANKL em todos os grupos, no entanto, houve maior expressão de todas as proteínas no Grupo 6. De acordo com a metodologia aplicada, foi possível concluir que as fresas avaliadas não causam aquecimento ósseo significativo em até 50 reutilizações, no entanto, causam maior trauma tecidual na 50ª fresagem... / The purpose of this study was evaluate the influence of high resistence drill reuse over bone heating, immediate bone-cell viability and drills wear after implant osteotomies in rabbit tibias. Therefore, 12 male white rabbits (Oryctolagus cunilicus, New Zealand) received 200 implant sequential osteotomies over the superior cortical of their tibia. Six groups were established (G1 to G6), according to the number of osteotomy of each drill (0, 10, 20, 30, 40 and 50). The implant sequence of drills was: spear drill, 2,0mm, 2.8mm, 3.0mm and 3.15mm helicoidal drills. The receptor-beds were collected to imunohistochemistry, the thermal changes were quantified and the drills received scanning electron microscopy analysis. In the results, a great correlation degree between drill wear and number of osteotomies was observed (Pearson’s Correlation Coefficient r = 0,984). Another result was that spear drill have more deformation than helicoidal drills (2:1 proportion). The bone heating analysis concluded that there was no statistical siginificance between number of osteotomy and bone heating (p > 0,05). However, the thermal changes during spear drilling was greater than during helicoidal drillings (3:1 proportion). Imunohistochemistry results showed a physiological balance of Osteoprotegerin and RANKL immunolabeling in all groups, however there was a greater immunolabeling of all proteins in the last group. According to this metodology, the conclusion is that the tested drills do not caused significant bone heating after 50 reuses, however, they caused more tissue trauma at 50th osteotomy. So, the studied system must be reused for 40 times, and the spier drill must be substituted after 20 osteotomies.

Made available in DSpace on 2015-04-17T15:03:04Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1319855 bytes, checksum: c9da3a0b2d2daa085371bfaec1a3a590 (MD5) Previous issue date: 2014-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The demand for fresh foods, with lower calories, high nutritional values and easily prepared has been increased the consumption of minimally processed vegetables. However, because the intense handling these products has been considered a potential problem to the microbiological safety, related mainly to the presence of psychrotrophic pathogenic and spoilage bacteria. The sanitization is considered a critical processing step for minimally processed vegetables, although some of the synthetic sanitizers allowed for the use in vegetables are cited as responsible for undesirable effects to consumers. In this context, as alternative to the synthetic sanitizers arise the essential oils, whose antimicrobial action mechanism is attributed, many times, to their majority compounds. Regarding these aspects, this study aimed to evaluate the potential application of carvacrol and 1,8-cineole against some strains of minimally processed vegetables contaminant bacteria (Listeria monocytogenes ATCC 7644, Aeromonas hydrophila INCQS 7966 and Pseudomonas fluorescens ATCC 11253), in mixed culture, by determining the values of Minimum Inhibitory Concentration (MIC), the index of Fractional Inhibitory Concentration (FIC), as well as by assessing the efficacy of the application of these compounds in inhibiting the growth and survival of the mixed bacterial inoculum in vegetable broth and in leafy vegetables. Moreover, it was performed the assessment of possible damage caused by the tested compounds in the morphology of the bacterial cells by the analysis of confocal microscopy and scanning electronic microscopy. MIC values of carvacrol and 1,8-cineole ranged from 1.25 e 40μL/mL, respectively. The FIC index against the mixed bacterial inoculum was 0.25, suggesting a synergic interaction between the tested compounds. The application of the compounds alone or combined in sub-inhibitory concentrations in vegetable broth caused a significant decrease (p <0.05) in counts (CFU/mL) of the tested bacteria over 24 h. The exposure of vegetables to the compounds for only 5 min also caused a significant reduction (p < 0.05) in counts of the tested bacteria. The observations of bacterial cell morphology suggest that the compounds carvacrol and 1,8-cineole alone or combined in sub-inhibitory concentrations, cause damage to the cell viability, and change the permeability of the cytoplasmic membrane and the cell surface, with the appearance of roughness appearance and like-vesicles structures. These results show that carvacrol and 1,8-cineole possess strong inhibitory effect of the growth and survival of bacteria associated with minimally processed vegetables when tested in mixed culture. Still, these data confirm that constituents of essential oils, with different molecular structures, when applied in combination can replace traditional synthetic sanitizing used in minimally processed vegetables allowing reaching the balance between the demand for microbiological safety and sensory acceptability of these products. / A demanda por alimentos frescos, de baixo teor calórico, elevados valores nutricionais e de fácil preparo, tem elevado o consumo de hortaliças minimamente processadas. Entretanto, devido a sua intensa manipulação estes produtos têm sido considerados um potencial problema de segurança microbiológica, envolvendo principalmente bactérias psicrotróficas patogênicas e deteriorantes. A sanitização é considerada uma etapa crítica do processamento deste de hortaliças minimamente processadas, embora alguns dos sanitizantes sintéticos permitidos para uso em vegetais sejam citados como responsáveis por efeitos indesejáveis para o consumidor. Neste sentido, como alternativa aos sanitizantes sintéticos, surgem os óleos essenciais, cujo potencial antimicrobiano é atribuído, muitas vezes, aos seus compostos majoritários. Diante deste contexto, este estudo objetivou avaliar o potencial da aplicação dos constituintes carvacrol e 1,8-cineol na inibição de cepas de bactérias contaminantes de vegetais minimamente processados (Listeria monocytogenes ATCC 7644, Aeromonas hydrophila ATCC 7966 e Pseudomonas fluorescens ATCC 11253), em inóculo misto, através da determinação dos valores de Concentração Inibitória Mínima (CIM), do índice de Concentração Inibitória Fracionada (CIF), bem como da influência da aplicação desses compostos na inibição do crescimento e sobrevivência do inóculo misto bacteriano em caldo vegetal e em vegetais folhosos. Além disso, foi realizada a avaliação dos possíveis danos causados pelos compostos testados à morfologia das células bacterianas através de análises de microscopia confocal e microscopia eletrônica de varredura. Os valores de CIM do carvacrol e do 1,8-cineol foram 1,25 e 40 μL/mL, respectivamente. O índice de CIF frente ao inóculo bacteriano misto foi 0,25, sugerindo uma interação sinérgica entre os compostos testados. A aplicação dos compostos isolados ou combinados em concentrações sub-inibitórias em caldo vegetal causou uma diminuição significativa (p < 0,05) na contagem das bactérias ensaiadas (UFC/mL) ao longo de 24 h. A exposição das hortaliças aos constituintes por apenas 5 min também causou uma redução significativa (p < 0,05) nas contagens dos micro-organismos testados. As observações morfológicas das células bacterianas sugerem ainda que os compostos carvacrol e 1,8-cineol, isolados ou combinados em concentrações sub-inibitórias, ocasionam danos à viabilidade celular, bem como alteração da permeabilidade da membrana citoplasmática e da superfície celular, com o aparecimento de rugosidades e estruturas semelhantes a vesículas. Estes resultados mostram que carvacrol e 1,8-cineol possuem considerável poder de inibição do crescimento e sobrevivência de bactérias contaminantes de hortaliças minimamente processadas, quando ensaiadas em inóculo misto. Ainda, confirmam que constituintes de óleos essenciais, com diferentes estruturas moleculares, quando aplicados em combinação podem substituir sanitizantes sintéticos clássicos utilizados em vegetais, possibilitando o alcance do equilíbrio entre a demanda pela segurança microbiológica e a aceitabilidade sensorial destes produtos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / For preservation of periodontal ligament cells after one tooth avulsion becomes necessary to determine a storage medium. The aim of this study was to analyze the preservation of human fibroblast cells and analyze the composition of root dentin of bovine teeth in different storage media. Immortalized human fibroblasts cells were grown in flasks containing Dulbecco's modified eagle medium (DMEM), and ffter reaching confluence the cells were trypsinized, counted by hemocytometer and plated. The culture medium was removed from each well and the cells were exposed at different times in the solutions: GLi - milk; GRl - ringer’s lactate; GPv - red propolis solution and GPd - Pedialyte. DMEM was considered positive control group and tap water was the negative control. After the experimental period of 15, 30, 45 and 60 minutes, we used the colorimetric method MTT formazan. For analysis of surface composition of root dentin, were using Fourier transform infrared (FTIR) where, were collected 60 bovine incisors. Samples were extracted from the cervical region of the root dentin in 3x3mm. The samples were randomly divided among experimental groups in differents times. Data were tabulated and statistically analyzed using analysis of variance for the storage media within each time and the time factor for each storage medium, followed by Dunnet test and Tukey's test (P <0.05 ). In 15 and 60 minutes, from the media types, the milk and Ringer's lactate showed better results when compared to the negative control. However, in 30 and 45 minutes only the milk showed satisfactory results. A comparison with DMEM only the milk showed similar statistical results. In assessing the viability of human fibroblasts, milk showed cell viability levels similar to the positive control and superior to other media tested. Within 60 minutes storage, the one most commonly used in clinical conditions, the ringer's lactate has higher performance than the red propolis and the pedialyte and these similar to tap water (negative control). / Para preservação das células do ligamento periodontal após um caso de avulsão dentária torna-se imprescindível a determinação de um meio de armazenagem. O objetivo deste estudo foi analisar os efeitos de diferentes meios de conservação na viabilidade celular e na composição iônica da dentina radicular de dentes bovinos em quatro períodos experimentais. Células de fibroblastos imortalizados humanos foram cultivadas em frascos contendo meio eagle modificado de Dulbecco (DMEM), e após atingir confluência as células foram tripsinizadas, contadas em hemocitômetro e plaqueadas. O meio de cultura foi removido de cada poço e as células foram expostas a diferentes tempos e meios de conservação: GLi - leite integral; GRl - ringer lactato; GPv - solução de própolis vermelho e GPd - pedialyte. DMEM foi considerado grupo controle positivo e, água de torneira, o controle negativo. Após os períodos experimentais de 15, 30, 45 e 60 minutos, foi aplicado o método colorimétrico MTT formazan para avaliar a viabilidade. Para análise de composição superficial da dentina radicular, foi utilizada a espectroscopia de infravermelho transformada de Fourier (FTIR) onde, foram coletados 60 incisivos bovinos para confecção de amostras de 3x3 mm de dentina radicular extraída da região cervical. As amostras foram divididas aleatoriamente entre grupos experimentais em diferentes tempos. Os dados foram tabulados e submetidos à análise estatística empregando análise de variância em fator único para os meios de armazenagem dentro de cada tempo, e o fator tempo para cada meio de armazenamento, seguido dos teste de Dunnet e Teste de Tukey (P<0,05). Nos tempos de 15 e 60 minutos, dentre os meios analisados, o leite integral e o ringer com lactato apresentaram resultados superiores quando comparados ao controle negativo. Entretanto, nos tempos de 30 e 45, minutos somente o leite integral apresentou resultados satisfatórios. Quando comparamos com o DMEM, somente o leite integral apresentou resultados estatísticos similares. Sendo assim, avaliando a viabilidade celular, o leite integral apresentou níveis de viabilidade celular similar ao controle positivo e superior aos demais meios testados. No período de 60 minutos de armazenagem, aquele mais comumente realizado nas condições clínicas, o meio Ringer com lactato apresentou desempenho superior ao própolis vermelho e ao pedialyte, e estes, similares à água de torneira (controle negativo).