<p>Normal hearing and balance relies on the detection of sound, orientation and acceleration by sensory hair cells (HCs) located in the inner ear. Once sound is detected, that information must be transmitted to the brain by sensory neurons. Damage to the HCs and/or neurons in the auditory or vestibular organs of the inner ear can result in hearing loss or balance disorders. In mammals, these disorders can be permanent, as HCs do not regenerate after damage. While hearing aids and cochlear implants can restore some ability to hear, there are currently no molecular therapies for hearing loss. By examining genes involved in HC development and innervation, basic science can identify candidate genes for potential molecular therapies. This dissertation focuses on molecular regulators involved in establishing and/or maintaining innervation in the chicken inner ear during embryonic development.</p><p>The basilar papilla (BP) is the auditory sensory organ in the chicken and is homologous to the mammalian organ of Corti (oC). The BP houses two types of sensory HCs – tall HCs and short HCs. On the neural side of the BP, tall HC receive primarily afferent innervation (neural-side identity). On the abneural side, short HC receive primarily efferent innervation (abneural-side identity). The patterning of these two identities along the radial axis is dependent upon the precise spatiotemporal expression of certain genes during embryonic development. One such gene is <i>Wingless/integrated (Wnt)9a</i>.</p><p>Previous work has shown that <i>Wnt9a</i>is expressed on the neural edge of the BP and is likely secreted in a gradient across the prosensory domain during crucial time points when proliferation, differentiation, and innervation are occurring. When <i>Wnt9a </i>was overexpressed, we observed an increase in the width of the BP as well as an expansion of the neural-side identity, likely at the expense of the abneural-side identity. RNA sequencing of <i>Wnt9a</i>-overexpressing and control BPs identified genes involved in the Wnt signaling pathway, cytoskeletal remodeling, and axon guidance signaling that were differentially expressed. This dissertation focuses on axon guidance genes, specifically those involved in Slit/Robo (Roundabout), Contactin (Cntn), and Semaphorin (Sema) signaling, that were differentially expressed in this RNA sequencing data set.</p><p>Slits typically act as repulsive cues for neurites expressing Robo receptors. RNA sequencing data indicates that <i>Slit2</i>transcripts increased by 1.2 fold when <i>Wnt9a </i>was overexpressed. When examining Slit2 spatial expression pattern in <i>Wnt9a-</i>overexpressing BPs, we did not observe an upregulation of <i>Slit2 </i>but rather an expansion of the <i>Slit2</i>-expression domain that is likely due to increased proliferation in response to <i>Wnt9a</i>. To better understand the role of Slit/Robo signaling in the developing BP, we examined the radial expression patterns of <i>Slit2</i>, <i>Robo1</i>, and <i>Robo2</i>. <i>Slit2 </i>is expressed on the anterior and posterior walls of the cochlear duct (CD). <i>Robo1</i>and <i>Robo2 </i>had graded expression in the prosensory domain of the BP, highest on the abneural side. <i>Robo1</i>is also present in the auditory ganglion. While only a small population of cochleovestibular ganglion neurites have been previously shown to respond to Slits, Slit-Robo has also been shown to activate TCF transcription factor by non-canonically activating β-catenin through Abl kinase. We examined Abl kinase-activated b-catenin in <i>Slit2-</i>and <i>Wnt9a-</i>overexpressing BPs but did not observe a change in phosphorylated b-catenin. We also overexpressed a dominant-negative Robo1. In some dominant-negative Robo1 overexpressing ears, we observed a reduction in ganglion size; however, this affect did not reliably replicate. These data suggests that Slit-Robo signaling could be involved in neuroblast delamination and/or migration.</p><p>RNA sequencing results indicate that <i>Contactin 6</i><i>Cntn6 </i>transcripts increased by 1.5 fold when <i>Wnt9a </i>was overexpressed. Contactins are cell adhesion molecules that have been previously shown to impact neurite outgrowth and innervation. In the auditory field, clinical studies have also shown that patients diagnosed with autism who also have mutations in <i>Cntn5 </i>and <i>Cntn6 </i>are more likely to exhibit increased sensitivity to sound. Based on RNA sequencing in the embryonic day (E)6 chicken ear, <i>Cntn6 </i>has low levels of expression in controls. We attempted to examine the spatial expression of <i>Cntn6 </i>but found that <i>in situ </i>hybridization is not sensitive enough to detect low levels of <i>Cntn6 </i>in control or <i>Wnt9a-</i>overexpressing BPs.</p><p>Class III Semaphorinsecreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. <i>Sema3D </i>and <i>Nrp2 </i>were downregulated in the presence of exogenous <i>Wnt9a</i>; however, the spatial expression of these transcripts did<i></i>not support their role in establishing or maintaining radial innervation patterns. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used <i>in situ </i>hybridization or immunohistochemistry to map the expression of <i>Sema3D</i>, <i>Sema3F</i>, Nrp1<i>, Nrp2</i>, and <i>PlxnA1 </i>in the chicken inner ear from E5 to E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. <i>Sema3D</i>expression flanking the sensory tissue in vestibular organs suggests that it may repel <i>Nrp2</i>- and <i>PlxnA1</i>-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8–E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, <i>Sema3D</i>in the future tegmentum vasculosum opposes Nrp1 and <i>PlxnA1 </i>in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. <i>Sema3D </i>immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.</p><p>To better understand innervation patterns in the avian BP, we also examined the developing efferent innervation patterns from E11 to E17 using NeuroVue lipophilic tracer dye. Our data suggest that efferents have already begun to penetrate the sensory epithelium at E11 and that efferents arrive to the ipsilateral BP earlier than the contralateral BP. By E12, many efferents appear to send back branches out to short HCs. At E15, many efferents appear to have reached the abneural edge of the BP, are innervating the hyaline cells, and are projecting apically.</p><p>In summary, this work suggests that Slit and Sema signaling are not involved in establishing radial innervation patterns but may have alternative roles in inner ear development. Additionally, while efferents appear to arrive to the ipsilateral BP sooner than the contralateral BP, both ears send projections across the radial axis and back branch around the same time.</p>

Doctor of Philosophy / Genetics Interdepartmental Program / Jesse A. Poland / A central question in modern biology is to understand the genotype-to-phenotype (G2P) link, that is, how the genetics of an organism results in specific characteristics. However, prediction of phenotypes from genotypes is a difficult problem due to the complex nature of genomes, the environment, and their interactions. While the recent advancements in genome sequencing technologies have provided almost unlimited access to high-density genetic markers, large-scale rapid and accurate phenotyping of complex plant traits remains a major bottleneck. Here, we demonstrate field-based complex trait assessment approaches using a commercially available light-weight Unmanned Aerial Systems (UAS). By deploying novel data acquisition and processing pipelines, we quantified lodging, ground cover, and crop growth rate of 1745 advanced spring wheat lines at multiple time-points over the course of three field seasons at three field sites in South Asia. High correlations of digital measures to visual estimates and superior broad-sense heritability demonstrate these approaches are amenable for reproducible assessment of complex plant traits in large breeding nurseries. Using these validated high-throughput measurements, we applied genome-wide association and prediction models to assess the underlying genetic architecture and genetic control. Our results suggest a diffuse genetic architecture for lodging and ground cover in wheat, but heritable genetic variation for prediction and selection in breeding programs. The logistic regression-derived parameters of dynamic plant height exhibited strong physiological linkages with several developmental and agronomic traits, suggesting the potential targets of selection and the associated tradeoffs. Taken together, our highly reproducible approaches provide a proof-of-concept application of UAS-based phenomics that is scalable to tens-of-thousands of plots in breeding and genetic studies as will be needed to understand the G2P and increase the rate of gain for complex traits in crop breeding.

xiii, 145 p. : ill. (some col.) / The goal of functional genomics is to elucidate the relationship between an organism's genotype and phenotype. A key characteristic of functional genomics is the use of genome-wide approaches as opposed to more traditional single-gene approaches. Genome-wide expression profiling is used to investigate the dynamic properties of transcriptomes, provides insights into how biological functions are encoded in genomes, and is an important technique in functional genomics. This dissertation describes the use of genome-wide expression profiling and other functional genomics techniques to address a variety of biological questions related to development and disease of the nervous system. Our results reveal novel and important insights into nervous system development and disease and demonstrate the power of functional genomics approaches for the study of nervous system biology. This dissertation also describes a novel technique called TUtagging that facilitates cell type-specific RNA isolation from intact complex tissues. The isolation of RNA from specific cell types within a complex tissue is a major limiting factor in the application of genome-wide expression profiling, and TU-tagging can be used to address a wide array of interesting and important biological questions. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Dr. John Postlethwait， Chair； Dr. Chris Doe， Advisor； Dr. Bruce Bowerman， Member； Dr. Patrick Phillips， Member； Dr. Tom Stevens， Outside Member

Glucocorticoids (GCs) have been widely used as a therapeutic agent for diverse inflammatory ocular diseases. However, a high percentage of patients undergoing this treatment develop high intraocular pressure (IOP), which if left unsupervised may lead to glaucoma. It is believed that the IOP elevation in response to GC treatment has a genetic determinant. In order to test this hypothesis, we analyzed in 52 patients the presence of single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor gene (GR), the principal mediator of GCs uptake by the cells. We studied six GR SNPs previously reported to be associated with sensitivity and resistance to GCs: GluArg22/23GluLys (codon 22-23), Asn363Ser (codon 363), IVS2+646C>G (intron 2/BclI), IVS3-46G>C (intron 3), IVS4-16G>T (intron 4), Asn766Asn (Codon 766). Nevertheless, the results of this preliminary study did not show any specific correlation between SNPs in the GR gene and IOP elevation. Therefore, we proceeded to perform a whole genome SNP screen with the DNA samples of these patients to search for possible target genes responsible for the elevated IOP after GC treatment. As a result, we identified forty-eight SNPs in thirty-three genes that correlate with the high IOP response. The gene showing the strongest association is a poorly known G-protein coupled receptor. In addition, four SNPs hit a single transporter gene. Other candidate genes identified are a translation elongation factor, an F-box protein, an oxysterol binding protein, and a solute carrier family gene. These results support our hypothesis that IOP elevation following GC treatment is a genetically determined response. GCs are a common treatment for innumerable medical conditions; we believe that a genetic association between GC treatment and its physiological response may be important for improving treatment management and drug development for retinal diseases as well as for other medical ailments. However, further studies need to be performed to analyze in depth the association between the candidate genes identified in this study and the steroid response.

The methods that fueled the microscale revolution (top-down design/fabrication, combined with application of forces large enough to overpower stochasticity) constitute an approach that will not scale down to nanoscale systems. In contrast, in nanotechnology, we strive to embrace nature’s quite different paradigms to create functional systems, such as self-assembly to create structures, exploiting stochasticity, rather than overwhelming it, in order to create deterministic, yet highly adaptable, behavior. Nature’s approach, through billions of years of evolutionary development, has achieved self-assembling, self-duplicating, self-healing, adaptive systems. Compared to microprocessors, nature’s approach has achieved eight orders of magnitude higher memory density and three orders of magnitude higher computing capacity while utilizing eight orders of magnitude less power. Perhaps the most complex of functions, homeostatis by a biological cell – i.e., the regulation of its internal environment to maintain stability and function – in a fluctuating and unpredictable environment, emerges from the interactions between perhaps 50M molecules of a few thousand different types. Many of these molecules (e.g. proteins, RNA) are produced in the stochastic processes of gene expression, and the resulting populations of these molecules are distributed across a range of values. So although homeostasis is maintained at the system (i.e. cell) level, there are considerable and unavoidable fluctuations at the component (protein, RNA) level. While on at least some level, we understand the variability in individual components, we have no understanding of how to integrate these fluctuating components together to achieve complex function at the system level. This thesis will explore the regulation and control of stochasticity in cells. In particular, the focus will be on (1) how genetic circuits use noise to generate more function in less space; (2) how stochastic and deterministic responses are co-regulated to enhance function at a system level; and (3) the development of high-throughput analytical techniques that enable a comprehensive view of the structure and distribution of noise on a whole organism level.

Stroke is a common disorder of later life with a complex etiology, including both environmental and genetic risk factors. The inherited predisposition is challenging to study due to the complexity of the stroke phenotype. Genetic studies in an isolated population have successfully identified a positional candidate gene for stroke, phosphodiesterase 4D (PDE4D). The aim of this thesis was to identify stroke susceptibility loci and positional candidate genes, taking advantage of low genetic variation in the northern Sweden population. All stroke cases were identified in a population-based stroke registry at the northern Sweden MONICA Centre. 56 families containing multiple cases of stroke and a follow up set of an additional 53 families were used for linkage studies. For association studies, 275 cases of first ever stroke together with 550 matched community controls were included. In paper I, we used a candidate region approach to investigate the PDE4D region on chromosome 5q. Linkage was obtained with a maximum allele-sharing LOD score of 2.06; P = 0.001. However, no significant association of ischemic stroke to the previously defined at-risk allele in PDE4D was observed. We next performed a genome wide linkage scan to explore new susceptibility loci for common forms of stroke (paper II). Non-parametric multipoint linkage analysis yielded allele-sharing LOD scores &gt; 1.2 at nine locations; 1p34, 5q13, 7q35, 9q22, 9q34, 13q32, 14q32, 18p11, 20q13. The highest allele-sharing LOD score was obtained on chromosome 18p (LOD = 2.14). Fine mapping resulted in increased allele-sharing LOD scores for chromosome 5q13 and 9q22. In the follow up analysis of the nine regions, including all 109 families, the highest allele-sharing LOD scores were obtained on chromosomes 5q, 13q and 18p although none reached the initial genome wide values. In paper III, we focused on the chromosome 5q region, and further mapping and haplotype analysis in the families was performed. A common 1 cM haplotype was found to be shared among affected members of five families. In this region only the regulatory subunit 1 of phosphatidylinositol 3-kinase (PIK3R1) gene was located. Association of three single nucleotide polymorphisms in the PIK3R1 gene to common stroke was obtained in the case-control material. Finally, in paper IV, an extended pedigree containing seven families connected to common founders eight generations back was identified by genealogical analysis, and submitted to a separate genome wide scan analysis. A significant allele-sharing LOD score of 4.66 (genome wide P &lt; 0.001) at chromosome 9q31-33 was obtained. Haplotype analysis identified a minimal common region of 3.2 cM, which was shared by four of the seven families. These four families contained all of the primary intracerebral hemorrhagic cases present in the extended pedigree. In conclusion we have replicated linkage of stroke susceptibility to the PDE4D region on chromosome 5q, but no significant association of ischemic stroke to PDE4D was observed. Linkage analysis of stroke did not identify any new major stroke loci, indicating that multiple minor susceptibility loci in addition to the previously known locus on chromosome 5q could contribute to the disease. In the chromosome 5q region a novel positional candidate gene for stroke was identified, the PIK3R1 gene. The PIK3R1 protein has several biological actions with potential roles in stroke susceptibility. Also a novel susceptibility locus for common forms of stroke at chromosome 9q was identified in a large pedigree, which may be of special importance for susceptibility to hemorrhagic stroke.

Recently, the pace of growth in the amount of data sources within Life Sciences has increased exponentially until pose a difficult problem to efficiently manage their integration. The data avalanche we are experiencing may be significant for a turning point in science, with a change of orientation from proprietary to publicly available data and a concomitant acceptance of studies based on the latter. To investigate these issues, a Network of Excellence (EMBRACE) was launched with the aim to integrate the major databases and the most popular bioinformatics software tools. The focus of this thesis is therefore to approach the problem of seamlessly integrating varied data sources and/or distributed research tools. In paper I, we have developed a web service to facilitate allergenicity risk assessment, based on allergen descriptors, in order to characterize proteins with the potential for sensitization and cross-reactivity. In paper II, a web service was developed which uses a lightweight protocol to integrate human endogenous retrovirus (ERV) data within a public genome browser. This new data catalogue and many other publicly available sources were integrated and tested in a bioinformatics-rich client application. In paper III, GeneFinder, a distributed tool for genome-wide association studies, was developed and tested. Useful information based on a particular genomic region can be easily retrieved and assessed. Finally, in paper IV, we developed a prototype pipeline to mine the dog genome for endogenous retroviruses and displaying the transcriptional landscape of these retroviral integrations. Moreover, we further characterized a group that until this point was believed to be primate-specific. Our results also revealed that the dog has been very effective in protecting itself from such integrations. This work integrates different applications in the fields of protein allergology, biotechnology, genome association studies and endogenous retroviruses. / EMBRACE NoE EU FP6

The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome. In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes. In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors. In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

Diseases that affect dairy cattle have serious economic and animal welfare implications. The inclusion of immune response (IR) traits in breeding indices has been suggested to improve inherent animal health, and decrease the use of antimicrobials. The objectives of this research were to (1) evaluate cell-mediated (CMIR) and antibody-mediated immune responses (AMIR) on 680 Holstein cows from 58 herds across Canada, (2) estimate genetic parameters of these traits (3) examine the associations with routinely evaluated traits as well as the incidence of mastitis, (4) evaluate the correlation of natural and specific antibody and (5) perform a genome-wide association study (GWAS) to determine genetic markers associated with high or low IR. In collaboration with the Canadian Bovine Mastitis Research Network cows were immunized with both a type 1 and type 2 test antigen to stimulate CMIR and AMIR, respectively. A cutaneous delayed-type hypersensitivity test to the type 1 test antigen was used as an indicator of CMIR, and serum antibody (IgG1 and IgG2) to the type 2 test antigen as an indicator of AMIR. IR phenotypes varied significantly by cow, herd and region in Canada. Heritability estimates were moderate, 0.19 for CMIR and 0.16-0.43 for AMIR depending on time in the immunization protocol and antibody isotype. Beneficial associations between AMIR and some reproductive traits were found. Using estimated breeding values, cows were classified as high, average or low responders. High AMIR cows had significantly lower incidence rates of clinical mastitis compared to average and low cows. No difference was found when cows were classified based on CMIR. Natural antibody was not genetically correlated with specific antibody nor was it associated with mastitis. The GWAS found 198 genetic markers significantly associated with AMIR, with the majority on chromosome 23 where the major histocompatability complex is located. Other significant genes involved in IR include those associated with the complement system, interleukin 17 and tumor necrosis factor. This research confirms the benefit of identifying high IR cows and gives a glimpse of current IR profiles in Canadian Holsteins. This was the first GWAS for IR traits in dairy cattle and suggests it may be possible to include IR traits in genomic selection indices. / This research was financed by grants to B.A. Mallard from National Sciences and Engineering Research Council of Canada, Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, DairyGen council of Canadian Dairy Network, Agriculture and Agri-Food Canada, Public Health Agency of Canada, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network. Kathleen Adele Thompson Crispi was funded by the Dairy Farmers of Ontario Doctoral Research Assistantship.

Genome-wide association studies (GWAS)s provide an unbiased means of exploring the landscape of complex genetic disease. As such, these studies have identified genetic variants that are robustly associated with a multitude of conditions. I hypothesize that these genetic variants serve as excellent tools for evaluation of the genetic interface between epidemiologically related conditions. Herein, I test the association between SNPs associated with either (i) plasma lipids, (ii) rheumatoid arthritis (RA) or (iii) diabetes mellitus (DM) and late-onset Alzheimer’s disease (AD) to identify shared genetic variants. Regarding the most significantly AD-associated variants, I have also attempted to elucidate their molecular function. Only cholesterol-associated SNPs, as a group, are significantly associated with AD. This association remains after excluding APOE SNPs and suggests that peripheral and or central cholesterol metabolism contribute to AD risk. The general lack of association between RA-associated SNPs and AD is also significant in that these data challenge the hypothesis that genetic variants that increase risk of RA confer protection against AD. Functional studies of variants exhibiting novel associations with AD reveal that the lipid-associated SNP rs3846662 modulates HMGCR exon 13 splicing differentially in different cell types. Although less clear, trends were also observed between the RA-associated rs2837960 and the expression of several BACE2 isoforms, and between the DM-associated rs7804356 and expression of a rare SKAP2 isoform, respectively. In conclusion, the overlap of lipid-, RA- or DM-associated SNPs with AD is modest but in several instances significant. Continued analysis of the interface between GWAS of separate conditions will likely facilitate novel associations missed by conventional GWAS. Furthermore, the identification of functional variants associated with multiple conditions should provide insight into novel mechanisms of disease and may lead to the identification of new therapeutic targets in an era of personalized genomic medicine.