abstract: Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by out-of-frame mutations in the dystrophin gene, and the absence of a functional dystrophin protein ultimately leads to instability of the sarcolemma, skeletal muscle necrosis, and atrophy. While the structural changes that occur in dystrophic muscle are well characterized, resulting changes in muscle-specific gene expression that take place in dystrophin’s absence remain largely uncharacterized, as they are potentially obscured by the characteristic chronic inflammation in dystrophin deficient muscle. The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression. I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

Tissue engineering is one of the fastest growing biotechnology industries. The overarching research goals of the tissue engineering industry are highly intertwined with the field of medicine. The tissue engineering field seeks to create a variety of technologies that can be used in a clinical setting for tissue repair therapy. These technologies include everything from cell-based trophic factor therapies to engineered tissue grafts, and potentially engineered tissues. At the forefront of tissue engineering research is the extensive use of adult stem cells. Adult stem cells are located throughout the body and have the ability to differentiate into a variety of different tissue types. This plasticity enables adult stem cells to be used in a wide range of tissue engineering research. Despite all of the advancements made in the tissue engineering field, some obstacles still need to be overcome before tissue engineering can be used extensively as a clinical therapy for tissue damage and repair. One of the largest obstacles is the creation of tissue engineering technologies that mimic the existing human body conditions as close as possible. The creation of fully biomimetic tissue engineering technologies will increase the quality and functionality of the engineered grafts and therapies. The research presented will discuss the development and implementation of a biomimetic perfusion bioreactor. The reactor was designed to incorporate a number of environmental factors that play a large role in controlling cellular behaviors into a culture system capable of supporting the growth of a human mesenchymal stem cell construct. Furthermore, the reactor was constructed to allow the tissue construct to be studied in a non-invasive manner. This characteristic provides a way to keep the integrity of the cell construct throughout the experimentation process so it remains functional and viable until it is potentially needed for tissue therapy. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2009. / July 2, 2009. / MRI, 3-D tissue construct, NMR, perfusion bioreactor, stem cells / Includes bibliographical references. / Teng Ma, Professor Directing Dissertation; Kenneth Goldsby, Outside Committee Member; Timothy Logan, Committee Member; Samuel Grant, Committee Member; Hong Li, Committee Member.

Oligosaccharides have various fundamental roles, ranging from providing nutritional and flavoring support to being involved in fertilization and parasite infection processes. Maltooligosaccharides are used heavily in the food and pharmaceutical industries, cellooligosaccharides have shown promise as prebiotic candidates, and cyclodextrins are potential candidates for drug delivery. Factors such as degree of polymerization and hydrogen bonding play key roles in the aforementioned processes and phenomena. Other factors such as anomeric configuration have been shown to influence bacterial binding and docking, while the glycosidic linkage is known to be a factor in binding processes such as those related to the interspecies spreading of avian influenza virus. Our group has pioneered the use of size-exclusion chromatography (SEC) in investigating solution conformational entropy (∆S) of select O-linked disaccharides and their monosaccharide constituents. I recently expanded the scope of these investigations to quantitate the ∆S of several homologous series of oligosaccharides, malto- and cellooligosaccharides and cyclodextrins. By using SEC and appropriate selection of hydrogen-bond accepting or non hydrogen-bond accepting solvents, I was able to isolate and quantitate the individual contributions of degree of polymerization, anomeric configuration, linearity versus cyclicity, and hydrogen bonding to the solution conformational entropy of the oligosaccharides. Due to the importance of oligosaccharides in biomolecular recognition phenomena, the next step was to study oligosaccharides at quasi-physiological conditions, i.e., in an aqueous solvent, at 37° C, and at a pH of 7.39. In the final step of the research I isolated the effects of glycosidic linkage on solution conformational entropy. To this effect, I compared homologous series of α-(1→6) and β-(1→3) oligomers, individually, to their α-(1→ 4) and β-(1→4) counterparts, respectively. The α-(1→6) oligomers were the isomaltosides with DP 2-7, which were compared to the maltooligosaccharides. The β-(1→3) series examined were the laminarbiosides with DP 2-7, compared to the previously studied cellooligosaccharides. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2009. / February 27, 2009. / Size-exclusion Chromatography, Conformational Entropy, Oligosaccharides / Includes bibliographical references. / André M. Striegel, Professor Directing Dissertation; Emmanuel Collins, Outside Committee Member; John Dorsey, Committee Member; Hugh Nymeyer, Committee Member.

p53 protein is considered a major player in maintaining the genomic integrity. It regulates cell cycle, recognizes damaged DNA, and promotes apoptosis of cells that are defective or developmentally programmed for removal. Most cancers, including breast cancer, lose genomic integrity and have impaired p53. Li-Fraumeni patients and BALB/c mice bearing germline mutations in one allele of the p53 tumor suppressor gene develop mammary tumors. The p53 pathway is also required for the protective effect of pregnancy on breast cancer. Therefore, identifying the cellular pathways that regulate p53 will provide improvements in assessing breast cancer risk in individuals as well as therapeutic targets. The function of p53 could be regulated directly by Mdm2 and Mdm4. While Mdm2 is clearly a negative regulator of p53, the roles of Mdm4 on p53 are still not fully defined. Transgenic mice which over-express Mdm4 specifically in mammary gland were used to investigate the effects of Mdm4 on p53 function. Ovarian hormones also regulate p53 activity, but through indirect mechanisms. Oligonucleotide-based transcriptional profiling was conducted to identify mechanisms, by which estrogen and progesterone enhance p53 activity in mammary epithelial cells. The results from these studies showed that Mdm4 is unlikely to be a primary regulator of p53 functions in mammary epithelium. In contrast, expression profiling revealed groups of genes that are associated with the sensitization of p53 by estrogen and progesterone as well as tamoxifen and progesterone. These results implicate a common pathway used to sensitize p53 which involves proteins in the extracellular matrix.

Onchocerciasis, or African River blindness caused by the filarial parasite Onchocerca volvulus is the second leading cause of infectious blindness world-wide, with over 18 million people infected. The parasite has a complex life cycle with developmental stages in both the black fly vector (L1, L2 and L3) and human host (infective L3, L4, adult, microfilaria). The parasite is protected by an external exoskeleton called the cuticle, which is a complex structure composed primarily of two classes of proteins: collagens and cuticlins. Of these two types of proteins, only the cuticlins are present in the parasite and not in the human host. Because the cuticle defines the host-parasite interface, characterization of these molecules is important in understanding the first line of parasite defense. More specifically, the composition of the O. volvulus cuticle during the dynamic process of the L3 to L4 molt may elucidate a mechanism for targeting the disruption of the infection cycle. Expressed sequence tag (EST) analysis of an O. volvulus molting L3 cDNA library reveals three distinct cuticlin gene family members present in the River Blindness Genome Project dataset. Although the three genes are most abundantly expressed in the molting L3 stage, PCR analysis indicates that they are differentially expressed in other O. volvulus life cycle stage cDNA libraries (L2, L3, adult female, adult male and microfilaria). Genomic copies of these three cuticlin gene family members have been obtained and the intron-exon structure determined for each gene. The genes contain between 5 and 9 exons ranging in size from 72 to 647 base pairs. The intron sizes of the three genes range from 84–1660 base pairs, with some of the largest O. volvulus introns represented. The cDNA copies of each gene, which range in size from 42–48 kDA, have been cloned in pieces (12–15 kDA) into a plasmid expression vector, and the highly insoluble protein fragments have been expressed. The proteins have been used for antibody production and patient serum ELISAs using both infected and putatively immune sera. Additionally, the proteins were identified in O. volvulus worms using immuno-light microscopy and immuno-gold electron microscopy localization.

Programmed cell death is essential for normal development and adult tissue homeostasis in almost all multicelluar organisms. Acheron gene was first isolated from the intersegmental muscles (ISMs) in Manduca sexta as a death-associated gene. Subsequently, we cloned human and mouse homolog of Acheron. Acheron encodes a novel protein that has not been previously characterized. Protein structure analysis revealed that Acheron proteins are structurally related to La proteins, but define a novel subfamily. Tissue expression analysis showed that mAcheron is widely expressed in most tissues at both the RNA and protein levels, with brain and heart displaying the highest levels. In mouse C2C12 cells, endogenous Acheron is constitutively expressed in cycling myoblasts and myotubes. Despite the presence of a putative nuclear localization site, the protein is localized predominantly in the cytoplasm. Analyses of the different Acheron transfected C2C12 cells suggested that Acheron is implicated in mediating differentiation and apoptosis in C2C12 cells by differentially regulating the expression of MyoD, Myf5 and Bcl-2. Acheron expression allows C2 C12 cells to up-regulate MyoD and differentiate into myotubes when the cells are induced to undergo differentiation. However, it does not support the myoblast self-renewal by specifically inhibiting the expression of Bcl-2, a key survival factor for ‘reserve’ cells in DM. Inhibition of Acheron activity by tAch (a putative dominant negative regulatory factor of Acheron) or antisense Acheron results in greatly increased ‘reserve’ cell population and decreased differentiation under differentiation condition. The mediation of differentiation and survival by Acheron may be achieved through its regulation on integrin—FAK signaling. To help determine how Acheron functions, we performed a yeast 2-hybrid screen with Acheron as the bait. A clone that contains partial cDNA of Ariadne was isolated from the screen. Ariadne contains RING finger domain and is known to bind to ubiquitin E2 conjugase. In vitro ubiquitination assay revealed that Ariadne has ubiquitin E3 ligase activity. We speculate that Ariadne may function as an E3 to target Acheron for ubiquitination and subsequent proteasome-dependent degradation.

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) are associated with chronic lung infections and are responsible for much of the mortality in CF. Little is known about interactions between these two, often co-infecting, species. When in competition, it is not known whether Bcc replaces the resident Pa or if the two species co-exist in the CF lung. Bacteriocins are potent toxins produced by bacteria. They have a quite narrow killing range in comparison to antibiotics and have been implicated in intra-specific and inter-specific bacterial competition brought on by limited nutrients or niche space. Both Pa and Bcc produce bacteriocins known as pyocins and cepaciacins, respectively. More than 90% of Pa strains examined to date produce one or more of three pyocin types: R, F, and S. A limited number of phenotypic surveys suggest that approximately 30% of Bcc also produce bacteriocins. The goals of my thesis study were to determine if clinical strains of Pa and Bcc produce bacteriocins and to determine whether these toxins play a role in mediating intra- and inter-specific bacterial interactions in the CF lung. The final goal was to identify novel bacteriocins from clinical Pa and Bcc strains. First, I designed a phenotypic bacteriocin survey to evaluate bacteriocin production in 66 clinical Pa (38) and Bcc (28) strains procured from CF patients. This study revealed that 97% of Pa strains and 68% of Bcc strains produce bacteriocin-like inhibitory activity. Further phenotypic and molecular based assays showed that the source of inhibition is different for Pa and Bcc. In Pa, much of the inhibitory activity is due to the well known S- and RF-type pyocins. S- and RF pyocins were the source of within species inhibitory activity while RF pyocins were primarily implicated in the between species inhibitory activity of Pa strains. In contrast, Bcc inhibition appeared to be due to novel inhibitory agents. Finally, I constructed genome libraries of B. multivorans, B. dolosa, and B. cenocepacia to screen for genes responsible for the inhibitory activity previously described in Bcc. ∼10,000 clones/genome were screened, resulting in fifteen clones with the anticipated inhibition phenotype. Of these fifteen, only five clones had stable inhibitory activity. These clones encoded proteins involved in various metabolic pathways including bacterial apoptosis, amino acid biosynthesis, sugar metabolism, and degradation of aromatic compounds. Surprisingly, none of Bcc clones possessed typical bacteriocin-like genes. These data suggest that, in contrast to all bacterial species examined in a similar fashion to date, Bcc may not produce bacteriocins. Instead, Bcc may be using novel molecular strategies to mediate intra- and inter-specific bacterial interactions.

Loss of p53 function can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53 +/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c-Trp53 +/−females, but are rare in C57BL/6-Trp53 +/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) is significantly more common among tumors arising in BALB/c-Trp53+/− mice compared to C57BL/6J-Trp53+/− mice. This increased LOH in BALB/c-Trp53+/− tumors was shown to be due to recombination events. The BALB/c strain has been shown to have a deficiency in non-homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis was that BALB/c-Trp53 +/− mice are more susceptible to mammary tumors due to impaired Homologous Recombination Repair (HRR) leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c-Trp53 +/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J- Trp53+/− MEFs. Similarly, co-localization of H2AX and the homologous recombination protein RAD51 remain at dsbs longer in BALB/c-Trp53+/− MEFs compared to C57BL/6-Trp53+/− MEFs. Palb2 , a gene that lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer, was chosen as an initial candidate gene. No coding SNPs or expression differences of Palb2 were found in the mammary glands between the two strains. Additional fine mapping and use of a filtering criteria in the SuprMam1 region yielded an additional 34 candidate genes. We demonstrate no significant differences in any of these genes in whole mammary glands and primary mammary epithelial cells between the two strains. Finally, using a congenic mouse strain, we demonstrate the lack of irradiation (IR) sensitivity alleles within the SuprMam1 region. These results suggest a possible defect in HRR in the BALB/c strain that is unlikely related to Palb2. The gene or genes responsible for increased mammary tumor incidence in the BALB/c-Trp53+/− remain to be identified.

Transcription regulation in eukaryotes is a complex process governed by the concerted action of different factors. The work in this thesis is focused on transcriptional regulation in Saccharomyces cerevisiae. I analyzed the regulation of the phospholipid biosynthetic gene INO1 , which has been a model gene for transcription studies for over three decades. Some major questions that I have addressed are: what kinds of cis regulatory sequences and trans factors are important for regulation of INO1? What is the sequence of events in this regulation? How is the recruitment of these trans factors consequential for INO1 transcription? I present my results here for the role of the basic helix loop helix transcription factor (bHLH) family in coordinated regulation of INO1 transcription. I report that the centromeric binding factor 1 (Cbf1p) together with two other members of the bHLH protein family, Ino2p and Ino4p, are required for efficient derepression of INO1 transcription. Together these bHLH transcription factors recruit the ISW2 chromatin-remodeling complex onto the INO1 promoter to drive productive transcription from the INO1 locus. My efforts in studying the regulation of INO1 led me to study the regulation of SNA3, a gene found in tandem upstream (→→) to the INO1 gene and regulated by the same environmental conditions as INO1. Studies on the mechanism of coregulation of adjacent genes in budding yeast have been largely speculative. I provide evidence that the same bHLH proteins which regulate INO1 also regulate SNA3, albeit differentially. Significantly, my results also show that the regulation of both SNA3 and INO1 is dictated from the intergenic region between the two genes. This is a novel mechanism of transcription regulation in yeast as regulation from downstream of ORF is unknown in yeast. Thus, my results with both SNA3 and INO1 provide novel details on how the process of transcription is regulated in response to an environmental cue.

Runx1 regulates the expression of several important target genes and plays critical roles in the process of hematopoiesis. Runx1 by itself is a poor regulator of transcription and instead nucleates transcription complexes through its C-terminus to transactivate or repress the expression of target genes. We generated a C-terminally deleted Runx1 construct (Runx1.d190), which lacks important co-factor sites, to further investigate the function of Runx1 in development. A potential role for Runx1 in regulating the expression of another potent transcriptional regulator, c-Myc, has been suggested by published studies, which show that Runx1 and c-Myc collaborate in oncogenesis. In these studies, we show that endogenous Runx1 binds to three Runx consensus sites upstream of the c-Myc transcriptional start site in Jurkat T cells and murine primary splenocytes. Retroviral transduction of Jurkat T cells with Runx1.d190 results in the increased transcription of c-Myc as determined by microarray analysis. In order to monitor c-Myc expression in response to early-acting and transient Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Treatment of murine primary splenocytes with TAT-Runx1.d190 protein results in a transient increase in the transcription of c-Myc and a corresponding increase in c-Myc protein levels. This effect is dependent on the ability of Runx1.d190 to bind to DNA. These data demonstrate that Runx1 directly regulates c-Myc expression in a C-terminally and DNA-binding dependent manner. In these studies, we also investigate the effects of the truncation of Runx1 C-terminus on hematopoietic stem cells (HSCs). We found that treatment of bone marrow cells enriched for HSCs with TAT-Runx1.d190 results in a 12.5 fold increase in hematopoietic precursors compared to untreated precursors in vitro as determined by Colony Forming Cell assays. We also show that hematopoietic precursors treated with TAT-Runx1.d190 are able to able to differentiate normally both in vitro and in vivo and thus represent functional hematopoietic precursors. Our findings show that we are able to transiently expand hematopoietic precursors ex vivo by treating the cells with a Runx1 construct that lacks the C-terminus. Collectively, this work demonstrates that Runx1 regulates the expression of c-Myc and the expansion of hematopoietic precursors in a C-terminally dependent manner.