BACKGROUND: Alopecia causes widespread psychological distress, but is relatively poorly controlled. The development of new treatments is hampered by the lack of suitable human hair follicle models. Although intermediate and vellus hair follicles are the main clinical targets for pharmacological therapy, terminal hair follicles are more frequently studied as smaller hair follicles are more difficult to obtain. OBJECTIVES: This investigation was designed to quantify in vivo morphological and in vitro behavioural differences in organ culture between matched intermediate and terminal hair follicles, in order to develop a new clinically relevant model system. METHODS: Microdissected terminal and intermediate hair follicles, from the same individuals, were analysed morphometrically (250 follicles; five individuals), or observed and measured over 9 days of organ culture (210 follicles; six individuals). RESULTS: Intermediate hair follicles were less pigmented and smaller, penetrating less below the skin surface (mean +/- SEM) (2.59 +/- 0.07 vs. 3.52 +/- 0.10 mm; P = 0.02), with smaller fibre (0.03 +/- 0.002 vs. 0.07 +/- 0.002 mm), connective tissue sheath (0.24 +/- 0.01 mm vs. 0.33 +/- 0.01 mm), bulb (0.19 +/- 0.01 vs. 0.31 +/- 0.01 mm) and dermal papilla (0.06 +/- 0.002 vs. 0.12 +/- 0.01 mm) diameters (P < 0.001). Intermediate hair follicle bulbs appeared 'tubular', unlike their 'bulbous' terminal follicle counterparts. In organ culture they also grew more slowly (0.044 +/- 0.002 vs. 0.067 +/- 0.003 mm per day; P < 0.001), remained in anagen longer (84 +/- 0.03% vs. 74 +/- 0.03% at day 9; P = 0.012) and produced less hair fibre (0.36 +/- 0.02 vs. 0.50 +/- 0.03 mm; P < 0.001) than terminal follicles. CONCLUSIONS: Smaller intermediate hair follicles showed major morphological differences from terminal follicles in vivo and retained significant, biologically relevant differences in vitro in organ culture. Therefore, intermediate hair follicles offer a novel, exciting, more clinically relevant, albeit technically difficult, model for future investigations into hair growth. This should be particularly important for developing new therapies.

A via AKT/PI3K apresenta-se geralmente alterada nos diversos tipos de cânceres humanos e a alteração dos componentes desta via ocorre através da ativação de oncogenes ou inativação de genes supressores tumorais levando a transformações celulares que podem promover a tumorigênese. No câncer de mama a via AKT/PI3K pode ser ativada por Erb-B2, receptores dos fatores de crescimento de insulina (IGF), receptores de estrógeno e perda da expressão do gene PTEN. mTOR (proteína alvo da rapamicina em mamíferos) é uma serina treonina quinase, membro da via AKT/PI3K que se encontra envolvida em múltiplas funções biológicas como controle da tradução, transcrição, degradação protéica e biogênese ribossomal. A ativação desta proteína resulta na fosforilação e ativação de seus principais substratos 4EBP1 e S6K1, requeridos para a biossíntese ribossomal e tradução de RNAms importantes para controle e progressão no ciclo celular. A rapamicina é uma droga com propriedades fungicidas, imunossupressoras e anticancerígenas que atua na inibição de mTOR afetando a expressão de genes envolvidos no metabolismo e síntese protéica. No nosso estudo avaliamos os elementos da via do AKT através de análise imunoistoquímica em fatias de tumores mantidos em cultura de órgão antes e depois do tratamento com rapamicina. A cultura de órgão mantém uma interação entre o epitélio mamário e estroma podendo-se preservar o microambiente que reconstitui o comportamento da célula tumoral. Nesta análise imunoistoquímica observamos uma diminuição significativa de 4EBP1 nas fatias dos tumores tratados com rapamicina em relação aos casos controles. Além disso, fizemos uma avaliação da mudança no perfil da expressão gênica nestas fatias tumorais sub-divididas em Erb-B2 positivos e negativos através da análise por microarray e observamos que a maioria dos genes afetados estavam envolvidos com as funções de transcrição e tradução celulares. Para confirmarmos os resultados obtidos por microarray fizemos uma análise por RT-PCR dos genes WWOX, EXT1 e GTF2E2 em amostras independentes e escolhidos aleatoriamente, conseguindo validá-los em 60% dos casos. Conclusão: A cultura de órgão representa um método simples para determinação dos efeitos da rapamicina. Utilizamos uma estratégia de análise do perfil gênico e novas proteínas que poderiam servir como possíveis marcadores de resposta aos inibidores da proteína mTOR foram identificadas / The AKT/ PI3K pathway are frequently disturbed in many human cancers and the alteration of the components of this pathway occurs through activation of oncogenes or inactivation of tumor suppresors leading to cellular transformation that can promove tumorigenesis. In breast cancer the AKT/PI3K pathway can be activated by ERb-B2, the insulin like growth factor (IGF), estrogen receptors and PTEN loss. mTOR (mammalian target of rapamycin) is a serine threonine kinase, member of the AKT/PI3K pathway, which is involved in multiple biologic functions such as transcription, translation, protein degradation and ribosome biogenesis. The activation of this protein results in phosphorilation and activation of S6K1 and 4EBP1, two downstream signaling elements that are required for ribosomal biosynthesis and mRNAs translation, which is important for cell cycle control and progression. Rapamycin is a potent fungicide, immunossupressive and anticancer agent that inhibits mTOR affecting the expression of genes involved in metabolism and protein synthesis. In the present study we examined some elements of AKT pathway by immunohistochemistry analysis in samples of breast cancer mantained in organ culture before and after treatment with rapamycin. The organ culture maintain an interaction between the mammary epithelium and stroma preserving the micro-environment and restoring the tumor cell behavior. In this immunohistochemistry analysis we noticed a significative decrease of 4EBP1 in the samples of tumors treated with rapamycin compared with the control cases. Besides this, we determined the variation of gene expression profile through microarray analysis in these samples subdivided in positive and negative Erb-B2 and we have identified that most part of the affected genes were mainly involved in cellular transcription and translation.To confirm the results obtained through microarray technique, we have performed the RT-PCR analysis of WWOX, EXT1, GTF2E2 genes and we were able to validate them in 60% of our cases. Conclusion: The organ culture represents a simple method to determine the effects of rapamycin. Using a strategic analysis of the gene profile, news proteins that possibly could be used as markers to the mTOR inhibitors were identifyied

A via AKT/PI3K apresenta-se geralmente alterada nos diversos tipos de cânceres humanos e a alteração dos componentes desta via ocorre através da ativação de oncogenes ou inativação de genes supressores tumorais levando a transformações celulares que podem promover a tumorigênese. No câncer de mama a via AKT/PI3K pode ser ativada por Erb-B2, receptores dos fatores de crescimento de insulina (IGF), receptores de estrógeno e perda da expressão do gene PTEN. mTOR (proteína alvo da rapamicina em mamíferos) é uma serina treonina quinase, membro da via AKT/PI3K que se encontra envolvida em múltiplas funções biológicas como controle da tradução, transcrição, degradação protéica e biogênese ribossomal. A ativação desta proteína resulta na fosforilação e ativação de seus principais substratos 4EBP1 e S6K1, requeridos para a biossíntese ribossomal e tradução de RNAms importantes para controle e progressão no ciclo celular. A rapamicina é uma droga com propriedades fungicidas, imunossupressoras e anticancerígenas que atua na inibição de mTOR afetando a expressão de genes envolvidos no metabolismo e síntese protéica. No nosso estudo avaliamos os elementos da via do AKT através de análise imunoistoquímica em fatias de tumores mantidos em cultura de órgão antes e depois do tratamento com rapamicina. A cultura de órgão mantém uma interação entre o epitélio mamário e estroma podendo-se preservar o microambiente que reconstitui o comportamento da célula tumoral. Nesta análise imunoistoquímica observamos uma diminuição significativa de 4EBP1 nas fatias dos tumores tratados com rapamicina em relação aos casos controles. Além disso, fizemos uma avaliação da mudança no perfil da expressão gênica nestas fatias tumorais sub-divididas em Erb-B2 positivos e negativos através da análise por microarray e observamos que a maioria dos genes afetados estavam envolvidos com as funções de transcrição e tradução celulares. Para confirmarmos os resultados obtidos por microarray fizemos uma análise por RT-PCR dos genes WWOX, EXT1 e GTF2E2 em amostras independentes e escolhidos aleatoriamente, conseguindo validá-los em 60% dos casos. Conclusão: A cultura de órgão representa um método simples para determinação dos efeitos da rapamicina. Utilizamos uma estratégia de análise do perfil gênico e novas proteínas que poderiam servir como possíveis marcadores de resposta aos inibidores da proteína mTOR foram identificadas / The AKT/ PI3K pathway are frequently disturbed in many human cancers and the alteration of the components of this pathway occurs through activation of oncogenes or inactivation of tumor suppresors leading to cellular transformation that can promove tumorigenesis. In breast cancer the AKT/PI3K pathway can be activated by ERb-B2, the insulin like growth factor (IGF), estrogen receptors and PTEN loss. mTOR (mammalian target of rapamycin) is a serine threonine kinase, member of the AKT/PI3K pathway, which is involved in multiple biologic functions such as transcription, translation, protein degradation and ribosome biogenesis. The activation of this protein results in phosphorilation and activation of S6K1 and 4EBP1, two downstream signaling elements that are required for ribosomal biosynthesis and mRNAs translation, which is important for cell cycle control and progression. Rapamycin is a potent fungicide, immunossupressive and anticancer agent that inhibits mTOR affecting the expression of genes involved in metabolism and protein synthesis. In the present study we examined some elements of AKT pathway by immunohistochemistry analysis in samples of breast cancer mantained in organ culture before and after treatment with rapamycin. The organ culture maintain an interaction between the mammary epithelium and stroma preserving the micro-environment and restoring the tumor cell behavior. In this immunohistochemistry analysis we noticed a significative decrease of 4EBP1 in the samples of tumors treated with rapamycin compared with the control cases. Besides this, we determined the variation of gene expression profile through microarray analysis in these samples subdivided in positive and negative Erb-B2 and we have identified that most part of the affected genes were mainly involved in cellular transcription and translation.To confirm the results obtained through microarray technique, we have performed the RT-PCR analysis of WWOX, EXT1, GTF2E2 genes and we were able to validate them in 60% of our cases. Conclusion: The organ culture represents a simple method to determine the effects of rapamycin. Using a strategic analysis of the gene profile, news proteins that possibly could be used as markers to the mTOR inhibitors were identifyied

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

No / Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown whether phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. We found that protein kinase C-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both of these events are necessary for the internalization of GluK2-containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation led to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Our results suggest a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.

Hair disorders cause psychological distress but are generally poorly controlled; more effective treatments are required. Despite the long-standing use of minoxidil for balding, its mechanism is unclear; suggestions include action on vasculature or follicle cells. Similar drugs also stimulate hair, implicating ATP-sensitive potassium (K(ATP)) channels. To investigate whether K(ATP) channels are present in human follicles, we used organ culture, molecular biological, and immunohistological approaches. Minoxidil and tolbutamide, a K(ATP) channel blocker, opposed each other's effects on the growing phase (anagen) of scalp follicles cultured in media with and without insulin. Reverse transcriptase-polymerase chain reaction identified K(ATP) channel component gene expression including regulatory sulfonylurea receptors (SUR) SUR1 and SUR2B but not SUR2A and pore-forming subunits (Kir) Kir6.1 and Kir6.2. When hair bulb tissues were examined separately, epithelial matrix expressed SUR1 and Kir6.2, whereas both dermal papilla and sheath exhibited SUR2B and Kir6.1. Immunohistochemistry demonstrated similar protein distributions. Thus, human follicles respond biologically to K(ATP) channel regulators in culture and express genes and proteins for two K(ATP) channels, Kir6.2/SUR1 and Kir6.1/SUR2B; minoxidil only stimulates SUR2 channels. These findings indicate that human follicular dermal papillae contain K(ATP) channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 164 pages. Includes vita. Includes bibliographical references.

Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F(2alpha)-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F(2alpha) analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.