published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

Male fertility is largely controlled by the hypothalamic-pituitary axis, a careful balance between stimulating and suppressing gene expression and the secretion of hormones. The critical factors for male fertility have in the past been thought to be limited to testosterone and the gonadotropins. Estrogen has only recently been demonstrated to be both a crucial requirement for fertility and a cause of infertility. Reports in the early 1990s demonstrated a decrease in mean sperm counts over the last 50 years. A hypothesis for this observation is the increase of xenoestrogens in the environment that are able to mimic and potential disrupt the natural estrogens involvement in fertility. Although the mechanisms of estrogens involvement are not yet defined, the Sertoli cells are a potential sites of action as they possess receptors for the hormone and are able to locally produce it. Sertoli cells both act to protect and provide for the male germ cells and the developing spermatozoa. Pyrethroids are common synthetic insecticides of which some have previously shown estrogenic activity. Therefore this investigation examined the effects of pyrethoids, whose estrogenicity was confirmed via the yeast assay, on the estrogen receptor expression in mouse Sertoli cells as a model for general effects of estrogenic chemicals on male fertility. The results first confirmed the estrogenicity of some pyrethroids and these pyrethroids when exposed to mouse Sertoli cells effected estrogen receptor mRNA expression however in a different way to the natural ligand 17β-estradiol.

Calcium is a component of signal transduction pathways that allow plants to respond to numerous endogenous and environmental signals during growth and development. Calcium-mediated signaling involves multiple components including: 1) channels, pumps, and exchangers that act in concert to generate a change in cytosolic calcium, 2) calcium-binding proteins that sense the calcium change, and 3) downstream target proteins that modify enzyme activity and gene expression needed for the subsequent response. One method for achieving specificity during calcium signaling is through regulation of the calcium-binding proteins that perceive changes in cytosolic calcium. These proteins can be regulated through differences in expression in response to stimuli, localization within the cell or plant, affinity for calcium, and interaction with downstream target proteins; all of which can result in specific cellular responses. My projects have focused on the Arabidopsis thaliana (Arabidopsis) CALCINEURIN B-LIKE10 (CBL10) calcium-binding protein, and specifically on understanding: 1) how post-transcriptional regulation of the CBL10 gene is used to modulate seedling growth in saline conditions (salinity), and 2) CBL10’s function in the flower during growth in salinity. In addition, 3) I have examined the roles of two putative CBL10-interacting proteins in plant growth and development. CBL10 is alternatively spliced into two transcripts; CBL10 encoding the characterized, full-length protein and CBL10 LONG A (CBL10LA) encoding a putative truncated protein due to a pre-mature termination codon within a retained intron. When seedlings are grown in the absence of salinity, both alternatively spliced transcripts are detected; however, in response to salinity, levels of the CBL10LA transcript are reduced. My data suggest a model in which the relative abundance of the two transcripts regulates the SALT-OVERLY-SENSITIVE (SOS) pathway involved in maintaining cellular sodium ion homeostasis. The presence of CBL10LA in the absence of salinity ensures that the SOS pathway is inactive. The removal of CBL10LA in response to saline conditions results in CBL10 activation of the SOS pathway to prevent sodium ions from accumulating to toxic levels in the cytosol. Successful fertilization during flowering requires the coordinated development of stamens and pistils. Stamens must elongate and anthers dehisce to release pollen onto the stigma while the pistil prepares to receive the pollen and promote growth and targeting of the female gametophyte. When the cbl10 mutant is grown in salinity, flowers are sterile due to decreased stamen elongation, reduced anther dehiscence, and abnormal pistil development. My studies demonstrated that the SOS pathway is not involved in maintaining flower development in salinity and indicate that CBL10 functions in different pathways to regulate vegetative and reproductive development during growth in saline conditions. An in silico search for Arabidopsis proteins that might interact with CBL10 resulted in the identification of two components of the Mediator complex involved in the regulation of transcription in eukaryotes. While additional studies I carried out suggest that interaction with CBL10 is unlikely, I have shown that these proteins are important for plant growth in high levels of chloride and in maintenance of growth in short-day conditions.

No description available.

Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis. / Dissertation