Genome-Wide Characterization of the Effects of Nucleic Acid Modifying Enzymes: Cytidine Deaminases and DNA Methylation

Activation-induced cytidine deaminase (AID) is essential for two processes of immunoglobulin diversification in germinal center B cells: somatic hypermutation (SHM), in which mutations are introduced into immunoglobulin (Ig) genes, and class-switch recombination (CSR), in which genomic constant regions are recombined to encode antibodies of different isotypes. Both of these processes require AID-catalyzed C-to-U lesions at the Ig loci, which are resolved to generate point mutations or double-stranded DNA breaks in the cases of SHM and CSR, respectively. Despite over a decade of intense study, a number of open issues remain surrounding AID. The diversity of findings regarding AID’s role in DNA demethylation raises the question of the scope of its involvement in this process. Additionally, while it is clear that AID-mediated damage occurs, the effects of this damage on the average B cell have not been characterized. Finally, the issue of whether AID is able to edit RNA in vivo has never been rigorously addressed in the literature. In each of these cases, the advent of high-throughput sequencing provides methods for genome-wide characterization of AID’s effects. This thesis presents the application of a number of genome-scale, sequencing-based methods to characterize the effects of AID deficiency and overexpression on the activated B cell: mRNA-Seq and miRNA-Seq allow for measurements of RNA expression and editing, while reduced-representation bisulfite sequencing (RRBS) assays DNA methylation. These analyses confirmed AID’s known role in immunoglobulin isotype switching, while also demonstrating that it has little other effect on gene expression. Additionally, no evidence of AID-dependent mRNA or miRNA editing could be detected. Finally, RRBS data failed to support a role for AID in the regulation of DNA methylation. Thus, despite evidence of its additional activities in other systems, antibody diversification appears to be AID’s sole physiological function in activated B cells. Following the conclusion of my studies of AID’s effects in B cells, I applied similar genomics tools to two amenable topics in nucleic acid modifications. First, I used mRNA-Seq to attempt to determine the substrate of the orphan cytidine deaminase Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 2 (APOBEC2). Next, I used whole-genome bisulfite sequencing to explore the distribution of 5-methylcytosine in Trypanosoma brucei. In both of these cases, results were inconclusive but suggest future directions for investigation

Lycium barbarum (wolfberry) polysaccharide facilitates ejaculatory behaviour in male rats

Poster Session AOBJECTIVE: Lycium barbarum (wolfberry) is a traditional Chinese medicine, which has been considered to have therapeutic effect on male infertility. However, there is a lack of studies support the claims. We thus investigated the effect of Lycium barbarum polysaccharide (LBP), a major component of wolfberry, on male rat copulatory behavior. METHOD: Sprague-Dawley rats were divided into two groups (n=8 for each group). The first group received oral feeding of LBP at dosage of 1mg/kg daily. The control group received vehicle (0.01M phosphate-buffered saline, served as control) feeding daily for 21 days. Copulatory tests were conducted at 7, 14 and 21 days after initiation of treatment. RESULTS: Compared to control animals, animals fed with 1mg/kg LBP showed improved copulatory behavior in terms of: 1. Higher copulatory efficiency (i.e. higher frequency to show intromission rather than mounting during the test), 2. higher ejaculation frequency and 3. Shorter ejaculation latency. The differences were found at all time points (Analyzed with two-tailed student’s t-test, p<0.05). There is no significant difference found between the two groups in terms of mount/intromission latency, which indicates no difference in time required for initiation of sexual activity. Additionally, no difference in mount frequency and intromission frequency was found. CONCLUSION: The present study provides scientific evidence for the traditional use of Lycium barbarum on male sexual behavior. The result provides basis for further study of wolfberry on sexual functioning and its use as an alternative treatment in reproductive medicine.postprintThe 30th Annual Meeting of the Australian Neuroscience Society, in conjunction with the 50th Anniversary Meeting of the Australian Physiological Society (ANS/AuPS 2010), Sydney, Australia, 31 January-3 February 2010. In Abstract Book of ANS/AuPS, 2010, p. 177, abstract no. POS-TUE-19

Study of Human Muscle Structure and Function with Velocity Encoded Phase Contrast and Diffusion Tensor Magnetic Resonance Imaging Techniques

The disproportionate loss of muscle force with aging and disuse atrophy compared to the loss of muscle mass is not yet completely understood. In addition to well-established neural and contractile determinants of force loss, remodeling of the extracellular matrix (ECM) has been recently shown in animal models to be another important contributor. In-vivo human studies exploring the structural remodeling of the ECM and its functional consequences are lacking due to the paucity of appropriate imaging techniques. This study focuses on the development and application of advanced Magnetic Resonance Imaging (MRI) methods to elucidate the mechanisms of loss of force with aging and disuse atrophy with the focus on ECM. Functional changes are investigated by strain and strain rate tensor mapping of muscle under different contraction paradigms using Velocity Encoded Phase-Contrast MRI. Methodological advances include improvements in hardware and software control of the dynamic studies. To overcome the limitation of long scan times, compressed sensing MR acquisition and reconstruction framework to reduce scan times to under a minute were developed. A multi-step automated analysis pipeline to extract 3D strain/strain rate tensors from the velocity images was implemented to process the large dynamic volumes. Strain indices reflecting the material properties of the ECM were shown to correlate with force loss leading to a hypothesis that shear strain may serve as a surrogate marker for lateral transmission of force. Diffusion tensor imaging has been applied previously to study skeletal muscle fiber architecture. The resolution of the images precludes direct inferences to be made about the microstructure. To address this limitation, bicompartmental and Random Permeable Barrier models of diffusion were applied to the diffusion data obtained with spin-echo and custom-developed stimulated echo echo-planar-imaging sequences respectively. Model derived parameters (fiber diameter, wall permeability) obtained from fitting time-dependent diffusion data were in physiologically reasonable range, with potential for tracking age related changes in muscle microstructure. The developed imaging and modeling techniques were applied to a cohort of young/senior subjects and to longitudinal tracking of disuse atrophy induced by Unilateral Limb Suspension. These studies may potentially provide the causal link between age- and disuse-related structural remodeling and its functional consequences

Population Declines and Genetic Variation: Effects of Serial Bottlenecks

Islands foster unique biodiversity, yet also present biogeographic limitations that impose increased risk for population extinction through demographic and genetic constraints and decreased probability of surviving a catastrophe. Of particular interest, especially with regard to endangered species, is the genetic response of insular species to severe population declines or translocations. Both types of events, considered population bottlenecks, are expected to reduce genetic variation, and correspondingly, adaptive potential. For these reasons, it is important to understand how bottlenecks interact with insular population dynamics to affect genetic diversity. I used a combination of a laboratory model experiment and population genetics study of an in situ bottleneck in an endangered species to investigate how quantitative and molecular genetic variation are affected during bottlenecks. I used a laboratory animal model (red flour beetle, Tribolium castaneum) to compare how quantitative genetic variation is affected if a serial bottleneck occurs in a novel versus familiar environment. The experiment was designed to model a founder event or translocation to a new island with a novel environment. I found that phenotypic and additive variance for a quantitative trait were larger following a bottleneck occurring in the novel environment, suggesting that the novel environment could improve adaptive potential in bottlenecked populations. Next, I used molecular genetic markers to assess variation and signatures of selection in the Laysan finch (Telespiza cantans), a Hawaiian honeycreeper endemic to a small Northwestern Hawaiian island. Laysan finches experienced a major bottleneck on Laysan in the early 20th century, followed by a translocation and series of founder events as populations were established on the islets of Pearl and Hermes Reef (PHR) in the 1960s – 70s. I found that, contrary to expectation, bottlenecked Laysan finch populations did not show declines in genetic variation and were not differentiated as a result of genetic drift. These results are potentially caused by insular demographic dynamics. I identified loci with extreme differentiation between modern populations, potentially indicating genomic signals of selection. These regions could be important for adaptation to the novel environment on PHR and are candidates for future study

Quantitative magnetic resonance angiography for flow quantification of carotid and intracranial stenosis

Carotid and intracranial stenosis are responsible for stroke, which is the third leading cause of death globally. Carotid and intracranial stenting are used in management of stenosis. In-stent stenosis occurs in 25-35% of patients. Stent related artifacts and artificial lumen narrowing are problems during MRI scanning. A potential solution is estimate the flow profile across stenosis. The goal of this project is to optimize and implement quantitative magnetic resonance angiography (QMRA) for flow encoding and flow velocity estimation for better characterizing stenosis and assessing its degrees. We conducted multiple phantom measurements (mimicking normal carotid and middle cerebral anatomy) using phase contrast sequence with implementing different degrees of stenosis. Subsequent volunteer and patients measurements were conducted. In 8 mm tubes, flow was stable till 75 % stenosis while in 4 mm tubes, decline starts at 65% stenosis. In subject measurements, there is flow asymmetry between paired ICAs and MCAs that is not pathologic but within certain range. Patients had reduced flow in stenotic vessels compared to the other side and compared to mean flow in our volunteer subjects. QMRA results demonstrated reduced flow on the exact side detected with conventional MRA, with a strong correlation between QMRA and conventional MRA (R2 =0.7942). The percentage of flow difference between sides varied with the degree of stenosis. QMRA can be used for non-invasive diagnosis of suspected stenosis even if the stenosis itself cannot be visualized

The Evolution of Gene Regulation in Drosophila.

Differences in gene expression drive phenotypic diversity. At the level of transcription, these differences are largely controlled by the complex interplay between trans-acting factors and the cis-regulatory sequences to which they bind. In this dissertation, I characterized how the regulation of gene expression has evolved both within and between several species of the Drosophila lineage. I began by describing current methodology to accurately quantify allele-specific expression (ASE) from RNA-seq data, comparing two different methods and highlighting sources of bias. Using this methodology, I measured allele-specific differences in F1 hybrids made by reciprocally crossing two strains of D. melanogaster. These two sets of genetically-identical hybrids differed only by which strain contributed the maternal or paternal allele, allowing me to test the hypothesis that D. melanogaster do not imprint their genome. Next, for that same intraspecific comparison as well as two interspecific comparisons, I measured total and allele-specific gene expression to categorize regulatory differences across divergence times ranging from 0.01-2.5 million years ago. This allowed me to test the hypothesis that cis-regulatory differences account for a higher proportion of the total regulatory differences between species, as well as to determine how patterns for inheritance of gene expression differ across an evolutionary timescale. Because all of these comparisons were made using female whole flies, I tested the prevalence of sex- and tissue-specific differences using gene expression data from female and male carcass and gonad tissues between D. pseudoobscura and its closely-related subspecies D. p. bogotana and their F1 hybrids. I determined that one must use caution when inferring patterns of regulatory divergence in whole flies, as the integration over all different tissue types can mask the complexity of gene regulation in individual tissues. The work in this dissertation expands our knowledge of how the regulation of gene expression differs across a well-characterized lineage and will continue to drive further studies of these phenomena in even more distantly-related species.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110328/4/Kraig_Stevenson_Dissertation.pd

Microbiome and aging: A study of microbial evolution and community structure across model organisms. Abtract and Metadata

Section I : Evolution of commensal gut microbes during host aging The animal intestine contains a large number of resident microorganisms (gut micro- biota), which has a significant impact on host physiology. The gut microbiota composition changes dramatically throughout the host lifespan as the gut microbes face multiple changes in their ecological niche. Age-associated changes in the host environment exert strong selective pressures on the microbial community, and whether and how gut microbes evolve during host life span and how this affects host age-associated phenotype is an open question. Specifically, novel genetic variants in the microbiome can confer new adaptive traits to the host or follow a virulent trajectory that affects host-microbiota homeostasis and delimit host lifespan. Previous studies demonstrated that the gut microbiome could rapidly adapt and evolve in a short timescale. However, less is known about the evolutionary dynamics of this complex microbial community during host aging within the same individual host. My research project aims to survey how the gut microbiota evolves during the host́s life span and whether evolutionary processes in the gut microbial communities affect microbial fitness. I leverage three different metagenomic sequencing strategies to reassemble more than 200 different metagenomic-assembled genomes from three different individual hosts. Using this genome catalog, I identified and tracked single- nucleotide variants that show signatures of selection during host life. Overall, I found that most new variants detected are impacted by purifying selection. However, I found that genetic variants in outer membrane proteins, carbohydrate-activate enzymes, and transposase genes show signatures of positive selection with no obvious beneficial effect on microbial fitness. The results obtained in this thesis will help understand the impact of host aging in determining microbiome evolution and whether evolution acts for the emergence of disease-promoting bacterial bacteria strains in the elderly. Section II: Characterization of the gut microbial community of the aging model Nothobranchius furzeri using short and long read metagenomics The African turquoise killifish (Nothobranchius furzeri) is an emerging model for aging research, thanks to its naturally short lifespan, easy husbandry under laboratory conditions, and the display of typical vertebrates hallmarks of aging. Recent work in this species has shown that gut microbiota transfer from young to middle-age killifish increases the recipients’ life span and delays age-related behavioral decline. The African turquoise killifish possesses a complex microbial diversity compared to other vertebrates (zebrafish, mouse, humans), which significantly decline during natural aging, with concurrent increase in the relative abundance of potentially pathogenic bacteria in older individuals. While the characterization of killifish-associated microbiota has focused on 16S amplicon sequencing, an account of the intestinal metagenome in this species is lacking. To fill this gap, I combine short-read with long-read sequencing technology to analyze the stool metagenome in captive turquoise killifish. I found that bacteria represent the majority of the stool microbiota (73%), with Proteobacteria as the most abundant Phylum (93%), followed by Actinobacteria (4%), Firmicutes (2%), and Bacteroidetes (2%). Viruses represent 0.04% of the total stool microbiome, followed by Archaea (0.02%) and Fungi (0.02%). Notably, 27% of the total stool metagenome does not map to any reference database (RefSeq) representing potentially novel host-associated species. Additionally, to generate a catalog of bacterial genomes associated with killifish gut, I assembled the genome of 8 of the most abundant bacterial species by culturing, isolating, sequencing, and hybrid assembly strategy, involving Illumina and long-read Nanopore sequencing technology. The analysis of the fully- assembled intestinal bacterial genomes revealed the presence of mobile elements, prophages, antibiotic-resistant genes, and other genetic elements that could potentially contribute to bacterial adaptations in the host. Together, metagenomics in the turquoise killifish represents a critical new resource for this species, which will help further develop this species as a naturally short-lived model to study host-microbiome interactions during aging

Microarray tools and analysis methods to better characterize biological networks

To accurately model a biological system (e.g. cell), we first need to characterize each of its distinct networks. While omics data has given us unprecedented insight into the structure and dynamics of these networks, the associated analysis routines are more involved and the accuracy and precision of the experimental technologies not sufficiently examined. The main focus of our research has been to develop methods and tools to better manage and interpret microarray data. How can we improve methods to store and retrieve microarray data from a relational database? What experimental and biological factors most influence our interpretation of a microarray's measurements? By accounting for these factors, can we improve the accuracy and precision of microarray measurements? It's essential to address these last two questions before using 'omics data for downstream analyses, such as inferring transciption regulatory networks from microarray data. While answers to such questions are vital to microarray research in particular, they are equally relevant to systems biology in general. We developed three studies to investigate aspects of these questions when using Affymetrix expression arrays. In the first study, we develop the Data-FATE framework to improve the handling of large scientific data sets. In the next two studies, we developed methods and tools that allow us to examine the impact of physical and technical factors known or suspected to dramatically alter the interpretation of a microarray experiment. In the second study, we develop ArrayInitiative -- a tool that simplifies the process of creating custom CDFs -- so that we can easily re-design the array specifications for Affymetrix 3' IVT expression arrays. This tool is essential for testing the impact of the various factors, and for making the framework easy to communicate and re-use. We then use ArrayInitiative in a case study to illustrate the impact of several factors known to distort microarray signals. In the third study, we systematically and exhaustively examine the effect of physical and technical factors -- both generally accepted and novel -- on our interpretation of dozens of experiments using hundreds of E. coli Affymetrix microarrays

Molecular genetic investigations of renal cell carcinoma predisposition

Renal Cell Carcinomas (RCC) are a diverse group of histologically and genetically distinct renal neoplasms accounting for 2.4% of all cancers worldwide. While a majority of RCC cases are sporadic in nature, a proportion are due to genetic predisposition caused by syndromic and non-syndromic conditions. Inherited renal cell carcinoma is associated with alterations in genes such as VHL, MET, FH, and FLCN and identification of these genes has been critical to understanding the molecular biology of both inherited and sporadic RCC, informing both clinical management and treatment. Despite the large number of known genes which are linked to RCC predisposition, most individuals with features of RCC predisposition do not harbour variants in known inherited RCC genes, suggesting additional unknown causes of heritability have yet to be uncovered. This study has utilised a range of genomic sequencing methodologies, scaling from single gene to whole genome sequencing, on individuals with features of renal cell carcinoma predisposition in order to identify novel causes of heritability associated with RCC. Multiple genomic sequencing approaches in these individuals has uncovered a range of potential genetic features that could be associated with predisposition to RCC, including genes not previously known to be associated with RCC, discovery of new molecular mechanisms of genetic inheritance for known RCC predisposition syndromes, and provided innovative methods for the identification and characterisation of molecular alterations in specific inherited RCC subtypes

Context matters:the power of single-cell analyses in identifying context-dependent effects on gene expression in blood immune cells

The human immune system is a complex system that we still do not fully understand. No two humans react in the same way to attacks by bacteria, viruses or fungi. Factors such as genetics, the type of pathogen or previous exposure to the pathogen may explain this diversity in response. Single-cell RNA sequencing (scRNA-seq) is a new technique that enables us to study the gene expression of each cell individually, allowing us to study immune diversity in much greater detail. This increased resolution helps us discern how disease-associated genetic variants actually contribute to disease. In this thesis, I studied the relation between disease-associated genetic variants and gene expression levels in the context of different cell types and pathogen exposures in order to gain insight into the working mechanisms of these variants. For many variants we learnt in which cell types and under which pathogen exposures they affect gene expression, and we were even able to identify changes in gene co-expression, suggesting that disease-associated variants change how our genes interact with each other. With the single-cell field being so new, much of my work was showing the feasibility of using scRNA-seq to study the interplay between genetics and gene expression. To set up future research, we created guidelines for these analyses and established a consortium that brings together many major scientists in the field to enable large-scale studies across an even wider variety of contexts. This final work helps inform current and future large-scale scRNA-seq research