Thesis (Ph. D.)--Florida State University, 2006. / Advisor: Geoffrey Strouse, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed June 9, 2006). Document formatted into pages; contains xiv, 163 pages. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 72-86). Also available in electronic version.

Thesis (M.S.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Douglas S. Cairns. Includes bibliographical references (leaves 106-107).

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 155-159). Available also in a digital version from Dissertation Abstracts.

The key to species survival depends on the ability to reproduce. In mammals ovulation requires a precisely timed luteinizing hormone (LH)-surge on the afternoon of proestrus. It is thought that this LH-surge requires the convergence of two signals one being a hormonal signal (i.e. the positive feedback of estrogen) and the other being a neural signal (i.e. a circadian signal). The circadian clock is known to be involved in regulating numerous biological functions, such as sleep/wake cycles, food intake, body temperature, hormonal regulation, etc. It does this through a series of transcriptional/translational feedback loops involving various clock genes (i.e. Clock, Bmal1, Per1, Per2, Cry1, Cry2, reverbα, etc.). CLOCK and BMAL1 bind to noncanonical E-boxes in the promoters of clock driven genes, which activates their own transcription and subsequent translation. PER and CRY are then translocated back into the nucleus to inhibit their own transcription and that of other E-box driven genes. There have been various published reports on clock mutant animals having impaired fecundity. Mammalian reproduction occurs through the hypothalamic-pituitary-gonadal (HPG) axis where GnRH (gonadotropin-releasing hormone) neurons, located in the hypothalamus, release GnRH in a pulsatile fashion. GnRH acts on the GnRH receptor (GnRHR) on the surface of anterior pituitary cells called gonadotropes. The GnRHR triggers the synthesis and release of LH and FSH, which act on the ovaries to produce estrogen and progesterone. Estrogen then feeds back on the hypothalamus and pituitary gland. In the present studies whole rat pituitaries were used to assess clock gene and protein expression at diestrus a.m./p.m. and proestrus a.m./p.m. It was found that mPer1 increased during the evening of both days, but peaked on the evening of proestrus. It was surprising that PER1 protein was also high during the morning of proestrus, which was not seen in mPer1 mRNA. Since LH is synthesized and secreted only in gonadotropes, which make up 3-5% of the entire pituitary, enriched primary gonadotropes were used to study clock gene expression. Using enriched primary gonadotropes it was seen that mPer1 is indeed expressed in these cells in a rhythmic fashion and also that GnRH induces mPer1 as well as LHβ. Because GnRH induces both mPer1 and LHβ, experiments were performed to determine whether the signaling pathway involved in the GnRH induction of mPer1 correlates to that of the GnRH induction of LHβ. Using various pharmacological approaches it was demonstrated that GnRH induces mPer1 and LHβ through the PKC/MAPK pathways. It has also been shown that the GnRH induction of LHβ requires the early growth response protein 1 (Egr-1). To ascertain the importance of Egr-1 in mPer1 expression, analysis of the mPer1 promoter was performed and an Egr-1 binding site was found. Using various techniques it was determined that Egr-1 is involved in the GnRH induction of mPer1 expression. GnRHR expression fluctuates in accordance with the frequency of GnRH pulses. Little is known about how this change in GnRHR expression occurs. E-boxes are required for circadian regulation of a gene. Through examination of the GnRHR promoter, seven noncanonical E-boxes were found proximal to the transcriptional start site (tss). Analysis of the putative clock gene regulation of the GnRHR was performed and it was found that clock genes are indeed important factors in controlling GnRHR expression. To reach the critical LH-surge, LHβ must be synthesized and released from the gonadotropes. To further the understanding of clock gene involvement in the HPG axis, the LHβ promoter was also examined and six noncanonical E-boxes were found proximal to the tss. Chromatin immunoprecipitation was performed, and it was seen that CLOCK and BMAL1 bind to these E-boxes in the LHβ promoter. To determine whether these elements play a role in the regulation of the LHβ each E-box was mutated to a nonsensical sequence using site-directed mutagenesis. The impact on LHβ expression was analyzed via luciferase reporter activity. Through these techniques it was established that clock genes play an important role in regulating LHβ. These data demonstrate a complex variety of ways by which the circadian clock is involved in the regulation of the reproductive neuroendocrine axis. / A Dissertation Submitted to the Department of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2009. / June 18, 2009. / Egr1, GnRH, Per1, HPG, Fertility, Circadian Rhythms / Includes bibliographical references. / James Olcese, Professor Directing Dissertation; Laura Keller, Outside Committee Member; Mohamed Kabbaj, Committee Member; Charles Ouimet, Committee Member.

Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Iδ and Iε (CKIδ/ε) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKIδ/ε while avoiding the embryonic lethality of CKIδ disruption in mice, we used CKIδ-deficient Per2Luc mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKIε (DN-CKIε) in the mutant MEFs. CKIδ-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKIδ loss. When CKIε activity was disrupted by the DN-CKIε in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKIδ/ε are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKIδ/ε was disrupted by overexpressing the CKIδ/ε binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKIε did not, suggesting that CKIδ/ε may have a noncatalytic role in stabilizing PER. Our results show that an essential role of CKIδ/ε is conserved between Drosophila and mammals, but CKIδ/ε and DBT may have divergent non-catalytic functions in the clockwork as well. Since reversible phosphorylation events in the circadian clock are thought to be involved in temporal regulation of clock proteins, a dynamic process of clock proteins mediated by protein kinases and phosphatases may be an essential feature in the time-keeping mechanism in mammals. To address these issues more definitively and extend findings that CKIδ/ε are essential for the clockwork, we proposed to explore the dynamics of reversible PER phosphorylation by studying CKIδ/ε conditional mutant mice / cells and by identifying protein phosphatases in targeting PER and characterizing them using genetic and biochemical approaches. We finally validated that CKIδ/ε are essential protein kinases to facilitate driving clockwork based on our findings that CKIδ/ε double KO cells have no circadian rhythms and x they are rescued by transducing CKIε. Moreover, PP1 is highly associated with PER dephosphorylation based on our results in genetic (dominant negative PP1) and chemical approaches (phosphatase inhibitors: OA vs. CA). Therefore, we propose that dynamic and reversible processes mediated by kinases and phosphatases are essential features in the timedriving/ keeping mechanism in mammals. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2010.. / October 15, 2010. / Mouse embryonic fibroblast, Dominant negative, Casein kinase, Circadian clock / Includes bibliographical references. / Choogon Lee, Professor Directing Dissertation; James Fadool, University Representative; Mohamed Kabbaj, Committee Member; James Olcese, Committee Member; Yoichi Kato, Committee Member.

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This dissertation explores various topics within the theme of nanostructured polymer electrolyte membranes having controlled morphology, and their resulting properties. Chapter 1 gives an introduction to the field of Polymer electrolyte membranes (PEM) in its current state, and an overview of the work done. In chapter 2, relatively inexpensive block copolymer ionomers of fluorinated poly(Isoprene)-block-sulfonated poly(Styrene) (FISS) with various sulfonation levels, in both the acid form and the cesium neutralized form, have been cast into membranes of desired random phase separated morphology. The morphology of these membranes were characterized by TEM and USAXS, as well as water uptake, proton conductivity and methanol permeability from 20 to 60°C. The transport properties increased with increasing sulfonation and temperature for all samples. The acid form samples absorbed more water than the cesium samples with a maximum swelling recorded at 60°C for the acid sample with 50mol% sulfonation. Methanol permeability for the latter sample was more than an order of magnitude less than Nafion 112 but so was the proton conductivity at 20°C within the plane of the membrane. Across the plane of the membrane this sample had half the conductivity of Nafion 112 at 60°C. In chapter 3, neutron and x-ray scattering techniques have been used to study the structural evolution of FISS materials as they have evolved from the dry state to the water soluble state. A dilation of the nanometer-scale hydrophilic domains have been observed as hydration has been increased, with higher swelling for the higher sulfonated sample or upon hydrating at higher temperatures. Furthermore a decrease in the order in these phase separated structures is reduced upon swelling. The glass transition temperature of the fluorinated blocks decreased upon hydration, and at the highest hydration levels loosely bound water was evident. Thermal and dynamic mechanical characterization of these materials have shown that there is a high degree of softening beyond the 45°C glass transition temperature. Finally highly sulfonated samples have shown the formation of spherical micelles, even at concentrations as low as 0.05 mg/ml. The sizes of these micelles range from 13–13.5 nm, with the higher concentration solutions having smaller radius of gyration, possibly due to crowding effects. In chapter 4, Ionomers from the cesium salt (20 mol%) of fluorinated Poly(Isoprene)-block-sulfonated Poly(Styrene) have been spun cast into membranes and annealed under an electric field of ∼40 V/um at 130°C for 24 hours. This resulted in the transformation of the morphology from a random phase separated state to one preferentially oriented in the direction of the electric field but with smaller domain sizes. The effect of this change in morphology was a 2.5 times increase in the ionic conductivity, as measured by electrochemical impedance spectroscopy, at all humidity conditions measured. This can be attributed to the increased connectivity of the ionic domains. In chapter 5, The applicability of electrospun nanostructure with high surface to volume ratios for PEM application is presented. To this end, sulfonated poly(ether ether ketone) has been electrospun and electrosprayed by varying concentration in DMF, yielding isotropic fibrous mats and beads. The glass transition temperatures of these materials have been shown to be higher those of the unsulfonated precursors and they increase with increasing sulfonation, due to hydrogen bonding induced rigidity. The presence of sulfonic acid groups on the surface has been confirmed by means of x-ray photoelectron spectroscopy, with sulfur representing 3% of the surface elemental composition. These acid groups on the surface of internal fibers, help to form a 3 dimensional network of conducting channels in the voids of the fibrous mats upon hydration. This in turn has led to an improvement of conductivity from 0.033 S/cm for void-less solution cast membranes to 0.040 S/cm for the electrospun fibrous mats.

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