Given the indispensable role of Ca2+ release in fertilization, the type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a key regulatory molecule in mediating the cross-talk between cell cycle progress and the Ca2+ release machinery in mammalian oocytes. The studies in this thesis addressed several important regulatory aspects of the Ca 2+ release activity at fertilization of mammalian eggs. In chapter I, we found that compromised functionality of IP3R1 underlay the defective IP3R1-mediated Ca2+ release in aging eggs. Further, our studies also indicated that modifications on the biochemical status of IP3R1 by incubation with caffeine have positive effects on the Ca2+ release activity and developmental fate of aging mouse eggs. Thus, these results may help facilitate the invention of a cure that could delay/reverse many of age induced detrimental changes thereby restoring the fertilizability of aged eggs. For the next two chapters we mainly focus on the regulatory role of IP3R1 as a significant participant in the early developmental event. Our completed results with the caspase-3 cleaved IP3R1 indicated that the truncated IP3R1 might not play a prominent role affecting the Ca2+ homeostasis during the aging process in mouse oocytes due to its rapid turnover rate. Regardless, we confirmed the leaky property of C-IP3R1 in mouse oocytes and found a novel protective proteolysis pathway in mouse oocytes. Finally development of a system by which we could selectively overexpress IP3R1 phosphorylation mutants allows us to probe important phosphorylation regulatory mechanisms underlying the [Ca2+]i oscillations in mammalian oocytes. Thus the experiments I have done provide valuable information regarding the extensive regulation of the PI pathway, regulation that in aged and non- aged MII eggs, results in a Ca2+ release program that is replete with information.

A newly discovered, nonoccluded baculovirus GSV has been reported to be a causative agent of sterility in adult corn ear-worms, Helicoverpa zea. Previous studies conducted by Hamm et al. (1996) and our laboratory indicated that it is an unusual insect virus with strict tissue tropism and the ability to establish persistent infections in vivo. After acquiring purified virus from infected adult insects, two permissive cell culture systems, TN-368 and Ld652Y were established for GSV replication in vitro. The cell culture-derived virus was confirmed to have the same morphology, biological activity and genetic identity as that of GSV recovered from insects. Using a cell culture system, several genetically pure GSV cloned isolates were acquired by plaque purification. The replication cycle of the virus including ultrastructural studies, viral DNA replication and virus specific protein synthesis were investigated in these two cell lines and interestingly, it was found that the exact same virus isolate had a different biology in the different permissive cell lines. Difference in the molecular biology of virus replication in these two cell lines was also observed. This suggests that host factors play an important role in determining the different host-viral interaction of the virus. In addition, biochemical properties of the GSV genome were investigated. The genome size was estimated using pulse-field gel electrophoresis to be 215-235 kb. CsCl-EtBr density gradient centrifugation indicated that GSV has a supercoiled, circular genome. Purified viral structural proteins, envelope proteins and glycoproteins were analyzed by SDS-PAGE and a total 16 of viral structural proteins were identified, three of them are glycosylated and five of these proteins are likely to be virus envelope or matrix components. Studies of GSV specific protein synthesis, DNA replication and transcription in the presence of specific inhibitors suggests that as with other most baculoviruses, GSV gene expression is temporally regulated and can be separated into early and late phases based on viral DNA replication and differential responses to the cellular RNA polymerase inhibitor, alpha-amanitin. That is, early gene expression is likely mediated by cellular RNA polymerase whereas a viral encoded or viral-modified host RNA polymerase likely mediates late viral gene expression. GSV persistent infection in vitro has been investigated using a persistently infected cell line, GSVP. GSV viral sequences and a very low level of infectious virus were detected from this normal-looking, persistently infected cell line. Co-culture of GSVP cells with another permissive cell line, Ld652Y, resulted in productive replication of GSV.

Family members with a heritable mutation in the BRCA1 or BRCA2 gene have up to an 85% life-long risk of contracting one or more of the cancers associated with a genetic alteration to these genes. The inheritance risks of a BRCA alteration to first and second-degree relatives of affected individuals are 50% and 25%, respectively. Currently, members from high-risk families can be tested for the presence of BRCA1 or BRCA2 mutations by gene sequencing of blood cell DNA. This test is expensive and generally is not reimbursed by insurance carriers. Over 85% of BRCA1 and BRCA2 mutations are reported to result in the production of a truncated protein, from which the carboxy terminal end sequences of their respective protein is missing. In preliminary studies, using antibodies specific for the amino acid terminal or the carboxy terminal of the BRCA1 protein, we demonstrated that immunohistochemical analysis of surgical specimens, consisting of matched ovarian tumor and normal uninvolved tissue, identified BRCA1 protein truncations in two matched samples. Each BRCA1 protein truncation was shown to be indicative of the presence of a BRCA1 germline mutation. Human buccal cells were then investigated as a minimally invasive cell source for this assay. Buccal cells were shown to express BRCA1 and BRCA2 protein immunohistochemically using distinct antibodies for the BRCA1 and BRCA2 protein terminals. Transcription of the BRCA1 and BRCA2 gene within buccal cells was also shown to occur by RT-PCR. A double blind study was then conducted using blood DNA and buccal cells collected from thirteen high-risk patients for carrying a germline BRCA1 or BRCA2 mutation. Mutation analysis (PCR, SSCP, and gene sequencing) was performed on DNA. Immunohistochemical analysis for the BRCA1 and BRCA2 protein was performed on cytospun deposited buccal cells. The buccal cell assay correctly predicted the presence of six germline BRCA1 mutations, three germline BRCA2 mutations, and three with no mutations. One predicted germline BRCA2 mutation did not correlate with DNA sequencing results. Based on these results, this assay exhibited a 100% negative predictive value and 90% positive predictive value.

Using reverse genetics, complementation analyses, and cell biological approaches with the moss Physcomitrella patens, I assessed the in vivo function of two actin turnover proteins: actin depolymerizing factor (ADF) and its binding partner actin interacting protein 1 (AIP1). My studies identify a single ADF and AIP1 in moss. Loss-of-function analyses reveal that ADF is essential for viability, and AIP1 is required to promote normal tip cell expansion. AIP1 and ADF are diffusely cytosolic proteins that function in a common genetic pathway to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin turnover, AIP1 knockout lines and plants silencing ADF accumulate F-actin bundles along the cortex. Quantitative analysis of time-lapse F-actin movies demonstrates that AIP1 promotes and ADF is essential for cortical F-actin dynamics. The development of a complementation assay permitted dissection of the physiological relevance of regulatory mechanisms that control ADF activity. Mutant complementation analyses reveal that phosphoregulation of ADF at a conserved, N-terminal serine is important for in vivo function. Phosphomimetic ADF mutants have severe tip growth defects, but remain viable, demonstrating that ADF is critical for tip growth. A gain-of-function ADF mutant with enhanced affinity for phosphatidylinositol 4,5-bisphosphate has minor defects in tip growth, suggesting that this phospholipid regulates ADF activity in vivo. Complementation analyses with ADF/cofilin proteins from other organisms reveal that moss ADF is functionally conserved with some, but not all ADF/cofilins. Interestingly, rescue is inversely proportional to pH-sensitivity, suggesting that pH-insensitive ADF activity is important for tip growth in moss. The complementation analysis has also facilitated the identification of two temperature-sensitive mutants in moss ADF. These temperature sensitive mutants, together with the AIP1 knockout lines, will be instrumental for identifying cellular processes in plants that require actin dynamics – an open question in plant biology.

The Cu$\rm\sb{A}$ center, located in subunit II of cytochrome c oxidase, is the primary site for electron entry from cytochrome c. The recent crystal structure confirms this site to be a binuclear copper center with a dithiolate, dihistidyl coordination. A methionine and the carbonyl oxygen of Glu$\sp{198}$ are also ligands. To further characterize the structure and function of the Cu$\sb{A}$ center, site directed mutagenesis has been implemented in subunit II of S. cerevisiae. Substitutions of direct ligands to the Cu$\rm\sb{A}$ site result in loss of cellular respiration. Cytochrome a from cytochrome c oxidase is not detected in the visible absorption spectrum. Furthermore, subunit II does not accumulate to wild-type levels as observed by immunodetection. These results suggest that the enzyme is not assembling, most likely due to the disruption of copper binding, and that subunit II is being degraded. In contrast, substitutions to Glu$\sp{198},$ whose carbonyl oxygen is a ligand to one of the coppers, merely results in cells with reduced respiratory function. In an attempt to perturb the Cu$\rm\sb{A}$ site without disrupting protein stability, nearby amino acids were substituted. Asp$\sp{158}$ is known to hydrogen bond to the Cu$\rm\sb{A}$ ligand His$\sp{161}$, and Ser$\sp{197}$ is adjacent to the Cu$\rm\sb{A}$ ligand Cys$\sp{196}$. Substitutions at these positions result in reduced cellular respiration and may perturb the Cu$\rm\sb{A}$ site. Substitutions to conserved residues Gly$\sp{194}$ and Gly$\sp{201}$ result in a loss of cellular respiration and the underaccumulation of subunit II, while substitutions at other conserved positions have no observable effect on respiration. These results are discussed in relation to the homologous quinol oxidases and nitrous oxide reductases. To begin to characterize residues which may be involved in binding cytochrome c, substitutions were made to various conserved carboxylates which had been implicated in binding cytochrome c. The recent crystal structure demonstrates that Glu$\sp{198}$ and Asp$\sp{158}$ are not available for direct interaction with cytochrome c. However, the crystal structure does point to other conserved carboxylates. Asp$\sp{112}$ may be at the periphery of a cytochrome c binding site since replacement of this residue by Arg results in only a small decrease in cellular respiration.

Programmed cell death (PCD) is an essential developmental process in all multicellular organisms. It serves multiple functions including selected removal of unneeded and/or deleterious cells, and regulation of cell numbers (reviewed in Milligan and Schwartz, 1996). The intersegmental muscles (ISMs) of the hawkmoth Manduca sexta provide an ideal model for studying PCD (reviewed in Schwartz, 1992). In response to a decline in the circulating titer of the steroid hormone 20-hydroxyecdysone (20-HE), these cells initiate a death program which includes both the up- and down-regulation of specific genes. Following eclosion, the ISMs undergo PCD that results in the complete destruction of the muscles during the subsequent 30 hours (Finlayson, 1956). This dissertation examines in detail the repression of actin and myosin heavy chain expression that occurs when the muscles become committed to die. At the protein level, actin expression was reduced by 84% at the time the muscles were committed to die, which presumably plays a role in the rapid dissolution of the muscles. When the ISMs became committed to die, there were dramatic increases in proteolytic activity that are correlated with an approximately eightfold increase in the absolute amounts of multicatalytic proteinase (MCP). At the time of commitment, four new MCP subunits were observed to be associated with the complex. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. Cells dying by PCD often display a characteristic set of features termed apoptosis. These features include chromatin condensation, DNA fragmentation, membrane blebbing and phagocytic removal of the dying cells. However, dying ISMs display few characteristics of apoptosis. Interestingly, apoptotic cell death does occur in Manduca embryogenesis. Evidently Manduca possesses the necessary biochemical machinery to undergo apoptosis and does so in specific developmental circumstances. These data suggest that more than one cell death mechanism is used during development.

During muscle differentiation in vertebrates, myoblasts initially form in somites and then migrate to proper locations in the trunk and limbs. Once there, these cells are faced with one of three choices: differentiate into myotubes, arrest as satellite cells, or initiate apoptosis and die. The molecular mechanisms that regulate the decision of myoblasts to die are poorly understood. To gain insight into this process, we have cloned death-associated genes from the intersegmental muscles of the moth Manduca sexta, a model system for developmentally regulated muscle cell death. One of the genes isolated in this screen was DALP (Death Associated LIM Only Protein), a protein that shares 52% similarity at the protein level with mammalian Hic-5. Ectopic expression of DALP in the skeletal muscles of the fruit fly Drosophila caused atrophy and disorganization of the contractile apparatus. To determine the role of DALP/Hic-5 in mammalian myogenesis, we took advantage of the mouse myoblast cell line C2C12. Ectopic expression of either DALP or Hic-5 blocked the ability of myoblasts to differentiate following serum withdrawal. These cells failed to express muscle differentiation markers such as MyoD or myosin heavy chain. In addition, these cultures displayed greatly enhanced rates of cell death. Hic-5 expression is restricted to mononucleated and apoptotic C2C12 cells in serum-depleted medium. The effects of ectopic DALP or Hic-5 expression could be prevented by contact with wild type C2C12 cells or by ectopic expression of MyoD. Gene profiling experiment demonstrated that the ectopic expression of Hic-5 results in enhanced expression of pro-apoptotic Bcl-2 family members and of polyubiquitin. Taken together, these data suggest that Hic-5 acts upstream of MyoD and functions as a negative-regulator of myoblast differentiation and may facilitate the initiation of apoptosis. In separate studies, the functional roles of another death-associated molecule, m56, were studied in Ratl fibroblasts. Our data strongly support the hypothesis that m56 is a proteasome subunit and misexpressing m56 can sensitize Ratl cells to apoptotic stimuli.

The pituitary gland is the major endocrine gland in the forebrain. The hormones secreted from this gland regulate vital processes such as reproduction, growth and stress response. Distinct endocrine cells arise from pituitary precursors cells. The endocrine cells are spatially organized along the anterior-posterior axis within the anterior lobe of the pituitary, the adenohypophysis. Several signaling molecules have been shown to play roles in the development of this endocrine gland. Previous work in our lab showed that Sonic Hedgehog (Shh) is required for induction and patterning of the adenohypophysis. Hedgehog (Hh) signaling is involved in many developmental processes including induction, patterning and differentiation of many tissues. In addition, independent studies show that Fibroblast growth factor (Fgf) signaling also plays a role in the development of zebrafish adenohypophysis. ^ One of the main aims of my dissertation was to determine how the Hh and Fgf signaling pathways specify the functional patterning of the adenohypophysis. Using small molecule inhibitors I show that high levels of Hh signaling are required for the formation of the anterior adenohypophysis, the pars distalis (PD) and high levels of Fgf signaling are required for the formation of the posterior adenohypophysis, the pars intermedia (PI). My dissertation work also shows that high Hh levels are required for differentiation of the endocrine cells in the PD, and in contrast high Fgf levels are required for differentiation of the endocrine cells in the PI. Using live-imaging of a transgenic zebrafish line, I show that the PD and PI originate from distinct regions. My analyses has revealed that graded Hh and Fgf signaling help pattern the adenohypophysis along anterior-posterior axis by guiding endocrine cell differentiation in a dose dependent manner. ^ A related aim of my research was to analyze the role of Hh signaling in zebrafish neural tube patterning. The transcriptional response to varying Hh levels is well characterized in chick and mouse neural tube, and this transcriptional response has been partially described in zebrafish. The analysis of the Hh transcriptional response in wild type, Hh mutant and Hh over-expressing embryos show that there is a conserved transcriptional response to Hh signaling in the zebrafish neural tube. My comprehensive analyses of the Hh transcriptional response in the zebrafish neural tube provides a useful tool for the characterization of Hh signaling in zebrafish. ^

Pollen tube elongation is a polarized cell growth process that directionally transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actin cytoskeleton is known to support this growth process and Rac-like GTPases have been shown recently to be important to regulate actin organization in elongating pollen tubes. Actin depolymerizing factor/cofilins (ADF/cofilins) are actinbinding proteins that increase actin dynamics by enhancing actin depolymerization. They are also responsible to regulate actin organization in Rac-mediated signaling. My thesis research focuses on establishing a signaling pathway from Rac GTPase to the actin cytoskeleton via the regulation of ADF/cofilins in tobacco pollen tubes. I have isolated and characterized cDNAs for tobacco pollen ADF, NtADFs, to study their function in pollen tube growth. First, I showed the activity of Rac-like GTPase is essential for pollen germination. Tobacco pollen germination and early pollen tube growth stimulates the activation of these small GTPases, the phosphorylation of NtADFs and an increase in the ratio of F- to G-actin. Moreover, over-production of a pollen-expressed Rac-like GTPase, NtRac1 from tobacco, induces increased ADF phosphorylation in transformed pollen and diminished the binding of GFP-tagged NtADF1 (GFP-NtADF1) to actin filaments in growing pollen tubes. These observations are consistent with the presence of a signaling pathway in pollen whereby Rac-like GTPase are stimulated by germination to activate a phosphorylation cascade that down regulates the activity of ADFs. This ultimately affects actin dynamics and growth characteristics in pollen tubes. Second, I also showed that NtADF activity is important for actin organization in pollen tube growth. When expressed in a moderate level in pollen tubes, GFP-NtADF1 associated prominently with a sub-apical actin mesh comprised of short dynamic actin filaments and with long dynamic actin cables in the shank. Over-producing NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes. Pollen tube growth was also inhibited by over-expressed NtADF1 in a dosage-dependent manner, suggesting proper regulation of actin turnover by NtADF1 is critical for the pollen tube growth process. In addition, NtADF1 activity is regulated by phosphorylation and pH. By creating mutants on the serine 6 residue on NtADF1, the result showed that the charge characteristics on serine 6 is important for NtADF1 interaction with actin and for its activity on pollen tube growth. By an in vitro depolymerization assay, recombinant NtADF1 depolymerizes actin more efficiently at pH 8 than pH6. This and localization of a NtADF1-rich actin mesh in the sub-apical region of elongating pollen tube, which is known to have a more alkaline cytoplasmic condition relative to the apex, suggest that interaction between NtADF1 and actin in this vicinity maybe critical for the pollen tube growth process. Finally, to examine a signaling pathway from Rac to ADF, I showed that overexpression of NtADF1 suppressed Rac-induced isotropic pollen tube growth. These observations demonstrating biologically that pollen ADFs mediate signaling activated by Rac-like GTPase to the actin cytoskeleton in pollen tube growth.* *This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.

Plants are often subjected to extreme environmental conditions and must adapt rapidly. The phytohormone abscisic acid (ABA) accumulates under abiotic stress conditions, signaling transcriptional changes that trigger physiological responses. Epigenetic modifications are also required to facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and environmentally induced expression. In maize (Zea mays), MEDIATOR OF PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent epigenetic pathway that regulates transcriptional silencing of loci across the genome. As critical regulators of gene expression, MOP1 and ABA pathways predictably regulate specific genes in a coordinated manner. In one project, the amino acid sequence of DNG103 and the gene promoter region were analyzed for conserved domains and cis-responsive elements, respectively. DNG103 is similar to the Arabidopsis ROS1, contains the conserved domains of a DNA glycosylase with DNA demethylase activity, and contains ABA-responsive elements (ABREs) in its promoter region. Transcript levels of Dng103, and the ABA-responsive gene Viviparous 1 (Vp1), were monitored in maturing embryos from two genotypes placed in culture under different conditions. Expression of both genes decreased after culture in hormone-free medium and was induced by ABA in Mop1 wildtype. Dng103 and Mop1 showed decreased expression in mop1-1 and dng103 mutants, respectively. Dng103 is not responsive to ABA in the mop1-1 mutant and Vp1 has reduced sensitivity. Therefore, DNG103, MOP1 and ABA might have common regulatory targets. Protoplast isolation and transfection protocols were standardized alongside the production of reporter constructs containing the Dng103 and Vp1 promoters. This technology will allow for the promoter characterization of genes of interest through quantifiable luciferase expression, using different conditions and genotypes. To identify genome-wide ABA-induced, MOP1-dependent and independent transcriptional responses, mop1-1 and Mop1 homozygous seedlings were subjected to exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes (DEGs) were identified in four pairwise comparisons. Overall, the loss of MOP1 exaggerated some ABA-induced changes in gene expression. The highest number of DEGs were identified in ABA-induced mop1-1 mutants, including many transcription factors. A gene regulatory network was used to predict relationships between DEGs; suggesting multifaceted regulatory scenarios including direct and indirect transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a hierarchical, cascading network of responsive genes. Additionally, a modest increase in CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in some genotypes, suggesting that MOP1 might be necessary to achieve environmentally induced transcriptional responses in maize. To understand the multistep ABA response of identified ABA-induced genes, an ABA-time course was carried out in another project using four different time points. The previously predicted ABA-responsive transcription factors Hb41 and Bzip4 showed an early induction to ABA and the late embryogenesis abundant Rab17 gene was induced during the transition from early to late response. Together, these results indicate that MOP1 and ABA act at multiple levels within complex, connected transcriptional networks to mediate tissue-specific growth and responses to some abiotic stresses. LIST OF SUPPLEMENTARY FILES 1. Significant differentially expressed genes in analysis groups I-VIII. 2. GO term enrichment in groups I, II, V and VI. 3. Arabidopsis and maize homologous transcription factors and target genes in ABA transcription factor hierarchical network and their corresponding gene expression. 4. Transcription factors and target genes in ABA transcription factor hierarchical network separated by transcriptional levels. 5. Group model parameters per gene. 6. Genome-wide siRNA changes in mop1-1 mutant. 7. TGS2 target genes. 8. Sequence Capture (SeqCap) DNA methylation ratios in all sequence contexts. 9. Promoter DNA methylation for Mop1 wildtype ABA-responsive DEGs. 10. MOP1-ABA targets with a loss of siRNA and DNA methylation at ABRE sites. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 6, 2019. / Abscisic acid (ABA), Epigenetics, Mediator of paramutation (Mop1), RNA-dependent DNA-Methylation (RdDM), siRNAs, Zea mays / Includes bibliographical references. / Karen M. McGinnis, Professor Directing Dissertation; Hong Li, University Representative; Henry W. Bass, Committee Member; Brian P. Chadwick, Committee Member; Jonathan H. Dennis, Committee Member.