QSpace Collection:

Generation and Optimization of the Self-Administered Bleeding Assessment Tool

Thu, 02 May 2013 04:00:00 GMT

Title: Generation and Optimization of the Self-Administered Bleeding Assessment Tool Authors: DEFOREST, MEGHAN Abstract: Defects in haemostasis, specifically in the quantitative or qualitative properties of von Willebrand factor (VWF), result in the most commonly inherited bleeding disorder, von Willebrand disease (VWD). Of the three types of VWD, type 1 is the most common and has the mildest phenotype. Type 1 VWD has a symptomatic prevalence of approximately 1 in 1000, but a much smaller number of patients who are actually diagnosed. This discrepancy is a result of a lack of standardized diagnostic criteria. The objective of the Self-Administered Bleeding Assessment Tool (Self-BAT) Study is to develop and optimize, a standardized, self-administered BAT to quantify bleeding history, and to distinguish between normal and abnormal bleeding. In Phase 1 of the study, the medical terminology in the expert-administered ISTH BAT was changed to a grade four reading level to produce the first version of the Self-BAT. Three subsequent versions of the Self-BAT were generated during optimization based on focus group feedback and statistical analysis. The Self-BAT was administered to a total of 38 control subjects and 20 subjects affected with type 1 VWD in Phase 1. Demographic information on all the subjects (gender, age) was collected and blood was drawn for determination of VWF antigen level, VWF ristocetin cofactor activity and factor VIII level. Bleeding scores calculated from the ISTH BAT and Self-BAT showed an excellent correlation of 0.869 in the final version. In Phase 2 of the study, the Self-BAT was administered to 27 control subjects and 23 affected subjects with the objective to simplify the questionnaire by identifying questions which can discriminate between unaffected and affected subjects. It was determined through category-total, category-category and item-item correlations that there was not sufficient evidence to reduce the Self-BAT. In conclusion, the Self-BAT functions as a simple and effective tool to distinguish between normal and abnormal bleeding and is suitable for adults to administer themselves. The Self-BAT has now moved on to prospective validation studies where it will be tested on subjects who have been referred to tertiary care, but not yet received a diagnosis, in order to test its ability to distinguish between normal individuals and those affected with type 1 VWD and, potentially, other mild bleeding disorders. Description: Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-04-29 17:04:36.196

Endothelial cell synthesis of Factor VIII

Wed, 13 Mar 2013 04:00:00 GMT

Title: Endothelial cell synthesis of Factor VIII Authors: Riches, Jonathan Jacob Abstract: Factor VIII (FVIII) is an essential blood-clotting protein and mutations in the FVIII gene are the cause of hemophilia A, a severe inherited bleeding disorder. FVIII synthesis has been observed in discreet endothelial sub-populations including liver sinusoidal endothelial cells and in selected microvascular beds. The mechanistic basis for this differential expression is unknown. Differences in shear stress are believed to play an important role in determining endothelial heterogeneity. In this study, we have evaluated the effect of various shear stress conditions on FVIII expression in blood outgrowth endothelial progenitor cells (BOECs) with an in vitro flow system. Under static conditions, BOECs do not express FVIII. In contrast, after exposure to laminar shear stress for 48 hrs, a significant increase in FVIII expression was documented by qRT-PCR, regardless of the magnitude of shear stress studied (1, 5, 15 and 30 dynes/cm2). To determine the effect of prolonged shear stress, laminar flow was applied over 120 hrs and FVIII mRNA levels returned to static levels. Induction of gene expression by laminar shear stress followed by repression after longer durations is common to other pro-coagulant genes induced by non-laminar or oscillatory flow (eg. tissue factor). BOECs exposed to 15 dyne/cm2 of shear stress, oscillating every 0.5 sec for 120 hrs, had FVIII mRNA levels 4.7-fold that of cells in static conditions. This was significantly higher than FVIII expression in BOECs exposed to 15 dyne/cm2 of laminar shear stress for the same duration. Expression of KLF2, a transcription factor that suppresses endothelial pro-coagulant gene expression under laminar shear stress, was significantly reduced in BOECs exposed to oscillatory as opposed to laminar shear stress. Finally, in BOECs exposed to oscillatory shear stress, FVIII protein was synthesized and co-localizes with its carrier protein VWF in Weibel-Palade bodies. These studies show that shear stress is a significant regulator of FVIII expression in BOECs, that FVIII expression is inversely correlated with that of KLF2, and that FVIII protein co-localizes with VWF in these cells. Description: Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-03-04 17:00:27.994

Defining clinically relevant subgroups of follicular lymphoma cases according to the functional status of the CDKN2A gene

Wed, 13 Mar 2013 04:00:00 GMT

Title: Defining clinically relevant subgroups of follicular lymphoma cases according to the functional status of the CDKN2A gene Authors: Alhejaily, Abdulmohsen Abstract: Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma (NHL). FL is clinically designated as an indolent disease with a long median survival of 8-10 years. However, the clinical and biological behavior of FL shows considerable variability, with some patients showing aggressive disease progression and very short survival. Because defects in the regulation of apoptotic cell death are fundamental in FL pathogenesis, we hypothesized that deregulated expression of components of the pRb signaling pathway may promote cell proliferation, thereby complementing antecedent anti-apoptotic mutations and producing more aggressive disease. In the present study we undertook an immunohistochemical (IHC) evaluation of expression of key cell-cycle regulatory proteins in diagnostic biopsies from 127 cases of FL using formalin-fixed, paraffin-embedded tissues (FFPE) in tissue microarray (TMA) sections immunostained for p53, pRb, p16INK4A and cyclin D3. Data analysis revealed that increased abundance of p53 or p16INK4A is associated with reduced overall survival (OS) (p=0.005 and p=0.014 respectively), and with conventional pathological markers of tumour aggressiveness including high histologic grade. Encouraged by this remarkable finding of a counterintuitive association between p16INK4A expression and clinical outcome, we analyzed CDKN2A gene deletion and methylation, as these are the most frequent mechanisms of the CDKN2A gene inactivation in NHL including FL. We determined the deletion and methylation status of CDKN2A in 105 FL cases. Laser-capture microdissection was used to enrich the samples for lymphoma cells. CDKN2A was deleted in 9 cases and methylated in 22 cases. The 29 cases (28%) with CDKN2A deletion or methylation had decreased overall survival (OS) (p=0.046) in all cases and in cases treated with rituximab (p<0.001). Our findings indicate that deleterious alterations of CDKN2A are relatively prevalent in FL at diagnosis and can predict poor clinical outcome. In summary, our data reveal novel insights into the pathogenesis of FL and suggest a relationship between increased p16INK4A expression and CDKN2A deletion or methylation and unfavorable clinical outcome in FL. We hope that the work presented herein will provide a useful prognostic tool for predicting the prognosis and choosing optimal treatment approaches to help patients suffering from FL. Description: Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-03-12 23:49:44.541

CYP26B1 limits inappropriate activation of RARgamma by retinoic acid during murine embryogenesis

Wed, 07 Nov 2012 05:00:00 GMT

Title: CYP26B1 limits inappropriate activation of RARgamma by retinoic acid during murine embryogenesis Authors: Pennimpede, Tracie Abstract: Proper embryonic patterning requires precise spatio-temporal regulation of retinoic acid (RA) activity. Morphogenesis can be regulated at the level of RA distribution, mainly via its synthesis and catabolism by the RALDH and CYP26 enzymes respectively, and at the level of RA-mediated transcription through activation of its cognate nuclear receptor, the retinoic acid receptors (RARs) α, β, and γ. Loss of Cyp26b1 leads to increased local levels of RA in tissues such as the limb and craniofacial structures, and results in neonatal lethality. Visible gross phenotypic defects in neonates include phocomelia (shortening of the limbs), adactyly (missing digits), micrognathia (shortened lower jaw), and open eyes at birth. In addition, these embryos exhibit cleft palate and have a paucity of vibrissal (whisker) and pelage (hair) follicles. We have previously shown that ablating the gene encoding RARγ in a Cyp26a1-null background was able to rescue the caudal abnormalities associated with improper RA exposure in these embryos by limiting aberrant RA signalling, and thus rescuing expression domains of target genes involved in caudal development. I show here that ablating Rarg in a Cyp26b1-null background is able to partially rescue the defects associated with loss of CYP26B1. These include a reduction in the severity of limb defects, rescued vibrissae, fused eyelids, and recovered aspects of axial skeletal development. This compound-null murine model illustrates that RARγ plays a specific role in transducing the RA signal within tissues that are affected by the loss of CYP26B1. Further molecular analysis of the pathways responsible for directing limb bud outgrowth and eyelid fusion provided insight into pathways regulated by RARγ in these rescued tissues. Description: Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-04-01 15:38:52.05