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It is routine for the forensic scientist to obtain a genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. In contrast, only a limited number of laboratories in the United States have the capability of performing Y-STR analysis in casework. In order to aid in facilitating the transfer of Y-STR technology to the crime laboratory community for operational use, a comparison between commercial products from three main vendors (Applied Biosystems AmpFLSTR Yfiler PCR Amplification Kit, Promega PowerPlex - Y System, Reliagene Y-PLEX 12) and two in-house Y-STR multiplexes (MPI and MPB) commenced. The main intention for this comparison was to ascertain whether commercial Y-STR kits are able to obtain a male profile from difficult samples which have been accomplished with our in-house Y-STR multiplexes; such as mixtures, post coital specimens, and environmental insults. To aid the crime laboratory community an in depth comparison of the three main commercial Y-STR kits began in hopes to glean information in circumstances where Y chromosome polymorphisms may need to be employed. For example, the ability to provide investigators with the numbers of semen donors in multiple rape cases, identification of the genetic profile of the male component in a male/female mixture, and identification of the genetic profile of the male component in an extended interval post-coital sample. The capability of typing Y-STR loci by the crime laboratory community could dramatically affect the admissibility of Y-STR evidence. Therefore, the comparison of commercially available kits is an imperative process by which the scientific community acquires the necessary information to assess the ability of a procedure to obtain reliable results, determine the conditions under which such results can be obtained and define the limitations of the procedure. Thus the information for the study could lend itself to a standard being established amongst Y-STR kits for operational use and/or the production of a new Y-STR kit. One example of how the comparison of the three main commercial Y-STR kits could directly impact a new standard being established is by examining post-coital samples and their extreme limits (>48 hrs) for each kit in which a full male genetic profile was observed and comparing it to other commercial Y-STR kit and in-house Y-STR multiplexes. This would help establish the types of cases where specific Y-STR kits would be most useful, and the parameters in which each kit is able to perform. Thus leading to the development of a highly sensitive Y-STR kit that would be more sufficient to perform with the variety of samples an operational crime laboratory would routinely analyze. The capability of typing Y-STR loci by the crime laboratory community could dramatically affect the admissibility of Y-STR evidence. Therefore, the comparison of commercially available kits is an imperative process in order to inform the forensic community of different Y-STR kits available and their performance through direct comparison using modified SWGDAM validation guidelines.

Meiosis is a specialized type of cell division characterized by a single round of DNA replication and two rounds of chromosome segregation, ultimately resulting in four haploid cells. During meiosis I, chromosomes align and reciprocal recombination results in the formation of a crossover, creating the tension required to properly segregate homologs during the first round of meiosis. Two mechanisms involved in regulating the occurrence of crossing over are assurance and interference. Crossover assurance describes the phenomenon that at least one crossover will form between each pair of homologous chromosomes during prophase I. Crossover interference, on the other hand, describes the nonrandom placement of crossovers between homologs, increasing the probability that a second crossover will occur at a discrete distance away from the first one. In addition to assurance and interference, chromosome size may play a role in the rate of meiotic recombination during prophase I. As a result of crossover assurance, small chromosomes receive a minimum of one crossover, the obligate crossover. Assuming chromosome size does not influence the rate of recombination, pairs of large chromosomes should experience the same number of crossovers per base pair as small chromosomes. Previous studies have been inconsistent: Kaback et al. (1999) saw decreased rates of crossing over between large chromosomes relative to small ones, suggesting that crossover interference acts across a larger distance on large chromosomes. Turney et al. (2004), however, saw no such effect, suggesting that these findings may be site- or sequence-specific. The current study used the Cre-loxP system to create translocated chromosomes, decreasing the size of chromosome VIII from 562 kb to 125 kb. The rate of crossing over was evaluated using nutrient marker genes that were inserted on the left arm of chromosome VIII to facilitate phenotypic detection of crossing over between homologous translocated chromosomes in comparison to crossing over between homologous nontranslocated chromosomes. Translocated strains were attempted, though further testing suggests that the translocation itself may be lethal. In the future, we plan to further investigate the potential lethal nature of the translocation. We also experienced difficulty in curing yeast cells of the Cre expression plasmid: as pSH47 was removed, translocated chromosomes reverted to nontranslocated chromosomes. In addition, crossing over in nontranslocated yeast, along with subsequent molecular analysis, revealed that one of the marker genes presumed to be on the left arm of chromosome VIII is, in fact, located on a different chromosome, preventing analysis of crossing over in this region. As a result, we were unable to proceed with current experimentation.

This dissertation unravels intricate biological dynamics in three distinct biological systems as the following. These studies combine mathematical models with experimental data to enhance our understanding of these complex processes. 1. Bipolar Spindle Assembly: Mitosis relies on the formation of a bipolar mitotic spindle, which ensures an even distribution of duplicated chromosomes to daughter cells. We address the issue of how the spindle can robustly recover bipolarity from the irregular forms caused by centrosome defects/perturbations. By developing a biophysical model based on experimental data, we uncover the mechanisms that guide the separation and/or clustering of centrosomes. Our model identifies key biophysical factors that play a critical role in achieving robust spindle bipolarization, when centrosomes initially organize a monopolar or multipolar spindle. These factors encompass force fluctuations between centrosomes, balance between repulsive and attractive inter-centrosomal forces, centrosome exclusion from the cell center, proper cell size and geometry, and limitation of the centrosome number. 2. Chromosome Oscillation: During mitotic metaphase, chromosomes align at the spindle equator in preparation for segregation, and form the metaphase plate. However, these chromosomes are not static; they exhibit continuous oscillations around the spindle equator. Notably, either increasing or decreasing centromeric stiffness in PtK1 cells can lead to prolonged metaphase chromosome oscillations. To understand this biphasic relationship, we employ a force-balance model to reveal how oscillation arises in the spindle, and how the amplitude and period of chromosome oscillations depend on the biological properties of spindle components, including centromeric stiffness. 3. Pattern Formation in Bacterial-Phage Systems: The coexistence of bacteriophages (phages) and their host bacteria is essential for maintaining microbial communities. In resource-limited environments, mobile bacteria actively move toward nutrient-rich areas, while phages, lacking mobility, infect these motile bacterial hosts and disperse spatially through them. We utilize a combination of experimental methods and mathematical modeling to explore the coexistence and co-propagation of lytic phages and their mobile host bacteria. Our mathematical model highlights the role of local nutrient depletion in shaping a sector-shaped lysis pattern in the 2D phage-bacteria system. Our model further shows that this pattern, characterized by straight radial boundaries, is a distinctive indicator of extended coexistence and co-propagation of bacteria and phages. Such patterns rely on a delicate balance among the intrinsic biological characteristics of phages and bacteria, which have likely arisen from the coevolution of cognate pairs of phages and bacteria. / Doctor of Philosophy / Mathematical modeling is a powerful tool for studying intricate biological dynamics, as modeling can provide a comprehensive and coherent picture about the system of interest that facilitates our understanding, and can provide ways to probe the system that are otherwise impossible through experiments. This dissertation includes three studies of biological dynamics using mathematical modeling: 1. Bipolar Spindle Assembly: Mitotic spindle is a bipolar subcellular structure that self-assembles during cell division. The spindle ensures an even distribution of duplicated chromosomes into two daughter cells. Certain perturbations can cause the spindle to assemble abnormally with one pole or more than two poles, which would cause the daughter cells to inherit incorrect number of chromosomes and die from the error. However, the cell is surprisingly good at correcting these spindle abnormalities and recovering the bipolar spindle. Here we build a model to explore how the cell achieves such recoveries and preferentially form a bipolar spindle to rescue itself. 2. Chromosome Oscillation: In mitotic metaphase, chromosomes are aligned at the spindle equator before they segregate. Interestingly, unlike the cartoon images in textbooks, the aligned chromosomes often move rhythmically around the spindle equator. We used a mathematical model to unravel how the chromosome oscillation arises and how it depends on the biological properties of the spindle components, such as stiffness of the centromere, the structure that connects the two halves of duplicated chromosomes. 3. Pattern Formation in Bacterial-Phage System: Phages are viruses that hijack their host bacteria for proliferation and spreading. In this study we developed a mathematical model to elucidate a common lysis pattern that forms when expanding host bacterial colony encounters phages. Interestingly, our model revealed that such a lysis pattern is a telltale sign that the bacterium-phage pair have achieved a delicate balance between each other and are capable of spreading together over a long period of time.

Recent advances in whole genome sequencing have led to many discoveries in the mechanisms involved in carcinogenesis. Genomic characterization of premalignant lesions in numerous cancers has led to new prevention strategies, early detection, and treatment options that have saved lives and improved the quality of life for the people suffering from these cancers. Prostate cancer (PCa) is the most common cancer in men in the United States (US) and the second leading cause of death in men from all cancers. However, in African American men (AAM) the mortality rate from PCa is 2.4 times higher than European American men (EAM). In addition, AAM are more likely to get PCa and have a higher PCa burden at diagnosis than their EAM counterparts. This may suggest that there are racial/ethnic differences in the mechanism of carcinogenesis in PCa. PCa and its premalignant lesion, high grade prostatic intraepithelial neoplasia (HGPIN), are one of the most heterogeneous and complex cancers for scientists to determine the mechanisms of carcinogenesis. Due to this complexity, research on HGPIN and PCa has been difficult to carry out and interpret. Projects have been undertaken and progress has been made in the discovery of some genes involved in PCa and potential drivers of initiation, progression and aggressiveness of PCa. However, these studies have mostly been conducted among EAM and have little ethnic diversity. Discovery of new prevention, early detection and treatment methods for PCa will not be possible without advances in the genetic characterization of the pathways of carcinogenesis of PCa among ethnically diverse study populations. To date there are no known genetic characterizations of HGPIN and PCa in AAM. This study aims to characterize chromosomal copy number aberrations (CNA) in paired HGPIN and PCa in AAM. By utilizing advanced microarray techniques, we will determine the degree to which HGPIN and PCa share CNA and identify CNA that may be involved in PCa progression. This study will lay the foundation for future research into CNA that may be used as potential biomarkers for early detection of neoplasms of high-risk for development into PCa in AAM. The discovery of biomarkers and the characterization of the mechanisms involved in PCa progression may lead to treatment options for the prevention of PCa and an overall better outcome for AAM suffering from PCa.

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