Calpains are calcium-dependent, intracellular, multi-domain cysteine proteases involved in many physiological functions regulated by calcium signaling, including cell motility. How calpains are activated in the cell is still unknown because the resting intracellular concentration of Ca2+ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Several stratagems by which calpains might overcome this Ca2+ concentration differential have been proposed. It is possible that post-translational modifications like phosphorylation, or accessory proteins that bind to calpain, might facilitate the enzyme’s activation at lower than optimal Ca2+ concentrations. Autoproteolysis (autolysis) and subunit dissociation are two other proposed activation mechanisms that could release constraints on the calpain core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, it was demonstrated that while the anchor helix is one of the first targets to be cut, several other potentially inactivating autolysis sites, particularly in Domain III, can also be cleaved within the first minute. Thus autolytic activation would go hand in hand with inactivation. By fractionating and identifying calpain 2 autolysis fragments, I show that the small subunit does not dissociate away from the large subunit, but is proteolyzed to a 40-45 k heterodimer of the penta-EF-hand Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit domain VI homodimer that would be produced by subunit dissociation. A calpastatin affinity column was constructed and used to capture recombinant calpain 2 from bacterial cell lysate. This affinity column provides a tool to screen for and capture calpain complexed to potential binding partners in the presence of Ca2+. Here I propose a model for calpain 2 activation in vitro that does not involve autolysis, subunit dissociation, or calpain activators. / Thesis (Master, Biochemistry) -- Queen's University, 2010-10-25 16:03:52.364

Understanding the intermolecular forces and dynamics of insulin self-assembly is crucial for devising formulations for the treatment of insulin-dependent diabetes. Insulin must dissociate from its hexameric storage form, through an intermediate dimer form, to the bioactive monomer before receptor binding. Specifically, the dimer dissociation is a pivotal step to control insulin dynamics and self-assembly. Steered molecular dynamics simulations were performed on native insulin to provide molecular insight into the insulin dissociation force spectroscopy experiment. Our simulation results of force-induced dimer dissociation revealed that the dimer dissociation occurs near the limit of extensibility of the B-chain with significant conformational changes to the monomer(s). These long-range interactions, consistent with our experiments, are due to stronger inter-monomer interactions across the anti-parallel β-sheet interface than any other intra-monomer interaction. Novel atomistic data played an important role in detailed structural characterization of multiple unfolding and dissociation pathways that depend on the relative strength of the inter-monomer interactions and the intra-monomer interactions. Comparative simulations of two rapid-acting insulin analogues (LysB28ProB29, AspB28) to native insulin were performed to investigate the effect of sequence on the dimer dissociation. The hypothesis is that site-specific alterations to the dimer-forming surface of two rapid-acting analogues will result in a weakening of the inter-monomer interactions, which would be reflected during force-induced dimer dissociation. The results revealed that these analogues dissociates with lower probability of long-range interactions and a corresponding reduction in B-chain extension. B-chain extensibility is thus a characteristic marker of inter-monomer interactions and multiple unfolding pathways. These data agree with the design strategies of sequence modifications to the weakened inter-monomer interface applied to the synthesis of rapid-acting insulin analogues. In contrast, the ligand-induced alteration to the strengthened inter-monomer interactions through a specific GluB13s-zinc bridge contributed to the unique unfolding force curves, so it can be applicable as design strategy to the development of a novel long-acting analogue. Overall, our force spectroscopy studies on insulin native and analogues have successfully provided atomistic insights into the dimer dissociation characteristics and control strategies of self-assembly. In addition, this study would provide a framework for the structure-dynamics-function relationships of insulin-insulin receptor binding.

Fatal earthquakes such as that which occurred in Christchurch on February 22nd 2011, can result in survivors having difficulties with cognitively processing the event, which may be the precursor to posttraumatic stress symptoms. Trauma related dissociation has been proposed to be a mechanism related to these cognitive processing difficulties. Most research focusing on information processing and dissociation post-trauma has conducted controlled analogue studies or has not focused solely on information processing and dissociation. There is also scant research on these constructs across therapy. In response to this gap in research, two studies were developed. An association was proposed between dissociation and information processing as demonstrated by an increase in conceptual processing and a reduction in dissociation. It was predicted that an improvement in these constructs would be related to a reduction in PTSD symptoms over therapy. Study1 applied a case-study design to 5 individuals who were attending therapy for post-traumatic stress disorder in response to the trauma they had experienced from the Christchurch earthquakes. Study 2 assessed information processing and dissociation (via self and observer report) in 20 individuals who had direct exposure to the effects of the earthquake. Earthquake information processing and dissociation were assessed as they were happening nearly two year’s post-quake using correlation analyses and hierarchical regressions. The hypotheses were partially confirmed, in that an increase in conceptual processing was not shown to be associated with a reduction in dissociation. However, an increase in conceptual processing was shown to be related to trauma symptom improvement particularly for re-experiencing symptoms. In addition, study 2 demonstrated a possible relationship between trait dissociation and arousal symptoms. These findings partially support the proposed role information processing and dissociation play in the recovery from PTSD. The findings suggest that trauma related difficulties should be assessed as early as possible to resolve issues related to a delay in symptom reporting.

Gas hydrates are known to form plugs in pipelines. Hydrate plug dissociation times can be predicted using the CSMPlug program. At high methane mole fractions of a methane + ethane mixture the predictions agree with experiments for the relative dissociation times of structure I (sI) and structure II (sII) plugs. At intermediate methane mole fractions the predictions disagree with experiment. Enthalpies of dissociation were measured and predicted with the Clapeyron equation. The enthalpies of dissociation for the methane + ethane hydrates were found to vary significantly with pressure, the composition, and the structure of hydrate. The prediction and experimental would likely agree if this variation in the enthalpy of dissociation was taken in to account. In doing the plug dissociation studies at high methane mole fraction a discontinuity was observed in the gas evolution rate and X-ray diffraction indicated the possibility of the presence of both sI and sII hydrate structures. A detailed analysis by step-wise modelling utilising the hydrate prediction package CSMGem showed that preferential enclathration could occur. This conclusion was supported by experiment. Salts such as tetraisopentylammonium fluoride form semi-clathrate hydrates with melting points higher than 30 ℃ and vacant cavities that can store cages such as methane and hydrogen. The stability of this semi-clathrate hydrate with methane was studied and the dissociation phase boundary was found to be at temperatures of about (25 to 30) K higher than that of methane hydrate at the same pressure.

Borderline and schizotypal personality constructs were compared as predictors of dissociation for 971 nonclinical university undergraduates who completed 11 self-report behavior inventories. Structural equation modeling and multiple regression were used to (a) test the hypothesis that the quantitative interaction of borderline and schizotypal constructs would be the strongest predictor of dissociation and (b) evaluate each construct alone as well as their additive effect for prediction of dissociation. The interaction hypothesis was rejected; the quantitative interaction was only a substantively trifling and statistically nonsignificant predictor of dissociation. In a commonality analysis, the additive borderline and schizotypal effect was equivalent to schizotypy alone and marginally larger than the borderline effect alone for the prediction of dissociation. However, all three effects separately were substantial predictors; thus, dissociation is an untenable discriminator for the borderline and schizotypal constructs. Finally, when schizotypy was compared directly to the borderline construct such that the common variance was distributed dependent upon comparative predictive power (i.e., beta weights), schizotypy was greatly superior to the borderline construct for dissociative predictability. Consequently, reconsideration of the existing paradigm that dissociation is more strongly associated with the borderline construct than with schizotypy is warranted. / Department of Psychological Science

The aim of this thesis was to investigate psychological factors influencing the development and maintenance of post-traumatic symptoms, with particular attention to the role of dissociation. The concept of dissociation is at present not well understood, and a secondary aim of this thesis was to clarify its meaning and structure and enable its assessment. Chapter 2 reports the development and testing of a questionnaire measuring trait dissociation, and the factor analysis of this questionnaire into seven sub-factors with low to medium intercorrelations, along with an analysis of the reliability and validity of this questionnaire, which was used in the subsequent studies. Chapters 3 and 4 consist of experimental studies investigating the relationship between dissociation and later intrusive memories using a videotape of scenes from road traffic accidents as a stimulus. The first study attempted to manipulate dissociation, while the second used extreme scorers on the trait dissociation questionnaire. The manipulation of dissociation proved difficult, but both studies showed a relationship between dissociation and later intrusions. Chapters 6 and 7 report on two prospective clinical studies following road traffic accident victims over the first six months following their accident, and investigates the role of dissociation and other psychological factors in the development of post-traumatic symptoms. Both state and trait dissociation, and the emotional numbing subfactor of dissociation in particular, predict the later development and maintenance of other post-traumatic symptoms. Persistent dissociation was more predictive of later symptoms than dissociation in the first few days after the accident. These results are discussed in relation to current thinking about the concept of dissociation and the DSM diagnoses of post-traumatic stress disorder and acute stress disorder. In particular it seems useful to distinguish between components of dissociation in terms of their relationship to PTSD. Possible future research and clinical implications are also addressed.

No description available.

Some animals have adapted to hypoxia by increasing their haemoglobin affinity for oxygen, but in vitro studies have not shown any change of haemoglobin affinity for oxygen in human high altitude natives or lowlanders acutely acclimatized to high altitude. We conducted the first in vivo study of the oxyhaemoglobin dissociation curve by progressively reducing arterial PO2 while maintaining normocapnia in lowlanders at sea level, lowlanders sojourning at 3600m for two weeks and native Andeans at the same altitude. We found that the in vivo PO2 at which haemoglobin is half-saturated (P50) is higher in lowlanders at sea level (32 mmHg) than that measured in vitro (27 mmHg) and that lowlanders and highlanders do significantly increase the in vivo affinity of their haemoglobin for oxygen with exposure to high altitude. These results indicate the value of an in vivo approach for studying the oxyhaemoglobin dissociation curve.

Some animals have adapted to hypoxia by increasing their haemoglobin affinity for oxygen, but in vitro studies have not shown any change of haemoglobin affinity for oxygen in human high altitude natives or lowlanders acutely acclimatized to high altitude. We conducted the first in vivo study of the oxyhaemoglobin dissociation curve by progressively reducing arterial PO2 while maintaining normocapnia in lowlanders at sea level, lowlanders sojourning at 3600m for two weeks and native Andeans at the same altitude. We found that the in vivo PO2 at which haemoglobin is half-saturated (P50) is higher in lowlanders at sea level (32 mmHg) than that measured in vitro (27 mmHg) and that lowlanders and highlanders do significantly increase the in vivo affinity of their haemoglobin for oxygen with exposure to high altitude. These results indicate the value of an in vivo approach for studying the oxyhaemoglobin dissociation curve.

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (p. 204-220). Also available via the World Wide Web.