Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), okadaic acid (OA), cycloheximide (CHX), actinomycin D (AcD) and tunicamycin (TuM) on plasminogen activator (PA) activity and maturation rate in bovine and porcine oocyte-cumulus cell complexes (BOCC and POCC, respectively) in vitro were determined. Plasminogen activator activity was measured by SDS-PAGE, casein-agar zymography and densitometry. Three plasmingen-dependent lytic zones (92-95, 71-73 and 49-51 kD) and one PA inhibitor (52 kD) were observed in BOCC. Immunoprecipitation and amiloride sensitivity suggested that the 49-51 kD protease is a urokinase type PA (uPA), the 71-73 and 92-95 kD proteases are a tissue type PA (tPA) and tPA-PAI complex, respectively, and the PAI is PAI-1. In POCC, two plasminogen activators (71-78 and 93-96 KD) were observed. Lack of amiloride sensitivity suggested that the 71 -78 kD band is a tPA and the 93-96 kD band is possibly a tPA-PAI complex. Increasing dbcAMP in the culture medium increased activity in both BOCC and POCC in dose-dependent fashion (P<0.05). In BOCC cultured with PMA, total PA activity increased, however high concentrations of PMA (10 and 100 ng/ml) decreased tPA activity in matured POCC. Plasminogen activator activity decreased in 6-DMAP, actinomycin D and cycloheximide and oocyte maturation was also inhibited in these treatments. When POCC were treated with 25 nM OA, uPA activity was observed. Plasminogen activator activity increased in either BOCC or POCC treated with up to 25 nM OA, however PA activity decreased at concentrations greater than 75 nM (P<0.05). Incubation of BOCC with tunicamycin reduced the molecular mass of tPA and tPA-PAI complex and PAI-1 by 5-10%, however PA activity was not inhibited. These data suggest that BOCC matured in vitro produce uPA, tPA and PAI-1 however POCC produce only tPA and PAI. The production of PA and PAI by either BOCC or POCC is associated with oocyte maturation and influenced by stimulators of the protein kinase A and C, modulators of intracellular phosphorylation and metabolic inhibitors. / Graduation date: 1993

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

Tissue engineering aims to regenerate, repair or replace organs or tissues which have become defective due to trauma, disease or age related degeneration. This engineering may take place within the patient's body or tissue can be regenerated in a bioreactor for later implantation into the patient. Regeneration of soft tissue is one of the most demanding applications of tissue engineering. Producing proper nutrient supply, uniform cell distribution and high cell density are the important challenges. Many experimental models exist for tissue growth in a bioreactor. It is important to put experiments into a theoretical framework. Mathematical modelling in terms of physical and biochemical mechanisms is the best tool to understand experimental results. In this work a mathematical model of convective and diffusive transport of nutrients and cell growth in a perfusion bioreactor is developed. A cell-seeded porous scaffold is placed in a perfusion bioreactor and fluid delivers the nutrients to the cells for their growth. The model describes the key features of the tissue engineering processes which includes the interaction between the cell growth,variation of material porosity, flow of fluid through the material and delivery of nutrients to the cells. The fluid flow through the porous scaffold is modelled by Darcy's law, and the delivery of nutrients to the cells is modelled by the advection-diffusion equation. A non-linear reaction diffusion system is used to model the cell growth. The cell diffusion depends on the cell density and growth of cells is modelled by logistic growth. The effect of shear stress on nutrient consumption and cell growth is also included in the model. COMSOL (a commercial finite element solver) is used to numerically solve the model. The results show that the distribution of cells and total cell number in the scaffold depends on the initial cell density and porosity. We suggest various seeding strategies and scaffold designs to improve the cell growth rate and total cell yield.

During spermatogenesis, developing germ cells must migrate across the blood-testis barrier (BTB) and enter the adluminal compartment for further development. Throughout this process, extensive junction restructuring occurs at Sertoli-Sertoli and Sertoli-germ cell interfaces. Cytokines are known to play crucial roles in regulating testicular cell junction dynamics at different regulatory levels. However, the mechanism of cytokine-mediated regulation on newly identified junction molecules remains unclear. In this dissertation, the molecular mechanisms on how cytokines regulate the junction proteins of immunoglobulin superfamily (IgSF) including coxsackievirus and adenovirus receptor (CAR), nectin-like molecule-2 (Necl-2) and Necl-4 in testicular cells were studied. CAR is expressed on Sertoli and germ cells. It mediates both homophilic and heterophilic interaction for Sertoli-germ cell adhesion. It was found that combined treatment of interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα) reduced CAR mRNA and protein levels, and caused the disappearance of CAR from germ cell interface. IFN-γ+TNFα promoted CAR protein degradation via ubiquitin-proteasome pathway. In addition, IFN-γ+TNFα reduced CAR mRNA through regulating the binding of NF-κB subunits and SP/KLF proteins to CAR promoter. Collectively, these results demonstrated for the first time the potential mechanism utilized by IFN-γ+TNFα to exert their effects during testicular inflammation. Necl-2 is exclusively expressed by spermatogenic cells in the testis. In this study, it was demonstrated that transforming growth factor-β1 (TGF-β1) down-regulated Necl-2 mRNA and protein levels, and caused the disappearance of Necl-2 from cell surface. Using inhibitors and shRNAs, it was found that TGF-β1 induced Necl-2 protein degradation through clathrin-dependent endocytosis. Endocytosis assay further confirmed that TGF-β1 accelerated the internalization of Necl-2 to cytosol. Moreover, TGF-β1 repressed Necl-2 gene transcription in Smad-dependent manner. Taken together, these results unraveled the mechanism of how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring during spermatogenesis. Necl-4 has been detected in Sertoli cells, but little is known about its regulation in the testis. It was found that TNFα down-regulated Necl-4 mRNA and protein levels. Inhibitor studies suggested that both caveolin-dependent endocytosis and ubiquitin-proteasome pathway were involved in TNFα-induced Necl-4 protein degradation. Co-immunoprecipitation indicated that Necl-4 was physically associated with Par3, Par6, aPKC and Cdc42 which are the major components of polarity complex in mouse testis. Further study was shown that TNFα reduced the expression of Par3, and altered the binding between Necl-4 and Par complex in Sertoli cells. These results suggested that Necl-4-mediated cell adhesion could be disrupted by TNFα via reducing its expression and altering its interaction with Par complex. Studies reported herein suggest that junction proteins of the IgSF are precisely regulated by cytokines at transcriptional and post-translational levels. These results further enrich current understanding on how junction dynamics are regulated during spermatogenesis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

No description available.

Centenarian population studies are one of several approaches currently used to study the aging process and characterize successful aging. I have described a methodology permitting the simultaneous generation of RNA, DNA, protein, and plasma samples, as well as fixed peripheral blood mononuclear cells (PBMC) and frozen blood aliquots, from a single 10- to 30-ml sample of peripheral blood obtained from donors of any age, and showed that although extremely old individuals are somewhat anemic, it is possible to obtain enough biological material from their blood to conduct aging studies. / I investigated the possibility of immortalizing B-lymphocytes from extremely old individuals, using the Epstein-Barr virus (EBV), and found that although extremely old individuals (90+ years) possess low levels of circulating B-lymphocytes, it is possible to immortalize B cells present in less than one milliliter of their blood using EBV. / Using biological material obtained from blood samples of individuals of all ages by the method for blood sample processing I have described, I studied the mRNA expression of cell death (specifically caspase) genes in nonagenarians and centenarians, successful models of aging who have survived or avoided age-associated diseases, as well as in their younger counterparts, to determine whether apoptotic genes may be part of the genetic determinants of longevity. I found that a population of extremely old individuals (90+) shows a unique pattern of caspase mRNA expression, characterized by high levels of caspase-1 and -3, and low levels of caspase-8, mRNA, while slightly less aged individuals (70--89) are characterized by high levels of caspase-8 mRNA expression. Furthermore, I showed that these changes in caspase mRNA do not appear to result from age-related changes in PBMC composition, such as decreases in CD24. Therefore, I suggest that unique patterns of caspase mRNA result from the regulation of message abundance on a per cell basis, via a putative regulation of caspase genes at the transcription or RNA processing level, rather than age-associated changes in immune profiles.

The seaweed fly, Coelopa frigida, lives in piles of rotting seaweed deposited on beaches by tides and winds. In all populations studied it is polymorphic for two gene arrangements on Chromosome I. A polymorphism at the alcohol dehydrogenase locus is strongly associated with this inversion and can be used to estimate karyotype frequencies. An extensive series of samples from natural populations has revealed a seasonal cycle in inversion frequencies but otherwise frequencies are remarkably constant both geographically and temporally. There is a consistent excess of heterokaryotypes in these samples. Three selective forces influencing inversion frequencies have been investigated. 1) An association between karyotype and development time, previously observed in the laboratory, has been demonstrated in conditions case to those in natural populations. 2) Viability differences between karyotypes have been examined. In natural populations there is some evidence that the excess of heterokaryotypes increases with larval density. In the laboratory heterokaryotypes are shown to have higher viability than either homokaryotype but the strong density dependence reported previously has not been observed. Viability differences are concentrated in the first two days of larval life and are probably related to the rate of supply of nutrients. 3) An association is demonstrated between karyotype and adult size - especially in males. Adult size is shown to correlate with longevity and fecundity of both sexes. Several experiments indicate that large males enjoy greater mating success than small males. The relationship between larval density, development time and adult size is described. The possibility that the effect of the inversion varies between populations or between alcohol dehydrogenase genotypes has also been investigated. A simulation has been used to study how these selective forces interact with one another, and with the changeable environment in which the flies live, and to examine the extent to which they can account for the observed karyotype frequencies.