Cheng Fu Yip Gheorghe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 97-116). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract (in English) --- p.III / Abstract (in Chinese) --- p.V / Table of Content --- p.VII / Symbols and Abbreviations --- p.XII / Scientific Names --- p.XIV / List of Tables --- p.XV / List of Figures --- p.XVI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropin / Chapter 1.1.1 --- Structure --- p.2 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.4 / Chapter 1.1.3.1 --- Neuroendocrine and endocrine regulation of GTHs --- p.4 / Chapter 1.1.3.1.1 --- Hypothalamic neuropeptides and neurotransmitters --- p.6 / Chapter 1.1.3.1.2 --- Gonadal steroids --- p.7 / Chapter 1.1.3.2 --- Paracrine regulation of GTH --- p.8 / Chapter 1.2 --- Activin / Chapter 1.2.1 --- Structure --- p.8 / Chapter 1.2.2 --- Function --- p.9 / Chapter 1.2.3 --- Regulation of activin activity --- p.12 / Chapter 1.2.3.1 --- Intracellular blockade of activin signaling by Smad7 --- p.12 / Chapter 1.2.3.2 --- Extracellular control of activin access --- p.13 / Chapter 1.2.3.2.1 --- Inhibin --- p.13 / Chapter 1.2.3.2.2 --- Activin-binding protein --- p.14 / Chapter 1.3 --- Follistatin / Chapter 1.3.1 --- Structure --- p.14 / Chapter 1.3.2 --- Function --- p.16 / Chapter 1.3.3 --- Regulation in the pituitary --- p.19 / Chapter 1.4 --- Objectives of the Present Study --- p.20 / Chapter Chapter 2 --- Cloning and Recombinant Production of Goldfish Follistatin / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Reagents --- p.26 / Chapter 2.2.2 --- Animal --- p.26 / Chapter 2.2.3 --- Extraction of total RNA and reverse transcription --- p.27 / Chapter 2.2.4 --- Cloning of full-length cDNA encoding goldfish follistatin --- p.27 / Chapter 2.2.5 --- Sequencing of the cDNA --- p.29 / Chapter 2.2.6 --- Distribution of follistatin mRNA in different tissues --- p.29 / Chapter 2.2.7 --- Production of rgFS --- p.30 / Chapter 2.2.8 --- RT-PCR of the rgFS-positive clones --- p.34 / Chapter 2.2.9 --- Extraction of genomic DNA from rgFS-positive clones --- p.34 / Chapter 2.2.10 --- Functional analysis of rgFS --- p.35 / Chapter 2.2.11 --- Data Analysis --- p.37 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Cloning and sequence analysis of goldfish follistatin --- p.37 / Chapter 2.3.2 --- Tissue distribution of follistatin mRNA in the goldfish --- p.39 / Chapter 2.3.3 --- Production and bioassay of rgFS --- p.43 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Function and Regulation of Follistatin in the Goldfish Pituitary; Evidence for an Intrinsic Activin/Follistatin Regulatory Feedback Loop / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Reagents --- p.57 / Chapter 3.2.2 --- Animals --- p.57 / Chapter 3.2.3 --- Primary culture of dispersed pituitary cells --- p.57 / Chapter 3.2.4 --- RNA extraction and reverse transcription --- p.58 / Chapter 3.2.5 --- Ovariectomy on pituitary follistatin expression --- p.5 9 / Chapter 3.2.6 --- Seasonal expression profile of follistatin --- p.59 / Chapter 3.2.7 --- Validation of semi-quantitative RT-PCR assays --- p.61 / Chapter 3.2.8 --- Real-time PCR for assay on follistatin and β-actin expression --- p.61 / Chapter 3.2.9 --- Data analysis --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Expression of follistatin in the goldfish pituitary --- p.64 / Chapter 3.3.2 --- Validation of semi-quantitative RT-PCR assay --- p.64 / Chapter 3.3.3 --- Activin regulation of pituitary follistatin --- p.64 / Chapter 3.3.4 --- Effects of sex steroids on pituitary follistatin expression --- p.69 / Chapter 3.3.5 --- Effect of GnRH on follistatin expression in the pituitary --- p.74 / Chapter 3.3.6 --- Effect of intracellular cAMP level on pituitary follistatin expression --- p.74 / Chapter 3.3.7 --- Seasonal variation profile of goldfish pituitary follistatin --- p.78 / Chapter 3.4 --- Discussion --- p.78 / Chapter Chapter 4 --- General Discussion / Chapter 4.1 --- Overview --- p.89 / Chapter 4.2 --- Contribution of the Present Study / Chapter 4.2.1 --- Cloning of full-length goldfish follistatin cDNA --- p.91 / Chapter 4.2.2 --- Establishment of stable cell line for expression of rgFS --- p.92 / Chapter 4.2.3 --- Evidence for the presence of intrinsic feedback loop of activin in the goldfish pituitary --- p.92 / Chapter 4.2.4 --- Modulation of follistatin expression in the pituitary by sex steroids --- p.93 / Chapter 4.2.5 --- Conclusions --- p.93 / Chapter 4.3 --- Future Prospects / Chapter 4.3.1 --- Production of rgFS --- p.95 / Chapter 4.3.2 --- Regulation of activin-follistatin system in the pituitary --- p.95 / Reference --- p.96

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The aim of this study was to investigate the powerful muscle regulator, myostatin, and its regulators in response to an acute bout of plyometric training. The participants were recruited and screened by characterization by means of isometric force production tests, baseline blood creatine kinase levels and VO2 max results. The selected individuals (n=15) were subjected to a baseline muscle biopsy for comparative purposes. The study made use of plyometric jumping, as source of eccentric exercise, to serve as an exercise intervention after which muscle biopsies (4 hours post and 24 hours post) and blood draw (4 hours post, 24 hours post and 48 hours post) samples were taken. Maximal voluntary isometric contractions of the knee extensors were also measured immediately after the exercise protocol and after 1 week recovery. Creatine kinase (CK) analysis on the serum samples was used to conclude muscle damage. The muscle biopsy samples were used for protein quantification (Western blot) and gene expression assessment (semi-quantitative and real-time PCR). The results showed decreased force production immediately after eccentric exercise (p < 0.05), while returning back to baseline values at 1 week post exercise and CK results showed a significant increases at 4 hours (p<0.05), 24 hours (p<0.001) and 48 hours (p<0.01) after exercise. There were no significant differences in myostatin precursor protein (43 kDa), phosphorylated Smad2,3, Smad7 or activin receptor IIb in response to eccentric exercise. However, the follistatin protein was increased at both 4 hours and 24 hours after exercise (p<0.01). RNA analysis of the extracellular matrix (ECM) protein, decorin, revealed the existence of the splice variants A1 and A2 in human skeletal muscle. The RT-PCR analysis (n=4) of these variants showed no significant difference when comparing pre- to post-exercise. The decorin core protein was also investigated by means of antibody probing and results revealed the need for ABC chondroitinase enzyme treatment before immunoblotting of human skeletal muscle samples. The results concerning knee extensor force reduction and circulating creatine kinase showed the effectiveness of plyometric jumping in producing skeletal muscle damage in the lower limbs of unfit individuals, unaccustomed to eccentric exercise. In conclusion, myostatin, and its associated signalling cascade, are not activated in early muscle regeneration, but follistatin is increased during this phase possibly aiding and initiating the muscle repair process. Future studies: Variants of decorin are expressed in human skeletal muscle, increasing the complexity that should be taken into account in studies concerning the regulation of decorin in a human model. Investigation into myostatin protein at different post-translational levels needs more clarification. Published methods and materials used in different laboratories are not consistent and investigators should attempt to standardise protocols in order to compare results between studies more effectively. Of importance, these results show that the myostatin at protein level report different results compared to mRNA analysis and that more investigation into myostatin regulatory factors, with special reference to follistatin and decorin, is needed in future human models. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die kragtige spiere reguleerder, miostatin, en sy reguleerders in reaksie op 'n akute aanval van pliometriese spronge te ondersoek. Die deelnemers is gewerf en gekeur deur karakterisering deur middel van isometriese krag produksie toetse, basislyn bloed kreatien kinase vlakke en VO2maks resultate. Die geselekteerde individue (N = 15) is onderhewig aan 'n basislyn spierbiopsie vir vergelykende doeleindes. Die studie het gebruik gemaak van pliometriese spronge (essentriese spier aksie) as die oefening intervensie waarna spierbiopsie (4 uur na en 24 uur na) en bloed (4 uur na, 24 uur na en 48 uur na) monsters geneem is. Isometriese kontraksies van die knieverlengers is ook gemeet onmiddellik na die oefening protokol en na 1 week se herstel. Kreatine kinase (KK) ontleding van die serum monsters is gebruik om spierskade aftelei. Die spierbiopsie monsters was gebruik vir proteïen kwantifisering (Western klad) en die assessering van geen uitdrukking (semi-kwantitatiewe en real-time PCR). Die resultate het gewys dat krag produksie afgeneem het onmiddellik na essentriese oefening (p <0.05), terwyl dit terugkeer na die oorspronklike waardes 1 week na oefening en KK resultate toon 'n beduidende toename by 4 uur (p <0,05), 24 uur (p <0,001) en 48 uur (p <0,01) na oefening. Daar was geen betekenisvolle verskille in Miostatien voorloper proteïen (43 kDa), gefosforileerde Smad2,3, Smad7 of Activin reseptoor IIb in reaksie op essentriese oefening. Dit is egter die follistatien proteïen wat verhoog by beide 4 uur en 24 uur na oefening (p <0,01). RNS ontleding van die ekstrasellulêre matriks (ESM) proteïen, decorin, het die bestaan van die splitsing variante A1 en A2 in menslike skeletspier, aan die lig gebring. Die RT-PCR analise (n = 4) van hierdie variante het geen betekenisvolle verskille getoon wanneer voor met na-oefening vergelyk is. Die decorin kern proteïen is ook ondersoek deur middel van teenliggaam afhanklike metodes en resultate het die behoefte aan ABC chondroitinase ensiem behandeling voor immunokladding van menslike skeletspier monsters gesteun. Die resultate aangaande knieverlenger krag vermindering en sirkuleerende kreatien kinase het die doeltreffendheid van pliometriese spronge in die vervaardiging van skeletspier skade in die onderste ledemate van individue ongewoond aan essentriese oefening verseker. Ten slotte, Miostatien, en sy verwante sein kaskade, is nie geaktiveer vroeg in spier herstelling, maar follistatien is tydens hierdie fase verhoog en help moontlik met die aanvang van die spier herstel. Toekomstige studies: variante van decorin word uitgedruk in menslike skeletspier, wat die kompleksiteit aangaande decorin verhoog en dit is iets wat in ag geneem moet word in studies wat handel oor die regulering van decorin in mens modelle. Ondersoek na miostatien proteïen op verskillende na-translasie vlakke moet meer duidelikheid verkry. Gepubliseer metodes en materiaal wat gebruik word in verskillende laboratoriums is nie konsekwent en ondersoekbeamptes moet probeer om protokolle te standaardiseer sodat resultate van studies meer effektief kan vergelyk word. Van belang is, die resultate wys dat miostatien op proteïen vlak verskillende resultate vertoon in vergelyking met boodskapper-RNS ontleding en dat meer ondersoek na miostatien regulerende faktore, met spesiale verwysing na follistatien en decorin, nodig is in toekomstige menslike modelle.

Nosso objetivo foi investigar o efeito da suplementação de leucina na via intracelular acionada por miostatina, uma proteína reguladora negativa da massa muscular, em um modelo de atrofia gerada por imobilização em ratos. Nossa hipótese inicial contemplava que a expressão de miostatina se elevaria com a imobilização e que a leucina poderia atenuar esse aumento. Nossos resultados, entretanto, mostraram uma regulação gênica no sentido de suprimir a via da miostatina. A suplementação com leucina, associada à imobilização, também provocou queda da expressão gênica de miostatina. Em ambos os grupos a expressão proteica permaneceu inalterada. Em contrapartida, a folistatina, inibidor endógeno da miostatina, apresentou acentuado aumento de expressão no grupo imobilizado e imobilizado/suplementados com leucina. Nossos resultados mostraram que o efeito protetor da leucina no modelo atrófico utilizado não se dá através da sinalização da miostatina. Os dados também sugerem uma resposta biológica coordenada entre o ligante, miostatina, e seu inibidor endógeno, a folistatina. / Our purpose was to investigate the effect of leucine supplementation upon the intracellular pathway triggered by myostatin, a negative regulator of muscle mass, in an atrophic model driven by immobilization in rats. Our initial hypothesis contemplated that myostatin expression would be elevated under immobilization and that leucine supplementation would be able to attenuate this raise. However our results showed a strong downregulation of myostatin gene expression in atrophy induced by cast. The immobilization associated with leucine supplementation also caused mRNA myostatin downregulation. Protein levels were unchanged. On the other hand, follistatin, a myostatin endogenous inhibitor, was markedly upregulated in both immobilized and immobilized/supplemented groups. Our results revealed that leucine protective effect in this atrophic model is not through myostatin signailing. Furthermore, these data suggest a coordinated biological response between ligand, myostatin, and its endogenous inhibitor, follistatin.

Abstract not available

<p>Horizontal cells are retinal interneurons that modulate the output from photoreceptors. Two horizontal cell (HC) subtypes are commonly identified in the vertebrate retina: axon-bearing and axon-less HCs. In this work, we have identified Isl1 as a novel HC marker and demonstrated that Lim1 and Isl1 distinguish axon-bearing and axon-less HCs, respectively. In the chick retina, axon-less HCs are furthermore split into two different subtypes based on the expression of GABA and TrkA.</p><p>We have demonstrated that during early chick retinogenesis, HCs expressing either Lim1 or Isl1 are generated consecutively as two equally large sub-groups at different time points. Moreover, these newborn HCs undertake an unexpected bi-directional migration before settling in their final laminar position. Different HC subtypes complete this migration at different times.</p><p>We investigated the role of activin signaling during HC subtype generation. Activin or its inhibitor follistatin was administrated during the main phase of HC generation and analyzed when HCs had completed migration. Activin caused a significant decrease in both HC subtypes and decreased the proliferation of retinal precursor cells. Follistatin increased the number of late born (Isl1+) HCs, which migrated to the HC-layer during a prolonged migration period. Both treatments affected retinal histology, but only activin influenced the generation of retinal populations other than HCs. These effects were most likely mediated by altered proliferation in certain retinal precursor cells.</p><p>The data on HC subtype ratios, birth-dates, migration, apoptosis and extrinsic activin modulation favor a scenario where the mature proportions of HC subtypes are generated sequentially from a specific HC-precursor cell lineage early in development and remain stable thereafter. These proportions are not adjusted by apoptosis, but rather by the combined actions of transcription factors and extrinsic signaling. Our studies on HC subtypes and their development promises to facilitate future studies on HC development, evolution and function.</p>

Horizontal cells are retinal interneurons that modulate the output from photoreceptors. Two horizontal cell (HC) subtypes are commonly identified in the vertebrate retina: axon-bearing and axon-less HCs. In this work, we have identified Isl1 as a novel HC marker and demonstrated that Lim1 and Isl1 distinguish axon-bearing and axon-less HCs, respectively. In the chick retina, axon-less HCs are furthermore split into two different subtypes based on the expression of GABA and TrkA. We have demonstrated that during early chick retinogenesis, HCs expressing either Lim1 or Isl1 are generated consecutively as two equally large sub-groups at different time points. Moreover, these newborn HCs undertake an unexpected bi-directional migration before settling in their final laminar position. Different HC subtypes complete this migration at different times. We investigated the role of activin signaling during HC subtype generation. Activin or its inhibitor follistatin was administrated during the main phase of HC generation and analyzed when HCs had completed migration. Activin caused a significant decrease in both HC subtypes and decreased the proliferation of retinal precursor cells. Follistatin increased the number of late born (Isl1+) HCs, which migrated to the HC-layer during a prolonged migration period. Both treatments affected retinal histology, but only activin influenced the generation of retinal populations other than HCs. These effects were most likely mediated by altered proliferation in certain retinal precursor cells. The data on HC subtype ratios, birth-dates, migration, apoptosis and extrinsic activin modulation favor a scenario where the mature proportions of HC subtypes are generated sequentially from a specific HC-precursor cell lineage early in development and remain stable thereafter. These proportions are not adjusted by apoptosis, but rather by the combined actions of transcription factors and extrinsic signaling. Our studies on HC subtypes and their development promises to facilitate future studies on HC development, evolution and function.

We have used the chicken retina as a model for investigating cell cycle regulation and cell fate commitment during central nervous system development. This thesis focuses on the characterization of and commitment to the horizontal cell fate in the retina. Horizontal cells are interneurons that provide intraretinal signal processing prior to information relay to the brain. We have identified molecular markers that selectively distinguish the three subtypes of horizontal cells, previously described in the chicken retina based on morphology. Subtype specific birth-dating revealed that horizontal cell subtypes are generated consecutively by biased progenitors that are sensitive to the inhibitory effects of follistatin. Follistatin stimulates proliferation in progenitors by repressing the differentiation signal of activin. Initially, injection of follistatin led to a decrease in committed horizontal cells but as the inhibitory effect dissipated it resulted in an increased number of horizontal cells. During development committed horizontal cell progenitors migrate to the vitreal side of the retina where they become arrested in G2-phase for approximately two days. When the arrest is overcome the horizontal cell progenitors undergo ectopic mitosis followed by migration to their designated layer. The G2-phase arrest is not triggered or maintained by any of the classic G2-arrest pathways such as DNA damage or stress. Nevertheless, we show that the cyclin B1-Cdk1 complex has a central role in maintaining this G2-phase arrest. Two transcription factors, FoxN4 and Ptf1a, are required for the generation of horizontal cells. We show that these factors are also sufficient to promote horizontal cell fate. Overexpression of FoxN4 and Ptf1a resulted in an overproduction of horizontal- and amacrine cells at the expense of ganglion- and photoreceptor cells. We identified Atoh7, a transcription factor required for the generation of ganglion cells, as a Ptf1a transcriptional target for downregulation. Our data support a common horizontal/amacrine lineage separated from the ganglion/photoreceptor lineage by the action of Ptf1a. In conclusion, these data describe several novel characteristics of horizontal cells enhancing our understanding of neural development and cell fate commitment.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 25, 2010). Vita. Thesis advisor: Christian Lorson. "July 2009" Includes bibliographical references.

Orientador: Eliana Pereira de Araujo / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T05:27:47Z (GMT). No. of bitstreams: 1 Gomes_FabianadeSouza_M.pdf: 1469055 bytes, checksum: 01ebfd4043c839e80e30bebeeb9bef95 (MD5) Previous issue date: 2013 / Resumo: O desenvolvimento de métodos que tem por objetivo acelerar e melhorar a qualidade do processo de cicatrização de feridas tem impacto positivo na condução de distúrbios de cicatrização associados a inúmeras condições médicas. Neste estudo, avaliamos os efeitos moleculares, celulares e clínicos da aplicação tópica de 5-azacitidina na cicatrização de feridas em ratos. De acordo com estudos pregressos, a 5-azacitidina reduz a expressão de folistatina, que é um regulador negativo das ativinas. Estas, por sua vez, promovem o crescimento de células em diferentes tecidos, incluindo a pele. Ratos Wistar machos com oito semanas de vida foram submetidos a um ferimento cutâneo com punch de oito milímetros na região dorsal. A seguir os ratos foram aleatoriamente separados em grupo controle (veículo) ou submetidos à aplicação tópica de 5-azacitidina (10 mM), uma vez por dia por até 12 dias, iniciando-se no terceiro dia após a lesão. A documentação fotográfica e coleta de amostras ocorreram nos dias 5, 9 e 15. O emprego desta droga resultou em aceleração da cicatrização da ferida, (99,7±7,0% versus 71,2±2,8% no dia 15, p <0,01). Este resultado clínico foi acompanhado pela redução de aproximadamente três vezes na expressão protéica de folistatina. O exame histológico da pele revelou re-epitelização eficiente com aumento da expressão de queratinócitos e aumento significativo na expressão do gene de TGF-? além da diminuição significativa de citocinas, tais como TNF-? e IL-10. Analisamos também a proliferação celular na lesão de pele através do método de incorporação de BrdU. O número de células positivas para BrdU aumentou significativamente quando comparado ao controle. No entanto, quando folistatina exógena foi aplicada na pele em paralelo ao tratamento tópico de 5-azacitidina a maioria dos benefícios do medicamento foi perdida. Assim, 5-azacitidina atua, pelo menos em parte, através da via folistatina/ativina para melhorar a cicatrização de feridas em ratos. Este trabalho pertence à linha de pesquisa Processo de Cuidar em Saúde e Enfermagem / Abstract: The development of new methods aimed at improving wound healing may have an impact on the outcomes of a number of medical conditions. Here we evaluate the molecular and clinical effects of topical 5-azacytidine, a compound used in myelodysplasia, on the wound healing in rats. According to previous studies, 5-Azacytidine decreases the expression of follistatin 1, which is a negative regulator of activins. Activins, in turn, promote cell growth in different tissues, including the skin. Eight-week old male Wistar rats were submitted to an 8 mm punchwound in the dorsal region. After three days, rats were randomly assigned to either control or topical application of a solution containing 5-azacytidine (10mM), once a day. Photo documentation and collection of samples occurred at days 5, 9 and 15. Overall, 5-azacytidine resulted on a significant acceleration of complete wound healing (99.7% ±0.7.0 vs. 71.2%±2.8 on days 15; n=10; p<0,01). This was accompanied by an up to 3-fold reduction in follistatin expression. Histological examination of the skin revealed efficient reepithelization with increase in gene expression of TGF-? and keratinocytes markers, involucrin and citokeratin, besides the significant decrease of cytokines such as TNF-? and IL-10. In addition, we analyzed cell proliferation in injured skin employing the BrdU incorporation method. The treatment with 5-azacytidine led to a progressive increase of BrdU positive cells. Finally when recombinant follistatin was employed in the skin in parallel to topical 5-azacytidine most of the benefits of the drug were lost. Thus, 5-azacytidine acts, at least in part, through the follistatin/activin pathway to improve wound healing in rats. This study belongs to online research process Caring in Nursing and Health / Mestrado / Enfermagem e Trabalho / Mestra em Ciências da Saúde

No description available.