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School of Biological Sciences

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https://hdl.handle.net/1853/70750

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College of Sciences

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Center for the Study of Systems Biology

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https://finding-aids.library.gatech.edu/agents/corporate_entities/1131

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Publication Search Results

Now showing 1 - 10 of 13

Development of algorithms for metagenomics and applications to the study of evolutionary processes that maintain microbial biodiversity

(Georgia Institute of Technology, 2012-12-20) Luo, Chengwei

Understanding microbial evolution lies at the heart of microbiology and environmental sciences. Numerous studies have been dedicated to elucidating the underlying mechanisms that create microbial genetic diversity and adaptation. However, due to technical limitations such as the high level of uncultured cells in almost every natural habitat, most of current knowledge is primarily based on axenic cultures grown under laboratory conditions, which typically do not simulate well the natural environment. How well the knowledge from isolates translates to in-situ processes and natural microbial communities remains essentially speculative. The recent development of culture-independent genomic techniques (aka metagenomics) provides possibilities to bypass some of these limitations and provide new insights into microbial evolution in-situ. To date, most of metagenomic studies have been focused on a few reduced-diversity model communities, e.g., acid mine drainage. Highly complex communities such as those of soil and sediment habitats remain comparatively less understood. Furthermore, a great power of metagenomics, which has not been fully capitalized yet, is the ability to follow the evolution of natural microbial communities over time and environmental perturbations, i.e., times-series metagenomics. Although the recent developments in DNA sequencing technologies have enabled (inexpensive) time-series studies, the bioinformatics approaches to analyze the resulting data have clearly fallen behind. Taken together, to scale up metagenomics for complex community studies, three major challenges remain: 1) the difficulty to process and analyze massive short read sequencing data, often at the terabyte level; 2) the difficulty to effectively assemble genomes from complex metagenomes; and 3) the lack of methods for tracking genotypes and mutational events such as horizontal gene transfer (HGT) through time. Therefore, developing efficient bioinformatics approaches to address these challenges represents an important and timely issue. This thesis aimed to develop novel bioinformatics pipelines and algorithms for high performance computing, and, subsequently, apply these tools to natural microbial communities to generate quantitative insights into the relative importance of the molecular mechanisms creating or maintaining microbial diversity. The tools are not specific to a particular habitat or group of organisms and thus, can be broadly used to advance our understanding of microbial evolution in different settings. In particular, the comparative whole-genome analysis of 24 Escherichia isolates form various habitats, including human and non-human associated habitats such as freshwater ecosystems and beaches, showed that organisms with more similar ecologies tend to exchange more genes, which has important implications for the prokaryotic species concept. To more directly test these findings from isolates and quantify the patterns of genetic exchange among co-occurring populations, three years of time-series metagenomics data from planktonic samples from Lake Lanier (Atlanta, GA) were analyzed. For this, it was first important to develop bioinformatics algorithms to robustly assemble population genomes from complex community metagenomes, identify the phylogenetic affiliation of assembled genome and contig sequences, and detect horizontal gene transfer among these sequences. Using these novel algorithms, in situ bacterial lineage evolution was quantitatively assessed, especially with respect to whether or not ecologically distinct lineages evolve according to the recently proposed fragmented speciation model (Retchless and Lawrence, Science 2008). Evidence in support of this model was rarely observed. Instead, it appeared that rampant HGT disseminated ecologically important genes within the population, maintaining intra-population diversity. By expanding the previous approaches to include methods to assess differential gene abundance and selection pressure between samples, it was possible to quantify how soil microbial communities respond to a decade of warming by 2 0C, which simulated the predicted effects of climate change. It was found that the heated communities showed significant shifts in composition and predicted metabolism, reflecting the release of additional soil carbon compared to the unheated (control) communities, and these shifts were community-wide as opposed to being attributable to a few taxa. These findings indicated that the microbial communities of temperate grassland soils play important roles in mediating the feedback responses to climate change. Collectively, the findings presented here advance our understanding of the modes and tempo of microbial community adaptation to environmental perturbations and have important implications for better modeling the microbial diversity on the planet. The bioinformatics algorithms and approaches developed as part of this thesis are expected to facilitate future genomic and metagenomic studies across the fields of microbiology, ecology, evolution and engineering.
Microbes and monitoring tools for anaerobic chlorinated methane bioremediation

(Georgia Institute of Technology, 2012-12) Justicia-Leon, Shandra D.

he chlorinated methanes carbon tetrachloride (CT), chloroform (CF), dichloromethane (DCM) and chloromethane (CM) are widespread groundwater pollutants that pose risks to human and ecosystem health. Although some progress has been made in elucidating the microbiology contributing to the aerobic degradation of DCM and CM, these efforts have had little impact on bioremediation practices aimed at restoring anoxic aquifers impacted by chlorinated methanes. Remaining knowledge gaps include the lack of understanding of the microbial mechanisms and pathways contributing to chlorinated methane transformations under anoxic conditions. Thus, the major goals of this research effort were to identify microbes that can contribute to the transformation of chlorinated methanes in the absence of oxygen, and to develop monitoring tools to assess anaerobic chlorinated methane bioremediation at contaminated sites. To accomplish these goals, freshwater and estuarine sediment samples from 45 geographically distinct locations, including 3 sites with reported chlorinated-methane contamination, were collected and screened for CT-, CF-, DCM- and/or CM-degrading activity. DCM degradation was observed in microcosms established with sediment materials from 15 locations, and the sediment-free, DCM-degrading enrichment culture RM was obtained from Rio Mameyes sediment. 16S rRNA-gene based community analysis characterized consortium RM, and identified a Dehalobacter sp. involved in DCM fermentation to non-toxic products. Organism- and process-specific monitoring tools were designed that target the 16S rRNA gene of the DCM-fermenting Dehalobacter sp. and the consortium’s specific 13C-DCM enrichment factor, respectively. Treatability studies using site materials that showed no chlorinated methane degradation activity demonstrated the feasibility of using CF- and DCM-degrading consortia for bioaugmentation applications. Collectively, this study expands our understanding of bacteria contributing to chlorinated methane degradation, provides new tools for monitoring anaerobic DCM degradation, and demonstrates that microbial remedies at chlorinated methane contaminated sites are feasible.
Comparative genomics reveal ecophysiological adaptations of organohalide-respiring bacteria

(Georgia Institute of Technology, 2012-11-13) Wagner, Darlene Darlington

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.
Dynamic stability of quadrupedal locomotion: animal model, cortical control and prosthetic gait

(Georgia Institute of Technology, 2012-11-13) Farrell, Bradley J.

The ability to control balance and stability are essential to prevent falls during locomotion. Maintenance of stable locomotion is challenging especially when complicated by amputation and prosthesis use. Humans employ several motor strategies to maintain stability during walking on complex terrain: decreasing walking speed, adjusting stride length and stance width, lowering the center of mass, and prolonging the double support time. The mechanisms of selecting these motor strategies by the primary motor cortex are unknown and cannot be studied directly in humans. There is also little information about dynamic stability of prosthetic gait with bone-anchored prostheses, which are thought to provide sensory feedback to the amputee through osseoperception. Therefore, the Specific Aims of my research were to (1) evaluate dynamic stability and the activity of the primary motor cortex during walking with different constraints on the base of support and (2) develop an animal model to evaluate mechanics and stability of prosthetic gait with a bone-anchored prosthesis. To address these aims, I developed a feline model that allows for investigating (1) the role of the primary motor cortex in regulation of dynamic stability of intact locomotion, (2) skin and bone integration with a percutaneous porous titanium implant facilitating prosthetic attachment, and (3) dynamic stability of walking on a bone-anchored prosthesis. The results of Specific Aim 1 demonstrated that the area and shape of the base of support influence the margins of dynamic stability during quadrupedal walking. For example, I found that the animal is dynamically unstable in the sagittal plane and frontal plane (although to a lesser degree) during a double-support by a forelimb and the contralateral hindlimb. Elevated neuronal activity from the right forelimb representation in the primary motor cortex during these phases suggests that the motor cortex may contribute to selection of paw placement location and thus to regulation of stability. The results of Specific Aim 2 on the development of skin-integrated bone-anchored prostheses demonstrated the following. Skin ingrowth into 3 types of porous titanium pylons (pore sizes 40-100 μm and 100-160 μm and nano-tubular surface treatment) implanted under skin of rats was seen 3 and 6 weeks after implantation, and skin filled at least 30% of available implant space. The duration of implantation, but not implant pore size (in the studied range) or surface treatment statistically influenced skin ingrowth; pore size and time of implantation affected the implant extrusion length (p<0.05). The implant type with the slowest extrusion rate (pore size 40-100 μm) was used in a feline model of prosthetic gait with skin-integrated bone-anchored prosthesis. The developed implantation methods, rehabilitation procedures and feline prostheses allowed 2 animals to utilize skin- and bone-integrated prostheses for dynamically stable locomotion. Prosthetic gait analysis demonstrated that the animals loaded the prosthetic limb, but increased reliance on intact limbs for weight support and propulsion. The obtained results and developed animal model improve the understanding of locomotor stability control and integration of skin with percutaneous implants.
Membrane effects of sex hormones on growth plate chondrocytes

(Georgia Institute of Technology, 2012-11-12) ElBaradie, Khairat Bahgat

Understanding and studying the normal bone growth and development is causal. Bone and cartilage tissue provide in addition to their mechanical support, they provide a protection for vital organs such as heart, lung and brain. Longitudinal growth is regulated by the activity of chondrocytes in the epiphyseal growth plates of long bones. Many hormones and growth factors are involved in the regulation of this process. Among these, sex steroids are of crucial importance, especially during puberty. In long bones, endochondral bone formation occurs at the growth plate, a region of developing cartilage located between the epiphysis and the metaphysic. The process of endochondral ossification is regulated in part by sex steroid hormones. Androgens stimulate endochondral bone growth and elongation, while estrogen is known to suppress longitudinal bone growth and accelerate growth plate closure. Studies using rat costochondral growth plate chondrocytes as a model show that the effects of 17β-estradiol (E₂) on apoptosis are found in both male and female cells and the same mechanism is involved. In contrast, E₂ causes rapid activation of PKC in female cells but not in male cells. Dihydroxytestosterone (DHT) also has direct effects on growth plate chondrocytes, increasing matrix synthesis including sulfated glycosaminoglycan production, and enhancing cell maturation by increasing alkaline phosphatase enzymatic activity. Short stature and abnormally slow increase in height is one of the main reasons for referral to endocrinologist. Excessive growth and abnormally tall is also a problem, especially because it increase risk for the trunk abnormalities. Furthermore until now a few growth-promoting therapies are available for clinical use. Therefore future therapies for treating the growth disorders are essential. The overall goal of this project is to investigate the sexual-dimorphic effect of the sex steroid hormone in rat growth plate chondrocytes, the cellular signaling pathways mediating these actions, and their physiological role. The information gleaned from this study will provide new information about the role of sex steroid hormones in chondrogenesis and has implications in the development of new therapies for the treatment of bone fracture healing, and growth plate disorders. The central hypothesis was that sex steroid would play an important and sex-specific role in regulating chondrocytes as a main regulator of longitudinal bone growth.
Evolution of Reproduction and Stress Tolerance in Brachionid Rotifers

(Georgia Institute of Technology, 2012-08) Smith, Hilary April

Stress can be a driving force for new evolutionary changes leading to local adaptation, or may be responded to with pre-existing, ancestral tolerance mechanisms. Using brachionid rotifers (microzooplankton) as a study system, I demonstrate roles of both conserved physiological mechanisms (heat shock protein induction) and rapid evolution of traits in response to ecologically relevant stressors such as temperature and hydroperiod. Rapid evolution of higher levels of sex and dormancy in cultures mimicking temporary waters represents an eco-evolutionary dynamic, with trait evolution feeding back into effects on ecology (i.e., reduced population growth). I also reveal that prolonged culture in a benign laboratory environment leads to evolution of increased lifespan and fecundity, perhaps due to reduction of extrinsic mortality factors. Potential mechanisms (e.g., hormonal signals) are suggested that may control evolvability of facets of the stress response. Due to prior studies suggesting a role of progesterone signaling in rotifer sex and dormancy, the membrane associated progesterone receptor is assayed as a candidate gene that could show positive selection indicating rapid divergence. Despite some sequence variation that may contribute to functional differences among species, results indicate this hormone receptor is under purifying selection. Detailed analyses of multiple stress responses and their evolution as performed here will be imperative to understanding current patterns of local adaptation and trait-environment correlations. Such research also is key to predicting persistence of species upon introduction to novel habitats and exposure to new stressors (e.g., warming due to climate change). Perhaps one of the most intriguing results of this dissertation is the rapid, adaptive change in levels of sex and dormancy in a metazoan through new mutations or re-arrangements of the genetic material. This suggests species may be able to rapidly evolve tolerance of new stressors, even if standing genetic variation does not currently encompass the suite of alleles necessary for survival.
Risk and resources in the plankton: effects on copepod population growth and zooplankton community dynamics

(Georgia Institute of Technology, 2012-07-03) Lasley, Rachel Skye

The focus of my thesis research is on the interplay between individual behavior, population dynamics and community-level processes within zooplankton communities in coastal Maine. The target organisms of my thesis work are marine copepods. Copepods are small (1-10 mm) crustaceans that perform the essential ecosystem function of consuming and assimilating primary production (phytoplankton) making it available to higher trophic levels such as commercially important fishes. Therefore, copepod population growth is of critical importance to marine food webs. Fertilization limitation has been suggested as a constraint on copepod population growth but field surveys describing the prevalence of fertilization limitation are lacking. During my doctoral research, I explored the in situ fertilization success of two marine copepod species, Temora longicornis and Eurytemora herdmani in coastal Maine. I collected monthly zooplankton samples and analyzed clutches from field-caught females using an egg-staining technique. My results indicate that both species exhibit fertilization limitation in nature and the factors correlated with their fertilization span population, community and ecosystem level factors. To determine a causal relationship between predator density and copepod mating success, I conducted laboratory experiments to assess the effects of a common mysid shrimp predator, Neomysis americana on Eurytemora herdmani mating success. I subjected males and females to predators or predator cues. I found that the presence of a mysid predator, or only a predator cue, reduced copulation frequency and spermatophore transfer leading to a 38-61% decrease in E. herdmani nauplii production. These results suggest that mysid predators can constrain copepod population growth through non-consumptive processes. To determine the effects that resources can impose on copepod behavior, I explored the behavioral and fitness consequences of Temora longicornis ingesting Alexandrium fundyense, a phytoplankton species that forms harmful algal blooms in coastal Maine. My results suggest that ingesting A. fundyense causes copepods to swim faster and with more directional persistence compared to control algae. Temora longicornis increased their average swimming velocity by 24%, which leads to a 24-54% increase in their theoretical encounter rate with predators. Therefore, these findings suggest behaviorally mediated copepod-algal interactions may have significant impacts on harmful algal bloom dynamics and the fate of toxins in marine food webs.
Computational algorithm development for epigenomic analysis

(Georgia Institute of Technology, 2012-07-03) Wang, Jianrong

Multiple computational algorithms were developed for analyzing ChIP-seq datasets of histone modifications. For basic ChIP-seq data processing, the problems of ambiguous short sequence read mapping and broad peak calling of diffuse ChIP-seq signals were solved by novel statistical methods. Their performance was systematically evaluated compared with existing approaches. The potential utility of finding meaningful biological information was demonstrated by the applications on real datasets. For biological question driven data mining, several important topics were selected for algorithm developments, including hypothesis-driven insulator prediction, unbiased chromatin boundary element discovery and combinatorial histone modification signature inference. The integrative computational pipeline for insulator prediction not only produced a list of putative insulators but also recovered specific associated chromatin and functional features. Selected predictions have been experimentally validated. The unbiased chromatin boundary element prediction algorithm was feature-free and had the capability to discover novel types of boundary elements. The predictions found a set of chromatin features and provided the first report of tRNA-derived boundary elements in the human genome. The combinatorial chromatin signature algorithm employed chromatin profile alignments for unsupervised inferences of histone modification patterns. The signatures were associated with various regulatory elements and functional activities. Both the computational advantages and the biological discoveries were discussed.
Chemically mediated competition, herbivory, and the structure of coral reefs

(Georgia Institute of Technology, 2012-07-03) Rasher, Douglas B.

Corals, the foundation species of tropical reefs, are in rapid global decline as a result of anthropogenic disturbance. On many reefs, losses of coral have coincided with the over-harvesting of reef herbivores, resulting in ecosystem phase-shifts from coral to macroalgal dominance. It is hypothesized that abundant macroalgae inhibit coral recovery and recruitment, thereby generating ecological feedback processes that reinforce phase-shifts to macroalgae and further diminish reef function. Notwithstanding, the extent to which macroalgae directly outcompete coral, the mechanisms involved, and the species-specificity of algal-coral competition remains debated. Moreover the capacity for herbivores to prevent vs. reverse ecosystem phase-shifts to macroalgae and the roles of herbivore diversity in such phenomena remain poorly understood. Here I demonstrate with a series of field experiments in the tropical Pacific and Caribbean Sea that multiple macroalgae common to degraded reefs directly outcompete coral using chemical warfare, that these interactions are mediated by hydrophobic secondary metabolites transferred from algal to coral surfaces by direct contact, and that the outcomes of these allelopathic interactions are highly species-specific. Using field observations and experiments in the tropical Pacific, I also demonstrate that the process of herbivory attenuates the competitive effects of allelopathic algae on corals by controlling succession of algal communities, and that the herbivore species responsible for macroalgal removal possess complementary tolerances to the diversity of chemical defenses deployed among algae, creating an essential role for herbivore diversity in reversing ecosystem phase-shifts to macroalgae. Lastly, I demonstrate with field experiments in the tropical Pacific that algal-coral competition simultaneously induces allelochemicals and suppresses anti-herbivore deterrents in some algae, likely due to trade-offs in the productions of defense metabolites with differing ecological functions. Together, these studies provide strong evidence that chemically mediated competitive and consumer-prey interactions play principal roles in coral reef degradation and recovery, and should provide resource managers with vital information needed for effective management of these ecologically and economically important but threatened ecosystems.
Exploiting phylogenetics to understand genome evolution in both modern and ancestral organisms

(Georgia Institute of Technology, 2012-07-02) Zhao, Ziming

Computational evolutionary analyses, particularly phylogenetics and ancestral reconstruction, have been extensively exploited under different algorithms and evolutionary models to better understand genome evolution from both small- and large-scale perspectives in order to assign genotypes based on assortment, resolve species relationships and gene annotation issues, further understand gene gain/loss within individual gene families, measure functional divergence among homologs, and infer ancestral character states. These evolutionary studies provide us with insights into biologically relevant issues including paleoenvironments inferred from resurrected proteins, developmental physiology associated with functional divergence of duplicated genes, viral epidemics and modes of transmission in attempt to better prepare, prevent and control diseases, evolution of lineage-specific pathogenicity, and attempts to create a synthetic ancient organism that would benefit the field of synthetic biology. Our work also provides us with greater insights into the accuracies and limitations of ancestral sequence reconstruction methods. In total, our work highlights the diverse questions that evolutionary studies attempt to address and the different biological levels that can be studied to answer these questions.