Human clinical genome-wide association studies (GWAS) have helped identify disease trait and pharmacogenomic loci without the need for biological under- standing. Molecular GWAS - associating genetic variation with traits such as gene expression - have been slow to fill the mechanistic gaps. While tissue specificity, lack of DNA resolution, and the need for better data integration are no dou bt important bottlenecks in molecular GWAS, there is also a very poor general understanding of which molecular phenotypes are important and how best to model them. Added to this is the clear need for a greater understanding of the strengths and weaknesses facing in vitro (and ex vivo) models as hypoth- esis generating and GWAS validation tools. The studies in this work focus on RNA expression in a popular human model: lymphoblastoid cell lines (LCLs). Chapters 2 and 3 examine microRNA (miRNA) and messenger RNA (mRNA) expression in a total of 300 genotyped human LCLs. The expression of only one miRNA could be associated with a nearby genetic variant. This result was observed in both the African and European samples studied, in a separate val- idation data set, and was technically validated with quantitative PCR. While limited genotype resolution and small sample sizes are likely to be important contributors to this low hit rate, the results strongly suggest experimental con- founders. Highly expressed miRNAs reflected the transformed nature of the cells, highly correlated miRNAs enriched for EBV and malaria associated tar- get mRNA genes, and several miRNAs that were differentially expressed be- tween the European and African samples suggested differential EBV transfer- mation. Chapter 4 presents a study on single cells from some of the same samples, to test the hypothesis that the lack of tissue spatial resolution is an important limiting factor in human genetic epidemiology. Experimental con- founders were also considered: sample growth was found to associate with the expression of several genes. Cell-to-cell gene correlations and distributions made it possible to propose how genes change their expression, functionally differ from each other, and are able to alter their behaviours without altered whole-tissue expression. The results suggest which type of genes are more likely to be susceptible to genetic effects, and propose promoter behaviours altered by genetic variants located near to 13 genes. From these whole-tissue and single cell results the broad conclusion is that, while LCLs are likely to be inappropriate for the study of miRNA genetics, their functional genomics at higher spatial resolution shows promise as a more mechanistic approach for the study of germline genetics.

This thesis has two overall themes, both of which involve the word functional, albeit in different contexts. The theme that motivates two of the chapters is the development of methods that enable a deeper understanding of the variability of functional data. The theme of the final chapter is the development of methods that enable a deeper understanding of the landscape of functionality across the human genome in different human tissues. The first chapter of this thesis provides a framework for quantifying the variability of functional data and for analyzing the factors that affect this variability. We extend functional principal components analysis by modeling the variance of principal component scores. We pose a Bayesian model, which we estimate using variational Bayes methods. We illustrate our model with an application to a kinematic dataset of two-dimensional planar reaching motions by healthy subjects, showing the effect of learning on motion variability. The second chapter of this thesis provides an alternative method for decomposing functional data that follows a Poisson distribution. Classical methods pose a latent Gaussian process that is then linked to the observed data via a logarithmic link function. We pose an alternative model that draws on ideas from non-negative matrix factorization, in which we constrain both scores and spline coefficient vectors for the functional prototypes to be non-negative. We impose smoothness on the functional prototypes. We estimate our model using the method of alternating minimization. We illustrate our model with an application to a dataset of accelerometer readings from elderly healthy Americans. The third chapter of this thesis focuses on functional genomics, rather than functional data analysis. Here we pose a method for unsupervised clustering of functional genomics data. Our method is non-parametric, allowing for flexible modeling of the functional genomics data without binarization. We estimate our model using variational Bayes methods, and illustrate it by calculating genome-wide functional scores (based on a partition of our clusters into functional and non-functional clusters) for 127 different human tissues. We show that these genome-wide and tissue-specific functional scores provide state-of-the-art functional prediction.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Equine laminitis is a debilitating disease that causes extreme sufferring in afflicted horses and often results in a lifetime of chronic pain. The exact sequence of pathophysiological events culminating in laminitis has not yet been characterized, and this is reflected in the lack of any consistently effective therapeutic strategy. For these reasons, we used a newly developed 21,000 element equine-specific whole-genome oligoarray to perform transcriptomic analysis on laminar tissue from horses with experimentally induced models of laminitis: carbohydrate overload (CHO), hyperinsulinaemia (HI), and oligofructose (OF). Samples were collected during the developmental (DEV) and Obel grade 1 (OG1) stages of laminitis for the CHO model. For the HI model, samples were collected at the Obel grade 2 (OG2) stage. For the OF model, samples were collected at the 12 h and 24 h time points. Appropriate control samples were obtained for all models. This is the first genome-wide transcriptome analysis of laminar tissue using an equine 21,000 70-mer long oligoarray approach in CHO, HI and OF induced laminitis. Overall, we identified the differential expression of genes encoding S100 calcium binding proteins, extracellular matrix proteins, glycoproteins, transporters, olfactory receptors, genes involved in signal transduction, body‟s homeostasis, apoptosis, and immune response. Between CHO and OF models of laminitis, there were more shared genes. We discovered several common differentially expressed genes (i.e., ADAMTS1, CYCS and CXCL14) among all three models that are likely important to the pathogenesis of equine laminitis. We also discovered what appear to be central roles of apoptosis, inflammatory response, and intracellular ion homeostasis molecular processes in CHO and OF models of laminitis. Pathway analysis detected the NOD-like receptor signaling pathway, which is involved in recognition of intracellular bacteria in both the CHO and OF models of laminitis. Genetic network analysis indicated convergent pathway core molecules present in equine acute laminitis: p38 MAPK and NF-κB. Most importantly, our results of overexpression of anti-microbial genes (i.e., DEFB4, PI3, and CXCL14) suggest the central involvement of these genes in the progression of early equine laminitis and will allow refinement of current hypotheses of disease pathogenesis.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Despite significant advances in chemotherapeutics since their initial application in the early 1900s, cancer remains a leading cause of death worldwide. Furthermore, issues encountered with the first chemotherapeutics, off-target toxicity, limited effective dosing time in the therapeutic window, multidrug resistance, and poor uptake in solid tumors persist today. This dissertation investigates two platforms to enhance chemotherapy efficacy, an implantable drug delivery depot affording tumor drug levels unachievable through systemic delivery, and a platform utilizing CRISPR knockout screening (CRISPRKO) to identify microRNA (miRNA) targets that synergize with chemotherapeutics using RNA interference (RNAi). First, we developed a flexible, implantable surgical buttress coated with a free and covalently-bound paclitaxel polymer blend with poly(1,2-glycerol carbonate)-graft-succinic acid-paclitaxel (PGC-PTX). Drug release is tunable between burst release of free paclitaxel and delayed, extended release of paclitaxel from PGC-PTX, delivering supratherapeutic levels of PTX locally at the tumor resection bed while avoiding systemic toxicity. Fabrication of paclitaxel-loaded film is scalable up to 8”x11” and well-tolerated in a porcine model wherein surgical technique and optimized film formulations reduce foreign body response and bowel adhesions. We test a similarly-designed film formulation with the novel chemotherapeutic, eupenifeldin, for treatment of lung cancer. Eupenifeldin-loaded films significantly prolong mice survival, although 60% of mice present issues with drug-related toxicity and wound healing. Second, despite the formidable ability of pooled CRISPRKO libraries to screen thousands of single-gene knockouts against a selection agent, minimal has been published on their use to determine novel targets for RNAi-based sensitization of chemotherapeutics. We utilize whole-genome CRISPRKO screening against a panel of lung cancers to identify miRNAs capable of synergizing with paclitaxel as well as two novel chemotherapeutics, eupenifeldin and verticillin A. Identified targets are validated for synergy with their respective chemotherapeutic in vitro, demonstrating a 50% or greater increase in cell death compared to drug-only treatment. In summary, this work presents two successful treatment platforms that address key issues preventing translation of chemotherapeutics to the clinic: 1) dose-limiting toxicity and 2) limited efficacy with poor safety/benefit ratio. Implantable, drug-loaded films serve as a platform to deliver increased doses of chemotherapeutic to tumor while avoiding off-target toxicity. As a second platform, we enhance tumor-specific chemotherapy efficacy utilizing miRNA targets identified in a CRISPRKO screening pipeline.

In the past decade, automatisation has led to an immense increase of data in biology. Next generation sequencing techniques will produce a vast amount of sequences across all species in the coming years. In many cases, identifying the function and biological role of a protein from its sequence can be a complicated and time-intensive task. The identification of a protein's interaction partners is a tremendous help for understanding the biological context in which it is involved. In order to fully characterise a protein-protein interaction (PPIs), it is necessary to know the three-dimensional structure of the interacting partners. Despite optimisation efforts from projects such as the Protein Structure Initivative, determining the structure of a protein through crystallography remains a time- and cost-intensive procedure. The primary aim of the research described in this dissertation was to produce a World Wide Web resource that facilitates visual exploration and validation (or questioning) of data derived from functional genomics experiments, by building upon existing structural information about direct physical PPIs. Secondary aims were (i) to demonstrate the utility of the new resource, and (ii) its application in biological research. We created a database that emphasises specifically the intersection between the PPIs-results emerging from the structural biology and functional genomics communities. The BISC database holds BInary SubComplexes and Modellable Interactions in current functional genomics databases (BICS-MI). It is publicly available at hyyp://bisc.cse.ucsc.edu. BISC is divided in three sections that deliver three types of information of interest to users seeking to investigate or browse PPIs. The template section (BISCHom and BISCHet) is devoted to those PPIs that are characterised in structural detail, i.e. binary SCs extracted from experimentally determined three-dimensional structures. BISCHom and BISCHet contain the homodimeric (13,583 records) and heterodimeric (5612 records) portions of these, respectively. Besides interactive, embedded Jmol displays emphasising the interface, standard information and links are provided, e.g. sequence information and SPOP classification for both partners, interface size and energy scores (PISA). An automated launch of the MolSurfer program enables the user to investigate electrostatic and hydrophobic correlation between the partners, at the inter-molecular interface. The modellable interactions section (BISC0MI) identifies potentially modellable interactions in three major functional genomics interaction databases (BioGRID), IntAct, HPRD). To create BISC-MI all PPIs that are amenable to automated homology modelling based on conservative similarity cut-offs and whose partner protein sequences have recrods in the UniProt database, have been extracted. The modellable interaction services (BISC-MI Services) section offers, upon user request, modelled SC-structures for any PPIs in BISC-MI. This is enabled through an untomated template-based (homology) modelling protocol using the popular MODELLER program. First, a multiple sequence alignment (MSA) is generated using MUSCLE, between the target and homologous proteins collected from UniProt (only reviewed proteins from organisms whose genome has been completely sequenced are included to find putative orthologs). Then a sequence-to-profile alignment is generated to integrate the template structure in the MSA. All models are produced upon user request to ensure that the most recent sequence data for the MSAs are used. Models generated through this protocol are expected to be more accurate generally than models offered by other automated resources that rely on pairwise alignments, e.g. ModBase. Two small studies were carried out to demonstrate the usability and utility of BISC in biological research. (1) Interaction data in functional genomics databases often suffers from insufficient experimental and reporting standards. For example, multiple protein complexes are typically recorded as an inferred set of binary interactions. Using the 20S core particle of the yeast proteasome as an example, we demonstrate how the BISC Web resource can be used as a starting point for further investigation of such inferred interactions. (2) Malaria, a mosquito-borne disease, affects 3500-500 million people worldwide. Still very little is known about the malarial parasites' genes and their protein functions. For Plasmodium falciparum, the most lethal among the malaria parasites, only one experimentally derived medium scale PPIs set is available. The validity of this set has been doubted in the the malarial biologist community. We modelled and investigated eleven binary interactions from this set using the BISC modelling pipeline. Alongside we compared the BISC models of the individual partners to those obtained from ModBase.

Activation-tagging is a functional genomics technique where strong enhancers are inserted randomly into target genomes to over-activate endogenous genes. Phenotypes of interest can be selected for investigation of genetic factors contributing to the mutant phenotype. From initial screens of a population of activation-tagged potato, a mutant with chocolate-coloured tuber skin has been identified. In this thesis, a novel sequence capture method for identifying T-DNA loci in activation tagged potato was used to characterize chocolate’s single T-DNA insertion locus. Transcriptome analysis of tuber periderm tissue was used to identify major processes occurring in the chocolate mutant. Our data suggest activation of a chitin-binding receptor-like kinase located 65 kb from T-DNA insert may cause activation of immune signaling pathways in chocolate. The present work explores a putative model of transcriptional and cellular responses involved in gain-of-function immune receptor activation. Selectively, these findings illustrate the periderm tissue as an important area of defense charged against biotic and abiotic stresses. Periderm development and anatomy are highly important for tuber storage. Further characterization of potato tuber periderm may contribute knowledge to model periderm systems and have implications for molecular breeding strategies to improve tuber storage quality. / Thesis (Master, Biology) -- Queen's University, 2012-09-27 11:45:16.478