Damage and/or loss of functional neurons can lead to detrimental cognitive and paralyzing effects in humans. Prime examples of such negative situations are well documented in patients with Parkinson's and Alzheimer's disease. In recent years, the utilization of neural stem cells and their derivation into neurons have been the focus of many research endeavors. The main reason for this is because neural stem cells are multi-potent and can differentiate into neurons, astrocytes, and oligodendrocytes. The research that will be detailed in this thesis involves the potential use of inorganic nanostructured materials to efficiently deliver bioactive molecules (i.e., retinoic acid, kinase inhibitors) to cells that can modulate the differentiation potential of certain cells into neurons. Specifically, PC12 (derived from rat pheochromocytoma) cells, as a neural model, was treated with select nanostructured materials with and without neuron inducers (molecules and ions) and the results were analyzed via biochemical assays and live-cell fluorescence microscopy. This thesis will include an in depth look into the cytocompatibility of the tested nanostructured materials that include silica nanoparticles, titanate nanotube microspheres, and carbon microparticles. / Bioengineering / Accompanied by two .avi files.

At the end of the 20th century it was known that cannabinoid drugs interact with two receptors, CB1 and CB2. Subsequent pharmacological studies have confirmed that there are other receptors interacting with cannabinoids. GPR55 is a transmembrane G protein coupled receptor, which together with the receptor GPR18 and GPR119 belong to a group of new cannabinoid receptors and is involved in the function of the endocannabinoid system. In addition to some of cannabinoid substances, it is stimulated primarily phospholipid lysofosfatidylinositolem. LPI-dependent signaling GPR55 plays an important role in the regulation of many physiological and pathological processes, such as pain, inflammation, cell proliferation, or endothelial function. It was found that LPI confers tolerance to ischemic brain damage and has a cytoprotective effect on the pyramidal cells. The aim of the study was to determine whether the application of five ligands induce phosphorylation of protein kinase ERK 1/2, Akt and activate the GTPase RhoA and whether activation of the receptor GPR55 has cytoprotective effect in model cell line PC12, in which hypoxic conditions were simulated by adding CoCl2. For working methods were used SDS-PAGE, Western bloting and colorimetric measurement. Pharmacological studies in recent years have shown...

A coenzima Q10 é uma provitamina lipossolúvel sintetizada endogenamente e naturalmente encontrada em alimentos como a carne vermelha, peixes, cereais, brócolis e espinafre. É comercializada como suplemento alimentar e utilizada em formulações cosméticas. Localiza-se na membrana de organelas celulares como retículo endoplasmático, vesículas e membrana interna da mitocôndria, onde atua como um cofator essencial na cadeia respiratória. Apresenta propriedades antioxidantes e potencial no tratamento de doenças neurodegenerativas e neuromusculares. O objetivo deste trabalho foi investigar os possíveis efeitos protetores de uma formulação hidrossolúvel de coenzima Q10 em células PC12 expostas à cisplatina, um fármaco antineoplásico que tem a neurotoxicidade como um dos fatores limitantes à sua utilização. A linhagem celular PC12 (feocromocitoma de ratos) utilizada nesta investigação é um reconhecido modelo in vitro para estudos neuronais. Os métodos empregados foram os ensaios do MTT, cometa, citoma micronúcleo com bloqueio da citocinese, crescimento de neuritos e análise da expressão do gene Tp53. Os resultados obtidos na avaliação da citotoxicidade da coenzima Q10 (0,1 - 20 µg/mL) mostraram que este antioxidante foi citotóxico às células PC12 na concentração de 20,0 µg/mL e não apresentou citotoxicidade em baixas concentrações. Para os ensaios do citoma e cometa, foram selecionadas três concentrações não citotóxicas de coenzima Q10 (0,1; 0,5 e 1,0 µg/mL) que não apresentaram mutagenicidade e genotoxicidade às células PC12. O efeito protetor da coenzima Q10 sobre a cisplatina no ensaio do citoma foi caracterizado pela diminuição da freqüência de micronúcleos e brotos nucleares, entretanto a proteção da coenzima Q10 não foi evidenciada no ensaio cometa. Alterações significativas na expressão do gene Tp53 não foram observadas no tratamento coenzima Q10 (1,0 µg/mL) associado à cisplatina (0,1 µg/mL). A coenzima Q10 (0,1 e 1,0 µg/mL) não foi neurotóxica em células PC12 indiferenciadas e diferenciadas após exposição ao fator de crescimento do nervo, e seu melhor efeito neuroprotetor foi observado na menor concentração avaliada. A coenzima Q10 reduziu a citotoxicidade da cisplatina (10,0 µg/mL) em células PC12 indiferenciadas e estimulou o crescimento de neuritos em células PC12 diferenciadas. A determinação dos efeitos citoprotetores da coenzima Q10 em um modelo neuronal é importante para elucidar possíveis estratégias de neuroproteção que poderiam ser aplicadas aos pacientes submetidos à quimioterapia. / Coenzyme Q10 is a liposoluble provitamin endogenously synthesized and naturally found in various foods items, such as meat, fish, cereals, broccoli and spinach. It is a dietary supplement in some countries and used in cosmetic formulations. Coenzyme Q10 is located in the membrane of cellular organelles such as endoplasmic reticulum, vesicles and inner mitochondrial membrane, where acts as an essential cofactor in the respiratory chain. It has antioxidant properties and potential in the treatment of neurodegenerative and neuromuscular diseases. The objective of this study was to investigate the possible protective effects of a water-soluble formulation of coenzyme Q10 in PC12 cells exposed to cisplatin, an anticancer drug that has neurotoxicity as a dose-limiting factor. The PC12 cell line (rat pheocromocytoma) used in this investigation is a recognized in vitro model for neuronal studies. The methods used were the MTT, comet, cytokinesis-block micronucleus cytome, neurite outgrowth assays and expression of Tp53 gene. The results obtained in the cytotoxicity of coenzyme Q10 (0.1-20 µg/mL) showed that this antioxidant was cytotoxic to PC12 cell at a concentration of 20.0 µg/mL and it was not cytotoxic at low concentrations. For the cytome and comet assays, were selected three non-cytotoxic concentrations of coenzyme Q10 (0.1, 0.5 and 1.0 µg/mL) without mutagenicity and genotoxicity PC12 cells. The protective effect of coenzyme Q10 in cytome assay was characterized by decreased frequency of micronuclei and nuclear buds induced by cisplatin, however the protection of coenzyme Q10 was not evidenced by the comet assay. No significant change in the Tp53 gene expression were observed in the coenzyme Q10 (1.0 µg/mL) plus cisplatin (0.1 µg/mL) treatment. Coenzyme Q10 (0.1 and 1.0 µg/mL) was not neurotoxic in undifferentiated and nerve growth factor differentiated PC12 cells and the lowest concentration evaluated showed the best neuroprotective effect. The coenzyme Q10 treatment reduced the citotoxicity of cisplatin (10.0 µg/mL) in undifferentiated PC12 cells and stimulated the neurite outgrowth in differentiated PC12 cells. Determination of the cytoprotective effects of the coenzyme Q10 in a neuronal model is important to elucidate possible strategies for neuroprotection that could be applied to patients undergoing chemotherapy.

Pode-se dizer que o câncer, ao lado da AIDS, é a doença que atinge, a níveis mundiais, maior clamor da sociedade por desenvolvimento de cura e melhor qualidade de vida ao paciente. Várias terapias são utilizadas para o tratamento do câncer, podendo-se destacar a importância da quimioterapia em inúmeros protocolos de tratamento. Um dos fármacos mais antigos e mais utilizados na quimioterapia é a cisplatina, que ao longo dos seus mais de 30 anos na prática clínica demonstrou sua eficácia e foi incorporada no protocolo de tratamento de uma variedade de tumores. Entretanto, ao lado se sua comprovada eficácia, é inerente o desenvolvimento de vários efeitos colaterais resultante de sua toxicidade à células sadias. Neste trabalho, destaque é dado a neurotoxicidade, que surge em um grande número de pacientes que recebem tratamento com a cisplatina. Inúmeros esforços têm sido realizados na tentativa de prevenir ou controlar os efeitos tóxicos induzidos pela cisplatina, com atenção especial à suplementação do tratamento quimioterápico com compostos antioxidantes. Nesse sentido, este estudo visa avaliar a associação da curcumina, um composto polifenólico com notável atividade antioxidante e neuroprotetora, ao tratamento com cisplatina, em um modelo in vitro com culturas de células PC12. A curcumina é reconhecidamente um composto polifenólico com um amplo espectro de atividades biológicas, com suas propriedades neuroprotetoras demonstradas em vários modelos estudados estando principalmente relacionadas com seu potencial antioxidante. Os múltiplos mecanismos de ação dos compostos fenólicos e suas propriedades neuroprotetoras atribuídas a capacidade de inibição das espécies reativas de oxigênio indicam um potencial de inibição da neurotoxicidade que surgem durante o tratamento quimioterápico com a cisplatina, visto a reconhecida capacidade de geração de espécies reativas de oxigênio da cisplatina. Como a geração de espécies reativas de oxigênio intracelular pode resultar em danos ao DNA pela ação direta das espécies reativas ou indiretamente via produtos de degradação da peroxidação lipídica, nosso estudo avaliou o potencial protetor da curcumina contra a genotoxicidade induzida pela cisplatina em culturas de células PC12, analisando a indução de micronúcleos e a análise de quebras de cadeia simples, cadeia dupla do DNA e sítios álcali-labeis pelo ensaio do cometa. Antes da avaliação antigenotóxica da curcumina, foi realizada uma avaliação citotóxica e genotóxica, em que pode-se observar que em concentrações elevadas a curcumina é citotóxica e genotóxica às células PC12. O efeito protetor da curcumina foi avaliado em pré-tratamento das culturas de células PC12 com concentrações não tóxicas. Nas três concentrações de curcumina estudadas não houve indícios de interferência com a citotoxicidade ou o efeito citostático da cisplatina. Embora o efeito protetor da curcumina contra os danos induzidos ao DNA de células PC12 não tenha sido evidente nos resultados obtidos pelo ensaio do cometa, a curcumina foi eficaz na redução de micronúcleos induzidos pela cisplatina, de maneira semelhante nas três concentrações avaliadas. Os resultados positivos obtidos nesse estudo juntamente com os dados pré-existentes a respeito de efeitos protetores da curcumina incentivam novas pesquisas em relação aos possíveis benefícios da utilização da curcumina em terapia combinada aos quimioterápicos. / Several therapies are used for treating cancer and the importance of chemotherapy can be emphasized in many protocols of treatment. One of the oldest and most used drugs in chemotherapy is the cisplatin which, with its more than thirty years of clinical practice, has demonstrated its effectiveness being incorporated into the protocol of a variety of tumor treatments. On the other hand, it is intrinsic the development of various side effects resulting from its toxicity to healthy cells. This research gives emphasis to the neurotoxicity which appears in a large number of patients receiving the cisplatin treatment. Numerous efforts are being made in attempt to prevent or even control the toxic effects induced by cisplatin with special attention being given to the supplementation of chemotherapy treatment with antioxidant compounds. In that sense, this study aims to assess the association of curcumin, a polifenolic compound with remarkable antioxidant and neuroprotective activity, to the cisplatin treatment in an in vitro neuronal model with PC12 cell cultures. The curcumin is admittedly a polifenolic compound with a broad spectrum of biological activities and their neuroprotective properties, demonstrated in several models, are strongly related to its antioxidant potential. The multiple mechanisms of action of the phenolic compounds and their neuroprotective properties credited to the ability of inhibition of the reactive oxygen species indicate a potential inhibition of neurotoxicity that takes place during the chemotherapy treatment with cisplatin, given its well known ability to generate reactive oxygen species. Since the generation of reactive oxygen species intracellular can result in DNA damage by direct action of reactive species or indirect, through degradation products of the lipid peroxidation, our studies evaluated the potential protector of curcumin against the genotoxicity induced by cisplatin in PC12 cell cultures by examining the induction of micronuclei and analyzing DNA single strand breaks, double strand breaks, and alkali-labeis sites through the comet test. Before the antigenotoxic evaluation of the curcumin, a cytotoxic and genotoxic test was conducted where it was observed that when in high concentrations the curcumin is cytotoxic and genotoxic to the PC12 cells. The protective effect of curcumin was evaluated at the pre-treatment of PC12 cell cultures with non-toxic levels of concentration. In the three studied curcumin concentrations there was no evidence of interference with the cytotoxicity or cytostatic effect of the cisplatin. Although the protective effect of the curcumin put against the damage caused to the DNA of cells PC12 was not evident in the results obtained by the comet test, the curcumin was equally effective in the reduction of micronuclei induced by the cisplatin in all three concentrations evaluated. The positive results obtained in this study combined with the pre-existing data about the protective effects of the curcumin encourage some more new research on possible benefits of using curcumin in combination to chemotherapy.

In der vorliegenden Arbeit konnte mit der PC12 Zellinie und dem MTT Viabilitätsassay Glypikan, ein Glykosylphosphatidylinositol-verankertes Heparan Sulfat, als Rezeptor identifiziert werden, der die toxischen Effekte von [beta]-Amyloid vermittelt. Kompetitive Substanzen im Medium, enzymatische Behandlung der Zelloberfläche und Block spezifischer zellulärer Synthesewege wurden eingesetzt. [beta]-Amyloid wird eine zentrale Rolle in der Pathogenese der Alzheimer'schen Krankheit zugeschrieben. Eine Intervention auf Ebene der [beta]-Amyloid-Rezeptor Interaktion könnte einen Therapieansatz für die Alzheimer'sche Krankheit darstellen. Weitere Experimente sind notwendig, um die Rolle von Glypikan bei der Vermittlung der [beta]-Amyloid Toxizität im Alzheimer-Gehirn zu untersuchen, z.B. in der Primärkultur von Neuronen und Mikroglia oder im lebenden Hirnschnitt. Eine ähnliche anatomische Verteilung der Glypikanexpression im Rattenhirn und der neurofibrillären Bündel im Alzheimergehirn wurde gefunden. Eine mögliche Rolle für Glypikan bei der Entstehung der neurofibrillären Bündel muß im Autopsiegehirn von Alzheimerpatienten überprüft werden. / [beta]-amyloid is thought to play a major role in the pathogenesis of Alzheimer's disease. In PC12 cell culture, competitive substances in cell culture medium, enzymatic cell surface treatment und block of specific cell synthesis pathways were used to identify glypican, a GPI- anchored heparan sulfate proteoglycan, as a receptor that mediates the toxic effects of [beta]-amyloid.

Pode-se dizer que o câncer, ao lado da AIDS, é a doença que atinge, a níveis mundiais, maior clamor da sociedade por desenvolvimento de cura e melhor qualidade de vida ao paciente. Várias terapias são utilizadas para o tratamento do câncer, podendo-se destacar a importância da quimioterapia em inúmeros protocolos de tratamento. Um dos fármacos mais antigos e mais utilizados na quimioterapia é a cisplatina, que ao longo dos seus mais de 30 anos na prática clínica demonstrou sua eficácia e foi incorporada no protocolo de tratamento de uma variedade de tumores. Entretanto, ao lado se sua comprovada eficácia, é inerente o desenvolvimento de vários efeitos colaterais resultante de sua toxicidade à células sadias. Neste trabalho, destaque é dado a neurotoxicidade, que surge em um grande número de pacientes que recebem tratamento com a cisplatina. Inúmeros esforços têm sido realizados na tentativa de prevenir ou controlar os efeitos tóxicos induzidos pela cisplatina, com atenção especial à suplementação do tratamento quimioterápico com compostos antioxidantes. Nesse sentido, este estudo visa avaliar a associação da curcumina, um composto polifenólico com notável atividade antioxidante e neuroprotetora, ao tratamento com cisplatina, em um modelo in vitro com culturas de células PC12. A curcumina é reconhecidamente um composto polifenólico com um amplo espectro de atividades biológicas, com suas propriedades neuroprotetoras demonstradas em vários modelos estudados estando principalmente relacionadas com seu potencial antioxidante. Os múltiplos mecanismos de ação dos compostos fenólicos e suas propriedades neuroprotetoras atribuídas a capacidade de inibição das espécies reativas de oxigênio indicam um potencial de inibição da neurotoxicidade que surgem durante o tratamento quimioterápico com a cisplatina, visto a reconhecida capacidade de geração de espécies reativas de oxigênio da cisplatina. Como a geração de espécies reativas de oxigênio intracelular pode resultar em danos ao DNA pela ação direta das espécies reativas ou indiretamente via produtos de degradação da peroxidação lipídica, nosso estudo avaliou o potencial protetor da curcumina contra a genotoxicidade induzida pela cisplatina em culturas de células PC12, analisando a indução de micronúcleos e a análise de quebras de cadeia simples, cadeia dupla do DNA e sítios álcali-labeis pelo ensaio do cometa. Antes da avaliação antigenotóxica da curcumina, foi realizada uma avaliação citotóxica e genotóxica, em que pode-se observar que em concentrações elevadas a curcumina é citotóxica e genotóxica às células PC12. O efeito protetor da curcumina foi avaliado em pré-tratamento das culturas de células PC12 com concentrações não tóxicas. Nas três concentrações de curcumina estudadas não houve indícios de interferência com a citotoxicidade ou o efeito citostático da cisplatina. Embora o efeito protetor da curcumina contra os danos induzidos ao DNA de células PC12 não tenha sido evidente nos resultados obtidos pelo ensaio do cometa, a curcumina foi eficaz na redução de micronúcleos induzidos pela cisplatina, de maneira semelhante nas três concentrações avaliadas. Os resultados positivos obtidos nesse estudo juntamente com os dados pré-existentes a respeito de efeitos protetores da curcumina incentivam novas pesquisas em relação aos possíveis benefícios da utilização da curcumina em terapia combinada aos quimioterápicos. / Several therapies are used for treating cancer and the importance of chemotherapy can be emphasized in many protocols of treatment. One of the oldest and most used drugs in chemotherapy is the cisplatin which, with its more than thirty years of clinical practice, has demonstrated its effectiveness being incorporated into the protocol of a variety of tumor treatments. On the other hand, it is intrinsic the development of various side effects resulting from its toxicity to healthy cells. This research gives emphasis to the neurotoxicity which appears in a large number of patients receiving the cisplatin treatment. Numerous efforts are being made in attempt to prevent or even control the toxic effects induced by cisplatin with special attention being given to the supplementation of chemotherapy treatment with antioxidant compounds. In that sense, this study aims to assess the association of curcumin, a polifenolic compound with remarkable antioxidant and neuroprotective activity, to the cisplatin treatment in an in vitro neuronal model with PC12 cell cultures. The curcumin is admittedly a polifenolic compound with a broad spectrum of biological activities and their neuroprotective properties, demonstrated in several models, are strongly related to its antioxidant potential. The multiple mechanisms of action of the phenolic compounds and their neuroprotective properties credited to the ability of inhibition of the reactive oxygen species indicate a potential inhibition of neurotoxicity that takes place during the chemotherapy treatment with cisplatin, given its well known ability to generate reactive oxygen species. Since the generation of reactive oxygen species intracellular can result in DNA damage by direct action of reactive species or indirect, through degradation products of the lipid peroxidation, our studies evaluated the potential protector of curcumin against the genotoxicity induced by cisplatin in PC12 cell cultures by examining the induction of micronuclei and analyzing DNA single strand breaks, double strand breaks, and alkali-labeis sites through the comet test. Before the antigenotoxic evaluation of the curcumin, a cytotoxic and genotoxic test was conducted where it was observed that when in high concentrations the curcumin is cytotoxic and genotoxic to the PC12 cells. The protective effect of curcumin was evaluated at the pre-treatment of PC12 cell cultures with non-toxic levels of concentration. In the three studied curcumin concentrations there was no evidence of interference with the cytotoxicity or cytostatic effect of the cisplatin. Although the protective effect of the curcumin put against the damage caused to the DNA of cells PC12 was not evident in the results obtained by the comet test, the curcumin was equally effective in the reduction of micronuclei induced by the cisplatin in all three concentrations evaluated. The positive results obtained in this study combined with the pre-existing data about the protective effects of the curcumin encourage some more new research on possible benefits of using curcumin in combination to chemotherapy.

A coenzima Q10 é uma provitamina lipossolúvel sintetizada endogenamente e naturalmente encontrada em alimentos como a carne vermelha, peixes, cereais, brócolis e espinafre. É comercializada como suplemento alimentar e utilizada em formulações cosméticas. Localiza-se na membrana de organelas celulares como retículo endoplasmático, vesículas e membrana interna da mitocôndria, onde atua como um cofator essencial na cadeia respiratória. Apresenta propriedades antioxidantes e potencial no tratamento de doenças neurodegenerativas e neuromusculares. O objetivo deste trabalho foi investigar os possíveis efeitos protetores de uma formulação hidrossolúvel de coenzima Q10 em células PC12 expostas à cisplatina, um fármaco antineoplásico que tem a neurotoxicidade como um dos fatores limitantes à sua utilização. A linhagem celular PC12 (feocromocitoma de ratos) utilizada nesta investigação é um reconhecido modelo in vitro para estudos neuronais. Os métodos empregados foram os ensaios do MTT, cometa, citoma micronúcleo com bloqueio da citocinese, crescimento de neuritos e análise da expressão do gene Tp53. Os resultados obtidos na avaliação da citotoxicidade da coenzima Q10 (0,1 - 20 µg/mL) mostraram que este antioxidante foi citotóxico às células PC12 na concentração de 20,0 µg/mL e não apresentou citotoxicidade em baixas concentrações. Para os ensaios do citoma e cometa, foram selecionadas três concentrações não citotóxicas de coenzima Q10 (0,1; 0,5 e 1,0 µg/mL) que não apresentaram mutagenicidade e genotoxicidade às células PC12. O efeito protetor da coenzima Q10 sobre a cisplatina no ensaio do citoma foi caracterizado pela diminuição da freqüência de micronúcleos e brotos nucleares, entretanto a proteção da coenzima Q10 não foi evidenciada no ensaio cometa. Alterações significativas na expressão do gene Tp53 não foram observadas no tratamento coenzima Q10 (1,0 µg/mL) associado à cisplatina (0,1 µg/mL). A coenzima Q10 (0,1 e 1,0 µg/mL) não foi neurotóxica em células PC12 indiferenciadas e diferenciadas após exposição ao fator de crescimento do nervo, e seu melhor efeito neuroprotetor foi observado na menor concentração avaliada. A coenzima Q10 reduziu a citotoxicidade da cisplatina (10,0 µg/mL) em células PC12 indiferenciadas e estimulou o crescimento de neuritos em células PC12 diferenciadas. A determinação dos efeitos citoprotetores da coenzima Q10 em um modelo neuronal é importante para elucidar possíveis estratégias de neuroproteção que poderiam ser aplicadas aos pacientes submetidos à quimioterapia. / Coenzyme Q10 is a liposoluble provitamin endogenously synthesized and naturally found in various foods items, such as meat, fish, cereals, broccoli and spinach. It is a dietary supplement in some countries and used in cosmetic formulations. Coenzyme Q10 is located in the membrane of cellular organelles such as endoplasmic reticulum, vesicles and inner mitochondrial membrane, where acts as an essential cofactor in the respiratory chain. It has antioxidant properties and potential in the treatment of neurodegenerative and neuromuscular diseases. The objective of this study was to investigate the possible protective effects of a water-soluble formulation of coenzyme Q10 in PC12 cells exposed to cisplatin, an anticancer drug that has neurotoxicity as a dose-limiting factor. The PC12 cell line (rat pheocromocytoma) used in this investigation is a recognized in vitro model for neuronal studies. The methods used were the MTT, comet, cytokinesis-block micronucleus cytome, neurite outgrowth assays and expression of Tp53 gene. The results obtained in the cytotoxicity of coenzyme Q10 (0.1-20 µg/mL) showed that this antioxidant was cytotoxic to PC12 cell at a concentration of 20.0 µg/mL and it was not cytotoxic at low concentrations. For the cytome and comet assays, were selected three non-cytotoxic concentrations of coenzyme Q10 (0.1, 0.5 and 1.0 µg/mL) without mutagenicity and genotoxicity PC12 cells. The protective effect of coenzyme Q10 in cytome assay was characterized by decreased frequency of micronuclei and nuclear buds induced by cisplatin, however the protection of coenzyme Q10 was not evidenced by the comet assay. No significant change in the Tp53 gene expression were observed in the coenzyme Q10 (1.0 µg/mL) plus cisplatin (0.1 µg/mL) treatment. Coenzyme Q10 (0.1 and 1.0 µg/mL) was not neurotoxic in undifferentiated and nerve growth factor differentiated PC12 cells and the lowest concentration evaluated showed the best neuroprotective effect. The coenzyme Q10 treatment reduced the citotoxicity of cisplatin (10.0 µg/mL) in undifferentiated PC12 cells and stimulated the neurite outgrowth in differentiated PC12 cells. Determination of the cytoprotective effects of the coenzyme Q10 in a neuronal model is important to elucidate possible strategies for neuroprotection that could be applied to patients undergoing chemotherapy.

Peptide CART (cocaine- and amphetamine- regulated transcript) is a neuropeptide acting in the hypothalamus to reduce food intake (anorexigenic peptide). Despite all efforts the receptor and the mechanism of action is still unknown. This peptide has two biologically active forms, CART(55-102) and CART(61-102). Peptide CART is able to bind to pheochromocytoma cells PC12. PC12 cells differentiated in neuronal phenotype with NGF (nerve growth factor) showed a higher number of binding sites (11250 ± 2520 binding sites/cell) compared to undifferentiated cells (3600 ± 570 binding sites/cell). PC12 cells differentiated by dexamethasone to chromaffin cells showed high non-specific binding. Peptide CART contains three disulfide bridges. To clarify the importance of each disulfide bridge to maintain biological activity, analogues with one (analogue 3, 4 and 5) or two (2, 6, 7 and 8) disulfide bridges and a peptide analogue of CART (61-102), which has methionin at position 67 replaced with norleucine were synthesized. We showed that biological activity was unchanged at analogue 1 and analogue 7 containing disulfide bridges in positions 74-94 and 88-101. When investigating cell signaling in PC12 cells, we tested if peptide CART activate of c-Fos, c-Jun, phosphorylated ERK1/2, CREB, JNK and p38. CART peptide...

Injuries to the central nervous system (CNS) have traumatic consequences such as irreparable disability due to the inability of the CNS to regenerate injured nerve fibres. The aim of the work presented here was to develop a scaffold which potentially provides guidance to axons in the injured spinal cord thus facilitating signal transduction. A poly-(lactic-co-glycolic acid) (PLGA, PLA:PGA ratio of 75:25) flat sheet membrane scaffold was created using phase inversion with N-methyl pyrrolidinone (NMP) as the solvent and water as the non-solvent for immersion precipitation. PLGA flat sheet membranes were exposed to surface treatments including aminolysis, peptide immobilisation and ozonation in order to achieve higher cell attachment of PC12 cells, a cell line which was cloned from a solid pheochromocytoma tumour of white rats, and used as a tool for measurement of regeneration. Cell attachment studies revealed no significant difference in cell attachment between modified and not-modified PLGA flat sheet membranes. However, the absence of foetal calf serum (FCS) resulted in fivefold higher cell attachment compared to medium supplemented with 10% FCS. A second scaffold was produced by electrospinning 10% (w/w) PLGA in a chloroform:methanol (CHCl3:MeOH) mixture in ratio of 3:1 resulting in a nanofibrous scaffold. Optimum settings for electrospinning were found to be 3 ml/h feeding rate, 15kV applied voltage and 11cm collector-to-needle distance. Random and aligned PLGA nanofibres were produced, with a fibre diameter of 530±140nm. PC12 cells attached and differentiated to the nanofibrous scaffold. When exposed to NGF these cells stopped dividing and extended neurites. On random fibres, neurite orientation was random, whereas on aligned fibres 63% of neurites grew with the fibre orientation ±15��ᵒ. After 7 days of exposure to NGF, cells had 1-4 neurites on random fibres, reaching a maximum length of 188μm, whereas on aligned fibres, cells had 1-2 neurites, reaching a maximum length of 400μm. PLGA nanofibres were also investigated as a delivery vehicle for bioactive molecules. For this, poly-L-lysine (PLL) was incorporated into electrospun PLGA nanofibres via emulsion electrospinning. PLGA-PLL nanofibres were significantly larger than PLGA nanofibres having a diameter of 830±190nm. In order to visualise the incorporation of PLL, FITC-PLL was electrospun und the resulting nanofibres fluoresced greed. Attachment of PC12s to PLGA-PLL nanofibres was not significantly different compared to PLGA nanofibres. Aligned PLGA-PLL nanofibres were shown to promote neurite outgrowth of PC12s with resulting neurites of up to twice the length compared to aligned PLGA nanofibres. The results suggest that PLGA nanofibres strongly influences neurite organisation, which is potentially useful for future therapeutic approaches. The work in this thesis has shown that electrospun PLGA nanofibre mats have the potential to be used as scaffolds for spinal cord repair addressing topographical guidance and delivery of bioactive molecules to the site of injury.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.