Gene regulatory networks analyze the relationships between genes allowing us to un- derstand the gene regulatory interactions in systems biology. Gene expression data from the microarray experiments is used to obtain the gene regulatory networks. How- ever, the microarray data is discrete, noisy and non-linear which makes learning the networks a challenging problem and existing gene network inference methods do not give consistent results. Current state-of-the-art study uses the average-ranking-based consensus method to combine and average the ranked predictions from individual methods. However each individual method has an equal contribution to the consen- sus prediction. We have developed a linear programming-based consensus approach which uses learned weights from linear programming among individual methods such that the methods have di↵erent weights depending on their performance. Our result reveals that assigning di↵erent weights to individual methods rather than giving them equal weights improves the performance of the consensus. The linear programming- based consensus method is evaluated and it had the best performance on in silico and Saccharomyces cerevisiae networks, and the second best on the Escherichia coli network outperformed by Inferelator Pipeline method which gives inconsistent results across a wide range of microarray data sets.

This research aimed to determine the effect of educating parents on Regulatory Sensory Processing Disorder (RSPD) and implementing an Ayers-SI sensory diet over a two-week period. Twelve infants who met the criteria for RSPD were subdivided into two groups: 7-12 and 13-24 months. The data was obtained from a telephonic interview and completion of the Infant Toddler Symptom Checklist (ITSC). Following the two-week intervention, the ITSC was completed again and results were compared using the Wilcoxin Signed Rank Test. Although the research sample was small, findings indicate that the Ayers-SI sensory diet was effective in reducing signs of fussiness. The research group showed a significant change (p=0.0024) over the two-week period, with a greater change evident in the 7-12 month group. Selfregulation and attachment showed the most significant change. Difficulties with sensory processing for example, tactile, vestibular and auditory sensitivities appeared to persist, indicating a possible need for direct sensory integrative therapy to address them. In addition, the findings indicate that there is a lack of referral for Ayers-SI occupational therapy. This research adds value to the body of research which indicates the need for early intervention for possible prevention of later developmental difficulties.

Superantigens are exotoxins produced by Staphylococcus aureus and induce extensive T cell proliferation and proinflammatory cytokines, leading to toxic shock syndrome at high concentrations. However, the role of superantigens produced at relatively low concentrations during asymptomatic colonization or chronic infection has not been well established. In this dissertation, we demonstrated that stimulation of human PBMCs with staphylococcal enterotoxin C1 (SEC1) at the dose inducing a half maximal T cell proliferation (suboptimal stimulation) induced immunosuppressive CD4+CD25+FOXP3+ and CD8+CD25+FOXP3+ T cells. The suppression of these cells was mainly mediated by the galectin-1. We found that suboptimal stimulation with SEC1 induced differential activation of PI3K-mTOR-Akt pathway, leading to expression of FOXP3 isoforms preferably localized to the nucleus and induction of PTEN that contributes to maintain stability and suppressive activity of regulatory T cells. Taken together, these results demonstrate the important role of superantigen produced at low concentration during asymptomatic colonization that induce immunosuppressive CD4+ and CD8+ regulatory T cells to promote survival, propagation, and colonization for S. aureus in the host.

Gene regulation is controlled by DNA-bound complexes of transcription factors (TFs) and indirectly recruited transcriptional cofactors (COFs). Understanding how and where these TF-COF complexes bind in the genome is fundamental to our understanding of the role of cis-regulatory elements (CREs) in gene regulation and our mechanistic interpretation of non-coding variants (NCVs) known to impact gene expression levels. In this thesis, I present three related array-based techniques for the high-throughput profiling of DNA-bound TFs and TF-COF complexes directly from cell nuclear extracts. First, I describe the nuclear extract protein-binding microarray (nextPBM) approach to profile TF-DNA binding using nuclear extracts to account for cell-specific post-translational modifications and cofactors. By analyzing cooperative binding of PU.1/SPI1 and IRF8 in monocytes, I demonstrate how nextPBM can be used to delineate DNA-sequence determinants of cell-specific cooperative TF complexes. Second, I present the CASCADE (Comprehensive ASsessment of Complex Assembly at DNA Elements) approach to simultaneously discover DNA-bound TF-COF complexes and quantify the impact of NCVs on their binding. To demonstrate applicability of CASCADE to screen NCVs, I profile differential TF-COF binding to ~1,700 single-nucleotide polymorphisms in human macrophages and discover a prevalence of perturbed ETS-related TF-COF complexes at these quantitative trait loci. Third, I present the human TF array (hTF array) as a general platform for surveying COF recruitment to a panel of 346 non-redundant consensus TF binding sites (TFBSs). Using the hTF array, one can examine the activity of a diverse panel of TFs by profiling TF-COF complexes in a cell state-specific manner. In addition to the hTF microarray design, I have developed analysis and visualization software that allows users to explore COF recruitment profiling results interactively. Collectively, nextPBM, CASCADE, and the hTF array represent a suite of new approaches to investigate TF-COF complex binding and their application will refine our understanding of CREs by linking NCVs with the biophysical complexes that mediate gene regulatory functions.

The solvent-producing clostridia are well known for their ability to produce acetone, butanol and isopropanol in industrial fermentation. Production of these compounds occurs in cells that have completed a metabolic switch under specific growth conditions. Knowledge of the regulation of the metabolic switch will make the industrial process more reliable. From an isopropanol-producing strain Clostridium beijerinckii NRRL B593, a gene which encodes a putative NtrC-like regulatory protein was cloned and sequenced. The gene codes for a polypeptide of 632 amino acids and has been designated the stc gene. Expression of the stc gene was confirmed by RT-PCR. The co-presence of the stc gene with the adh gene which encodes a primary/secondary alcohol dehydrogenase in isopropanol-producing clostridia suggests that the stc gene may be functionally related to isopropanol production. From C. beijerinckii NRRL B592, a region which encompassed the solvent-production genes ald (aldehyde dehydrogenase), ctfA and ctfB (acetoacetate: butyrate/acetate CoA-transferase) and part of adc (acetoacetate decarboxylase) was cloned and sequenced. The organization of these genes was similar to that in C. beijerinckii NRRL B593. Northern analysis indicated that these four genes were co-transcribed on the same messenger RNA in C. beijerinckii NRRL B593. Therefore, in C. beijerinckii, the sol operon consists of the ald -ctfA-ctfB-adc genes, which differs from the sol operon in Clostridium acetobutylicum. / Master of Science

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus. / Master of Science / Brucella abortus is a bacterial pathogen that primarily infects cattle but is also transmitted to humans. Human disease most commonly results from the consumption of unpasteurized milk and milk products. Human brucellosis has very limited treatment options, with a high incidence of disease relapse. B. abortus survives and replicates within immune cells, which create a harsh environment. However, the bacteria are able to sense and adapt to survive and replicate within these immune cells, maintaining a chronic infection. A better understanding of the adaptation process B. abortus utilizes to survive within the human host can lead to improvement of treatment options. The present work characterizes a novel regulatory small RNA- VcrS, which was found required for survival and replication inside immune cells

Indiana University-Purdue University Indianapolis (IUPUI) / Complex biological systems are composed of many regulatory components, which can be measured with the advent of genomics technology. Each molecular assay is normally designed to interrogate one aspect of the cell state. However, a comprehensive understanding of the regulatory mechanism requires characterization from multiple levels such as genome, epigenome, and transcriptome. Integration of multi-omics data is urgently needed for understanding the global regulatory mechanism of gene expression. In recent years, single-cell technology offers unprecedented resolution for a deeper characterization of cellular diversity and states. High-quality single-cell suspensions from tissue biopsies are required for single-cell sequencing experiments. Tissue biopsies need to be processed as soon as being collected to avoid gene expression changes and RNA degradation. Although cryopreservation is a feasible solution to preserve freshly isolated samples, its effect on transcriptome profiles still needs to be investigated. Investigation of multi-omics data at the single-cell level can provide new insights into the biological process. In addition to the common method of integrating multi-omics data, it is also capable of simultaneously profiling the transcriptome and epigenome at single-cell resolution, enhancing the power of discovering new gene regulatory interactions. In this dissertation, we integrated bulk RNA-seq with ATAC-seq and several additional assays and revealed the complex mechanisms of ER–E2 interaction with nucleosomes. A comparison analysis was conducted for comparing fresh and frozen multiple myeloma single-cell RNA sequencing data and concluded that cryopreservation is a feasible protocol for preserving cells. We also analyzed the single-cell multiome data for mesenchymal stem cells. With the unified landscape from simultaneously profiling gene expression and chromatin accessibility, we discovered distinct osteogenic differentiation potential of mesenchymal stem cells and different associations with bone disease-related traits. We gained a deeper insight into the underlying gene regulatory mechanisms with this frontier single-cell mutliome sequencing technique.

Regulatory T cells (TRegs) are stable markers of immune functioning, acting to suppress inflammation. TRegs are important during implantation and early pregnancy where they suppress immune-mediated rejection of the embryo. Given the role of TRegs in the maintenance of pregnancy, their depletion can be associated with obstetric complications. Through the completion of two studies, this thesis seeks to identify the role of TRegs in two forms of perinatal pathology: depression and arterial thickening. The first study examines whether decreased TReg levels during pregnancy are associated with an increase in depressive symptoms, and if this relationship is mediated by maternal stress. We predicted that the TReg-depression relationship would be unique to pregnancy, and not occur in the postpartum. In the second study we assessed if decreased TRegs were inversely correlated with carotid arterial thickness. TReg samples were obtained from women between 24 and 32 weeks gestation (N=16), and at 12 weeks postpartum (N=19). Depression was assessed using the Edinburgh Perinatal Depression Scale (EPDS) and the Mongomery-Asberg Depression Rating Scale (MADRS) , and stress with the Perceived Stress Scale (PSS). TRegs were measured using flow cytometry. In the first study, we showed that lower TRegs were associated with increased levels of depression in pregnancy, and that this association was mediated by perceived stress. In the postpartum period, TRegs were not associated with changes in mood. In the second study, we found no relationship between TRegs and carotid arterial thickness. Our results suggest that TReg changes in pregnancy may be associated with maternal mood in pregnancy, but not in the postpartum period. Despite the fact that we failed to find a correlation between TRegs and carotid arterial thickness during pregnancy, our limited sample size leads us to recommend that the presence of an inverse correlation between these two markers not be ruled out, but suggest that these links be further examined using a larger sample and more precise imaging. Together, these two studies may provide very early insights into the role of TRegs in perinatal mood disorders and cardiovascular health and highlight the need for further research. / Thesis / Master of Science (MSc)

No description available.

Salmonella enterica is an enteric pathogen with a broad host tropism that can cause disease ranging from self-limited gastroenteritis to enteric fever. The evolution of S. enterica as a pathogen is driven by the horizontal acquisition of genes that promote virulence and survival within host immune cells, as well as the coordinated regulation of these and ancestral genes by two-component systems (TCS). TCS integrate environmental cues with the transcriptional reprogramming of bacteria, and in the case of Salmonella, result in niche-specific gene expression in response to anti-bacterial cues produced by the host. The TCS SsrA-SsrB in S. enterica is considered the master regulator for intracellular virulence, where SsrA is a sensor kinase that triggers the activation of the DNA binding protein SsrB. The full suite of genes regulated by SsrB in S. enterica, as well as the cues that activate this TCS, have not been fully characterized. Here, we demonstrated that horizontally acquired and ancestral genes in the S. enterica genome have evolved to be regulated by SsrB, and the repression of a set of ancestral genes involved in flagellar motility promotes evasion of the host immune system. Additionally, we identified the production of reactive oxygen species (ROS) by host immune cells as a signal that can activate a cluster of genes regulated by the SsrA-SsrB TCS, likely mediated by SsrA sensing of these ROS. Together, these results expand our understanding of the complex interplay between the pathogen S. enterica and the host that results in bacterial infections. / Thesis / Doctor of Philosophy (PhD) / Salmonella enterica (S. enterica) is a species of bacteria that can cause food poisoning in various animals, including humans, through consumption of contaminated food and water. During an infection, host cells activate numerous defense mechanisms to prevent disease. S. enterica has evolved to turn specific genes on or off in response, resulting in modifications to bacterial and host cell behaviour that promote infection. The timing of these genetic changes is controlled by proteins that can sense specific environmental signals and adjust gene expression accordingly. The specific signals sensed by S. enterica that allow for adaptive gene expression within the host, and the types of genes that are regulated to promote survival, have not been fully identified. Here, we show that S. enterica evolved to repress genes involved in flagellar motility to hide from the host immune response. We further demonstrate that S. enterica can sense anti-bacterial molecules produced by the host, called reactive oxygen species, to trigger specific changes in gene expression. Together, this work reveals novel aspects for the molecular basis of Salmonella enterica pathogenesis.