The study of cell physiology and functional genomics has seen an explosion of interest stemming from the development and commercialization of DNA sequencing technologies that allow upwards of several billion molecules to be probed simultaneously. However, of the three most abundant biomolecules in the cell—DNA, RNA, and protein—the dynamic and ever-changing quantities of RNA and proteins in a cell dictate much of the phenotypic variation observed from tissue-to-tissue, organ-to-organ, and cell-to-cell. Though much work has been done to measure RNA quantities in cells, and even to model their temporal dynamics from a single time-point measurement, the focus of this thesis will be on the development of methods to measure proteins within cells to draw conclusions about their physiological implications for the larger organism. In the outlined work, we couple protein measurements to DNA readouts that allow us to leverage commercial sequencing platforms to determine phenotypic outcomes through different methodologies. This thesis will proceed in two parts. Chapter 2 highlights the development of our method (Quantum Barcoding 2; QBC2) which uses DNA-barcoded antibodies to simultaneously quantify the expression of dozens of proteins on single cells. We demonstrate through head-to-head comparisons between our method and the traditional diagnostic gold standard of flow cytometry that we can accurately distinguish cell types and readily capture rare phenotypes that are otherwise too costly or labor intensive to probe using traditional methods. Chapter 3 discusses a deep mutational scanning (DMS) study conceived and developed during the COVID-19 pandemic, which reveals a detailed understanding of the 3CL protease of the SARS-CoV-2 virus, one of the critical components of the virus replication machinery. This technique is similarly applied to DNAJB6 to evaluate its ability to function as a chaperone protein. By leveraging comprehensive mutagenesis with methods of probing gene function en masse, we were able to evaluate the fitness effect of all amino acid substitutions within the 3CL protease and a large portion of DNAJB6, giving us valuable insight into their mechanisms of action. As a whole, this thesis presents a multi-faceted view of how new tools can be developed to measure protein expression and function, with the potential to generalize to other currently unexplored modalities.

Soil fungi release up to three-fold more carbon dioxide (CO2) to the atmosphere than human activity through the decomposition of dead organic matter (i.e. litter) in soil. Ecologists have frequently observed a pattern of fungal community assembly during litter decomposition, wherein different fungal taxa dominate different stages of decay in individual ecosystems. However, we still lack a complete understanding of how these diverse fungal communities generate decomposition. My dissertation helps to fill this knowledge gap by elucidating the cross-ecosystem taxonomic patterns of fungal decomposer succession and identifying biological features that correlate with these lineage-specific patterns. I conducted a meta-analysis of fungal decomposer succession in 22 ecosystems to identify the taxonomic patterns that were consistent across ecosystems and test environmental correlates of those patterns. I found a phylogenetic signal to succession and observed that the relative abundance of Ascomycetes was negatively correlated with observed peak decay stage, the relative abundance of Zygomycetes was positively correlated with peak decay stage, and the relative abundance of Basidiomycetes remained relatively constant throughout decay. I also found that plant litter type and climate factors were correlates of peak decay stage. Next, I performed laboratory culture experiments to test two longstanding hypotheses: that innate potential activity of plant cell wall-degrading enzymes and intrinsic growth rate underlie organisms’ peak decay stage. I found that potential cellulase activity and growth rate correlated negatively with peak decay stage and that growth rate explained more than a third of the phylogenetic signal to peak decay stage. Finally, I completed a comparative genomics study of fungi that have different observed decay stages. I found that protein domains related to plant C-degrading enzymes and growth-related biochemical pathways are part of the biology that underlies the succession of fungi and that transcriptional regulation and stress response may also underlie succession. This dissertation generates novel insights into the biology that underlies fungal decomposer community turnover during plant litter decay. This work provides direction for future research into the drivers of the assembly of decomposer communities and has important implications for efforts to incorporate fungal biology into predictive models of decomposition and terrestrial C-cycle dynamics.

No description available.

Precision medicine is defined as the use of genomics and big data approaches to health to better tailor the diagnosis and treatment of disease to patients. Precision medicine was conceived in the National Research Council’s 2011 report Towards Precision Medicine and was picked up by the Obama Administration in its 2015 launch of the Precision Medicine Initiative. Central to this is the All of Us Research Program, which seeks to sequence the genomes and conduct a longitudinal study of 1 million individuals to advance knowledge about various health outcomes. Precision medicine has been taken up by governments and organizations around the world, notably in China, where the term was incorporated in national plans in 2016 such as the 13th Five Year Plan and Healthy China 2030. Precision medicine became a “key strategy”, and a large amount of funding was pledged to finance the start of precision medicine projects at a range of research organizations, such as the Chinese Academy of Sciences and BGI. My dissertation investigates why precision medicine attracted the attention of scientists, policymakers, and clinicians in the 2010s. It also traces how the precision medicine bandwagon gained so many allies globally, and what precision medicine means for stakeholders located at different positions in the emerging field. To answer these questions, I apply the concepts of global field and scientific capital to trace the emergence of precision medicine at the global and national levels. My argument analytically distinguishes between global scientific capital and national scientific capital in order to show how varying combinations of scientific capital orients actors towards different goals and priorities of precision medicine. More generally, I demonstrate how hybrids and “off-label” forms of science appear in the process of scientific globalization. In the introduction of the dissertation, I look to Bourdieu’s writing on scientific fields to lay out my theoretical framework of fields and capitals as it applies to global science. The dissertation is then organized into three substantive chapters. In Chapter 1, I trace the emergence of the global field of precision medicine drawing on two sources of data: first, a bibliometric analysis of scientific publications in precision medicine, and a further analysis of the key institutions and actors behind its global push. This chapter charts the contours of the global scientific field of precision medicine and the logics of accruing global scientific capital. In Chapter 2, I examine the differentiation of the national field of precision medicine in China from the global field, and trace the logics of accumulation for a national scientific capital. In this chapter, I draw on documentary analysis to tell the recent history of genomics in China, as well as interviews with scientists and participant observation of scientific conferences. In doing this, I shed light on two hybrid forms of precision medicine in China: Chinese Precision Medicine or the use of genomics to identify “Chinese DNA” and to cure “Chinese diseases”, and Precision Chinese Medicine or the use of genomics to open the “black box” of traditional Chinese medicine. In Chapter 3, I take the case of genetic talent testing in China to show how precision medicine is understood by the public. Making use of social media data, and a content analysis of news articles and marketing material, I argue against the “deficit model” of science used to paint parents who use genetic talent tests as scientifically illiterate. Instead, I show how this “off-label” use of genomics responds to broader social, political, and economic pressures of parenting in contemporary China, and argue that scientific capital continues to shape the circulation of genetic talent testing as it encounters the public. I conclude with notes on how the imaginary of precision medicine is affecting the practice of precision governance in China and observations of how the ongoing U.S.-China uncoupling may shape global science.

The classic view of evolution as "shifting gene frequencies" in the Modern Synthesis literally means that evolution is the modulation of existing variation ("standing variation"), as opposed to a "new mutations" view of evolution as a 2-step process of mutational origin followed by acceptance-or-rejection (via selection and drift). The latter view has received renewed attention, yet its implications for evolutionary causation still are not widely understood. We review theoretical results showing that this conception of evolution allows for a role of mutation as a cause of nonrandomness, a role that could be important but has been misconceived and associated misleadingly with neutral evolution. Specifically, biases in the introduction of variation, including mutational biases, may impose predictable biases on evolution, with no necessary dependence on neutrality. As an example of how important such effects may be, we present a new analysis partitioning the variance in mean rates of amino acid replacement during human-chimpanzee divergence to components of codon mutation and amino acid exchangeability. The results indicate that mutational effects are not merely important but account for most of the variance explained. The challenge that such results pose for comparative genomics is to address mutational effects as a necessary part of any analysis of causal factors. To meet this challenge requires developing knowledge of mutation as a biological process, understanding how mutation imposes propensities on evolution, and applying methods of analysis that incorporate mutational effects.

Neuroblastoma (NBL), the most common non-Central Nervous System (CNS) solid tumor of childhood, characteristically displays heterogeneous clinical presentation and biological behavior. Previous work has studied the genetic basis of the disease and revealed a low somatic mutation burden. In order to identify novel therapeutic targets and better understand the biology of high-risk NBLs, I investigated whole transcriptome profiles of two cohorts of metastatic NBLs using RNA sequencing. First, I studied changes in splicing pattern in a cohort of 29 patients. V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplified NBLs showed a distinct splicing pattern affecting multiple cancer hallmarks. Six splicing factors have altered expression patterns in MYCN-amplified tumors and cell lines, and binding motifs for these factors were significantly enriched in differentially-spliced genes. ChIP-seq analysis showed direct binding of MYCN to promoter regions of splicing factors PTBP1 and HNRNPA1, demonstrating that MYCN regulates splicing by directly regulating expression of key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage 4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, a pro-tumor-growth isoform, resulted in repression of NBL cell growth. Second, I used whole transcriptome sequencing in a cohort of 150 patients to assess expressed mutations, fusion genes, and gene expression including long non-coding genes to provide clinically-relevant classification and to offer insights into NBL tumor biology. Twenty-four genes including ALK, ATRX and MYCN were recurrently mutated in NBL transcriptomes. In-frame FOXR1 fusions were detected in 4 samples, including 3 cases or 14% of stage 4S NBLs. Unsupervised gene expression analysis revealed four molecular subgroups. MYCN and tumor microenvironment were the primary discriminating signatures in these molecular subgroups. Fifty-eight percent of MYCN-not-amplified samples showed high MYCN signatures, which were potentially contributed by various genomic events such as MYCN activating mutations and FOXR1 fusions. High MYCN signature was significantly associated with poor overall survival in MYCN-not-amplified tumors (p=0.0017). In addition, the tumor microenvironment including stromal and immune cell infiltration significantly contributed to the NBL transcriptional landscape and tumor progression.

The mycolic acid bacteria are a distinct suprageneric group of asporogenous Grampositive, high GC-content bacteria, distinguished by the presence of mycolic acids in their cell envelope. They exhibit great diversity in their cell and morphology; although primarily non-pathogens, this group contains three major pathogens Mycobacterium leprae, Mycobacterium tuberculosis complex, and Corynebacterium diphtheria. Although the mycolic acid bacteria are a clearly defined group of bacteria, the taxonomic relationships between its constituent genera and species are less well defined. Two approaches were tested for their suitability in describing the taxonomy of the group. First, a Multilocus Sequence Typing (MLST) experiment was assessed and found to be superior to monophyletic (16S small ribosomal subunit) in delineating a total of 52 mycolic acid bacterial species. Phylogenetic inference was performed using the neighbor-joining method. To further refine phylogenetic analysis and to take advantage of the widespread availability of bacterial genome data, a computational framework that simulates DNA-DNA hybridisation was developed and validated using multiscale bootstrap resampling. The tool classifies microbial genomes based on whole genome DNA, and was deployed as a web-application using PHP and Javascript. It is accessible online at http://cbrc.kaust.edu.sa/dna_hybridization/ A third study was a computational and statistical methods in the identification and analysis of a putative minimal mycolic acid bacterial genome so as to better understand (1) the genomic requirements to encode a mycolic acid bacterial cell and (2) the role and type of genes and genetic elements that lead to the massive increase in genome size in environmental mycolic acid bacteria. Using a reciprocal comparison approach, a total of 690 orthologous gene clusters forming a putative minimal genome were identified across 24 mycolic acid bacterial species. In order to identify new potential drug candidates against the pathogenic mycolic acid bacteria, a drug target analysis of the protein-coding genes, that are conserved across 15 pathogens, identified a total of 175 potential broad spectrum drug candidates. The activity of the predicted targets can be modified by a total of 306 chemical compounds, 34 of which have been approved by the Food and Drug Administration (FDA), for human treatment.

Bats asymptomatically host a number of viruses that are the cause of recently emergent infectious diseases in humans. While the mechanisms underlying this asymptomatic infection are currently not known, studies of sequenced bat genomes help uncover genetic adaptations in bats that may have functional importance in the antiviral response of these animals. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of the Egyptian rousette bat (ERB; Rousettus aegyptiacus), a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We used this genome to understand the evolution of immune genes and gene families in bats, and describe several observations relevant to defense against viruses. We observed an unusual expansion of the NKG2/CD94 natural killer (NK) cell receptor gene families in Egyptian rousette bats relative to other species, and found genomic evidence of unique features and expression of these receptors that may result in a net inhibitory balance within bat NK cells. The expansion of NK cell receptors is matched by an expansion of potential major histocompatibility complex (MHC) class I ligands, which are distributed both within and, surprisingly, outside the canonical MHC loci. We also observed that the type I interferon (IFN) locus is considerably expanded and diversified in the ERB, and that the IFN-ω subfamily contributes most to this expansion. To understand the functional implications of this expansion, we synthesized multiple IFN-ω proteins and examined their antiviral effects. Members of this subfamily are not constitutively expressed but are induced after viral infection, and show antiviral activity in vitro, with different antiviral potencies observed for different IFN-ω proteins. Taken together, these results show that multiple bats, including the ERB, have expanded and diversified numerous antiviral loci, and potentially developed unique adaptations in NK cell receptor signaling, and type I IFN responses. The concerted evolution of so many key components of immunity in the ERB is strongly suggestive of novel modes of antiviral defense that may contribute to the ability of bats to asymptomatically host viruses that are pathogenic in humans.

Down Syndrome is a human condition caused by an extra copy of a #21 chromosome. At least one to two percent of free (not translocated) trisomy 21 cases are mosaics, i.e., they have two or more distinct cell lines. Usually, one cell line is 47 ,XX or XY ,+21 while the other cell line is normal 46,XX or 46,XY. The purpose of the study was to establish the etiologies of the separate cell lines by determining whether the zygote was trisomic or normal. Meiotic non-disjunction in the formation of a gamete could lead to a trisomic zygote; loss of a #21 chromosome during a later mitotic division could then lead to a chromosomally normal cell line. Alternatively, a mitotic error in a normal embryo can produce a trisomy 21 cell line.

No description available.