The medial geniculate body (MGB) is the target of excitatory and inhibitory inputs from several neural sources. Among these, the inferior colliculus (IC) is an important nucleus in the midbrain that acts as a nexus for many auditory pathways and projections, ascending and descending, throughout the rest of the central auditory system and provides both excitatory and inhibitory projections to the MGB. In addition, the thalamic reticular nucleus (TRN) is a major source of inhibition to the MGB, particularly in rodents. Finally, the auditory cortex (AC) is a major source of descending input to the MGB, providing direct excitation and indirect inhibition via the TRN. In our study, we assessed the relative contribution from these excitatory and inhibitory projection sources to the MGB of the auditory system in mice. Using retrograde tract tracing with CTβ -Alexa Fluor 594 injected into the MGB of the mouse, we quantitatively mapped the projections from both the ipsilateral and contralateral IC, the TRN, and the AC to the ipsilateral MGB. Our results indicate significant GABAergic projections from the IC and TRN to the MGB and excitation from the AC that play an overlooked role in shaping auditory processing. These results complement prior studies in other species, which suggests that these pathways are important factors in the regulation of neuronal activity in the auditory forebrain.

No description available.

No description available.

Escherichia coli biofilm formation is believed to be a significant cause of chronic equine endometritis, but limited studies exist to support this theory. In total 130 equine reproductive tract E. coli were collected, with 35 isolates showing strong in vitro biofilm formation (OD570 > 0.4). Changing incubation conditions did not significantly alter the distribution of E. coli between biofilm-forming groups, and strong biofilm formation began as early as 4 hours after initiation of incubation. The collected uterine isolates could be screened for biofilm formation using an adapted crystal violet (CV) assay consisting of chromogenic agar incubation and polyvinyl chloride conical tubes, with results demonstrating an in-house technique for screening isolates for biofilm formation. The strong biofilm-forming uterine E. coli showed a 7- to 11-fold increase over corresponding planktonic state in antibiotic resistance to ampicillin, ticarcillin, ceftiofur, gentamicin, amikacin, tobramycin, polymixin B and enrofloxacin. The exposure of certain biofilm-forming uterine E. coli isolates to 12.5% and 25% sub-MIC ceftiofur concentrations resulted in a significant dose-dependent increase in biofilm biomass after 24 hours incubation. All E. coli isolates evaluated (total = 101) were shown to carry the genes for Type-1 fimbriae, S-fimbriae, curli and the PGA polysaccharide, all of which are associated with biofilm formation. When exposed to endometrial explants E. coli adherence occurred rapidly and early stages of biofilm formation were visible on endometrial surfaces using SEM. Attempts to demonstrate uropathogenic E.coli associated cellular internalization were unsuccessful, but instead showed that adhered E. coli acquire biofilm associated antibiotic resistance rapidly after adhering to endometrial surfaces. In conclusion these experiments have shown that approximately 30% of equine reproductive tract E. coli can form strong in vitro biofilm, and show significant resistance to commonly used equine reproductive antibiotics. Also there is potential for increased biofilm formation to occur after exposure to low-level antibiotic exposure. The CV assay is an important screening assay for biofilm formation and can be adapted for use by clinicians in equine practice. The results of these experiments have shown the need to include biofilm-screening assays in equine practice for effective treatment of chronic endometritis.

Functional genomics uses high throughput genome-wide technologies to investigate the functional consequences of genetic variants on gene expression and protein products. In the context of disease, using integrative functional genomic approaches to understand the genetic variation which underlies disease susceptibility and aetiology may contribute to better diagnosis, treatment and prevention. This thesis investigated genetic determinants of variation in response to infection by applying a functional genomics approach to investigate three clinical cohorts: patients with severe sepsis, an influenza challenge study and patients with common variable immune deficiency disorders. The transcriptomic response to severe sepsis is reported here for the largest known adult severe sepsis community acquired pneumonia cohort. Two clusters within the cohort, based on gene expression signatures, which have different survival rates and identify individuals with distinct immune responses to sepsis, highlight the value of functional genomics for identifying heterogeneity within patient cohorts. This was further complemented by the resolution of gene expression signatures in healthy individuals following influenza challenge which identified individuals with moderate to severe disease. Shared gene expression signatures between the cohorts highlighted components of the immune response to viral infection important across multiple clinical settings and may assist with the identification of viral infections in the sepsis patients. Gene expression was mapped as a quantitative trait (eQTL). Comparison to data sets for healthy individuals and from innate immune stimulated cells identified regulatory variants specific to the context of sepsis. Whole genome sequencing for a cohort of patients with common variable immune deficiency disorders was performed. This identified novel variants and pathways which may play a role in the underlying immunopathogenesis of disease. Integration with RNA-seq for a small number of patients allowed prioritisation of non-coding variants based on evidence of altered gene expression. Comparison to the sepsis cohort analysis identified key immune related genes involved in rare and common responses to bacterial infection. This thesis has demonstrated the value of integrating multiple functional genomic techniques to further our understanding of the mechanisms underlying variation in the response to infection.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.

Epithermal Growth Factor Receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC). Cetuximab is the first and the only anti-EGFR monoclonal antibody which received approval from FDA for the treatment of HNSCC. However, most patients either do not respond to cetuximab or develop acquired resistance. The aim of my D.Phil. study was to investigate the role of ADAM10 and 17 in resistance mechanisms of cetuximab in HNSCC. Chronic exposure to cetuximab led to an activation of HER receptors and downstream signalling pathways in HNSCC cell lines. Higher levels of ADAM10 and 17 and their substrates, BTC and NRG-1 were found in cetuximab resistant cells compared to their parental cells, suggesting the involvement of ADAM-mediated ligands’ release in reactivation of HER receptors. Inhibition or knockdown of ADAM10 and 17 enhanced cetuximab response and reversed cetuximab resistance in HNSCC cells. In addition, results from this study showed that the combination of cetuximab with EGFR-TKIs had greater effect in parental cells and reversed cetuximab resistance in HNSCC cells. Upregulation of ADAM10 and 17 also was observed in HNSCC TMAs compared to normal head and neck TMAs. High nuclear ADAM10 and cytoplasmic ADAM17 expression levels were associated with shorter DFS. By evaluating tumour excision samples from HNSCC patients who underwent a cetuximab window study high ADAM10 and 17 expression levels were found to be associated with poor response to cetuximab. In conclusion cetuximab-induced ADAM-mediated ligands’ release is a potential mechanism of resistance to cetuximab in HNSCC. Thus, targeting ADAM10/17 or subsequent HER activations may represent an important strategy in overcoming resistance to cetuximab in HNSCC. Results from this study also suggest the implication of ADAM10 and 17 as potential prognostic and predictive biomarkers in HNSCC although further validation is required.

The tyrosine kinase 2 (TYK2) gene was first implicated in autoimmune disease in 2009 when a nonsynonymous single nucleotide polymorphism (nsSNP) in TYK2 was reported to be associated with multiple sclerosis (MS). The immunological function of TYK2, as a kinase involved in signal transduction downstream of numerous different cytokine receptors, further strengthened the candidacy of the gene as an MS-relevant risk factor. More recently, this nsSNP has been associated with several other autoimmune conditions. In addition, another three different SNPs in the region have been found to be associated with a number of autoimmune diseases, sometimes in opposing directions. Considering the complex genetic association pattern that is emerging for the TYK2 region across autoimmune conditions, it was hypothesised that this complexity reflects shared but also distinct pathogenic mechanisms, with the consequences of disease-associated SNPs being unlikely to all be restricted to genotype-dependent effects influencing TYK2. Therefore, the main aim of the work presented in this thesis has been to address this hypothesis by investigating the functional consequences of the disease-associated SNPs in the TYK2 gene region. Using an in vitro cell line system and primary human immune cells, obtained from a genotype-selectable donor cohort, protection at the MS-associated nsSNP was found to correlate with reduced TYK2-mediated signalling downstream of the type I interferon receptor. However, other cytokine signalling pathways were not affected, indicating a greater specificity to the function of TYK2 than has previously been appreciated. For the other SNPs in the region, substantial effects on TYK2 were not observed but immune cell subset-specific correlations with RNA-level expression of other genes in the region were identified. Thus, this is the first study to support the concept that a careful cross-comparative analysis of SNP association patterns in a single genomic region across multiple autoimmune diseases can have significant implications for enabling the delineation of pathways common or specific to different conditions, and this is of particular importance for drug repositioning strategies.

Patients admitted to the Intensive Care Unit (ICU) are critically ill and often at extremely high risk of death. These patients receive aggressive interventions to prolong their lives. Despite these measures, many patients still succumb to their illness. Although ICU physicians are good at predicting which patients have a high risk of mortality, they are still offering interventions that do not prolong life, but potentially cause more suffering at the end of life. This is because there is a lack of high quality and early communication to discuss prognosis and establish patients' goals of care. This gap in communication is even more profound when patients are transferring from rural hospitals to busy tertiary care centers. This dissertation discusses the utilization of tele-video conferencing to enhance early communication with family members/loved ones of critically ill patients prior to their transfer from a rural hospital to a tertiary care center. It begins with a description of telemedicine and its uses in the ICU to date. Chapter 2 discusses the poor prognoses of patients receiving high intensity interventions such as cardiopulmonary resuscitation (CPR). The extremely dismal outcomes underscore the importance of early, thorough discussions regarding prognosis and goals of care in these patients. The next chapter describes a pilot study utilizing telemedicine to conduct formal unstructured telemedicine conferences with family members prior to transfer. This study demonstrated that palliative care consultations can be provided via telemedicine for critically ill patients and that adequate preparation and technical expertise are essential. Although this study is limited by the nature of the retrospective review, it is evident that more research is needed to further assess its applicability, utility and acceptability. Chapter 4 describes an investigation into the barriers and facilitators of conducting conferences via telemedicine and the perceptions of clinicians regarding the use of telemedicine for this purpose. This chapter identified unique barriers and facilitators to the use of telemedicine that will need to be addressed when designing a telemedicine intervention for conducting family conferences. This thesis describes the importance and process of implementation of telemedicine for the novel purpose of enhancing early communication among physicians and family members of critically ill loved ones. Further studies are needed to refine and investigate patient and family centered clinical outcomes utilizing this intervention.

Etiological models of complex disease are elusive[46, 33, 9], as are consistently replicable findings for major genetic susceptibility loci[54, 14, 15, 24]. Commonly-cited explanations invoke low-frequency genomic variation[41], allelic heterogeneity at susceptibility loci[33, 30], variable etiological trajectories[18, 17], and epistatic effects between multiple loci; these represent among the most methodologically-challenging issues in molecular genetic studies of complex traits. The response has been con- sistently reactionary—hypotheses regarding the relative contributions of known func- tional elements, or emphasizing a greater role of rare variation[46, 33] have undergone periodic revision, driving increasingly collaborative efforts to ascertain greater numbers of participants and which assay a rapidly-expanding catalogue of human genetic variation. Major deep-sequencing initiatives, such as the 1,000 Genomes Project, are currently identifying human polymorphic sites at frequencies previously unassailable and, not ten years after publication of the first major genome-wide association find- ings, re-sequencing has already begun to displace GWAS as the standard for genetic analysis of complex traits. With studies of complex disease primed for an unprecedented survey of human genetic variation, it is essential that human geneticists address several prominent, problematic aspects of this research. Realizations regarding the boundaries of human traits previously considered to be effectively disparate in presentation[44, 39, 35, 27, 25, 12, 4, 13], as well as profound insight into the extent of human genetic diversity[23, 22] are not without consequence. Whereas the resolution of fine-mapping studies have undergone persistent refinement, recent polygenic findings suggest a less discriminant basis of genetic liability, raising the question of what a given, unitary association finding actually represents. Furthermore, realistic expectations regarding the pattern of findings for a particular genetic factor between or even within populations remain unclear. Of interest herein are methodologies which exploit the finite extent of genomic variability within human populations to distinguish single-point and cumulative group differences in liability to complex traits, the range of allele frequencies for which common association tests are appropriate, and the relevant dimensionality of common genetic variation within ethnically-concordant but differentially ascertained populations. Using high-density SNP genotype data, we consider both hypothesis-driven and agnostic (genome-wide) approaches to association analysis, and address specific issues pertaining to empirical significance and the statistical properties of commonly- applied tests. Lastly, we demonstrate a novel perspective of genome-wide genetic “background” through exhaustive evaluation of fundamental, stochastic genetic processes in a sample of matched affected and unaffected siblings selected from high- density schizophrenia families.