X chromosome inactivation (XCI), the mammalian form of dosage compensation, is a canonical example of epigenetic regulation and involves the transcriptional repression of nearly an entire chromosome (Xi) while preserving the transcriptional activity of its homologue (Xa) in females. Since the initial report describing a dense nuclear cytological feature in female feline neurons and Mary Lyon’s subsequent hypothesis of random X chromosome inactivation as a means of compensating for the lack of two X chromosomes in males, XCI has yielded decades of insights into the mechanisms of epigenetic regulation. This dissertation focuses on the three-dimensional organization of the Xi and the functional potential of large tandem repeats. The large X-linked tandem repeat, DXZ4, adopts a euchromatic conformation on the Xi in contrast to the largely heterochromatic chromosome and is able to form CTCF-dependent interactions with other euchromatic repeats exclusively on inactive X chromosome in females. We demonstrate here that DXZ4 has a critical role in maintaining the three-dimensional organization of the Xi as well as the separation of multi-megabase domains containing different types of heterochromatin. While characterizing the genomic interval of DXZ4, we uncovered transcriptional activity corresponding to two novel, long non-coding RNAs (lncRNAs) which originate on opposite sides of the DXZ4 and are transcribed antisense to one another. Both of these lncRNAs traverse the array in human embryonic stem cells (hESCs). Developmentally associated transcription suggests a potential connection between their transcription activity and maintenance of a heterochromatic DXZ4 on the Xa and male X prior to differentiation. Mouse and human genomes largely share the same gene content in accordance with Ohno’s law; however, the mouse genome has undergone rearrangements involving large syntenic blocks and has acquired several multi-megabase, lineage-specific regions of repetitive DNA that are absent in human. To further our understanding of the mouse inactive X chromosome and highlight another difference between human and mouse XCI, we characterized an approximately 20-Mb repeat that, similar to DXZ4, displays marks of euchromatin on the Xi and conversely displays marks of heterochromatin on the Xa. Overall, this work gives new insight into the function and epigenetic regulation of macrosatellites as well as the relationship between higher-order chromatin organization and heterochromatin maintenance of the Xi. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2017. / December 4, 2017. / Includes bibliographical references. / Brian P. Chadwick, Professor Directing Dissertation; Myra M. Hurt, University Representative; Karen M. McGinnis, Committee Member; Wu-Min Deng, Committee Member; Hank W. Bass, Committee Member.

The examination and processing of sexual assault evidence within the forensic science community has presented many challenges. Sexual assault evidence is often submitted as a heterogeneous mixture that requires separation of cell types for further analysis. The utilization of differential extractions provides a separation technique based on structural differences between epithelial cells (E-cells) and spermatozoa. Differential extraction does not separate cell types completely, as there may be carry over in both fractions. A protocol using several proteases was designed to separate cell types, making use of structural differences between spermatozoa and epithelial cells. The purpose of this study included optimizing the protease extraction process to produce the greatest DNA yield with focus on the following variables: concentration of enzymes, concentration of semen, (plus/ minus) addition of the ZyGEM Buffer BLUE, addition of proteases together or separately at the start of the thermal cycler program, reduction of final reaction volume, and digestion time of both enzymes. When initiated, the total process to prepare DNA from sperm was 90 minutes; this time was reduced to 45 minutes. The protocol is capable of use over a wide range of semen concentrations; a final serial dilution including 9 concentrations ranging from 1:50 to 1:3200 was prepared with DNA extracted from each concentration. For this protocol to be further utilized the epithelial cell digest optimization is also needed. An additional concern when processing sexual assault evidence is the ability to locate spermatozoa quickly and efficiently after their separation from the evidentiary substrate. Of the numerous cytological and immunohistochemical staining protocols it is important to find a quick and efficient way to fluoresce sperm heads. The current fluorescent techniques require many wash steps with long incubation times. Using digitonin, tris(2-carboxyethyl)phosphine (TCEP), and the thiol-reactive probe N-(7-Dimenthylamino-4-Methylcoumarin-3-yl)) Maleimide (DACM), a tentative protocol for fluorescently labeling sperm heads has been produced. Future work with this protocol will include optimization of the reagent concentrations, time of incubation, and sufficient control of sperm pelleting through the entirety of the procedure. / 2018-11-03T00:00:00Z

Periodontium consists of gingiva, alveolar mucosa, alveolar bone, cementum and periodontal ligament (PDL). PERIOSTIN, which is expressed in fibroblastic cells in PDL and in osteoblastic cells on the alveolar bone surface, is a key extracellular matrix protein that maintains homeostasis of periodontal tissue. During the process for FAM20C protein purification, we discovered that PERIOSTIN is co-purified with FAM20C. Immunolocalization of both FAM20C and PERIOSTIN were observed in the PDL extracellular matrix of murine periodontal tissues. PERIOSTIN bound with FAM20C in vitro. We further identified the binding domain of FAM20C in PERIOSTIN, which mapped within Fasciclin (FAS) I domain 1-4 (RD 1-4) of PERIOSTIN. PERIOSTIN has four conserved S-X-E motifs in both human and mouse FAS domain, which allowed us to use the murine model determine whether PERIOSTIN is phosphorylated by FAM20C and whether this phosphorylation might contribute to the integrity of periodontium tissues. / 2019-04-12T00:00:00Z

The increased sensitivities of recently developed polymerase chain reaction (PCR) and separation techniques have afforded forensic deoxyribonucleic acid (DNA) analysts an opportunity to detect low template deoxyribonucleic acid (LT-DNA) samples. However, with LT-DNA samples stochastic effects become more prevalent, compromising the reliability and robustness of these techniques. In addition, these innovations have presented analysts with an increased incidence of higher-order mixtures. These types of mixtures, confounded by LT-DNA effects, continue to test the interpretation step of the DNA analysis pipeline. The combination of allele drop-out, allele drop-in and allele sharing create such complex samples that it necessitates the transition from traditional, threshold-based, interpretational methods to a probabilistic approach. Therefore, this study has two objectives: 1) to optimize the computational tool NOCIt, designed to provide a probability distribution on the number of contributors (NOC) to a sample and 2) to investigate the source of the variability present in quantitative real-time polymerase chain reaction (qPCR) in hopes of minimizing variations observed when determining the quantity of an unknown DNA sample. The NOCIt graphical user interface (GUI) was validated during its developmental phase. With no current forensic guidelines for the validation of computational tools, the protocol was designed based on a guide developed by the Center for Devices and Radiological Health for the Food and Health Administration (FDA). The protocol required using a variety of test types and detailed documentation of the methods used, the inputs, outputs and results. A total of 325 tests were completed across 11 different software distributions. As a critical software system, NOCIt’s settings also had to be optimized because of its ability to substantially influence the interpretation, the statistical conclusions and the accuracy of the results. Two different settings (Condition 3 and Condition 4) were tested on AmpFlstr® Identifiler® Plus and PowerPlex® 16 HS PCR amplified samples. The differences between the two conditions were the parameter Number of Samples in Batch and the Multiplicative Factor. All other settings were kept the same. The reproducibility and accuracy were examined to determine which condition was more reliable. Both settings had similar results, with both performing better in different categories. As with interpretation, quantification is an integral part of the human identification pipeline. Previous studies have shown that the Quantifiler® recommended protocol of generating a standard curve for every quantification (referred to in this study as the Recommended Method) introduces additional sources of variability compared to protocols that utilize one, external, validated curve (referred to in this study as the Experimental Method). To identify the major source of variability inherent in the quantitative polymerase chain reaction (qPCR) process, these two methods of determining the quantity of a DNA sample were investigated using the Quantifiler® Duo and Quantifiler® Trio DNA Quantification assays. Four tenfold serial dilutions were quantified in five independent runs using the Quantifiler® Duo DNA Quantification kit and five independent runs using the Quantifiler® Trio DNA Quantification kit. Quantification with Quantifiler® Duo reported less variability using the Experimental Method than the Recommended Method. Conversely, quantification with Quantifiler® Trio exhibited approximately equal variability between both methods. To assess whether the errors associated with generating a calibration played a substantive role in introducing additional variability, the test samples were also quantified using digital polymerase chain reaction (dPCR). The data for the more dilute samples were indistinguishable from the noise associated with the instrument. The more concentrated samples showed less variability than the samples quantified with Quantifiler® Duo and approximately the same as those amplified with Quantifiler® Trio. This suggests that both qPCR and dPCR processes can be used to quantify DNA amounts, however, fundamental differences in the ways each determines the values suggests that noise is an inherent and measurable part of dPCR. Thus, for purposes of DNA quantification signal thresholds will need to be determined prior to implementation.

Alzheimer’s disease results from an accumulation of aggregated amyloid beta peptide into oligomeric forms. Soluble oligomers are neurotoxic species, which are believed to be the pathophysiological cause of Alzheimer’s neurodegeneration. Amyloid β species (Aβ) are formed via normal physiological cleavage of amyloid precursor protein by β and γ secretases. Cleaved isoforms aggregate further to form oligomeric configurations of Αβ peptide. To target toxic soluble Aβ oligomers, monoclonal antibodies have been synthesized. Experimental analysis demonstrates the ability of these antibodies to recognize synthetic and endogenous oligomers. In transgenic mice designed to overexpress oligomeric isoforms of Aβ, the antibodies were able to reduce the cerebral amyloid load with proceeding improvements in cognitive abilities. However, large-scale clinical trials corroborated results indicating diminished amyloid load, but failed to produce observable improvements in clinical outcome in patients with Alzheimer’s disease. Simply put, the removal of amyloidogenic species was insufficient in alleviating the associated neurodegeneration and elicited no improvement in cognitive ability, suggesting that Aβ might not be the responsible pathogen in Alzheimer’s. The successes of antibodies in in vitro and transgenic mice studies suggest the potential of antibodies in the treatment of Alzheimer’s, but the inability of these drugs to produce marked improvements in clinical trials questions the role of amyloid in the pathophysiology of the disease.

Late SV40 Factor (LSF) is a ubiquitously expressed mammalian transcription factor and an oncogene in Hepatocellular Carcinoma (HCC), a deadly cancer that currently has poor treatment options. LSF is expressed at low levels in normal hepatocytes but is overexpressed in HCC making it a key target for treating HCC. Small molecule inhibitors called Factor Quinolinone Inhibitors (FQIs) that specifically target LSF have been developed as possible chemotherapeutics to treat HCC. FQI1, a lead inhibitor, reduces HCC tumor sizes in mouse models without any evident toxicity in other organs. The focus of many recent studies regarding LSF has been to characterize it as an oncogene in HCC, however little is known about how it functions in normal hepatocytes. Understanding the role of LSF in hepatocytes could provide more insight into how it drives HCC as well as give more insight into how to target it in HCC. Here, FQI1 was used as a tool to uncover phenotypes in immortalized fetal hepatocytes (FH-B) that result from inhibiting the low levels of LSF expressed in this cell line. Rapid morphological phenotypes resulting from FQI1 treatment suggested a disruption in a non-transcriptional v process, which was unexpected since LSF is a transcription factor. Further investigation revealed that LSF and a-tubulin interact in immortalized human fetal hepatocytes and that this interaction at least partially disrupted upon short treatment with FQI1 in FH-B cells. These findings suggest mechanisms for non-transcriptional roles that LSF may have in addition to its roles as a transcription factor. / 2020-02-20T00:00:00Z

The Endoplasmic Reticulum (ER), which serves as a site for protein biogenesis in budding yeast, contains a quality control system that ensures that only proteins that have attained a native conformation are deployed to other destinations in the cell. In order to gain insight into the mechanisms that encompass the quality control system, two studies were conducted. First, I tested whether the host of chaperones and secretion machinery that is induced by the Unfolded Protein Response during ER stress can have a positive impact on protein biogenesis. My results indicate that degradation of misfolded proteins, rather than refolding, seems to be one of the major mechanisms activated by the Unfolded Protein Response that the cell uses to reduce the burden on the ER. Packaging of certain proteins into ER-derived vesicles seems to increase in order to counter balance the load in the ER during stress. Finally, the Unfolded Protein Response seems to play a role in the processing of proteins after the stress is removed; however this rescue does not appear to be dependent on the ER membrane expansion component of the Unfolded Protein Response but rather in other players like chaperones, ER-associated degradation and forward traffic. Second, a genome-wide screen was conducted to identify novel players involved in ER protein retention and export. For this purpose, extracellular secretion of the ER resident protein, Kar2p, was monitored in strains of the yeast gene deletion collection. We identified 73 strains in which deletion of a particular gene causes increased secretion of Kar2p. Secretion of Kar2p in some of these strains depended on an intact Unfolded Protein Response and moreover, deletion of some genes was synthetic lethal with deletion of HAC1, placing these genes as prime candidates to be involved in protein biogenesis. Further characterization of these strains revealed novel candidates involved in protein glycosylation, glycosylphosphatidylinositol-anchored protein maturation and quality control. These results represent a strong starting point to gain further insight in how the processes necessary for proper ER homeostasis are interrelated.

Accurate DNA replication is essential for proper cellular growth and requires an adequate and balanced supply of dNTPs. In Saccharomyces cerevisiae, de novo dNTP synthesis through nucleotide reduction by the Ribonucleotide reductase (RNR) enzyme is the sole method of production. Hence, RNR activitity is highly regulated via allosteric control, transcriptional control, differential localization of subunits, and direct inhibition of the large subunit, Rnr1, by Sml1. Loss of RNR regulation results in increased mutation due to an imbalance or an absolute change in the dNTP levels in cells. In this study, I describe how mutants in dNTP regulation, including sml1∆, play a role in telomere length homeostasis. Reduction in total dNTP concentration results in a modest decrease in telomere length, while altering the ratios between the four dNTPs has a much more pronounced effect. The altered telomere lengths correlate with the relative amount of dGTP and are dependent on telomerase. At reduced levels of relative dGTP, telomerase repeatedly stalls and dissociates from telomeres, thereby leading to shorter telomeres. Conversely, with elevated relative dGTP levels, telomerase is able to processively add nucleotides and even shows low levels of repeat addition processivity. The correlation between telomerase activity and dGTP is conserved in human telomerase, which shows increased repeat addition processivity at increased dGTP concentrations. Thus, telomere length homeostasis is also sensitive to dNTP regulation in the cell via a conserved dependence on dGTP. RNR regulation is, however, relaxed in the cell following DNA damage to allow for an increase in dNTP levels to repair the damage. In response to various forms of damage, Rad53 and Dun1 are activated and then phosphorylate numerous downstream targets, including the Rnr1 inhibitor Sml1. In this study, it was shown that the phosphorylation of Sml1 triggers its ubiquitylation by the Rad6-Ubr2-Mub1 ubiquitin ligase complex and subsequent degradation by the 26S proteasome. Furthermore, I was able to identify novel genes involved in the degradation of Sml1. Of the genes identified, many are involved in the spindle assembly checkpoint (SAC), cohesin establishment, and kinetochore integrity. The loss of SML1 in mutants of these genes resulted in synthetic growth defects that were not due to the loss of dNTP regulation, indicating a second dNTP-independent function for Sml1. Analysis of the double mutants revealed elevated chromosome loss and aberrant spindle dynamics, pointing to a role for Sml1 in the spindle/kinetochore. Through analysis of kinetochore assembly kinetics, Sml1 was found to be the functional human Mis18α ortholog involved in timely establishment of the kinetochore. Thus, Sml1 has a novel structural function at the kinetochore in addition to its role in dNTP regulation.

Sequencing studies performed in recent decades have revealed that the cells of most eukaryotic organisms express a diverse repertoire of small RNAs. Genetic and biochemical investigation of these molecules has demonstrated that many small RNAs, most notably the microRNA subclass, possess the capacity to influence the expression of other genes, adding substantial complexity to our understanding of genetic regulatory networks. In the years since their initial discovery, microRNAs have been implicated in nearly every aspect of metazoan biology, including medically relevant processes such as the development of mammalian immune cells and the oncogenic transformation of such cells into leukemias and lymphomas. Even in these well-studied systems, however, the function of only a small fraction of microRNAs is understood, and novel RNAs may yet remain undiscovered. Thus, in order to better define the microRNA landscape of both normal mature B cells and their oncogenic counterparts, we undertook sequencing of their small RNA transcriptomes. In addition to microRNAs, these studies unexpectedly identified a class of RNA fragments whose sequences matched to transfer RNA, but whose size distribution resembled that of microRNAs. Deep sequencing of small RNAs from a panel of normal mature B cells and B cell lymphomas reveals that these cell types express thousands of unique transfer RNA-derived fragments, with highly distinct expression profiles in each biological subtype. We hypothesized that these fragments might be derived from direct processing of tRNA but nonetheless function as microRNA, and sought to experimentally characterize one representative sequence of this class, cloned from human mature B cells and designated CU1276. The resulting data demonstrate that CU1276 does indeed possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Specifically, CU1276 represses endogenous expression of RPA1, a gene with critical functions in many aspects of DNA dynamics, including replication and repair. CU1276 is abundantly expressed in normal mature B cells but strongly downregulated in B cell-derived lymphomas, while its target, RPA1, is overexpressed in lymphomas. Furthermore, enforced expression of CU1276 in a lymphoma cell line results in an RPA1-dependent impairment of both proliferation and DNA damage repair. These results suggest that relief from CU1276-mediated repression of RPA1 may confer a selective advantage to lymphoma cells, and they shed light on a possible regulatory role for transfer RNA-derived microRNA in the in the maturation of normal B cells. Taken together with published data, these results suggest that in a broad spectrum of organisms and tissues, transfer RNAs act as a previously unrecognized substrate for the biogenesis of microRNA, with substantial implications for the future study of small RNA-mediated gene regulation.

The Mdm2-p53 axis is an important pathway in cells that is frequently misregulated in cancer. Under basal conditions, Mdm2 suppresses p53 through multiple mechanisms. However, when stress is encountered, this suppression is lifted and p53 transactivates the expression of many target genes to effect outcomes such as cell cycle arrest and apoptosis. One type of stress that can activate p53 is ribosomal stress, also called nucleolar stress. Ribosomal stress occurs when mishaps occur in ribosomal biogenesis, and various ribosomal proteins (RPs) have been shown to signal to Mdm2 and activate p53. This thesis presents two studies in the regulation of the Mdm2-p53 axis by ribosomal proteins. In the first study, three ribosomal proteins are newly linked to the Mdm2-p53 axis. RPL37, RPS15, and RPS20 are shown to bind to Mdm2, inhibit its E3 ubiquitin ligase activity towards itself and p53, upregulate various p53, and cause both G2 arrest and apoptosis. Additionally, they downregulate levels of MdmX, a homolog of Mdm2 that also suppresses p53 activity. In the second study, a novel extra-ribosomal function has been identified for RPL36A. Unlike other ribosomal proteins that interact with and activate the Mdm2-p53 axis, RPL36A represses it. RPL36A enhances the E3 ubiquitin ligase activity of Mdm2, downregulates p53 levels, and inhibits the response to ribosomal stress.