Mediation of kinetochore-microtubule interactions through the Ndc80 complex component Hec1

2011 Fall.Includes bibliographical references.To view the abstract, please see the full text of the document

Doctor of Philosophy

dissertationI will describe a low-pressure flow-through 129Xe polarizer and report its performance by examining both the output 129Xe and in situ Rb polarization. The 129Xe polarization was made using standard NMR techniques, and the Rb polarization measurement was made using optically detected electron paramagnetic resonance. I compared the results of these measurements to a one-dimensional numerical model of the system. While we qualitatively understand the behavior of the system, the comparison between measurement and model reveals several inadequacies in our understanding of many important physical mechanisms. I will discuss the relevant physics necessary to qualitatively understand the system's behavior and suggest what mechanisms may cause the discrepancies in the modeled and measured behavior. I will demonstrate the utility of this Xe polarizer by measuring xenon's chemical shift dependence on the concentration of Bovine Pancreatic Trypsin Inhibitor (BPTI) and some of its mutants. Mutants Y23A and F45G have measured dependence of 0.56±0.05 ppm/mM and 0.47±0.07 ppm/mM, respectively, which is consistent with relatively strong, manufactured binding sites in the structure. Wild type BPTI has a measured dependence of only 0.15±0.02 ppm/mM, suggesting that there exists no specific binding site to which Xe can bind. Finally, the mutant Y35G has a dependence of 0.10±0.07 ppm/mM. This, with previous data, suggests that a large fraction of solution-phase Y35G does not exist in a conformation that allows Xe access to its binding cavity

Optimization of a Fluorescent In Situ Hybridization Protocol in the Chloroplast Hosting Mollusc Elysia chlorotica

The symbiosis between the mollusc Elysia chlorotica and chloroplasts of its algal prey, Vaucheria litorea, has challenged the understanding of chloroplast biology. E. chlorotica feeds on and retains the algal chloroplasts within its digestive cells. The chloroplasts remain photosynthetically active for months in the animal’s cells devoid of the algal nucleus which encodes approximately 90% of the proteins required for the chloroplasts. To help explain the ability of E. chlorotica to maintain functionally active chloroplasts, horizontal gene transfer (HGT) of V. litorea nuclear genes to the E. chlorotica genome has been hypothesized. However, the extent of HGT, as well as the transcriptional viability of putatively transferred genes, is still largely unknown. To better understand the genetic composition of E. chlorotica, optimization of a fluorescent in situ hybridization (FISH) procedure was performed on both V. litorea and E. chlorotica to develop a direct molecular tool to understand the genetic components involved in this symbiosis. Successful FISH analysis of the chloroplast Rubisco gene (rbcL) was accomplished using whole-mounts of both E. chlorotica and V. litorea. Whole-mount FISH analysis of the E. chlorotica nuclear β-actin gene was accomplished in aposymbiotic E. chlorotica larvae. Sectioned, adult E. chlorotica tissue was successfully hybridized for both the rbcL and β-actin genes. The development of the FISH technique for use on whole-mount sea slugs and intact heterokont algal filaments is the first direct application of FISH and can help elucidate the genetic components required to maintain this remarkable symbiosis

Demonstrating the effect of live weight on heifer pregnancy rates in northern Queensland

A group of beef producers north of Charters Towers identified low heifer pregnancy rates as a consistent and significant problem, aligning with the regional median from the Cash Cow project of 67%. Results from their Producer Demonstration Site (PDS) over three years monitoring >1,300 heifers clearly demonstrated a highly significant (p<0.001) relationship between proportion pregnant and weight. To achieve rates of more than 75% in high-percentage Brahman type heifers, live weights of at least 400 kg at pregnancy diagnosis (end of mating) was required. Ovarian scanning demonstrated a pre-mating live weight of approximately 340 kg was required for 80% of heifers to be at or near puberty. Neither pestivirus nor vibriosis were shown to be contributing factors in this study. Use of fewer, better bulls (<2% v 4%; selected on scrotal circumference and semen traits) significantly reduced business costs without reducing pregnancies. A 2% bull: female ratio was subsequently adopted by the host property across the entire breeding herd. This project’s findings are relevant to the entire northern beef industry and highlighted the importance of managing for high heifer growth prior to their first mating

Growth of Brahman cross heifers to 2 years of age in the dry tropics

Growth of 1368 Brahman cross heifers from 6 year cohorts was monitored over the 2 years post-weaning in Australia’s northern forest, a low-animal-growth dry tropical environment. Heifers weighing 47–266 kg at weaning were managed in groups weighing 199 kg during the post-weaning dry season. Weaner heifers were allocated to receive 300 g/day of a protein meal during the dry season or to basic nutritional management to sustain health. Heifers in three cohorts were allocated to first mating at 1 or 2 years of age, in four cohorts to vaccination against androstenedione, and in a small proportion of two cohorts to ovariectomy post-weaning. Growth was highly variable between seasons and years; average cohort liveweight by the start of 2-year-old mating was 256–319 kg. Heifer groups not receiving protein supplementation gained –16 to 21 kg (2 kg average) during 6-month dry seasons, and 49–131 kg (101 kg average) during wet seasons to reach an average of two-thirds of mature liveweight (445 kg) and 95% of mature hip height (1350 mm) by the start of mating at 2 years. Average body condition score (1–5) fluctuated by 1–2 units between seasons. Hip height gain continued, irrespective of season, commencing at ~0.60 mm/day at 6 months of age, and decelerating by ~0.00075 mm/day through to 2.5 years of age. Standard errors of predicted means across analyses were ~0.015 for average daily weight gains, 0.4 mm for average monthly height gain and 0.06 score units for average seasonal body condition score change. Post-weaning dry-season supplementation increased gains in liveweight, height and body condition score by an average of 0.1 kg/day, 0.1 mm/day and 0.5 units, respectively, during the supplementation period. Periods of poor nutrition or high nutritional demand secondary to reproduction suppressed daily gains in liveweight and hip height, at which times body condition score was also reduced. Subsequent to this, partial to full compensation occurred for all measures. Ovariectomy had negative effects on growth. Androstenedione vaccination had no effect on growth. The main conclusion is that heifer growth in Australia’s dry tropical northern forest region is highly variable between seasons and years, thus limiting significant proportions of some cohorts from reaching target weights for mating at 2 years of age, even after compensatory growth

Consultation etiquette in general practice: A qualitative study of what makes it different for lay cancer caregivers

Background: It is commonplace for lay caregivers to overlook their own health concerns when supporting someone with advanced cancer. During this time, caregivers’ needs as patients are often marginalised by health professionals, including General Practitioners (GPs), who may miss the breadth of caregivers’ needs by focusing on the practicalities of caregiving. GPs traditionally rely on patients to raise their concerns, and then respond to these concerns, but caregivers as patients may be disinclined to cue their GP. The norms of engagement when caregivers consult their GP are less defined, and how they interact with their GP regarding their own health is under-explored. This sub-study investigates the norms, assumptions and subtleties which govern caregiver-GP consultations, and explores factors affecting their interaction regarding caregivers’ own health concerns. Methods: We conducted semi-structured interviews with six lay caregivers and 19 health professionals in Brisbane, Australia, and analyzed the interview transcripts thematically. Results: Traditional norms of engagement are subjected to assumptions and expectations which caregivers and GPs bring to the consultation. Practice pressures also influence both parties’ capacity and willingness to discuss caregivers’ health. Nonetheless, some GPs monitor caregivers’ health opportunistically. Their interaction is enhanced by the quality of the caregiver-GP relationship and by the GP’s skills. Conclusions: Caregivers are caught in a paradox whereby their health needs may become subsumed by the care recipient’s needs in a setting where patient needs are normally scrutinised and supported. Caregivers may not raise their health concerns with their GP, who instead may need to cue them that it is timely and safe to do so. The routine use of a prompt may help to address caregivers’ needs systematically, but it needs to be complemented by GPs’ desire and capacity to engage with patients in a caregiving role. The potential difference GPs can make to the health of these patients is substantial