Investigation of mechanisms for restricting the activity of cyclic-AMP dependent protein kinase

Cyclic AMP (cAMP) is an ancient second messenger that is essential for many cellular processes including synaptic plasticity and control of heart rate and contractility. Cyclic AMP-dependent protein kinase (PKA) is the major intracellular receptor for cAMP. PKA consists of dimeric regulatory (R) subunits that bind and inhibit catalytic (C) subunits. PKA is activated upon binding of cAMP to the R subunits, which leads to the release of C subunits, and phosphorylation of intracellular protein substrates. An enduring challenge in cAMP research is to understand how PKA activity is directed to specific substrates, as the C subunits exhibit only limited substrate specificity in vitro. Elevations of cAMP are controlled in both space and time in the cell. This is achieved by the co-localization of enzymes for both the synthesis (cyclases) and breakdown (phosphodiesterases) of cAMP. Anchoring proteins are also essential for directing PKA to substrates in their immediate vicinity. However, a mechanism is yet to be established to explain how the activity of the C subunit of PKA is restrained following its dissociation from R subunits. This thesis details three parallel investigations that apply novel approaches with the shared aim of understanding how C subunit restraint is achieved. First, using quantitative immunoblotting in conjunction with purified PKA subunits, I investigated PKA subunit stoichiometry, finding that PKA R subunits typically outnumber C subunits by ~15-fold. Second, I developed a novel approach for monitoring R subunit isoform-specific association with C subunits in cells, with temporal precision. Comparative experiments using this approach and measurements with a fluorescent reporter of PKA activity show that only a small portion of C subunits need be dissociated to achieve high PKA activity. Third, I applied and developed a novel cross-linking coupled to mass spectrometry (XL-MS) protocol for analysis of the structure of PKA complexes. Insights include the likely orientation of PKA complexes that contain type II R (RII) subunits towards the membrane, and identification of a possible conformational change in PKA upon binding an anchoring protein. Together these experiments illuminate several aspects of PKA to show how the activity of this critical signalling enzyme is restrained within cells

Collaboration and competition: ethics in toxicology

From animal research through adverse events in clinical trials to health scares around food contamination, toxicology has frequently been a focus of scientific and societal concern. As these concerns shift with each new drug, new technology or public health scare, how can toxicology stay current, relevant and ethical? Two of the biggest ethical challenges in pharmaceutical toxicology are the use of animals in testing and the high safety-related attrition rates in new drug development. Both of these require progress in the discipline that will only be driven by research funding. Yet, very little is invested in these two fields compared with investment in new efficacy models, new disease targets and new technologies. How can this be addressed? Here, we explore current paradigms in toxicology that may have the potential for perceived or actual unethical ramifications. We discuss the underpinnings of such practices and make recommendations for change around peer review, resourcing, transparency and data sharing. These ideas build on the analysis presented in the 2004 Paton Prize lecture (Purchase, 2004) where issues around conflict of interest (COI), collaboration and competition in the context of ethical behaviours were highlighted. These areas are clearly relevant to many aspects of scientific research but here we focus on toxicology and specifically toxicology in the pharmaceutical industry

Phase-space views into dye-microcavity thermalised and condensed photons

We have observed momentum- and position-resolved spectra and images of the photoluminescence from thermalised and condensed dye-microcavity photons. The spectra yield the dispersion relation and the potential energy landscape for the photons. From this dispersion relation, below condensa- tion threshold, we nd that the e ective mass is that of a bare cavity photon not a polariton. Above threshold, we place an upper bound on the dimensionless two-dimensional interaction strength of ~ g . 1

Breast cancer in young women: prevalence of LOH at p53, BRCA1 and BRCA2

Breast cancer in young women: prevalance of LOH at p53, BRCA1 and BRCA

Functional analysis of altered Tenascin isoform expression in breast cancer

Background: Cellular interactions with the extracellular matrix (ECM) control many aspects of cell function. The complex ECM protein Tenascin-C (TN), which exists as multiple isoforms, is upregulated in breast cancer. We previously have identified a change in the TN isoform profile in breast cancer, with detection of two additional isoforms — TN16 and TN14/16 — not seen in normal breast [1]. The purpose of this study was to investigate directly the effects of these tumour-associated TNC isoforms on breast cancer cell behaviour

Emplacement of inflated Pāhoehoe flows in the Naude’s Nek Pass, Lesotho remnant, Karoo continental flood basalt province: use of flow-lobe tumuli in understanding flood basalt emplacement

Physical volcanological features are presented for a 710-m-thick section, of the Naude’s Nek Pass, within the lower part of the Lesotho remnant of the Karoo Large Igneous Province. The section consists of inflated pāhoehoe lava with thin, impersistent sedimentary interbeds towards the base. There are seven discreet packages of compound and hummocky pāhoehoe lobes containing flow-lobe tumuli, making up approximately 50% of the section. Approximately 45% of the sequence consists of 14 sheet lobes, between 10 and 52-m-thick. The majority of the sheet lobes are in two packages indicating prolonged periods of lava supply capable of producing thick sheet lobes. The other sheet lobes are as individual lobes or pairs, within compound flows, suggesting brief increases in lava supply rate. We suggest, contrary to current belief, that there is no evidence that compound flows are proximal to source and sheet lobes (simple flows) are distal to source and we propose that the presence of flow-lobe tumuli in compound flows could be an indicator that a flow is distal to source. We use detailed, previously published, studies of the Thakurvadi Formation (Deccan Traps) as an example. We show that the length of a lobe and therefore the sections that are ‘medial or distal to source’ are specific to each individual lobe and are dependent on the lava supply of each eruptive event, and as such flow lobe tumuli can be used as an indicator of relative distance from source

Age at death estimation from bone histology in Malaysian males.

Estimation of age from microscopic examination of human bone utilizes bone remodeling. This allows 2 regression equation to be determined in a specific population based on the variation in osteon turnover in different populations. The aim of this study was to provide age estimation for Malaysian males. Ground undecalcified cross sections were prepared from long limb bones of 50 deceased males aged between 21 and 78 years. Ten microstructural parameters were measured and subjected to multivariate regression analysis. Results showed that osteon count had the highest correlation with age (R = 0.43), and age was estimated to be within 10.94 years of the true value in 98% of males. Cross validation of the equation on 50 individuals showed close correspondence of true ages with estimated ages. Further studies are needed to validate and expand these results

Discovery of a tyrosine-rich sporocyst wall protein in Eimeria tenella.

BACKGROUND: Eimeria is an important genus of apicomplexan parasites. A defining feature of these parasites is the oocyst, which is transmitted into the environment via the faeces of definitive hosts. The oocyst wall contains cross-linked, tyrosine-rich proteins and protects eight infectious sporozoites, housed in pairs within a second walled structure, the sporocyst. The biochemical basis for sporocyst wall formation is not known. FINDINGS: Here, we report the discovery of a novel tyrosine-rich protein, EtSWP1, in Eimeria tenella. Like the tyrosine-rich proteins of the oocyst wall, EtSWP1 is an intrinsically disordered protein with the tyrosine residues concentrated in a specific region of the protein, located immediately following the region of intrinsic disorder. We engineered E. tenella to express mCherry-tagged EtSWP1 and showed that the tagged protein localises specifically to sporocyst walls, indicating that the biochemistry of sporocyst wall assembly is analagous to that of oocyst walls. CONCLUSIONS: Tyrosine-rich proteins are known to be key components of the oocyst wall and we now demonstrate, using gene and protein analyses combined with genetic manipulation, that a novel tyrosine-rich protein is specific for the sporocyst wall. This finding is important because it shows that the biochemistry of these two distinct walls is similar and, hence, brings targeted disruption of sporulation and, therefore, potential neutralisation of oocysts in the environment, a step closer