Search

The pro-inflammatory anaphylatoxin C5a exerts its biological actions via the C5a receptor (C5aR), a G-protein-coupled-receptor (GPCR). Cholesterol, a crucial component of biological membranes, has previously been shown to regulate expression and function of numerous GPCRs. As statin therapy is widely used to reduce serum cholesterol levels, it was hypothesised that statins can exert anti-inflammatory effects by down regulation expression and/or function of the C5aR. This thesis first investigated how basal human C5aR expression was regulated. It was shown that the majority of the -2Kbp promoter region is dispensable for transcription of the C5aR and that the main regulatory domains are localised in the first 200 bp of the promoter region. Furthermore CCAAT and NFAT motifs are important for the transcriptional control of the human C5aR, however the transcription factors which bind to these sites could not be identified. A model system to measure C5aR expression and function was set up using two pro-monocytic U937 sub-cell lines, which demonstrated that dibutyryl-cyclic-AMP was the best inducer of the C5aR, but only in one of the cell lines. Induction of the C5aR made these cells more responsive to C5a induced intracellular calcium-release and IL-8 and MMP-9 secretion. Investigating possible effects of cholesterol and modulation of cholesterol on C5aR expression and function showed that statins did not affect expression and function of basal levels of C5aR in monocytes or U937 cells, but reduced induced C5aR expression. Concomitantly C5a induced release of intracellular calcium and secretion of IL-8 reduced, however C5a induced MMP-9 secretion increased. This reduction was due to inhibition of isoprenoid biosynthesis rather than inhibition of cholesterol biosynthesis. Using sucrose gradient floatation it was shown that the C5aR is unlikely to reside in a lipid raft region of the plasma membrane making it less likely to be susceptible to membrane cholesterol content.

The work presented in this thesis is concerned with the characterisation of the UL32 gene of herpes simplex virus type 1 (HSV-1). UL32 encodes an essential 596 amino acid cysteine-rich, zinc-binding protein that is highly conserved throughout the herpesviruses. The UL32 protein is essential for the cleavage of concatemeric viral DNA into monomeric genomes and their packaging into preformed capsids. Conservation is highest at the C-terminus and three CxxC motifs are present in almost all known herpesvirus UL32 sequences The UL32 antibodies available in the laboratory at the beginning of the project were incapable of detecting small amounts of UL32 protein and so new rabbit antisera were created. Soluble extracts from insect cells infected with a UL32-expressing baculovirus (AcUL32) were fractionated by anion exchange chromatography and the UL32-containing fractions used to immunise rabbits. The resultant antisera successfully recognised UL32 from transfected, HSV-1 infected and baculovirus infected cells on western blots, and UL32 in transfected cells by immunofluorescence. I performed random mutagenesis of the UL32 gene in an effort to examine structure-function relationships within this protein, and generated a panel of 37 mutants containing 5 amino acid insertions at distinct positions. The abilities of these mutants to complement the DNA packaging and growth defects of a virus lacking a functional copy of UL32 (the null mutant hr64) were examined and 15 of the mutants retained functionality in both assays. A complete correlation was found between the ability of mutants to support growth and DNA packaging, suggesting that the key functions of UL32 are confined to the DNA packaging pathway. There was also good correlation with the degree of amino acid conservation within UL32, with most of the mutants which abolished functionality being located in the highly conserved regions, and the functional mutants in less conserved regions. A number of site-specific mutants were also created, in which the paired cysteine residues were replaced with serines (i.e. CxxC to SxxS). Mutation of the first and third cysteine pairs (from the N-terminus) completely abrogated growth and packaging, whereas significant functionality was maintained following mutagenesis of the central pair. Finally, removal of the C-terminal 4 amino acids also resulted in generation of a non-functional protein. Generation of an HA-tagged UL32 construct and the introduction of this into HSV-1 allowed the localisation of UL32 in infected cells to be studied. In contrast to previous reports, I detected UL32 predominantly in the nuclei of infected cells, co-localising with ICP8 in replication compartments. DNA packaging has previously been shown to occur within the replication compartments and a number of the other packaging proteins also localise to these sites. It was previously reported that UL32 played a role in the localisation of capsids to the replication compartments. However, work presented in this thesis shows this not to be the case, and that capsid proteins VP5 and VP19C were correctly localised in replication compartments during infections with the UL32 mutant hr64. I found no evidence of UL32 interaction with the UL6, UL25 or UL17 DNA packaging proteins in HSV-1 infected or transfected cells, or using immunoprecipitation from baculovirus-expressed cells. Immunofluorescence studies of co-transfected cells showed that UL15 could direct the partial re-localisation of UL32 from the cytoplasm to the nucleus. The addition of the other terminase subunits UL28 and UL33 caused the complete re-localisation of UL32 to the nucleus, suggesting that UL32 might interact with the terminase complex. Fifteen of the insertional mutants were completely re-localised to the nucleus in the presence of UL15, UL33 and UL28, with eleven further mutants showing an intermediate phenotype of partial nuclear localisation. The ability to at least partially co-localise with the terminase complex appeared necessary for the ability of the mutants to support virus growth and DNA packaging, suggesting that this interaction may be essential for the function of UL32. However, no interaction could be demonstrated between UL32 and any of the individual terminase subunits using immunoprecipitation from insect cells. A series of experiments was undertaken to further characterise the UL32 protein. A new UL32 mutant virus (Δ32EP) was generated by insertion of a kanamycin resistance cassette in place of a large portion of the UL32 gene. This mutant had an indistinguishable phenotype from hr64, confirming that the main function of UL32 is within DNA packaging. The functional conservation between HSV-1 UL32 and the homologues from HCMV and VZV was examined, but neither protein could support the growth of Δ32EP. DNA fragments from replicated concatemeric DNA from Δ32EP infected cells behaved similarly to wt HSV-1 fragments in PFGE, suggesting that UL32 is not involved in the resolution of branched structures within the genome prior to packaging. UL32 had previously been reported to bind zinc, and this was confirmed using a zinc-release colourimetric assay. The amount of zinc bound to soluble baculovirus-expressed UL32 was quantified, showing that UL32 bound zinc in a 1:1 molar ratio. UL32 does not share all of the characteristics of a zinc finger motif, but the results of the mutagenesis experiments suggest that the outer CxxC/CxxxC motifs may be important for zinc binding. Because of its zinc-binding properties and potential interaction with the terminase complex, the DNA binding properties of UL32 were also investigated. It was found that UL32 did not bind to dsDNA containing either the minimal packaging sequence (Uc-DR1-Ub) or an unrelated non-HSV-1 sequence using an electrophoretic mobility shift assay (EMSA).

Developmental and Epileptic Encephalopathies (DEE) are characterised by severe early-onset seizures and have poor developmental outcomes, significant co-morbidities, premature mortality and substantial psychosocial and economic impacts on families and society. Most DEE have a Mendelian genetic cause. Clarifying the underlying molecular diagnosis can permit targeted therapeutics and accurate genetic counselling. Massively parallel sequencing (MPS) has the potential to be a transformative technology for diagnostic evaluation of DEE. This study set out to (i) evaluate the diagnostic yield, cost-effectiveness and clinical utility of MPS panels and exome sequencing (ES), to guide clinical utilization, (ii) explore the potential of the more expensive but more comprehensive whole genome sequencing (WGS) to improve diagnostic yield for DEE,and, (iii) delineate novel genetic causes of DEE. Using a trio ES approach, we first analysed a cohort of 30 children with DEE with negative comprehensive first-tier testing. The diagnostic yield was 47% (14/30) and ES was demonstrated to be cost-effective when integrated into a diagnostic pathway for DEE. Next, we evaluated the role of WGS in diagnosis of DEE by analysis of 15 children remaining undiagnosed after the ES study and 15 additional children undiagnosed after MPS panel. In the 15 ES negative patients 8 additional diagnoses were made (cumulative diagnostic rate 73%) including 3 patients with complex or copy neutral structural variants. A diagnosis was confirmed or suspected in 11 of the 15 panel negative patients (73%) with several additional novel findings under evaluation. Most (n=10) were in genes excluded from the panel, 1 variant was missed by the panel for technical reasons. This work led to the identification and clinical characterisation of 4 novel causes of DEE: variants in KCNT2, ARV1, PUM1 and ATN1. Our study demonstrates ES or MPS gene panels are cost-effective for the diagnosis of DEE and that WGS further increases diagnostic yield. The importance of reanalysis and collaborative multidisciplinary evaluation of novel findings are demonstrated. Approaches to further optimise diagnostic yield and ways that genomic diagnoses can be leveraged to allow a targeted therapeutic and management approach are discussed.

Supplemental dietary yeast products are beneficial during times of stress. Calves experience increased levels of stress during birth, weaning, and the post-weaning receiving period. Therefore, 3 experiments were conducted to determine how yeast supplementation affects growth performance and health during this time. In experiment 1, 2 truckloads of highly stressed calves (n = 175; initial body weight [BW] = 226 ± 24.5 kg) were assigned randomly to 1 of 3 treatments; unsupplemented control, or supplementation with 2 commercial yeast products (Y1 and Y2). Average daily gain (ADG) was not different (P = 0.99) during the 28 d receiving period; nor was there any difference (P = 0.29) in the percentage of calves treated for bovine respiratory disease. In experiment 2, heifer calves (n = 95; initial BW = 165 ± 27 kg) were randomly assigned to pastures and pastures were randomly assigned to treatment: 1) no yeast (CON), or 2) addition of yeast product (YP). Heifers were supplied treatments for 35 d prior to weaning and through a 42-d backgrounding period. Average daily gain prior to weaning was not different (P ≥ 0.45) between treatments. However, CON had increased (P = 0.01) ADG compared to YP from weaning to the end of the backgrounding period. Microbiome analysis found that supplemental yeast did not dramatically change α or β diversity nor was there a difference in community structure for rumen bacteria; fecal α or β diversity were different on d 34. In experiment 3, late gestation cows (n = 97) were supplemented YP approximately 45 d prior to parturition. At parturition colostrum and blood samples (n = 30) were collected to determine the effect on passive transfer. Supplementation ended 22 d after the last calf was born (d 85). Body weight on d 85 was greater (P = 0.01) for YP calves compared with CON. Cows that were supplemented YP had a lower (P = 0.03) neutrophil:lymphocyte at hour 0 and 48 after parturition. Similarly, calves on YP treatment had a lower (P = 0.02) neutrophil:lymphocyte at hour 48. Overall, effects of yeast supplementation have been variable between the 3 experiments.

The sibling bond is likely the longest, most resilient relationship in one’s life. Subsequently, the sibling relationship is a strong predictor of outcomes across the lifespan, including positive intimate relationships, feelings of self-worth, and social skills. The purpose of this dissertation was to use Bowen Family Systems Theory (BFST) to examine individual’s perception of their sibling relationships and the connection with interpersonal and intrapersonal factors, and to explore the congruence of sibling and romantic relationship distance regulation. Online research listservs were used to recruit participants (N=476). A cross-sectional descriptive survey design was used to explore quantitative survey items. The Lifespan Sibling Relationship Scale (LSRS) was used to assess the sibling relationship in childhood and adulthood. The Social Connectedness Scale-Revised (SCSR) and the Separation-Individuation Test of Adolescence (SITA) were used to measure distance regulation. Interpersonal and intrapersonal factors included depression, general perceptions of health, and couple relationship satisfaction. BFST was used to explore four hypotheses: 1) The “closest” siblings will be same-sex dyads, with sisters being the closest among all the dyads; 2) Siblings that are 2.5 years apart in age; 3) Individuals’ perceptions of the sibling relationship in childhood and adulthood are related; and 4) The sibling relationship in childhood and adulthood are predictive of inter/intrapersonal factors. In addition, three research questions were explored: 1) Are there common characteristics of the selected “influential sibling” for participants with more than one sibling?; 2) What is the relationship between sibling and couple distance regulation?; and 3) To what extent does sibling and couple distance regulation mediate the influence of the sibling relationship on inter/intrapersonal factors?Correlations confirmed a significant relationship between the sibling relationship in childhood and adulthood (r(476)=.45, pF(3, 458)= 7.87, pF(3, 458)=5.38, p=.001). There were not significant differences between siblings > or n=338) to test the conceptual model using structural equation modeling. First, a direct effects model assessed the influence of the adult and sibling relationship on depression, general perceptions of health, and couple relationship satisfaction. Results showed significant paths from the child sibling relationship to depression (ß=-.14, pß=.21, pX2(56)=76.41 (p=.036); NFI=.974;CFI=.993; RMSEA=.033. There were significant indirect effects of the paths from the child sibling relationship, through couple distance regulation, to general perceptions of health (ß=.04, pß=-.02, pß=.29, p

Lydia Maria Child is of historical significance because of her leadership in the abolitionist movement. She is of literary significance because of the early Romantic novels she wrote. Further, her influence as a transcendentalist has not been fully recognized. Her philosophical leadership in the feminist movement needs also to be acknowledged. This study examines these three aspects of her life: Child’s influence as a transcendentalist, Child’s leadership in the feminist movement, and Child as an emblem of the nineteenth-century American character. The complexities of Child’s character in some ways parallel the complexities in the national character of the time.