Indiana University-Purdue University Indianapolis (IUPUI) / This thesis is aimed at creating a pipeline for analyzing DNA methylation epigenetics data and creating a data model structured well enough to store the analysis results of the pipeline. In addition to storing the results, the model is also designed to hold information which will help researchers to decipher a meaningful epigenetics sense from the results made available. Current major epigenetics resources such as PubMeth, MethyCancer, MethDB and NCBI’s Epigenomics database fail to provide holistic view of epigenetics. They provide datasets produced from different analysis techniques which raises an important issue of data integration. The resources also fail to include numerous factors defining the epigenetic nature of a gene. Some of the resources are also struggling to keep the data stored in their databases up-to-date. This has diminished their validity and coverage of epigenetics data. In this thesis we have tackled a major branch of epigenetics: DNA methylation. As a case study to prove the effectiveness of our pipeline, we have used stage-wise DNA methylation and expression raw data for Lung adenocarcinoma (LUAD) from TCGA data repository. The pipeline helped us to identify progressive methylation patterns across different stages of LUAD. It also identified some key targets which have a potential for being a drug target. Along with the results from methylation data analysis pipeline we combined data from various online data reserves such as KEGG database, GO database, UCSC database and BioGRID database which helped us to overcome the shortcomings of existing data collections and present a resource as complete solution for studying DNA methylation epigenetics data.

Indiana University-Purdue University Indianapolis (IUPUI) / Solid organ transplantation is limited by available donor allografts. Pig to human transplantation, xenotransplantation, could potentially solve this problem if physiologic and immunologic incompatibilities are overcome. Genetic modifications of pigs have proven valuable in the study of xenotransplantation by improving pig to human compatibility. More genetic targets must be identified for clinical success. First, this study examines platelet homeostasis incompatibilities leading to acute thrombocytopenia in liver xenotransplantation. Mechanisms for xenogeneic thrombocytopenia were evaluated using liver macrophages, Kupffer cells, leading to identification of CD18, beta-2 integrin, as a potential target for modification. When disruption of CD18 was accomplished, human platelet binding and clearance by pig Kupffer cells was inhibited. Further, human and pig platelet surface carbohydrates were examined demonstrating significant differences in carbohydrates known to be involved with platelet homeostasis. Carbohydrate recognition domains of receptors responsible for platelet clearance Macrophage antigen complex-1 (CD11b/CD18) and Asialoglycoprotein receptor 1 in pigs were found to be different from those in humans, further supporting the involvement of platelet surface carbohydrate differences in xenogeneic thrombocytopenia. Second, immunologic incompatibilities due to antibody recognition of antigens resulting in antibody-mediated rejection were studied. Identification of relevant targets was systematically approached through evaluation of a known xenoantigenic protein fibronectin from genetically modified pigs. N-Glycolylneuraminic acid, a sialic acid not found in humans, was expressed on pig fibronectin and was identified as an antigenic epitope recognized by human IgG. These studies have provided further insight into xenogeneic thrombocytopenia and antibody-mediated rejection, and have identified potential targets to improve pig to human transplant compatibility.

Indiana University-Purdue University Indianapolis (IUPUI) / Sequestration at the aggresome and degradation through autophagy are two approaches by which a cell can counteract the toxic effect of misfolded proteins. Tuberous sclerosis (TS) and cancer cells can become dependent on autophagy for survival due to the high demand for protein synthesis, thus making protein clearance a potential therapeutic target. Because of its histone deacetylase (HDAC) inhibitory activity, we hypothesized that 4-phenylbutyrate (4-PBA) inhibits HDAC6 and aggresome formation to induce TS cell death. We found that 4-PBA treatment increases cell death and reduces bortezomib-induced aggresome formation. To link these results with HDAC inhibition we used two other HDAC inhibitors, trichostatin A (TSA) and tubastatin, and found that they also reduce bortezomib-induced protein aggregation. Because tubulin is a target of HDAC6, we next measured the effect of the HDAC inhibitors and 4-PBA treatment on tubulin acetylation. As expected, tubastatin increased tubulin acetylation but surprisingly TSA and 4-PBA did not. Because 4-PBA did not significantly inhibit HDAC6, we next hypothesized that 4-PBA was alternatively inducing autophagy and increasing aggresome clearance. Surprisingly, autophagy inhibition did not prevent the 4-PBA-induced reduction in protein aggregation. In conclusion, we found 4-PBA to induce cell death and reduce aggresome levels in TS cells, but we found no link between these phenomena. We next hypothesized that loss of the Ral guanine nucleotide exchange factor Rgl2 induces cell death via autophagy inhibition in pancreatic adenocarcinoma (PDAC) cells. KRas is mutationally activated in over 90% of PDACs and directly activates Rgl2. Rgl2 activates RalB, a known regulator of autophagy, and Rgl2 has been shown to promote PDAC cell survival. We first confirmed that loss of Rgl2 does increase cell death in PDAC cells. Initial experiments using doubly tagged fluorescent p62 and LC3 (autophagy markers) suggested that loss of Rgl2 inhibited autophagosome accumulation, but after developing a more sophisticated quantitation method we found loss of Rgl2 to have no effect. We also measured endogenous LC3 levels, and these experiments confirmed loss of Rgl2 to have no effect on autophagy levels. Therefore, loss of Rgl2 increases cell death in PDAC cells, but does not have a significant effect on autophagy.

Indiana University-Purdue University Indianapolis (IUPUI) / The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.

Indiana University-Purdue University Indianapolis (IUPUI) / Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.

Indiana University-Purdue University Indianapolis (IUPUI) / Previous research has found that the clarity of information provided to raters about women managers’ performance affects ratings of their competence, likeability, and overall evaluation. The current study sought to contribute to this literature by examining whether individual differences of raters can explain the reason for differential performance evaluations of male and female managers, despite them both performing equally. For this purpose, the current research extended the findings of Heilman and colleagues by replicating their methodology while introducing a moderator variable, the rater’s Implicit Person Theory (IPT). The IPT differentiates people into either entity theorists (that is, those who believe that behavior is trait-based and therefore fixed and stable) and incremental theorists (those who believe that behavior is situationally mediated and hence, changeable). Specifically, it was proposed that the effects found in the previous study would be stronger when the rater possessed an entity theory as opposed to an incremental theory. In doing so, this research attempted to provide an understanding of why male and female managers might be given different ratings, all other things being equal. Analyses revealed results that were consistent with, as well as some that were quite inconsistent with, previous findings. Rater IPT was found to have a significant effect on ratings provided by male participants but not those of female participants. Other findings and implications are discussed and limitations and future research directions are stated.

Spirituality is a neglected dimension in psychology, but has recently been growing in importance. Depression is a widespread phenomenon, worldwide, although presenting differently in different cultures, including South Africa. The aim of this study was to give an opportunity to those who have experienced spirituality and depression to voice their experiences in their respective contexts. The ontological framework was postmodernism and the guiding epistemology was social constructionism. Qualitative research was the method of research selected, because it suited the aims of the research. The specific method of analysis was a "categorical content analysis". In depth interviews with eight participants from various faith backgrounds were conducted. The participants' stories were interpreted by the researcher in the form of themes. Recurring and pertinent themes were compared and discussed with other voices in the field, such as the literature and societal and cultural discourses. The study facilitated rich, detailed and complex descriptions of people's experience of spirituality and depression. The researcher hopes the voice of this research will lead to new understandings of these phenomena in a South African context. / Psychology / D. Litt. et Phil. (Psychology)

Forests in the Drakensberg Mountains, although generally small and fragmented, represent a wealth of globally significant biodiversity. This is especially true of the invertebrate fauna, which includes a range of localized endemic species. Ground dwelling, flightless invertebrates living in forests show relative immobility and poor dispersal abilities compared to birds, butterflies and other animal taxa. The naturally fragmented state of Afrotemperate forests also geographically isolates these taxa. No quantified, systematic surveys of flightless invertebrates have been conducted in Afrotemperate forests in the Drakensberg Mountains. This means that, to date, invertebrates have not been considered in the management of these habitats. However, invertebrates are vitally important in ecosystem functioning and maintenance and must be included in management plans. This study quantified flightless invertebrate diversity in Afrotemperate forest patches in the Drakensberg by investigating spatial patterns and seasonal changes in invertebrate diversity. The broad objectives were to: 1) Determine the appropriate spatial scale at which conservation of flightless invertebrates should be implemented; 2) Determine which season, taxa and sampling methods are most suitable for biodiversity assessment and monitoring in Afrotemperate forest; and 3) Investigate methods of prioritizing Afrotemperate forests for conservation of flightless invertebrate diversity. Seventeen forest patches in six valleys in four Drakensberg reserves (Rugged Glen Nature Reserve, Royal Natal National Park, Cathedral Peak and Injisuthi) were sampled in the summer of 2004/2005. In addition, three forests at Injisuthi were sampled in March, June, September and December 2004 to enable seasonal comparisons. Sampling methods included soil samples, leaf litter samples, pitfall traps, active search quadrats and tree beats. The study focused on terrestrial molluscs (Class Gastropoda), earthworms (Class Annelida), onychophorans (Class Onychophora), centipedes (Class Chilopoda), millipedes (Class Diplopoda) and ants (Class Insecta). Target taxa were sorted to morphospecies and then identified to species by taxonomic experts. Seventy-two species and a total of 5261 individual specimens from the six target taxa were collected. Species composition of sites varied along the north-south gradient, and species turnover (beta diversity) was related to the distance between sites. Flightless invertebrate species richness and community structure fluctuated seasonally. Therefore, I recommend that sampling should take place during the wet season (summer months). Molluscs were the most suitable taxon among those surveyed to represent flightless invertebrate diversity and leaf litter samples and active search quadrats are the most suitable sampling methods for flightless invertebrates in forest. This study compared approaches to prioritizing Afrotemperate forests in terms of their invertebrate diversity using ranked species richness with complementarity indices of species presence/absence, taxonomic distinctness (orders, families and genera) and endemicity. There was no consistent spatial trend in the priority ranking of forests based on species richness. Complementarity based on species richness only required eight out of 17 forests to represent all 62 species. Although complementarity based on taxonomic distinctness and endemicity required fewer sites, not all species were represented. The minimum set of sites identified using complementarity based on species richness and augmented with information on taxa of conservation importance (local endemic and threatened species) was the most rigorous approach to prioritizing Afrotemperate forest patches in the Drakensberg for flightless invertebrate conservation. Urgent conservation interventions are required because invertebrates play a critical role in ecosystem functioning. As many forest patches and invertebrate populations as possible should be protected to conserve the full complement of invertebrate species of the region. Special management attention should be paid to the eight forests identified as priority sites in the complementarity analyses. Forest patches cannot survive in isolation, so it is important to manage the grassland, riverine vegetation, forest ecotone and forests holistically. Provisional targets were set for the conservation of flightless invertebrates, based on estimates of the requirements for persistence of invertebrates in Afrotemperate forest, made within the constraints of available information and expert opinion. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Balancing increasing elephant numbers with biodiversity conservation in small reserves has become a concern for many protected area managers. Elephants are considered important agents of disturbance creating heterogeneity and thus contributing to the maintenance of biodiversity. However elephants also damage vegetation through their destructive feeding habits, and this has led to pressure to reduce elephant populations in many reserves. Quantitative data on the impact of elephants on invertebrates, the main component of biodiversity at the species level, are lacking. The aim of this project was to assess the effect that habitat alteration by elephants has on the diversity of selected ground-dwelling invertebrates (ants, centipedes, millipedes, spiders, scorpions and termites) through the provision of logs and dung as a potential refuge niche for these invertebrate communities, and to determine the effect of spatial (vegetation types) and temporal (season and age of dung) variation on the invertebrates using these refugia. Variation in impacts was considered important because savanna is not homogenous and the impact of the refugia is likely to be dynamic in terms of seasonal trends in invertebrate populations, and in terms of changes in the environmental conditions offered by the refugia. Elephant impact on vegetation, quantity of refugia (logs and dung) produced and invertebrate diversity associated with refugia were determined for 115 transects within Madikwe Game Reserve in the North Western Province, South Africa. Invertebrate abundance, species richness and diversity were always higher under refugia than in areas without refugia. Vegetation utilisation, frequency of refugia production and invertebrate diversity showed strong temporal variation (seasonal); elephant impact and production of logs were higher in winter than in summer because elephants are more likely to feed on woody vegetation in winter when grass nutrient levels are low. Invertebrate diversity under the logs was higher in summer than in winter, and this probably reflected the higher abundance and diversity of invertebrates that are usually associated with the warmer, wetter summer months. The effect of adding refugia to three vegetation types on invertebrate diversity was tested experimentally at Makalali Private Game Reserve in the Limpopo Province, South Africa. Logs and elephant dung were set out in five plots each measuring 20m x 20m within Govender - iii mixed bushveld, riverine and mopane woodland. Significant differences were observed in invertebrate abundance, species richness and diversity between the refugia and control plots that lacked refugia and between the three vegetation types sampled. Similarity between invertebrate communities utilising the different refugia types and between the three different vegetation types were tested using the Jaccard similarity coefficient. The three vegetation types shared fewer than 50% of their species, as did the logs, dung and control sites. However the results obtained do illustrate a higher degree of similarity between the refugia substrates (logs and dung) than the control sites and between the more heterogeneous vegetation types (mixed bushveld and riverine) than the mopane veld. This indicated that invertebrate communities associated with refugia were not uniform, but were influenced by vegetation type. An experimental test of temporal changes in invertebrate community composition illustrated the importance of elephant dung as a microhabitat for different invertebrate groups over different ages of dung (three days, two, four, 12 and 32 weeks old). Colonisation of the dung, by dung beetles was immediate but as the microclimate of the dung changed with time, the new conditions were ideal for other invertebrate taxa. Over a period of eight months, the change of invertebrate communities utilising the dung included dung beetles, followed by millipedes and [mally ant and termite communities. The results of this study illustrated the importance of refugia (logs and dung) produced by elephants for ground-dwelling invertebrate species in the savanna environment. The extent of the influence of the refugia varied both spatially and temporally and this should be considered in future monitoring or in measuring impacts. While further research on a broader range of organisms and at larger scales is necessary, elephants do have a positive impact on at least some components of biodiversity, through the process of facilitation of refugia. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.