The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and β-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of β-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that β-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of β-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of β-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Made available in DSpace on 2016-05-02T13:55:49Z (GMT). No. of bitstreams: 1 TelcioParreiraMinare- dissertacao.pdf: 1033418 bytes, checksum: 5214b443feda2d64e44b38ae6d5613b4 (MD5) Previous issue date: 2013-05-16 / Most of the previous studies aiming to improve superovulation evaluated only differences in the number of corpora lutea (CL) observed and of embryos recovered. Therefore, differences in total follicular population before hormonal stimulation and in response parameters associated to follicle growth were frequently neglected, causing bias in the conclusion. The aim of the present study was to characterize individual variation in both relative and absolute efficiency of superovulation, based on ovarian follicular population. Non-lactating Gir breed cows (n=2) and heifers (n=15), kept under the same management, were used. Four days before the start of superovulatory treatment, follicular growth was synchronized by the insertion of an intravaginal device of progesterone and estradiol benzoate application. Superovulations started on D1 with the injection of 200 UI FSHp, following a conventional protocol. The number and diameter of the follicles present before (D0) and during superovulation (D5 to D8), as well as the number of CL at flushing (D12), were evaluated by ultrasonography. Follicular population was ranked according to size (&#8804;4 mm, 5-7 mm, &#8805;8 mm), and changes in the percentage of follicles in each size class were used to calculate relative efficiency. The absolute efficiency was determined by the ratio number of embryos recovered : number of follicles &#8804;4 mm on D5. Results are shown as mean±SEM. As expected, there was a great individual variation in the superovulation outcomes, both considering the number of CL (0 to 28, mean 12.6±2.1; CV=68.2%) and embryos collected (0 to 15, mean 5.1±1.1; CV=92.4%). There was no increase (P>0.05) in total follicular population during treatment, and the correlation between the number of follicles during superovulation and the further number of CL or embryos remained relatively constant between D5 and D8 (r=0.56 to 0.65 and r=0.70 to 0.79, respectively; P<0.01). FSH treatment induced a progressive (P<0.05) but partial mobilization of small follicles to larger size classes. The relative efficiency of the follicle growth stimulation was 41.9±5.5% (0 to 75.6%), and this was the endpoint with the largest correlation (R=0.80; P<0.0001) with the absolute efficiency of the process (12.2±2.1%, ranging from 0.0 to 25.0%). Retrospective analysis demonstrated that donors with relative efficiency >50% had a number of follicles &#8804;4 mm on D1 similar to those with efficiency <50% (41.6±6.8 vs 42.1±3.1; P<0.001), but produced more CL and embryos (17.8±2.5 and 7.6±1.7 vs 6.9±2.1 and 2.4±0.9, respectively; P<0.001). In conclusion, individual differences in follicular population and in the follicle response to FSH are important sources of variation in superovulation results, and shall be taken into account for experimental design. Acknowledgement: FAPEMIG, CAPES, Biotran LTDA. / A maioria dos estudos que objetiva otimizar a técnica de superovulação em bovinos considera como parâmetros de avaliação apenas o número de corpos lúteos (CL) formados e de embriões recuperados. Desse modo, variações na população folicular no início do tratamento e parâmetros de resposta relacionados ao crescimento folicular são frequentemente desconsiderados, gerando vieses de interpretação. O objetivo deste trabalho foi caracterizar as variações individuais na eficiência relativa e absoluta da superovulação, com base na população folicular ovariana. Foram utilizadas vacas não lactantes (n=2) e novilhas (n=15) da raça Gir, mantidas em condições semelhantes de manejo e alimentação. Quatro dias antes do início do tratamento superovulatório, o crescimento folicular foi sincronizado pela inserção de um dispositivo intravaginal de progesterona e aplicação de benzoato de estradiol. As superovulações foram iniciadas no D1 pela administração de 200 UI de FSHp, segundo protocolo convencional. O número e o tamanho dos folículos presentes no dia anterior ao início (D0) e durante a superovulação (D1 a D4), assim como o número de CL presentes no dia da coleta (D12), foram avaliados por ultrassonografia. A população folicular foi classificada em função do tamanho (&#8804;4 mm, 5-7 mm, &#8805;8 mm), e nas variações do percentual de folículos entre as classes de tamanho utilizadas para cálculo de eficiência relativa. A eficiência absoluta foi calculada pela razão entre o número de embriões recuperados e de folículos &#8804;4 mm presentes no D1. Os resultados são apresentados como média±EPM. Como esperado, observou-se uma grande variação individual de resposta, tanto considerando o número de CL formados (0 a 28, média de 12,6±2,1; CV=68,2%) quanto de embriões recuperados (0 a 15, média de 5,1±1,1; CV=92,4%). Não houve aumento (P>0,05) no número total de folículos presentes ao longo do tratamento, e a correlação entre população folicular total e o número de CL ou de embriões permaneceu relativamente constante entre o D1 e o D4 (r=0,56 a 0,65 e r=0,70 a 0,79, respectivamente; P<0,01). O tratamento superovulatório resultou em uma mobilização progressiva (P<0,05), porém parcial, da população de folículos pequenos para classes de tamanho superiores. A eficiência relativa do tratamento estimulatório foi de 41,9±5,5% (0 a 75,6%), e este foi o indicador parcial com maior correlação (R=0,80; P<0,0001) com a eficiência absoluta do processo (12,2±2,1%, variando de 0,0 a 25,0%). Em análise retrospectiva, demonstrou-se que doadoras com eficiência relativa acima de 50% apresentavam um número de folículos &#8804;4 mm no D1 semelhante àquelas com eficiência abaixo de 50% (41,6±6,8 vs. 42,1±3,1; P<0,001), mas produziram mais CL e embriões (17,8±2,5 e 7,6±1,7 vs. 6,9±2,1 e 2,4±0,9, respectivamente; P<0,001). Conclui-se que diferenças individuais na população folicular inicial e na resposta ao FSH são importantes fontes de variação na resposta superovulatória, e devem ser consideradas nos delineamentos experimentais. Agradecimentos: FAPEMIG, CAPES, Biotran LTDA.