The purpose of this thesis is to investigate the link between advanced paternal ages (APA) (i.e., APA ≥ 35 years and APA ≥ 50 years) and genetic diseases and congenital anomalies. Currently, the relationship between both advanced paternal ages and genetic diseases and congenital anomalies remains unclear. However, there is room for improvement to systematically investigate the relationship between specific congenital anomalies in newborns and advanced paternal ages. More recently, the link between advanced paternal age (as opposed to existing studies analyzing advanced maternal age alone) and genetic diseases has been recognized by researchers, epidemiologists, and various health experts. Thus, this study serves to examine the effect of advanced paternal ages on the likelihood of birth defects using a new dataset intended to discover those relationships. I create three different datasets and utilize 12 statistical models to analyze the relationship between advanced paternal ages (APA ≥ 35 years and APA ≥ 50 years) (while including advanced maternal age or AMA [AMA ≥ 35 years]) and genetic diseases and congenital anomalies. I focus on Down syndrome, cleft lip with or without cleft palate, and meningocele/spina bifida and explore the relationship between both advanced parental ages. I explore whether (a) the advanced paternal ages and (b) the advanced maternal age increase the likelihood of newborn reproductive defects: (a) Down syndrome, (b) cleft lip with or without cleft palate, and (c) meningocele/spina bifida. This study includes all U.S. births between 2016 and 2019 using the CDC Natality Registry[1] database (2020). I perform the analyses using logistic regression models (to estimate odds ratios) that provide explanations of the relationship between each birth defect and advanced paternal ages. Analysis results suggest that advanced paternal ages (APA ≥ 35 years and APA ≥ 50 years) are positively associated with Down syndrome, whereas advanced paternal age (APA ≥ 35 years) is negatively associated with cleft lip with or without cleft palate. The results from the advanced paternal ages models do not suggest any causal relationship/effect on spina bifida. The results of this study are expected to offer some insight of the following reproductive defects: (a) Down syndrome, (b) cleft lip with or without cleft palate, and (c) meningocele/spina bifida. [1] Collection of data for all variables used in this research are obtained with full permission from: United States Department of Health and Human Services (US DHHS), Centers for Disease Control and Prevention" "(CDC), National Center for Health Statistics (NCHS), Division of Vital Statistics, Natality public-use data 2016-2019, on CDC" WONDER Online Database, October 2020. Accessed at http://wonder.cdc.gov/natality-expanded-current.html on Jun 6, 2021, 1:24:47 PM;" United States Department of Health and Human Services (US DHHS), Centers for Disease Control and Prevention" "(CDC), National Center for Health Statistics (NCHS), Division of Vital Statistics, Natality public-use data 2016-2019, on CDC" "WONDER Online Database, October 2020. Accessed at http://wonder.cdc.gov/natality-expanded-current.html on Jun 6, 2021, 1:29:36 PM;" And United States Department of Health and Human Services (US DHHS), Centers for Disease Control and Prevention""(CDC), National Center for Health Statistics (NCHS), Division of Vital Statistics, Natality public-use data 2016-2019, on CDC" "WONDER Online Database, October 2020. Accessed at http://wonder.cdc.gov/natality-expanded-current.html on Jun 6, 2021, 1:07:36 PM."

Recent years have seen an explosion of interest in the use of freely available DNA present in aquatic systems, otherwise known as environmental DNA (eDNA), as a tool for monitoring aquatic organisms. However, much remains unknown about the behavior of eDNA over a range of environmental conditions. This is particularly true in high gradient headwater streams, which have received less attention than other types of water bodies. In the summer of 2011, a headwater stream system with well established species distributions was sampled using eDNA techniques. Though species were detected where known to be present, detections also occurred where traditional techniques failed to detect species. This suggests that a cautious approach to positive eDNA detections is advisable. In 2012 a second study was conducted to better understand the dynamics of eDNA concentration in lotic systems. Caged brook trout (Salvelinus fontinalis) were introduced into two otherwise fishless headwater streams, and eDNA samples were collected at evenly spaced intervals downstream of the cage. This was repeated 19 times from mid-summer through autumn, over flows ranging from approximately 1 to 96 l/sec. Quantitative PCR was used to relate DNA copy number to distance from source for each of these 19 sampling events. In all cases, DNA was detectable at 239.5 m from the cage. Increasing flows generally decreased eDNA copy number near the cage but had relatively little effect at downstream sites. Additionally, the presence of leaf biomass during the fall period had the potential to completely erase otherwise high DNA levels.

In this work, we study error correcting codes and generalize the concepts with a view toward a novel application in the study of DNA sequences. The author investigates the possibility that an error correcting linear code could be included in the human genome through application and research. The author finds that while it is an accepted hypothesis that it is reasonable that some kind of error correcting code is used in DNA, no one has actually been able to identify one. The author uses the application to illustrate how the subject of coding theory can provide a teaching enrichment activity for undergraduate mathematics.

Plethodon yonahlossee is the largest eastern Plethodontid salamander. It has been classified as a species of greatest conservation need by the Tennessee Wildlife Resources Agency (TWRA). Found only in mountainous areas along the borders of Tennessee, North Carolina, and Virginia, populations of the yonahlossee are considered to be rare and local throughout their range. Genetic differentiation among populations of any species is usually attributable to long-standing, extrinsic barriers to gene flow. Because of their disjunct population structure and some observed morphological variation, genetic differentiation among yonahlossee populations is expected. A genetic structure study of yonahlossee was conducted to identify any genetically differentiated populations as conservation units. One mitochondrial DNA marker as well one nuclear DNA marker were amplified using polymerase chain reaction. After analysis, both markers show genetic differentiation suggesting geographic isolation. This information can be used by management agencies for the protection and conservation of the species.

Group II Introns are small segments of DNA that reside in the chromosome of bacteria or the organelles of primitive eukaryotes. These elements have some very interesting properties. First, they are retrotransposons that can move from one location to a new location in DNA via a reverse transcription mechanism. Second, they form a large ribozyme that mediates self-splicing of the intron from pre-mRNA. A Group II Intron type protein with similarity to reverse transcriptase was discovered in the thermophilic bacterium Geobacillus stearothermophilus strain 10 (Vellore et al., 2004, Appl. Environ. Microbiol. 70: 7140-7147). Numerous copies of the intron, designated Gs. Int1, are present in the chromosome of strain 10 but absent from a related strain ATCC 12980. Experiments to detect the in vivo splicing of intron Gs.Int1 from G. stearothermophilus cells did not work. Plasmids to that will over-express the Gs. Int1 intron to detest splicing in vivo in Escherichia coli have been constructed.

The genome of mammalian cells is under constant attack from DNA-damaging agents. To maintain genomic integrity, cells activate an array of pathways primarily consisting of DNA repair and DNA damage checkpoints. Human replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is essential for almost all DNA metabolic pathways. However, the role of RPA in nucleotide excision repair (NER), a DNA repair pathway for removing bulky DNA lesions, remains elusive. In this study, the binding of RPA to a battery of well-defined ssDNA substrates has been systematically examined using fluorescence spectroscopy. The results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA, and there was no direct contact between RPA residues and the lesion itself. These findings will help define the roles of RPA in DNA damage recognition in NER. In cells, RPA undergoes hyperphosphorylation in the N-terminus of RPA32 subunit after DNA damage. In this study, the hyperphosphorylation-induced conformational changes of RPA have been probed using mass spectrometry-based protein foot-printing, fluorescence spectroscopy and limited proteolysis. The data show that upon hyperphosphorylation RPA undergoes a subtle structural change involving its DNA-binding domain B (DBD-B), reducing its affinity for short ssDNA. These results suggest that hyperphosphorylation may modulate RPA functions by altering DBD-B-mediated RPA-DNA/protein interactions. Cellular accumulation of DNA damage has been widely implicated in premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is caused by defective maturation of lamin A and linked to accumulation of DNA double-strand breaks (DSBs). However, how lamin A dysfunction leads to genome instability and premature aging is not understood. Here evidence showed that in HGPS and RD fibroblasts DNA damage checkpoints are persistently activated and recruitment of repair factors to DSBs was impaired. Strikingly, xeroderma pigmentosum group A (XPA), a unique NER protein, formed foci and colocalized with the unrepairable DSBs in the patient cells. RNAi knockdown of XPA in HGPS cells significantly restored DSB repair. These results indicate that XPA dysfunction may play an important role in accumulating DSBs in HGPS, implicating a potential strategy for treatment of these premature aging diseases.

The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

Like many angiosperms, Brassica rapa underwent several rounds of whole genome duplication during its evolutionary history. Brassica rapa is particularly valuable for studying genome evolution because it also experienced whole genome triplication shortly after it diverged from the common ancestor it shares with Arabidopsis thaliana about 17-20 million years ago. While many B. rapa genes appear resistant to paralog retention, close to 50% of B. rapa genes have retained multiple, paralogous loci for millions of years and appear to be multi-copy tolerant. Based on previous studies, gene function may contribute to the selective pressure driving certain genes back to singleton status. It is suspected that other factors, such as gene expression patterns, also play a role in determining the fate of genes following whole genome triplication. Published RNA-seq data was used to determine if gene expression patterns influence the retention of extra gene copies. It is hypothesized that retention of genes in duplicate and triplicate is more likely if those genes are expressed in a tissue-specific manner, as opposed to being expressed globally across all tissues. This study shows that genes expressed specifically in flowers and roots in B. rapa are more resistant to fractionation than globally expressed genes following whole genome triplication. In particular, there appears to have been selection on genes expressed specifically in flower tissues to retain higher copy numbers and for all three copies to exhibit the same flower-specific expression pattern. Future research to determine if these observations in Brassica rapa are consistent with other angiosperms that have undergone recent whole genome duplication would confirm that retention of flower-specific-expressed genes is a general feature in plant genome evolution and not specific to B. rapa.

Huntington disease (HD) is a fatal neurodegenerative disease caused by CAG tract expansion in the huntingtin (HTT) gene, which results in production of mutant huntingtin (mtHTT) protein. Although mtHTT is expressed throughout life, onset of HD symptoms typically begins in mid-life, around 35 to 50 years of age. Characteristic HD symptoms include motor, cognitive, and psychiatric abnormalities. The emergence of symptoms in adulthood suggests that aging may play a role in HD pathogenesis. Furthermore, markers of accelerated aging can be observed in HD patients, including telomere attrition, epigenetic alterations, and mitochondrial dysfunction. Our lab has previously observed that induction of age-like changes by treatment with progerin, the mutant protein that causes Hutchinson-Gilford Progeria Syndrome, enhances HD phenotypes and contributes to pathogenesis in HD neurons. Taken together, these findings suggest a link between aging and HD, with implications for potential therapeutic benefits from anti-aging treatment. Our lab has conducted a young blood anti-aging trial in which aged HD and wild-type (WT) mice were injected with plasma from young WT or HD mice. Previous work in our lab confirmed that cortical aging markers decline with age at the protein level and are differentially affected by young blood treatment. In this study, we observed a significant effect of age on striatal expression of aging markers, Grin1 and Lmnb1. Varying effects of young blood anti-aging treatment were observed on the genes of interest.

Chromosomal structure variations (SV) including insertions, deletions, inversions, and translocations occur within the genome and can have a significant effect on organismalphenotype. Some of these effects are caused by structural variations containing genes. Modern sequencing using short reads makes the detection of large structural variations (> 1kb) very difficult. Large structural variations represent a significant amount of the genetic diversity within a population. We used a global sampling of Drosophila melanogaster (Ithaca, Zimbabwe, Beijing, Tasmania, and Netherlands) to represent diverse populations. We used long-read sequencing and optical mapping technologies to identify SVs in these genomes. Because the average read length used for these approaches are much longer than traditional short read sequencing, these maps facilitate the identification of chromosomal SVs of greater size and with more clarity. We found a wide diversity of structural variations in each of the five strains. These structural variations varied greatly in size and location, and significantly affected exonic regions of the genome. Structural variations accounted for a much larger difference in number of base pairs between strains than single nucleotide polymorphisms (SNPs).