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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 168

Experiments to optimize the ribose-seq protocol

(Georgia Institute of Technology, 2023-07-31) Bahl, Smriti

Ribonucleoside monophosphates (rNMPs), the units of RNA, are the most abundant non-standard nucleotides found in genomic DNA. They can be incorporated by DNA polymerases during DNA replication and repair, by hydroxyl radicals during oxidative stress or during incomplete maturation of Okazaki fragments. rNMPs have profound consequences on genome stability, DNA structure, function, and various cellular processes. To better understand these effects, the Storici lab developed the ribose-seq protocol which is a systematic technique for capturing and analyzing rNMPs in genomic DNA. The aim of this study is to optimize the ribose-seq protocol by enhancing the efficiency and accuracy of rNMP detection while minimizing the required amount of starting DNA, thereby enabling easier acquisition also for possible clinical applications. We systematically investigated three key steps of the protocol: (1) adaptor ligation, (2) self-ligation using Arabidopsis thaliana tRNA ligase (AtRNL), and (3) degradation of linear single-stranded DNA (ssDNA) using exonuclease. Through rigorous experimentation and analysis, we observed that modifying the adaptor ligation conditions resulted in approximately a 30% increase in ligation efficiency of the adaptor to the fragmented DNA. The use of AtRNL with an extended incubation period at lower temperature enabled improved circularization of DNA containing the rNMPs, resulting in more abundant ribose-seq library product. Furthermore, novel exonucleases were evaluated as potential replacements for T5 exonuclease in order to effectively eliminate the remaining linear ssDNA following AtRNL self-ligation and protect the circular ssDNA structures containing rNMPs from exonuclease-mediated degradation. To validate the findings of this project, ribose-seq libraries were constructed using Saccharomyces cerevisiae DNA, demonstrating the potential to reduce the starting DNA amount by up to 50%. These findings present a significant advancement in the ribose-seq methodology, enabling researchers to investigate ribonucleotide-mediated genomic processes with enhanced sensitivity and reduced resource requirements.
Mechanisms of Coherence and Incoherence Between GWAS and Single-Cell eQTL Effects in Crohn's Disease

(Georgia Institute of Technology, 2023-05-02) Collins, Jared Blake

The integration of expression quantitative trait loci with GWAS data has proven invaluable in the exploration of mechanisms through which genetic variants influence complex traits. However, it has also highlighted instances of incoherence in which the eQTL effects of GWAS risk variants seemingly contradict observed case and control expression. Patterns of incoherence may indicate variants associated with disease via protection, but due to the highly heterogenous nature of varying cell-types, may also indicate cell-type specific associations with disease that are convoluted by bulk RNA sequencing. Here, we conduct exploratory analysis integrating sceQTL and GWAS data associated with Crohn’s Disease to assess patterns of coherence and incoherence, using both bulk RNA-seq and predicted single-cell gene expression for case-control expression. We show that integration of GWAS summary statistics with single-cell eQTL data is a promising approach for uncovering cell type specific patterns of coherence and incoherence, and may suggest functional mechanisms underlying these associations.
Changes of community structure of seagrass-associated elasmobranchs and teleosts in Florida’s “Big Bend” ecosystem

(Georgia Institute of Technology, 2023-05-02) Rackley, Piper M.

Sharks play a significant ecological role as predatory species in the world’s oceans. While they have been around for millions of years, they face many threats today, such as fishing and habitat destruction (via pollution and coastal development), that diminish their populations, and some species are on the verge of extinction. Over the past four decades, ocean water temperatures have risen significantly and have severely altered marine ecosystems. Florida’s “Big Bend” ecosystem is a diverse seagrass ecosystem and is characterized by the gradient of freshwater influence that enters the system from major rivers in its northern region. This study took place from 2009 to 2021 and covered four sections of the “Big Bend” that varied in abiotic factors, such as salinity, water clarity, temperature, etc. We hypothesize that the abundance and distribution of these elasmobranch and large teleost species are associated with differences in abiotic factors between our sampling sites. Specifically, salinities and water temperature will play a significant role in the habitat usage of these organisms, and we expect there to be significant impacts of these factors that indicate habitat preferences amongst these species.
Nitrate and Phosphate Loads, but not Light Availability, Impact Freshwater Phytoplankton Diversity via Tradeoffs Between Dominant Species

(Georgia Institute of Technology, 2022-12-01) Southard, Michael Kelton

Eutrophication of freshwater ecosystems, mainly caused by nitrogen (N) and phosphorus (P) pollution, causes significant economic damages every year in the U.S. Excess N and P deposition in lakes can result in harmful algal blooms, reduced biodiversity, and increased greenhouse gas production, but we still do not fully understand how and why phytoplankton communities react to nutrient enrichment under varying conditions. Several theories – including the niche dimension hypothesis, biomass-driven competition hypothesis, nitrogen detriment hypothesis, and benthic model – are currently being explored in both terrestrial and aquatic producer communities in attempt to better understand the biological mechanisms effecting these systems, and the goal of this study was to determine which models are most applicable to freshwater phytoplankton. Using five-species microcosms of green algae, we found that N and P enrichment significantly reduced diversity (independent of light availability), which was likely mediated through nutrient tradeoffs between the two dominant species, Ankistrodesmus falcatus and Selenastrum capricornutum. Additionally, we observed a significant decrease in monoculture carrying capacity across all species with high N addition in low P concentrations, indicating that high N:P ratios may be physiologically harmful to green algae. These findings suggest that the niche dimension and nitrogen detriment hypotheses may be the most applicable to freshwater phytoplankton communities and could be useful for protecting and mitigating economic losses from these systems.
Characterization of chromatinized hydrogen peroxide biosensor in cancer cells

(Georgia Institute of Technology, 2022-06-02) Pfliger, Jessica M.

Hydrogen Peroxide (H2O2) is an important eukaryotic signaling molecule regulating cellular processes. As one of the most abundant reactive oxidative species (ROS), H2O2 can cause oxidative stress and cytotoxicity at elevated concentrations leading to DNA damage and programmed cell death. Despite the recent advancements in modulating ROS responses for potential cancer therapies, little is known about nuclear ROS temporal emergence and dynamics. To investigate the role of nuclear ROS and H2O2 in chromatin, we established a genetically engineered chromatin-targeted biosensor for H2O2, H2B-HyPer, by fusion of HyPer, a specific H2O2 biosensor, with core histone H2B. In this thesis, I utilized fluorescent microscopy and fluorescent recovery after photobleaching (FRAP) to study the H2O2 dynamics within the chromatin of HCT116 colon cancer cells containing genetically integrated H2B-HyPer. I demonstrate that H2B-HyPer is localized in the nucleus of HCT116/H2B-HyPer cells with an average residence time and mobile fraction of 9.14 minutes and 20.59%, respectively, comparable to the core histones in chromatin. I then show that H2B-HyPer is sensitive to changes in H2O2 levels post addition of H2O2 or DTT in culture medium. Further analysis of H2B-HyPer kinetics revealed a rapid increase and recovery of H2B-HyPer signal intensity in HCT116/H2B-HyPer cells upon treatment with and the removal of H2O2. Altogether, these studies establish H2B-HyPer as an effective biosensor for real-time spatio-temporal tracking of chromatin-proximal H2O2 dynamics.
Trophic structure in the Western Tropical North Atlantic Ocean using Stable Isotope Abundances (δ15N and δ13C)

(Georgia Institute of Technology, 2022-05-06) Salter, Shannon Marie

We investigated the biomass concentration and natural abundance of nitrogen and carbon stable isotopes (δ15N and δ13C) of size-fractionated zooplankton collected on two cruises to the Amazon River Plume (ARP) region. Mean animal biomass concentration was 3.34 mg/m2 during the day and 2.68 mg/m2 on Cruise EN614. The mean biomass on Cruise EN640 was 7.22 mg/m2 during the day and 9.60 mg/m2 at night. . Sampling stations were classified into habitat types based on biologically relevant physical and chemical parameters: the young plume core (YPC), old plume core (OPC), outer plume margin (OPM), western plume margin (WPM), and oceanic seawater (OSW). In general, zooplankton δ15N varied markedly and significantly among habitats for both cruises, with generally higher values in the YPC, OPC, WPM, and OSW habitats relative to the OPM habitat. Zooplankton δ15N values reflect the sources of nitrogen supporting biological production, and trophic processing within the food web. Suspended particles collected from these habitats showed lower δ15N values than the zooplankton, but we did not find a consistent increase in animal δ15N with size. Vertical migration led to diel shifts in zooplankton δ15N at the surface, with generally higher δ15N values at night than during the day during both cruises. Zooplankton δ13C values varied less than their δ15N, with no significant differences among habitats or with animal size.
Phylotranscriptomics points to multiple independent origins of multicellularity and cellular differentiation in the volvocine algae

(Georgia Institute of Technology, 2021-12-14) Lindsey, Charles

The volvocine algae, which include the single-celled species Chlamydomonas reinhardtii and the colonial species Volvox carteri, serve as a model in which to study the evolution of multicellularity and cellular differentiation. Studies reconstructing the history of this group have by and large relied on datasets of one to a few genes for phylogenetic inference and ancestral character state reconstruction. As a result, volvocine phylogenies lack concordance depending on the number and/or type of genes (i.e., chloroplast vs nuclear) chosen for phylogenetic inference. While multiple studies suggest that multicellularity evolved only once in the volvocine algae, that each of its three colonial families is monophyletic, and that there have been at least three independent origins of cellular differentiation in the group, other studies call into question one or more of these conclusions. An accurate assessment of the evolutionary history of the volvocine algae requires inference of a more robust phylogeny. We performed RNA sequencing (RNA-seq) on 55 strains representing 47 volvocine algal species and obtained similar data from curated databases on 13 additional strains. We then compiled a dataset consisting of transcripts for 40 single-copy, protein-coding, nuclear genes and subjected the predicted amino acid sequences of these genes to maximum likelihood, Bayesian inference, and coalescent-based analyses. These analyses show that multicellularity independently evolved at least twice in the volvocine algae and that the colonial family Goniaceae is not monophyletic. Our data further indicate that cellular differentiation arose independently at least four, and possibly as many as six times, within the volvocine algae. Altogether, our results demonstrate that multicellularity and cellular differentiation are evolutionarily labile in the volvocine algae, affirming the importance of this group as a model system for the study of major transitions in the history of life.
The impact of copper oxide nanoparticles on bacterial community assembly

(Georgia Institute of Technology, 2021-11-22) Wang, Jiong

A growing amount of copper oxide nanoparticles (CuO NPs) has been released into the environment. CuO NPs are known to be toxic to various organisms, but their impacts on the assembly of ecological communities are poorly understood. We examined the role of CuO NPs in influencing the assembly of laboratory bacterial assemblages consisting of three bacterial species. CuO NPs had significant negative effects on the growth rate and carrying capacity of all three species. Alpha diversity was significantly reduced by CuO NPs, regardless of assembly history. CuO NPs enhanced the inhibitive priority effects of early arriving species on later arriving species during community assembly. By so doing, CuO NPs promoted the importance of assembly history in shaping bacterial community structure, resulting in greater beta diversity between communities subjected to different histories. Moreover, communities in the copper ion treatment tended to have similar alpha and beta diversity as those in the controls, suggesting that copper ions may not be responsible for the effect of CuO NPs on community assembly. Our results indicate that CuO NPs could modify bacterial community structure by changing the importance of community assembly history and thus the strength of priority effects.
Assay development for and evaluation of sphingomyelinase D and associated activities in venoms from Loxosceles reclusa and Kukulcania hibernalis and in isolated soil bacteria

(Georgia Institute of Technology, 2021-08-02) Lachmayr, Hannah L.

Since a sphingomyelinase D (SMase D) enzyme is presumably the major agent in the necrosis and toxicity of the brown recluse spider venom by cleaving sphingomyelin (SM) to ceramide 1,3-cyclic phosphate (Cer(1,3)P), a rigorous but simple assay would aid in detecting the presence of this sphingomyelinase activity. Several Sicariidae spiders, such as the brown recluse spider Loxosceles reclusa, and some pathogenic bacteria contain phospholipase D (PLase D) enzymes that act on phospholipid substrates. PLases D that cleave SM to release the headgroup choline are also called SMases D. Although the lipid product of the L. reclusa SMase D was initially thought to be ceramide 1-phosphate (Cer1P), a transphosphatidylation mechanism by L. reclusa SMase D has been shown to produce Cer(1,3)P. For this thesis, fluorescent nitrobenzoxadiazole (NBD) analogs of products that are known to be made by various types of SMases (i.e., ceramide, Cer1P and Cer(1,3)P) were synthesized and an elution solvent system was identified that fully resolves these compounds as well as the substrate NBD-SM using silica gel thin-layer chromatography plates. When venom from L. reclusa was assayed, the product was the expected NBD-labeled Cer(1,3)P. Venom from Kukulcania hibernalis, a spider that has been suggested to have SMase D but whose products have not yet been determined, was assayed as well and NBD-Cer(1,3)P was found to be produced. To explore additional biomedically relevant applications of these methodologies, such as to discover bacterial enzymes that can degrade sphingolipids, we attempted to isolate bacteria from the soil with enzymatic activity to degrade SM and/or cyclic Cer(1,3)P. Single isolates of soil microorganisms were selected based on their ability to grow on the restrictive carbon sources SM or Cer(1,3)P and then examined for their cleavage of NBD-SM and/or NBD-Cer(1,3)P. Of four candidate isolates, genome sequencing of two isolates was taxonomically assigned to Klebsiella variicola, a gram-negative bacterium and opportunistic human pathogen. Unfortunately, in the timeframe of this thesis, the conditions to assay the degradatory enzymes were not found. In summary, this work has developed a facile assay for SMase D, characterized for the first time Cer(1,3)P as a breakdown product of the action of K. hibernalis venom on SM, and expanded the methodologies that are available for analysis of activities that produce and degrade Cer(1,3)P.
The Role of Cu, Zn Superoxide Dismutase (SOD1) in Muscle Stem Cell Function

(Georgia Institute of Technology, 2021-05-04) Ergun, Utku

Age-related loss of muscle mass and function often referred to as sarcopenia, dramatically affects the quality of life in the elderly population and predisposes them to an increased risk of morbidity, disability, and mortality. The etiology of sarcopenia is a multi-factorial process that involves both intrinsic and extrinsic factors. However, mounting evidence from both animal and human studies suggests that uncontrolled production of reactive oxygen species (ROS) and subsequent oxidative stress and oxidative modifications to key macromolecules such as DNA, RNA, proteins, and lipids are key underlying mechanisms that exacerbate sarcopenia. Cu, Zn superoxide dismutase (SOD1) is one of the essential antioxidant enzymes that are indispensable for redox homeostasis and prevention of oxidative damage. To maintain skeletal muscle homeostasis and repair damaged muscle, a population of dedicated muscle stem cells (MuSC), called satellite cells, activate, express myogenic transcription factors, migrate, proliferate, and fuse with existing myofibers or form de novo myofibers to complete regeneration. However, increased oxidative stress in muscle stem cells may harm the muscle regeneration process by impairing the interactions between existing myofibers with newly regenerated myoblasts and muscle stem cells. Previous studies showed that the whole body-deletion of Sod1 increases the age‐associated sarcopenia in mice; however, effects of deletion of Sod1 specifically in muscle stem cells and how it affects the muscle regeneration processes are still not known. We aimed to fill this gap by investigating the change in myogenesis and muscle regeneration of muscle stem cell-specific Sod1 knock-out (MuSC-Sod1KO) mice. Our in-vivo results revealed that the muscle regeneration and MuSC function are severely compromised in the MuSC-Sod1KO mice. Furthermore, our in-vitro studies show that the proliferation, differentiation, and fusion were impaired in the Sod1KO due to the increased number of apoptotic death in the myogenic progenies. In summary, we provide direct evidence that oxidative stress in muscle stem cells deteriorates and impairs muscle stem cell function by increasing the apoptotic response in muscle stem cells. We hope our study will open the path for better understanding the redox regulation in stem cells and aid in developing strategies to enhance stem cell-based therapies against muscle-wasting conditions, such as sarcopenia.