Ovaries maintain and produce functional female gametes, oocytes, for fertilisation. Oocytes develop inside cellular assemblies, the ovarian follicles, before birth and can reside there for up to 50 years in the human. Despite recent inroads, the precise mechanisms of initial follicle recruitment and growth remain unclear. Although the pituitary gonadotrophins play a role in this developmental process, locally produced factors have been implicated strongly in initiation of follicle growth. It is known that fibroblast growth factor 2 (FGF2) is a powerful mitogen for follicular granulosa cells in culture and initial studies undertaken in this project were successful in detecting FGF2 gene expression in ovarian biopsies from fertile healthy women. To further elucidate which cells were expressing FGF2, laser microdissection was employed to isolate differentially staged follicle populations. Real-time RT-PCR was used to quantify mRNA in relation to follicle development. Decreasing levels of FGF2 expression were detected as follicles developed. Non-radioactive in situ hybridisation confirmed FGF2 mRNA localisation in granulosa cells of preantral follicles. FGF2 protein localisation was assessed with immunohistochemistry; two primary antibodies raised against different fragments of human FGF2 were used. Both antibodies detected FGF2 in the oocyte cytoplasm of putative non-growing follicles, whereas only one of the antibodies showed additional reactivity to the basement membrane region of these same follicles. These results suggest different isoforms of FGF2 may localise specifically to different cellular sites. Follicle stimulating hormone receptor (FSHR) gene expression was also investigated in follicles using laser microdissection, real-time RT-PCR and in situ hybridisation. FSHR mRNA was detected in all follicle populations, including the smallest putative non-growing follicles. Disparity to other published works was attributed to the position of primer annealing, and thus the ability to detect alternatively spliced transcripts. In conclusion, the work presented here provides evidence that FGF2 and FSHR are present in small follicles and that their actions may be stimulatory or inhibitory to initial follicle recruitment.

Fibroblast Growth Factor (FGF) 21 is a liver-derived endocrine factor with multiple metabolic effects on glucose and lipid homeostasis in animals. The adipose tissue has been proposed as a major target of FGF21, where it enhances glucose uptake and modulates lipolysis as well as thermogenesis. However, the molecular mechanisms underlying the pleiotropic effects of FGF21 in adipocytes and the physiological roles of FGF21 in regulating energy homeostasis remain poorly characterized. Therefore, the present study aimed to investigate: 1) the signal transduction pathway whereby FGF21 enhances glucose uptake in white adipocytes; 2) the role of FGF21 in lipolysis in both mouse and human white adipose tissues (WAT) and its underlying mechanisms involved; 3) the phenotypes of FGF21 knockout (KO) mice with respect to energy expenditure and adiposity under both standard chow and high fat diet. Key findings: 1. In vitro studies demonstrated that extracellular signal-regulated kinases (ERK1/2) play an obligatory role in mediating FGF21-induced upregulation of glucose transporter-1 (GLUT1) expression and hence elevation of glucose uptake in 3T3-L1 adipocytes. 2. Chromatin immunoprecipitation assay revealed that Serum Response Factor (SRF) and ETS-like protein-1 (Elk-1), the two transcription factors which are known as the downstream targets of ERK1/2, were recruited to the endogenous GLUT1 promoter in adipocytes. A conserved binding motif for these two transcription factors was also identified in the GLUT1 promoter responsive to FGF21 stimulation in 3T3-L1 adipocytes by site-directed mutagenesis and luciferase assay. 3. In WAT of diet-induced obese mice, FGF21-evoked downstream signaling events, including the phosphorylation of ERK1/2 and SRF/Elk-1, the upregulation of GLUT1, and the increased glucose uptake, were markedly blunted compared to lean controls, suggesting the existence of “FGF21 resistance” in obesity. 4. In vivo and ex vivo studies on fasted wild type and FGF21 KO mice demonstrated that FGF21 acutely suppressed basal and forskolin-stimulated lipolysis in WAT. 5. FGF21-inhibited lipolysis was mediated by Akt-dependent reduction of cyclic adenosine monophosphate (cAMP) levels in both mouse and human WAT. 6. FGF21 KO mice were resistant to diet- and aging-induced obesity, which was attributed to decreased fat mass. The increased lipolysis and fatty acid oxidation in FGF21 KO mice may explain in part the lean phenotype of FGF21 KO mice. Conclusions: These results collectively suggest FGF21 as a key modulator of glucose and lipid metabolism in WAT, by activation of ERK1/2 kinase and Akt respectively. FGF21 and its signaling components may represent potential targets for the future development of new strategies for treating obesity and its medical complications. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Florida State University, 2004. / Advisor: Dr. Michael Blaber, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Sept. 21, 2004). Includes bibliographical references.

Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2009. / Includes vita. Includes bibliographical references (leaves 95-107).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Sep. 9.

No description available.

Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviridae is the only known virus family that encodes genes with homology to vertebrate and invertebrate fibroblast growth factors (fgfs), key regulators of developmental processes affecting cell growth, differentiation, and motility. The role of viral fgfs during infection is not known. In this study, we investigated gene regulation and function of the Autographa californica M nucleopolyhedrovirus (AcMNPV) fgf during infection of permissive insect cells. We demonstrated that the AcMNPV fgf, vfgf, was transcribed as a 0.6-kb mRNA at early times post infection, but as part of a 1.4-kb bicistronic mRNA at late times. To determine its function, we examined common characteristics between vFGF and other well-characterized FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate chemokinesis of different types of insect cells. We also constructed a recombinant of AcMNPV lacking a functional vfgf and analyzed it in two insect cell lines. The kinetics of budded virus production were similar in the parental and vfgf-deficient viruses in two cell lines and at both high and low multiplicities of infection. In addition, we observed no obvious differences in the viral DNA synthesis and the protein kinetic profiles of cells infected with the mutant and parental viruses. Finally, coinfection of vfgf-containing and -deficient viruses and their passage for several generations did not reveal a consistent growth advantage for either virus. We propose that vFGF is the signal that directs the motility of uninfected tracheal or blood cells to infected tissues, enabling the virus to infect additional cells and spread systemically in the insect host. This proposal may explain a dispensable role for vfgf during virus infection in cell culture; nonetheless, we expect a distinct phenotypic difference between vfgf-deficient and vfgf-containing viruses during infection in the insect host.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains x, 148 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 136-148).