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ItemStudies in microrna function and gene dysregulation in ovarian cancer(Georgia Institute of Technology, 2014-11-18) Hill, Christopher G.Ovarian cancer results from the dysregulation, in normal ovarian epithelial cells, of genes responsible for the control of critical biological processes. Since their discovery 20 years ago, microRNAs have increasingly been implicated in that dysregulation due to their role mediating gene expression; changes in microRNA expression levels in cancer have been linked with tumor growth, proliferation and metastasis. Their imputed involvement in cancer has led to the possibility of their use as biomarkers and to their potential clinical use. Using mRNA and microRNA microarray analysis to compare human gene expression in normal ovarian surface epithelial (OSE) cells and epithelial ovarian cancer (EOC) cells, we explored the interactions between microRNAs and genes. First, we validated in silico predictions of microRNA targets by comparing them with in vitro evidence after exogenous microRNA transfection. We found that pairs of microRNAs with identical 7-nt (nucleotide) seed regions shared 88% of their predicted targets and 55% of their in vitro targets, confirming the importance of the seed as a targeting mechanism. But more importantly, we found that even a single nucleotide change in the seed region can result in a significant shift in the set of targeted genes, implying strong functional conservation of the seeds and their corresponding binding sites. Next, we discovered a 3-element network motif which explains the upregulation of nearly 800 genes in ovarian cancer which, as predicted microRNA targets, might be expected to be down- regulated. This model shows that, under certain circumstances, repressor genes which are down- regulated in cancer can apparently override the repressive effects of microRNAs, resulting in the upregulation of predicted microRNA targets. Finally, we developed a phenomenological network model, based on the Pearson correlation of microarray gene expression data, to identify subnetworks dysregulated in cell cycle and apoptosis. While our methodology reported many genes previously associated with ovarian cancer, it significantly suggested potentially oncogenic genes for further investigation. This network model can easily be extended to identify dysregulated genes in other cancers.
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ItemActive methane oxidizing bacteria in a boreal peat bog ecosystem(Georgia Institute of Technology, 2014-11-14) Esson, Kaitlin ColleenBoreal peatlands are important ecosystems to the global carbon cycle. Although they cover only 3% of the earth's land surface area, boreal peatlands store roughly one third of the world's soil carbon. Peatlands also comprise a large natural source of methane emitted to the atmosphere. Some methane in peatlands is oxidized before escaping to the atmosphere by aerobic methane oxidizing bacteria. With changing climate conditions, the fate of the stored carbon and emitted methane from these systems is uncertain. One important step toward better understanding the effects of climate change on carbon cycling in peatlands is to ascertain the microorganisms actively involved in carbon cycling. To investigate the active aerobic methane oxidizing bacteria in a boreal peat bog, a combination of microcosm experiments, DNA-stable isotope probing, and next generation sequencing technologies were employed. Studies were conducted on samples from the S1 peat bog in the Marcell Experimental Forest (MEF). Potential rates of methane oxidation were determined to be in the range of 13.85 to 17.26 μmol CH₄ g dwt⁻¹ d⁻¹. After incubating with ¹³C-CH₄, DNA was extracted from these samples, separated into heavy and light fractions with cesium chloride gradient formation by ultracentrifugation and needle fractionation, and fractions were fingerprinted with automated ribosomal intergenic spacer analysis (ARISA) and further interrogated with qPCR. Based on ARISA, distinct banding patterns were observed in heavy fractions in comparison to the light fractions indicating an incorporation of ¹³C into the DNA of active methane oxidizers. This was further supported by a relative enrichment in the functional gene pmoA, which encodes a subunit of the particulate methane monooxygenase, in heavy fractions from samples incubated for fourteen days. Within heavy fractions for samples incubated for 8 and 14 days, the relative abundance of methanotrophs increased to 37% and 25%, respectively, from an in situ abundance of approximately 4%. Phylogenetic analysis revealed that the methanotrophic community was composed of both Alpha and Gammaproteobacterial methanotrophs of the genera Methylocystis, Methylomonas, and Methylovulum. Both Methylocystis and Methylomonas have been detected in peatlands before, however, none of the phylotypes in this study were closely related to any known cultivated members of these groups. These data are the first to implicate Methylovulum as an active methane oxidizer in peatlands, though this organism has been detected in another cold aquatic ecosystem with consistent methane emissions. The Methylovulum sequences from this study, like Methylocystis and Methylomonas, were not closely related to the only cultivated member of this genus. While Methylocystis was dominant in ¹³C-enriched fractions with a relative abundance of 30% of the microbial community after an eight-day incubation, Methylomonas became dominant with a relative abundance of approximately 16% after fourteen days of incubation. The relative abundance of Methylovulum was maintained at 2% in ¹³C- enriched fractions after eight and fourteen days.
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ItemComputational analysis of gene expression in complex disease(Georgia Institute of Technology, 2014-08-21) Huang, Andrew DouglasCardiovascular disease (CVD) causes 45% of on-duty firefighter fatalities, a high fraction even when compared to the risk of CVD found in other first-responder professions like police work and emergency medical services. Monitoring and managing firefighter cardiac health is important for both individual health and public safety. In our study, we are interested in assessing the utility of the most commonly used risk assessment scoring, known as the Framingham Risk Score, in evaluating the atherosclerotic risk in asymptomatic firefighters. To this end we determined FRS for 159 male firefighters from Gwinnett County, Georgia, and compared their risk categorization against their known atherosclerotic burden as determined by CIMT and CAC. While the 20% FRS threshold, corresponding to medium risk, had a high specificity for both CAC and CIMT, it also had a low sensitivity (17% and 40%, respectively), indicating that a large percentage of individuals with clinically significant atherosclerosis are being misclassified. By adjusting the FRS threshold downward, we were able to raise the sensitivity greatly with only a modest loss of specificity. Following percutaneous transluminal coronary angioplasty for the treatment of coronary artery disease, stents are commonly implanted at the treatment site to prevent recoil and negative remodeling. To combat in-stent restenosis, an arterial healing response that results in luminal loss in stented arteries, anti-restenotic drugs like sirolimus (SES) and zotarolimus (ZES) are commonly eluted by stents to suppress cell proliferation at the treatment site. While comparative studies have revealed significant difference between bare metal stents (BMS), SES, and ZES in both clinical and histological arterial response, the molecular basis of these differences remains poorly understood. We conducted a comparative gene expression profiling study using microarrays to examine differences in gene expression and pathway function in coronary arteries exposed to ZES, SES, and BMS in a porcine animal model. These molecular profiles suggest a model of delayed restenosis, resulting from a drug-induced suppression of inflammatory responses and proliferative processes, rather than an elimination of restenosis. microRNAs play a regulatory role in metastasis-related epithelial to mesenchymal transitions and mesenchymal to epithelial transitions in ovarian cancer cells. We previously showed that over-expression of miR-429 in ovarian cancer cells drove a transition from mesenchymal phenotypes to epithelial phenotypes both in morphology and expression of markers like ZEB1, ZEB2, and E-cadherin. Our study represents the first time course analysis of miR-429-induced MET in ovarian cancer cells. We transfected Hey cells with miR-429 and assayed gene expression over the course of 144 hours at regular intervals. The cell morphology and gene expression of our transfected cells changed to become more epithelial-like at 24 and 48 hours and then became more mesenchymal-like by 144 hours. By 144 hours the average gene expression levels for 98.6% of our genes were not significantly different from the levels they started from at 0 hours when we adjusted for baseline expression changes observed in our negative control treated cells. This suggests the use of microRNAs as cancer therapies and driving cancer cells to a more drug susceptible state.
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ItemTranscriptomics of malaria host-pathogen interactions in primates(Georgia Institute of Technology, 2014-08-21) Lee, Kevin JosephMalaria is a pernicious disease that has greatly impacted and continues to affect the human population. While much research has been performed to understand the underlying nature of this disease, gaps in the knowledge-base persist. In order to address these deficiencies, a multi-disciplinary, multi-institutional project has been funded to study the systems biology of the host pathogen interaction during malaria infection in both humans and non-human primates. In the course of investigating the transcriptome during two 100-day experiments in Macaca mulatta, this work elucidated many of the underlying molecular pathways of the host and parasite that are affected by antimalarial drugs, as well as through host-pathogen interactions. The malaria-disease-related host pathways are related to, not surprisingly, immune-associated signalling and hematopoesis, and the altered parasite pathways demonstrate an association between disease severity and parasite life stage abundance. Continuing integration of this research with other data-types collected during the course of these experiments will improve our understanding of malaria systems biology and improve targeted malaria therapies.
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ItemCalibration of phenol oxidase measurement in acidic wetland environments(Georgia Institute of Technology, 2014-07-22) Chanton, PatrickPhenol oxidases mediate the degradation of recalcitrant compounds, polyphenolics, in wetland soils and are considered to play a key role in the microbial carbon cycle of peatlands which predominate in boreal biomes. In order to validate a method for quantification of oxidative enzyme activity in acidic wetland environments, the relationship between pH and substrate oxidation was studied using the standard enzyme tyrosinase and in soils collected from six freshwater wetlands including three marshes in north Florida and peatlands of northern Minnesota. Phenol oxidase (PO) activity was quantified with two commonly used assay substrates, ABTS (2,2'-azino-bis(3-ethylobenzthiazoline-6-sulfonic acid) and L-DOPA (L-3,4-dihydroxyphenylalanine), across a pH range of 4 to 7 which matched the in situ pH range of the studied wetlands. The PO assay is sensitive and activity could be detected with either substrate across a pH range of 4 to 7. However, with the standard enzyme tyrosinase, it was shown that a large change or threshold in oxidation rates occurred at pH 5. At pH < 5, L-DOPA oxidation rates were greatly diminished and ABTS oxidation was at a maximum. Above pH 5, ABTS oxidation occurred at much slower rates and L-DOPA oxidation was at a maximum. The pH response of PO activity in wetland soils corroborated observations made with tyrosinase. Thus, ABTS is recommended to be an effective substrate for the quantification of PO activity at an in situ pH of < 5, while L-DOPA is recommended at an in situ pH of > 5. In soils collected from a northern Minnesota peatland, assays conducted at an in situ pH of 4 showed one to two orders of magnitude higher rates of PO activity in solid phase peat in comparison to porewater, indicating that the majority of PO activity is associated with the peat. At three Minnesota peatland sites, PO activity was shown to attenuate with depth in agreement with the activities of other enzymes and with rates of peat decomposition.
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ItemRoles of protein sequence and cell environment in cross-species prion transmission and amyloid interference(Georgia Institute of Technology, 2014-07-08) Bruce, Kathryn LynProteinaceous infectious particles, termed 'prions' are self-perpetuating protein isoforms that transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Each conformational variant of a prion protein is faithfully propagated to a homologous protein in the same cell environment. However, a reduction in the efficiency of prion transmission between different species is often observed and is termed "species barrier". Prion transmission to a heterologous protein may, in some cases, permanently change the structure of the prion variant, and divergent proteins may interfere with prion propagation in a species-specific manner. To identify the importance of both protein sequence and the cell environment on prion interference and cross-species transmission, we employed heterologous Sup35 proteins from three Saccharomyces sensu stricto species: Saccharomyces cerevisiae (Sc), Saccharomyces paradoxus (Sp), and Saccharomyces bayanus (Sb). We performed our experiments in two different cell environments (Sc and Sp). Our data show that Sup35 from one species can form a prion in another, and we employed a transfection procedure to perform cross-species transfer of the prion. Using a shuffle procedure, we demonstrate that the specificity of prion transmission is determined by the protein itself rather than the cell environment. Interestingly, we noted that variant-specific prion patterns can be altered irreversibly during cross-species transmission through S. bayanus module II. We further show that prion interference does not always correlate with cross-species prion transmission, and the identity of particular regions or even a specific amino acid, rather than the overall level of PrD homology is crucial for determining cross-species transmission and interference. Lastly we provide evidence to suggest that prion interference is specific to the cell environment.
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ItemFunctional Analysis Of The Inhibition Of Topoisomerase II Alpha By Linker Histone H1(Georgia Institute of Technology, 2014-07-02) Hamdan, HibaIn higher eukaryotes, DNA is progressively packaged into chromatin. In these varying levels of compaction, linker histone H1 is a key player for mediating chromatin folding. As a result, linker histone H1 is involved in regulating cellular activities, such as gene transcription. Also essential for multiple cellular processes, topoisomerase II alpha (Top2a) is an enzyme that regulates DNA topology and thus is a target in cancer therapeutics. Previous studies in our lab have identified Top2a as an H1.3 binding partner by immunoprecipitation in embryonic stem cells. Further studies have shown that mouse histone H1.3 inhibits Top2a enzymatic activity in vitro. In this thesis, I pursued a functional analysis to confirm and analyze the inhibitory effect of human H1.3 on Top2a in vivo. We found that overexpression of hH1.3 significantly suppressed the growth of HeLa cancer cells and resulted in an increased cell population with abnormal mitotic chromosomes. These analyses may lead to a better understanding of the role of H1 and Top2a in chromatin structure and function.
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ItemDetection and characterization of gene-fusions in breast and ovarian cancer using high-throughput sequencing(Georgia Institute of Technology, 2014-06-26) Mittal, Vinay K.Gene-fusions are a prevalent class of genetic variants that are often employed as cancer biomarkers and therapeutic targets. In recent years, high-throughput sequencing of the cellular genome and transcriptome have emerged as a promising approach for the investigation of gene-fusions at the DNA and RNA level. Although, large volumes of sequencing data and complexity of gene-fusion structures presents unique computational challenges. This dissertation describes research that first addresses the bioinformatics challenges associated with the analysis of the massive volumes of sequencing data by developing bioinformatics pipeline and more applied integrated computational workflows. Application of high-throughput sequencing and the proposed bioinformatics approaches for the breast and ovarian cancer study reveals unexpected complex structures of gene-fusions and their functional significance in the onset and progression of cancer. Integrative analysis of gene-fusions at DNA and RNA level shows the key importance of the regulation of gene-fusion at the transcription level in cancer.
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ItemExperimental studies of the causes and consequences of biodiversity over ecological and evolutionary timescales(Georgia Institute of Technology, 2014-06-17) Tan, JiaqiThis dissertation presents four microbial microcosm-based experimental studies addressing questions related to the causes and consequences of biodiversity. All four studies adopted an approach that integrates ecology and evolutionary biology. Two studies explored the utility of knowledge on species phylogenetic relationships for understanding community assembly (chapter 1) and invasibility (chapter 3). The other two studies investigated the impacts of important ecological factors, including competition (chapter 2) and temporal niches (chapter 4), on adaptive radiation, using the rapidly diversifying bacterium Pseudomonas fluorescens SBW25 as the model organism. The first study, described in Chapter 1, examined how phylogenetic relatedness between competing species affected the strength of priority effects and ecosystem functioning during community assembly. Strong priority effects emerged only when competing bacterial species were phylogenetically most closely related, resulting in multiple community states associated with different assembly histories. In addition, the phylogenetic diversity of bacterial communities effectively predicted bacterial production and decomposition. The second study, described in Chapter 2, explored the role of competition in the adaptive radiation of P. fluorescens. The adaptive radiation was generally suppressed by competition, but its effect was strongly modulated by the phylogenetic relatedness between the diversifying and competing species and their immigration history. The inhibitive effect of competition on adaptive radiation was strongest when phylogenetic relatedness was high and when competitors were introduced earlier. The third study, described in Chapter 3, evaluated the relative importance of phylogenetic relatedness between resident and invading species and phylogenetic diversity of resident communities for invasibility. Laboratory bacterial communities containing a constant number of resident species with varying phylogenetic diversity and relatedness to invaders were challenged by nonresident bacterial species. Whereas invader abundance decreased as phylogenetic relatedness increased as predicted by Darwin's naturalization hypothesis, it was unaffected by phylogenetic diversity. The final study, described in Chapter 4, presented the first experimental demonstration of the maintenance of biodiversity that emerged from adaptive radiation in the presence of temporal niches. Only when provided with temporal niche opportunities were multiple derived phenotypes of P. fluorescens able to coexist as a result of negative frequency-dependent selection. When temporal niche was absent, the specialized phenotypes either did not emerge or were predominated by one superior phenotype.
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ItemAdaptation of locomotor control in able and impaired human walking(Georgia Institute of Technology, 2014-06-13) Toney, MeganExtensive research has documented the stereotypical kinematic and kinetic patterns in healthy human walking, but we have a limited understanding of the neuromechanical control principles that contribute to their execution. Furthermore, the strategies used to adapt human walking to morphological or environmental constraints are poorly understood. After a traumatic injury, like amputation, regaining independent mobility is a primary goal of rehabilitation. Without a clear understanding of the neuromechanical principles governing locomotion, monitoring and quantitatively improving gait rehabilitation outcomes is challenging. The purpose of this doctoral work was to identify controlled variables in able and impaired human walking and to compare the control strategies used to adapt to a novel walking environment both with and without amputation. I apply an uncontrolled manifold (UCM) analysis to test whether likely goal variables of human walking are selectively stabilized through step-to-step variability structure. I found that both able-bodied subjects and subjects with an amputation maintain consistent whole body dynamics and leg power production by exploiting inherent motor abundance. Consistent leg power production is accomplished primarily through step-to-step leg force corrections that are driven by variable timing of ankle torque production. Covariance between ankle and knee torques enable robust motor control in able-bodied individuals, but this stabilizing mechanism is absent in individuals with a transtibial amputation. This coordinated joint torque control also appears to assist able-bodied short-term adaptation, invoked by split-belt treadmill walking. However, loss of ankle motor control and distal sensory feedback due to amputation appears to limit reactive, feedback driven adaptation patterns in subjects with an amputation. Ultimately, this work highlights the role of intact distal sensorimotor function in locomotor control and adaptation. The major findings I present have substantial implications for gait rehabilitation and prosthetic design.