Master of Arts, The Ohio State University, 1915, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Arts, The Ohio State University, 1917, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry Program, Ohio State

This dissertation evaluates the current methods utilized in label-free bottom-up proteomics to quantify proteins and describes best practices for analyzing this type of data and applying the methodology to perform multi-omics experiments across different biological data. Chapter 1 introduces background that will be expanded upon in the later application chapters. It also introduces key concepts in proteomics that will be the groundwork of the first two chapters of this dissertation. Chapter 2 addresses the missing value problem common in peak abundance analysis by evaluating the performance of different imputation methods. The most common sources of missing values in proteomics experiments are: 1) the biology and/or technical sample preparation, 2) actual presence below the instrument's limit of detection (LOD) and 3) presence above the LOD but error in data preprocessing. This chapter presents a case of the use of hybrid left-censored missing value imputation approaches common in proteomics data. Chapter 3 is an analysis of public data with immunoprecipitations of EZH2 and SUZ12, components of the repressive PRC2 complex. A co-interaction analysis was performed, combining the results from spectral counting and peakabundance. This type of analysis is extremely powerful for isolating protein interactions shared by co-interaction partners with much higher confidence than either alone. Secondly, mining the data in this manner can generate hypothesis testing and novel validation targets, as evidenced by the results. Chapters 4 and 5 are multi-omics projects that are an amalgamation of all of the interdisciplinary techniques and informatics skills that I have accumulated over the last 5 years in the laboratory of Dr. Michael A. Freitas. Chapter 4 describes the histone methyltransferase, EZH2, and the downstream effects of mutations occurring within the active site and substrate-binding channel of the protein on histone modifications and transcription. We also (open full item for complete abstract)

Committee: Michael Freitas PhD (Advisor); Robert Baiocchi MD, PhD (Committee Member); Brandon Biesiadecki PhD (Committee Member); Kotaro Nakanishi PhD (Committee Member); Mark Parthun PhD (Committee Member) Subjects: Biochemistry; Bioinformatics

Master of Science (MS), Wright State University, 2020, Biological Sciences

Microtubule-based structures are an essential part of eukaryotic cells as they are involved in a number of processes such as phagocytosis, chromosome separation, intracellular transport, and cell motility. Transport along microtubules is accomplished by kinesin superfamily proteins (KIFs), which are motor proteins that bind cargo and use ATPase activity to move along microtubules in an anterograde fashion. One of the most common kinesin complexes is the heterotrimeric Kinesin 2 complex which is composed of KIF3 subunit dimers and the Kinesin Associated Protein 3 (KAP3). This complex is known as the KIF3 complex and functions along microtubules in cilia and flagella. A KAP3 ortholog in Schmidtea mediterranea (Smed-KAP3) was identified as a candidate gene with enriched expression in the planarian reproductive system. Whole-mount in situ hybridization analyses confirmed preferential expression of Smed-KAP3 in planarian testes. Functional analysis by RNA interference (RNAi) followed by immunofluorescence revealed loss of flagella in elongating spermatids of Smed-KAP3 knockdown animals. Additionally, Smed-KAP3(RNAi) planarians exhibited an inchworm-like movement rather than the continuous gliding seen in negative control knockdowns. The inchworm-like phenotype is indicative of dysfunctional epidermal motile cilia, which line the outside of the organism and propel planarian movement. This was confirmed by transmission electron microscopy analysis, which showed fewer and shorter epidermal cilia on Smed-KAP3(RNAi) animals. From these data we conclude that Smed-KAP3 is required for assembly and/or maintenance of planarian motile cilia and flagella. Broader implications of this research include information that can be applied towards understanding asthenozoospermia and ciliopathies caused by motile cilia disfunction.

Committee: Labib Rouhana Ph.D. (Advisor); Scott Baird Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member) Subjects: Biology; Developmental Biology

Master of Science (MS), Wright State University, 2020, Biological Sciences

The molecular processes underlying the control of external stimuli on development of the reproductive system remain to be understood. The Transient Receptor Potential superfamily of proteins (TRPs) consists of cation channels that respond to external stimuli and are abundant in the somatosensory as well as reproductive systems. Mammalian TRP-Melastatin 3 (TRPM3) channels are activated by heat and the neurosteroid pregnenolone. Here we characterize an ortholog of TRPM3 in the planarian flatworm Schmidtea mediterranea (Smed-TRPM3). Smed-TRPM3 was hypothesized to play a role in germline development due to enriched expression in the reproductive system of sexual planarians. In situ hybridization analysis revealed that Smed-TRPM3 is preferentially expressed in planarian testes and ovaries. Functional analysis by RNA interference (RNAi) revealed that Smed-TRPM3 promotes spermatogenesis, as sexual Smed-TRPM3(RNAi) planarians had fewer and less-developed testes compared to control knockdowns. Asexual Smed-TRPM3(RNAi) animals had fewer nanos+ clusters compared to controls which may indicate that the observed spermatogenesis defects are due to the loss of male germline stem cells. We hypothesize that Smed-TRPM3 responds to the hormone pregnenolone or close derivatives to ultimately regulate gonad development. Alternatively, Smed-TRPM3 may be regulating reproductive development in response to temperature.

Committee: Labib Rouhana Ph.D. (Advisor); David Goldstein Ph.D. (Committee Member); Scott Baird Ph.D. (Committee Member) Subjects: Biology; Developmental Biology

Doctor of Philosophy, Case Western Reserve University, 2020, Biochemistry

RNA binding proteins have essential roles in post-transcriptional gene regulation, evidenced by the fact that abnormal expression of these proteins is linked to many diseases. The main focus of this dissertation is the germ cell-restricted RNA binding protein DAZL (Deleted in Azoospermia Like), a member of the DAZ family (DAZ, DAZL, and BOULE). These proteins were first shown to be essential for spermatogenesis in humans and mice over two decades ago. Thus, while the biological importance of the DAZ proteins is clear, many critical questions have remained, including the identities of their direct RNA targets, how these RNAs are regulated, and why loss of this regulation results in germ cell defects. Here, we generated high-resolution, transcriptome-wide maps of DAZL-RNA interactions in mouse testes. These maps reveal Dazl binding to thousands of mRNAs, predominantly through sequence-specific interactions near the polyA tail. Using Cre-lox technology in transgenic mice and fluorescence activated cell sorting (FACS), we isolated Dazl knockout germ cells and used RNA-Seq to identify mRNAs sensitive to DAZL ablation. Intersection of the RNA-Seq and DAZL-RNA interaction datasets revealed that DAZL enhances expression of a subset of directly-bound transcripts, namely mRNAs for a network of essential cell cycle regulatory genes and a number of genes critical for spermatogenesis. We also demonstrate that DAZL's polyA-proximal binding is facilitated by interactions between PABPC1 and the mRNA polyA tail, uncovering a novel mechanism for recruitment of an RNA-binding protein to mRNA. Our observations reveal the critical function of DAZL as a master regulator of a specific mRNA program necessary for germ cell survival in mammals. Given the functional conservation of DAZ family proteins, these findings also provide important insights into the molecular basis for azoospermia in 10-15% of infertile men with Y chromosome deletions affecting the DAZ gene. Overall, the work prese (open full item for complete abstract)

Committee: Donny Licatalosi (Advisor) Subjects: Biochemistry; Molecular Biology

PHD, Kent State University, 2019, College of Arts and Sciences / School of Biomedical Sciences

Gametogenesis involves the interplay of a number of signaling processes and regulatory proteins. YWHA or 14-3-3 proteins are key regulatory proteins found in eukaryotic cells that, among other functions, are central in regulating the cell cycle. The 14-3-3 proteins form homodimers or heterodimers and act as adaptor proteins that affect protein localization, confirmation, stability, or activity. The family of 14-3-3 proteins, encoded by seven different genes, includes YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ, YWHAZ, and SFN). Transgenic mice containing LoxP sites to remove exon 2 from the 14-3-3 eta (YWHAH) and exons 3 and 4 from 14-3-3 epsilon (YWHAE) mice were used in this study to define roles for these two isoforms in germ cells during spermatogenesis and oogenesis. The transgenic mice were bred with ACTB Cre mice for global knockout, Stra8 Cre mice for the male germ cell-specific knockout, or Zp3 Cre mice for the oocyte-specific knockout. Confirmation of the transgenic genotypes was accomplished through PCR amplification using specific primers targeted to the coding region. The absence of the protein in sperm and oocytes was confirmed by using both western blot and immunohistochemical staining techniques. In vivo breeding tests and in vitro fertilization indicate that, in the absence of 14-3-3 epsilon, males are infertile. However, mice lacking 14-3-3 eta were normal and fertile. Females lacking either of the two 14-3-3 isoforms or both does not appear to alter oogenesis, oocyte maturation or fertility. Low sperm count with higher abnormal sperm was seen in 14-3-3 epsilon knockout mice. Using the Computer Assisted Semen Analysis (CASA) system, the motility of 14-3-3 epsilon knockout sperm was seen to be significantly lower compared to the control sperm. A decrease in the phosphorylation of both glycogen synthase kinase 3 (GSK3) and Protein Phosphatase1γ2 (PP1γ2), the signal enzymes essential for male fertility, were seen in sperm from 14-3-3 epsilon knockout mice, sugg (open full item for complete abstract)

Committee: Douglas Kline PhD (Advisor); Srinivasan Vijayaraghavan PhD (Advisor); Gail Fraizer PhD (Committee Member); Kristy Welshhans PhD (Committee Member); Soumitra Basu PhD (Other) Subjects: Biology; Biomedical Research; Developmental Biology; Molecular Biology; Physiology

PhD, University of Cincinnati, 2019, Medicine: Cancer and Cell Biology

Germ cell development is a remarkable story. Developing germ cells undergo large-scale, dynamic programs of gene expression that are essential for fertility, unlike any found in somatic cells. The rewiring of transcription networks takes place when germ cells enter meiosis, a critical period for preparing the gamete genome and the acquisition of totipotency following fertilization. In meiosis, it is thought that dynamic forms of germline chromatin organization support diverse forms of epigenetic programming and gene regulation, yet it is poorly understood how the spatiotemporal organization of germ cell chromatin facilitates the epigenomes and transcriptomes necessary for the next generation of life. To address this, I undertook two major research projects: The first seeks to understand the molecular mechanisms of meiotic sex chromosome inactivation, an essential event in male germ cell development, through genetic analyses of a protein network that directs epigenetic regulation of the sex chromosomes. The second project employs Next-Generation Sequencing technologies to understand how the spatiotemporal organization of male germ cell chromatin facilitates and relates to vitally important transcriptomes and epigenomes. In the first project, first published in 2016 (PMID: 27760317), I and a small team analyze meiosis in eight mouse models deficient for various DNA damage response (DDR) factors, including Fanconi anemia (FA) proteins. We reveal a network of FA and DDR proteins in which FA core factors FANCA, FANCB, and FANCC are essential for FANCD2 foci formation, whereas BRCA1 (FANCS), MDC1, and RNF8 are required for BRCA2 (FANCD1) and SLX4 (FANCP) accumulation on the sex chromosomes during meiosis. Furthermore, FA proteins modulate distinct histone marks on the sex chromosomes. Our data suggest that RNF8 integrates the FA- BRCA pathway. We reveal distinct functions for FA proteins and illuminate the male sex chromosomes as a model to dissect the function of t (open full item for complete abstract)

Committee: Satoshi Namekawa Ph.D. (Committee Chair); Paul Andreassen Ph.D. (Committee Member); Artem Barski Ph.D. (Committee Member); Chunying Du Ph.D. (Committee Member); Carolyn Price Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2019, Biochemistry

Alternative RNA processing is a critical factor in increasing transcriptome complexity during cell differentiation and tissue development. Remarkably, spermatogenic cells express more alternatively processed transcripts compared to most whole tissues. While alternative mRNA processing is known to be prevalent in the testis, the full extent of its regulation and biological impact remain unclear. Here, we describe distinct stage-specific programs of pre-mRNA alternative splicing in male germ cells, revealing that the greatest number of splicing changes occur between mitosis and meiosis. Further, we identify an essential function for the RNA binding protein, Ptbp2, in regulating stage-specific alternative splicing. Using high-throughput sequencing technologies, we demonstrate that Ptbp2 regulates the alternative splicing of over 200 genes and inhibits splicing of alternative exons by directly binding near 3' splice sites. Strikingly, Ptbp2 regulates the alternative splicing of a network of genes important for germ-Sertoli cell communication, protein transport, and mitochondria dynamics. Indeed, using ultrastructural approaches, we observed cellular protein trafficking defects and increased mitochondria fission in the absence of Ptbp2. Using polyA-seq, we revealed stage-specific regulation of alternative polyadenylation during male germ cell development. In contrast to the regulation of alternative splicing during spermatogenesis, the majority of differences in 3'-end formation occur in post-meiotic germ cells and are accompanied by significant 3'UTR shortening. Collectively, the data reveal that alternative pre-mRNA processing is highly regulated during spermatogenesis and offers important insights into the physiological functions of alternative splicing in the mammalian germline. 

Committee: Donny Licatalosi (Advisor) Subjects: Biochemistry; Molecular Biology

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein-serine kinase regulating key cellular functions. In mammals GSK3 is expressed as two isoforms, GSK3a and GSK3ß, encoded by distinct genes. The catalytic domains of the two isoforms are 98% identical. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3ß is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. That is, 25% of the total GSK3 catalytic activity appears to be sufficient to maintain normalcy in cells and tissues. We have previously shown that both GSK3 isoforms are present in bovine, primate, and mouse sperm and suggested that they may play a role in epididymal initiation and regulation of sperm motility. Using genetic approaches, here we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3a and GSK3ß are expressed at high levels in testis coincident with the onset of spermatogenesis. Mice harboring a conditional knock out of GSK3ß in developing germ cells in testis are normal and fertile. By contrast, conditional knock out of GSK3a in developing testicular germ cells results in male infertility. Mice lacking one allele each of GSK3a and GSK3ß, i.e. heterozygous for both isoforms, are fertile. Despite overlapping expression and localization of the two isoforms in testis, GSK3ß does not substitute for loss of GSK3a. GSK3a is essential and irreplaceable in testis and sperm. Loss of GSK3a impairs sperm hexokinase activity resulting in low ATP levels. Low ATP and net adenine nucleotide levels in caudal sperm lacking GSK3a resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1¿2 with known protein interactors, which occurs during sperm maturation in the epididymis, is impaired in sperm lacking GSK3a. Localization of GSK3a is predominant in the principal piece suggesting this protein kinase binds t (open full item for complete abstract)

Committee: Srinivasan Vijayaraghavan PhD (Committee Chair); Douglas Kline PhD (Committee Member); Gary Koski PhD (Committee Member); Hamza Balci PhD (Committee Member); Songping Huang PhD (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

MA, Kent State University, 2017, College of Arts and Sciences / Department of Anthropology

Macaque monkeys live in multimale-multifemale social groups, and the males exhibit some of the largest testis:body weight ratios among the primates. They are believed to experience intense levels of sperm competition. Several spermatogenesis genes are located on the Y-chromosome and, interestingly, occasional hybridization has led to the introgression of the rhesus macaque (Macaca mulatta) Y-chromosome deep into the range of the cynomolgus macaque (Macaca fascicularis). These observations have led to the hypothesis that the successful introgression of the rhesus Y-haplotype is due to selectively advantageous functional differences in sperm genes compared to those of the native cynomolgus Y-haplotype. The hypothesis is examined here at four Y-chromosomal genes. The genes were surveyed in representative animals from north of, south of, and within the rhesus x cynomolgus introgression zone. Amino acid differences were uncovered in some genes; however, statistical analyses did not detect a definite signal of positive selection. Yet, because such differences yield distinct protein folding structures, they may indeed present selective advantages to the rhesus Y-haplotype. Implications of these results are discussed, as are directions for future study.

Committee: Anthony Tosi Ph.D. (Advisor); Richard Meindl Ph.D. (Committee Member); Mary Ann Raghanti Ph.D. (Committee Member) Subjects: Biology; Evolution and Development; Genetics; Physical Anthropology

PHD, Kent State University, 2017, College of Arts and Sciences / Department of Biological Sciences

This thesis sheds light on the role of two main Ser/Thr phosphatases in sperm function: PP1¿2, and Calcineurin (PP2B). The results show for the first time that spermatogenesis in the testis can be supported by the somatic PP1 isoform PP1¿1, and that the unique male germ cell phosphatase “PP1¿2” has probably evolved in mammals along with the evolution of the mammalian epididymis for the sole purpose of supporting sperm epididymal maturation. We suggest that calcineurin has high activity in caput sperm due to the high levels of intracellular calcium. And that the effect of high calcium levels along with high phosphatase activity in caput sperm, have an inhibitory effect on sperm motility, opposing the effect of calcium on motility during sperm hyperactivation in the female reproductive tract. We propose that calcium signaling in sperm function has a biphasic role depending on which calcium dependent proteins are selectively activated at each developmental stage of the sperm journey. Calcieurin may also have a role in regulating mitochondrial activity. A shift in energy production pathways and substrate utilization occurs during sperm transition from the caput to the cauda epididymis, and calcineurin is believed to play a role in this process. With PP1¿2 shown to be irreplaceable by PP1¿1 during sperm epididymal maturation, the following step would be comparing the phosphoproteome of PP1¿2 bearing sperm with those of PP1¿1 bearing sperm. A wide phosphoproteome analysis will reveal the specific substrates of PP1¿2 that PP1¿1 are unable to dephosphorylate, highlighting proteins with an essential role in epididymal maturation and sperm motility regulation. There is a possibility that the phenotype of PP1¿1 rescue males is caused by the effect of the transgenes incorporation into a random region 145 of the mouse genome. The only way to test this hypothesis would be the generation of a new transgenic model using a gene knock-in technique, where the end (open full item for complete abstract)

Committee: Srinivasan Vijayaraghavan Dr. (Advisor); Douglas Kline Dr. (Committee Member); Jennifer Marcinkiewicz Dr. (Committee Member); Fayez Safadi Dr. (Committee Member); Soumitra Basu (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1976, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1975, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 1974, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1973, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1972, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1957, Graduate School

Committee: Not Provided (Other) Subjects: Agriculture

Doctor of Philosophy, The Ohio State University, 1957, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Master of Science, University of Toledo, 2015, Biology (Cell-Molecular Biology)

Centrosomes are highly conserved organelles that are composed of two microtubule-based centrioles surrounded by an amorphous protein cloud of pericentriolar material (PCM), which is able to nucleate astral microtubules. They serve as microtubule organizing centers during cell division and are important for fertilization. During fertilization, upon fusion with the ovum, the sperm contributes modified centrioles and a haploid set of genetic material. These modified centrioles recruit maternal PCM proteins and then form the microtubule sperm aster. These microtubules extend to find the female pronucleus and facilitate its movement towards, and eventual fusion with, the male pronucleus. This process creates a complete genome, and allows the first zygotic cell division to take place. During spermatogenesis, the centrioles undergo a variety of changes such as elongation, duplication, and separation, all of which precede centrosome reduction. This is the process which creates modified centrioles in the mature sperm to be contributed to the oocyte. During this phenomenon, the centrosome loses its astral microtubule nucleating function, PCM, and many centriolar proteins. By using a forward genetic approach, we have created a random mutagenesis screen for centriolar mutants in the testes, with the overall goal of finding those with centrosome reduction defects. We have chosen ethyl methylsulfonate (EMS) as our mutagen. Using Ana1-GFP or Asl-GFP, which label the centrioles and PCM respectively, we dissect and visualize Drosophila testes using fluorescence microscopy. In total we have examined 1436 mutants, finding many defects including those in testes morphology, centriole length, spermatid nucleus morphology, and one mutant of particular interest with Asl-GFP labeling in the mature sperm, indicating a defect in centrosome reduction.

Committee: Tomer Avidor-Reiss (Committee Chair); Deborah Chadee (Committee Member); Scott Leisner (Committee Member); John Plenefisch (Committee Member) Subjects: Biology; Cellular Biology; Developmental Biology