Indiana University-Purdue University Indianapolis (IUPUI) / LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.

Indiana University-Purdue University Indianapolis (IUPUI) / The tight-binding model that has been used for many years in condensed matter physics, due to its analytic and numerical tractability, has recently been used to describe light propagating through an array of evanescently coupled waveguides. This dissertation presents analytic and numerical simulation results of light propagating in a waveguide array. The first result presented is that photonic transport can be achieved in an array where the propagation constant is linearly increasing across the array. For an input at the center waveguide, the breathing modes of the system are observed, while for a phase displaced, asymmetric input, phase-controlled photonic transport is predicted. For an array with a waveguide-dependent, parity-symmetric coupling constant, the wave packet dynamics are predicted to be tunable. In addition to modifying the propagation constant, the coupling between waveguides can also be modified, and the quantum correlations are sensitive to the form of the tunneling function. In addition to modifying the waveguide array parameters in a structured manner, they can be randomized as to mimic the insertion of impurities during the fabrication process. When the refractive indices are randomized and real, the amount of light that localizes to the initial waveguide is found to be dependent on the initial waveguide when the waveguide coupling is non-uniform. In addition, when the variance of the refractive indices is small, light localizes in the initial waveguide as well as the parity-symmetric waveguide. In addition to real valued disorder, complex valued disorder can be introduced into the array through the imaginary component of the refractive index. It is shown that the two-particle correlation function is qualitatively similar to the case when the waveguide coupling is real and random, as both cases preserve the symmetry of the eigenvalues. Lastly, different input fields have been used to investigate the quantum statistical aspects of Anderson localization. It is found that the fluctuations in the output intensity are enhanced and the entropy of the system is reduced when disorder is present in the waveguides.

Indiana University-Purdue University Indianapolis (IUPUI) / Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.

Indiana University-Purdue University Indianapolis (IUPUI) / MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis.

Microtubule targeting agents (MTAs) are chemotherapeutics commonly used in the treatment of breast, ovarian, lung, and lymphoma cancers. There are two main classes of MTAs based upon their effects on microtubule stability. The two classes are the destabilizing agents, which include the drug vincristine, and the stabilizing agents, which include paclitaxel and epothilone B. These drugs are highly effective antineoplastics, but their use is often accompanied by several side effects, one of which is peripheral neuropathy. Peripheral neuropathy can be characterized by burning pain, tingling, loss of proprioception, or numbness in the hands and feet. In some patients, the MTA-induced peripheral neuropathy is debilitating and dose-limiting; however, there are no effective prevention strategies or treatment options for peripheral neuropathy as the mechanisms mediating this side effect are unknown. The goal of this work was to investigate MTA-induced effects on neuronal activity and morphology in order to elucidate the underlying mechanisms involved in the development of MTA-induced peripheral neuropathy. As an indicator of sensory neuronal activity, the basal and stimulated release of the putative nociceptive peptide, calcitonin gene-related peptide (CGRP), was measured from sensory neurons in culture after exposure to the MTAs paclitaxel, epothilone B, and vincristine. Neurite length and branching were also measured in sensory neuronal cultures after treatment with these MTAs. The results described in this thesis demonstrate that MTAs alter the stimulated release of CGRP from sensory neurons in differential ways depending on the MTA agent employed, the CGRP evoking-stimulus used, the concentration of the MTA agent, the duration of exposure to the MTA agent, and the presence of NGF. It was also observed that MTA agents decrease neurite length and branching, independent of the concentration of NGF in the culture media. Thus, this thesis describes MTA-induced alterations of sensory neuronal sensitivity and neurite morphology and begins to elucidate the underlying mechanisms involved in MTA-induced alterations of sensory neurons. These findings will undoubtedly be used to help elucidate the mechanisms underlying MTA-induced peripheral neuropathy.

Indiana University-Purdue University Indianapolis (IUPUI) / There are many advantages of mobile agent such as delegation of tasks, asynchronous processing, adaptable service in interfaces, and code shipping. Mobile agents can be utilized in many areas such as electronic commerce, information retrieval, network management, etc. The main problem with mobile agents is security. The three basic security design goals of a system are confidentiality, integrity, and availability. The goal of this thesis concerns the property of secure purchasing by mobile agents. First present Jalal's anonymous authentication protocol. Next, we construct our single mobile agent protocol based on Jalal's authentication technique. Also, we add some addition cryptography techniques to make the data more secure during its migration. Lastly, we build a multiple mobile agent protocol based on the single mobile agent protocol. Here, the multiple mobile agents are capable to make the decision and purchase the item for user.

Indiana University-Purdue University Indianapolis (IUPUI) / Every year, more than 200 schools of social work graduate thousands of Master of Social Work (MSW) students. These graduates enter the world of work and continue on their journey toward becoming professional social workers. Surprisingly, very little is known in social work about the transition from student to professional, especially for MSW graduates. Related literature in nursing and education is reviewed in order to have a foundational knowledge of the transition process for similar professionals. The research questions for this study were: (a) What is the process of transition from student to employee like for new MSW graduates? (b) What are the factors that influence this transition during the first 18 months for MSW graduates? and (c) Are there critical junctures in the processes of transitioning from being a student through the first 18 months of MSW employment that are related to satisfaction and/or professional growth? This dissertation used qualitative, constructivist grounded theory methodology in order to study this relatively unknown subject. The conceptual model that emerged in this study is called Riding the Waves, and illustrates the transition process for new graduates learning to become a professional social worker. There are five stages of this model: Testing the Waters, Jumping In, Sinking or Swimming, Treading Water, and Riding the Waves. In Testing the Waters, critical issues of finding a job, negotiating a salary and licensure are salient. In Jumping In, new graduates experience orientation, examine preparedness from school and encounter the real world of work as opposed to their expectations. In Sinking or Swimming, new graduates negotiate not knowing, supervision, dealing with emotions and difficult work situations. In Treading Water, new graduates explore finding a balance between self-care and compassion fatigue and articulate job and compassion satisfaction. In the last stage, Riding the Waves, new graduates are more stable, gain confidence, find their voice and discuss what is ahead for them. Implications for social work students, educators, and employers are discussed including better preparing students for the transition, improving orientation and supervision, and providing the support that these new professionals require and deserve.

Indiana University-Purdue University Indianapolis (IUPUI) / A recent review of randomized controlled trials found that trivalent chromium (Cr3+) supplementation significantly improved glycemia among patients with diabetes, consistent with a long-standing appreciation that this micronutrient optimizes carbohydrate metabolism. Nevertheless, a clear limitation in the current evidence is a lack of understanding of Cr3+ action. We tested if increased AMP-activated protein kinase (AMPK) activity, previously observed in Cr3+-treated cells or tissues from Cr3+-supplemented animals, mediates improved glucose transport regulation under insulin-resistant hyperinsulinemic conditions. In L6 myotubes stably expressing the glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation increased GLUT4myc translocation by 69% and glucose uptake by 97%. In contrast, the hyperinsulinemic state impaired insulin stimulation of these processes. Consistent with Cr3+’s beneficial effect on glycemic status, chromium picolinate (CrPic) restored insulin’s ability to fully regulate GLUT4myc translocation and glucose transport. Insulin-resistant myotubes did not display impaired insulin signaling, nor did CrPic amplify insulin signaling. However, CrPic normalized elevated membrane cholesterol that impaired cortical filamentous actin (F-actin) structure. Mechanistically, data support that CrPic lowered membrane cholesterol via AMPK. Consistent with this data, siRNA-mediated AMPK silencing blocked CrPic’s beneficial effects on GLUT4 and glucose transport regulation. Furthermore, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) protected against hyperinsulinemia-induced membrane/cytoskeletal defects and GLUT4 dysregulation. To next test Cr3+ action in vivo, we utilized obesity-prone C57Bl/6J mice fed a low fat (LF) or high fat (HF) diet for eight weeks without or with CrPic supplementation administered in the drinking water (8 µg/kg/day). HF feeding increased body weight beginning four weeks after diet intervention regardless of CrPic supplementation and was independent of changes in food consumption. Early CrPic supplementation during a five week acclimation period protected against glucose intolerance induced by the subsequent eight weeks of HF feeding. As observed in other insulin-resistant animal models, skeletal muscle from HF-fed mice displayed membrane cholesterol accrual and loss of F-actin. Skeletal muscle from CrPic-supplemented HF-fed mice showed increased AMPK activity and protection against membrane cholesterol accrual and F-actin loss. Together these data suggest a mechanism by which Cr3+ may positively impact glycemic status, thereby stressing a plausible beneficial action of Cr3+ in glucose homeostasis.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to different environmental stresses, phosphorylation of eIF2 (eIF2P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. In this study, we also show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter resulting in its transcriptional repression. The LIP isoform of C/EBPβ, but not the LAP version is regulated following UV exposure and directly represses ATF4 transcription. Loss of the LIP isoform results in increased ATF4 mRNA levels in response to UV irradiation, and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2P and translational control, combined with transcription factors regulated by alternative signaling pathways, can direct programs of gene expression that are specifically tailored to each environmental stress.