Ankyrin-B (Ank-B) is a crucial scaffolding protein regulating expression and localization of contractile machinery in the cardiac muscle. Recent genetic investigations in the First Nations Community, the Gitxsan of Northern BC, identified a mutation in Ank-B (p.S646F c.1937 C>T) associated with a cardiac arrhythmia, Long QT Syndrome Type 4 (LQTS4). Distinct from other LQTS4 subtypes, individuals harbouring the p.S646F variant exhibit development deficits including cardiomyopathies and accessory electrical pathways. How p.S646F interferes with the development of the heart is unknown due to a fundamental lack of understanding regarding Ank-B proteostasis and its role in cardiac differentiation. Initial in silico analyses predicted the p.S646F mutant to be deleterious to the Ank-B protein. Using in vitro techniques, I determined p.S646F mutant reduced levels of Ank-B in H9c2 rat ventricular cardiomyoblasts. Furthermore, haploinsufficiency in mice was previously shown to result in developmental cardiac deficits. I, therefore, hypothesized that p.S646F interferes with Ank-B proteostasis, thereby affecting cardiomyocyte development. I showed that p.S646F destabilized Ank-B in cardiomyoblasts, due to increased degradation via the proteasome. Furthermore, overexpression of p.S646F Ank-B had a significant impact on cellular behaviour including reduced cell viability, and altered expression of cellular differentiation markers. Together these data address critical knowledge gaps with regards to Ank-B protein homeostasis and the role of Ank-B in cardiomyocyte viability and development. These findings inform the diagnosis and treatment of patients with the p.S646F variant, creating potential targeted pathways of intervention, and furthering our understanding of the role of the Ank-B in the development of the heart. / Graduate / 2019-04-26

Memoria para optar al título profesional de Bioquímico / La migración de los linfocitos a los órganos linfoides secundarios es un paso crucial en la iniciación de la respuesta inmune adquirida. Este fenómeno es caracterizado por múltiples eventos que requieren de interacciones entre el endotelio y el linfocito, e involucra la participación de diversas moléculas que favorecen la migración de los linfocitos a través de las células endoteliales columnares (HEC). La transmigración es un proceso activo que depende del estado de activación y diferenciación de los linfocitos y de señales específicas de las células endoteliales. Diversos estudios han demostrado que los linfocitos T de memoria migran selectivamente a través de células endoteliales a sitios de inflamación y/o infección. Además, ciertas evidencias sugieren que la expresión de moléculas MHC-II por células endoteliales facilitan el reclutamiento de linfocitos T antígeno específicos, hacia un tejido. Por otra parte, en nuestro laboratorio hemos demostrado que la adhesión de linfocitos B a células endoteliales columnares derivadas de amígdalas humanas (HUTEC) induce la activación endotelial, conduciendo a la producción de diversas citoquinas y quimioquinas. En esta tesis determinamos que la adhesión de linfocitos B a HUTEC permite la presencia de moléculas MHC-II en la superficie endotelial, además de la producción de un patrón de expresión diferencial de quimioquinas, las cuales podrían estar involucradas en la transmigración de los linfocitos. Los resultados muestran que la presencia de moléculas MHC-II en la superficie de las células endoteliales depende de la adhesión de los linfocitos B, pero no de linfocitos T. Sin embargo, MHC-II no se induce a nivel transcripcional, sugiriendo que las HUTEC adquieren moléculas MHC-II de los linfocitos B humanos durante el proceso de adhesión celular. Además, el contacto de los linfocitos B a HUTEC induce la expresión del mRNA de las quimioquinas CCL2, CCL3, CCL5, CCL7, CXCL8, entre otras. Demostramos que el efecto de la adhesión de linfocitos B a HUTEC induce la transmigración de linfocitos T de memoria central. Por otra parte, investigamos el efecto de factores soluble secretados por HUTEC y/o linfocitos B, después del contacto, en la migración transendotelial. Nuestros resultados muestran que no son necesarios los factores solubles para un incremento en la transmigración, aunque estos factores permiten una migración más eficiente de los linfocitos T. En conclusión, estos resultados sugieren que la interacción de linfocitos B con células endoteliales modula la respuesta inmune, favoreciendo la transmigración preferencial de linfocitos T CD4+ de memoria central / Lymphocytes migration to secondary lymphoid organs is a crucial step in the initiation of the acquired immune response. This phenomenon is characterized by multiple events that require interactions between the endothelium and lymphocytes, and involves diverse molecules that favor the migration of T lymphocytes across the high endothelial cells (HEC). Transmigration is an active process that depends on the state of activation and differentiation of T lymphocytes and specific signals of endothelial cells. Diverse studies have demonstrated that memory T lymphocytes selectively migrate across endothelial cells to sites of inflammation and/or infection. In addition, certain evidences suggest that expression of MHC-II molecules by endothelial cells facilitate the recruitment of antigen specific T lymphocytes towards a tissue. On the other hand, in our laboratory we have demonstrated that the adhesion of B lymphocytes to high endothelial cells derived from human tonsils (HUTEC) induces endothelial activation, leading to the production of several cytokines and chemokines. In this thesis we determined that the adhesion of B lymphocytes to HUTEC cells allows the presence of MHC-II molecules on the endothelial surface, in addition to the production of a differential expression pattern of chemokines, which could be involved in the lymphocytes’ transmigration. These results show that the presence of MHC-II molecules on the surface of endothelial cells depends on the adhesion of B lymphocytes, but not T lymphocytes. However, MHC-II is not induced at transcriptional level, suggesting that HUTEC cells acquire MHC-II molecules from human B lymphocytes during the process of cellular adhesion. In addition, the contact of B lymphocytes to HUTEC cells induces the mRNA expression of chemokines such as CCL2, CCL3, CCL5, CCL7, CXCL8, among others. We demonstrated that the effect of the adhesion of B lymphocytes to HUTEC cells induces the memory central T lymphocytes transmigration. In the other hand, we investigated the effect of soluble factors secreted by HUTEC cells and/or B lymphocytes, after the contact between them, in the transendotelial migration. Our results show that although soluble factors are not necessary to increase the transmigration, these factors allow a more efficient migration of T lymphocytes. In conclusion, these results suggest that the interaction of B lymphocytes with endothelial cells modulates the immune response, favoring the preferential transmigration of central memory CD4+ T lymphocytes

This thesis defends the diagnostic accuracy and political usefulness of the claim that women are complicit in their sexual objectification. Feminists have long struggled to demarcate the appropriate limits of feminist critiques of sexual objectification, particularly when it comes to objectifying practices which women both consent to and experience as empowering. These struggles, I argue, are the result of a fundamental misdiagnosis of what happens when women are sexually objectified, whereby the abstract notion of 'treating as an object' is called upon to explicate the kind of phenomena which can only be properly understood in light of a more general set of social norms of masculinity and femininity. A more accurate diagnosis of sexual objectification, I argue, is provided by Catharine MacKinnon's radical feminist theory, according to which sexually objectifying acts are manifestations of the social process through which women are made into objects of male sexual gratification. One important implication of this account is that women themselves play a role in perpetuating the norms through which sexually objectifying treatment of women is enabled: insofar as they participate in the re-constitution of the social context which facilitates their sexual objectification, they are complicit in it. Although this idea lacks intuitive appeal from a feminist perspective, I argue that understanding the nature of the contribution women make to perpetuating their objectification enables a better understanding of what practices of resistance are necessary for effectively combatting the sexual objectification of women. I defend the explanatory power of the complicity account of objectification in light of two pressing debates in contemporary feminist philosophy: the question of how women can disidentify from femininity given the strong attachments they develop to it, and the question of how feminism can continue to appeal to the motif of solidarity considering the anti-essentialist commitments of recent feminist theory.

No description available.

B-lymphocytes are lymphoid cells, which are a part of the adaptive/innate immune system and generate antibodies. Recently, many studies have supported hypothesis that different rather minor B-lymphocyte subpopulations may play a direct and indirect role in immunopathogenesis in human pathologies such as Crohn's disease (CD). The aim of current study was therefore to investigate distribution of frequencies of B lymphocyte subpopulations (from transient to mature effector B cell stages) in peripheral blood of healthy subjects (CO), patients with Crohn's disease (CD) and ulcerative colitis (UC). Thus, using 11-colour flow cytometry we have analysed 30 blood samples of individuals, including 14 healthy controls, 11 patients with Crohn's disease and 5 with UC. In 6 patients with CD we have had an opportunity to analyze blood samples collected 2 hours after an administration of anti-TNF therapy. Higher frequencies of memory B-lymphocytes (CD19+ CD27+ , CD19+ CD20+ CD27+ and CD19+ CD20+ CD27+ IgM+) were found in patients with CD as compared to COs. (20.06±13.58%; 17.61±13.48%; 88.60±20.56% vs. 11.75±26.47%; 11.25±26.50%; and 66.82±22.60%), in case of CD19+CD20-CD27-IgM+ B-lymphocytes the difference was statistically significant (57.15±17.21% in CD vs. 19.59±31.79% in CO; p=0.0341), which is in accordance...

No description available.

Abstract Mammalian peroxisomes contain two parallel multifunctional enzymes (MFE), MFE type 1 and MFE type 2 (MFE-2), which are responsible for the degradation of fatty acids. They both catalyze the second and third reactions of the β-oxidation pathway, but through reciprocal stereochemical courses. MFE-2 possesses (2E)-enoyl-CoA hydratase-2 and (3R)-hydroxyacyl-CoA dehydrogenase activities. In addition, the carboxy-terminal part is similar to the sterol carrier protein type 2 (SCP-2). The purpose of this work was to study the structure-function relationship of functional domains of mammalian MFE-2 by recombinant DNA technology, enzyme kinetics and X-ray crystallography. The work started with the identification of conserved regions in MFE-2. This information was utilized when dehydrogenase, hydratase-2 and/or SCP-2-like domain were produced as separate recombinant proteins. Subsequently, both dehydrogenase and SCP-2-like domains were crystallized and their crystal structures were solved. The structure of the dehydrogenase region of rat MFE-2 contains the basic α/β short-chain alcohol dehydrogenase/reductase (SDR) fold and the four-helix bundle at the dimer interface, which is typical of dimeric SDR enzymes. However, the structure has a novel carboxy-terminal domain not seen among the known structures. This domain lines the active site cavity of the neighbouring monomer, reflecting cooperative behaviour within a homodimer. The monomeric SCP-2-like domain of human MFE-2 has the same fold as rabbit SCP-2. The structure includes a hydrophobic tunnel occupied by an ordered Triton X-100 molecule, demonstrating the ligand-binding site. Compared to the unliganded rabbit SCP-2 structure, the position of the carboxy-terminal helix is different. The movement of this helix in the liganded human SCP-2-like domain resulted in the exposure of a peroxisomal targeting signal, suggesting ligand-assisted protein import into peroxisomes. The roles of conserved protic residues in the hydratase-2 region of human MFE-2 were studied by mutating them to alanine. In the first step, the ability of mutated variants to utilize oleic acid in vivo was tested with Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2). Subsequently, in vitro characterization of the mutant enzymes revealed two amino acid residues, Glu366 and Asp510, vital for hydratase-2 activity. The results indicate that the acid-base catalysis is valid for hydratase-2.

Abstract Gelatinases (MMP-2 and MMP-9) play a key role during invasion and metastazising of malignant cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumours. However little is known about their role in hematological malignancies. In the present work, gelatinase expression and its clinicopathological correlations were studied with immunohistochemical staining in 10 cases representing normal bone marrow aspirate smears, 123 cases representing diagnostic bone marrow samples of patients with different leukaemias (35 AML, 7 CLL, 6 CML, 75 ALL), 67 diagnostic paraffin-embedded lymph node biopsies from patients with Hodgkin's lymphoma and 57 biopsies from patients with non-Hodgkin's lymphomas. The lymphoma samples were also stained with factor VIII antibody to evaluate the extent of new vessel formation and the non-Hodgkin's lymphoma cases also with tissue inhibitor of metalloproteinases -1 (TIMP-1) antibody. CLL did not express either of the MMP enzymes, while CML in the chronic phase expressed strongly both of the enzymes. In ALL, gelatinase expression was weak and detectable in pediatric cases in only 12.7% and in the adults in 65% of the cases. In adult ALL, MMP-2 expression correlated strongly with an extramedullary and invasive pattern of disease presentation. In AML MMP-2 positivity had markedly favorable prognostic and predictive power. In lymphoma studies, no correlations could be detected between gelatinase expression and the clinical parameters of invasion. MMP-9 positivity was related to the presence of B symptoms, which difference was statistically significant in Hodgkin's lymphoma. In Hodgkin's lymphoma, strong MMP-9 expression also implicated decreased neovascularization. In both lymphoma types, strong MMP-9 expression correlated with unfavorable prognosis, which difference was statistically significant in non-Hodgkin's lymphomas and remained as a tendency in Hodgkin's lymphoma. MMP-2 had statistically significant association with a favorable prognosis in Hodgkin's lymphoma. Combination of the results of both stainings further increased prognostic power. All together these findings implicate that gelatinases could be used as prognostic tools in AML and lymphomas albeit this needs to be verified in larger materials.

The recent years have witnessed an increased interest in e-learning platforms that incorporate adaptive learning and teaching systems that enable the creation of adaptive learning environments to suit individual student needs. The efficiency of these adaptive educational systems relies on the methodology used to accurately gather and examine information pertaining to the characteristics and needs of students and relies on the way that information is processed to form an adaptive learning context. The vast majority of existing adaptive educational systems do not learn from the users’ behaviours to create white-box models to handle the high level of uncertainty and that could be easily read and analysed by the lay user. The data generated from interactions, such as teacher–learner or learner–system interactions within asynchronous environments, provide great opportunities to realise more adaptive and intelligent e-learning platforms rather than propose prescribed pedagogy that depends on the idea of a few designers and experts. Another limitation of current adaptive educational systems is that most of the existing systems ignore gauging the students' engagements levels and mapping them to suitable delivery needs which match the students' knowledge and preferred learning styles. It is necessary to estimate the degree of students’ engagement with the course contents. Such feedback is highly important and useful for assessing the teaching quality and adjusting the teaching delivery in small and large-scale online learning platforms. Furthermore, most of the current adaptive educational systems are used within asynchronous e-learning contexts as self-paced e-learning products in which learners can study in their own time and at their own speed, totally ignorant of synchronous e-learning settings of teacher-led delivery of the learning material over a communication tool in real time. This thesis presents novel theoretical and practical architectures based on computationally lightweight T2FLSs for lifelong learning and adaptation of learners’ and teachers’ behaviours in small- and large-scale asynchronous and synchronous e-learning platforms. In small-scale asynchronous and synchronous e-learning platforms, the presented architecture augments an engagement estimate system using a noncontact, low-cost, and multiuser support 3D sensor Kinect (v2). This is able to capture reliable features including head pose direction and hybrid features of facial expression to enable convenient and robust estimation of engagement in small-scale online and onsite learning in an unconstrained and natural environment in which users are allowed to act freely and move without restrictions. We will present unique real-world experiments in large and small-scale e-learning platforms carried out by 1,916 users from King Abdul-Aziz and Essex universities in Saudi Arabia and the UK over the course of teaching Excel and PowerPoint in which the type 2 system is learnt and adapted to student and teacher behaviour. The type-2 fuzzy system will be subjected to extended and varied knowledge, engagement, needs, and a high level of uncertainty variation in e-learning environments outperforming the type 1 fuzzy system and non-adaptive version of the system by producing better performance in terms of improved learning, completion rates, and better user engagements.

This thesis work provides a new way to detect and track the displacement of flexor tendons within the human arm, using a non-invasive, ultrasound-based, speckle tracking technique. By tracking the tendons in the arm, it provides a way to monitor a person’s intention to move their hands and fingers. This has application to hand prosthetic control, as well as tendon injury assessment, which has significant contributions to the medical and rehabilitation community. The system works by capturing and processing a sequence of B-scan ultrasound images, to detect and track the flexor tendon motion (excursion) in the wrist, as the user flexes their muscles. Given the biomechanics of the hand, tendon displacement is correlated to the user’s intention to move their finger. Several speckle tracking techniques using B-scan ultrasound image sequences are developed in this work, including: auto-location of the tendon, a stationary ROI (region of interest), and novel use of similarity measures such as FT (Fisher Tippett), and hybrid methods. As well, work is done to investigate various speckle tracking parameters, and their effects on tracking accuracy. The different speckle tracking techniques are developed using data obtained from cadaver hands, and human volunteers undergoing regular surgery. The tracking techniques are compared in terms of successfully detecting the tendon, accurately tracking tendon displacement, successfully tracking multiple tendons, successfully detecting and tracking the onset of low tendon displacement, and computational efficiency of the algorithms. Another major aspect of this work is the design of a novel quad-array transducer that can collect image sequences from up to four tendons simultaneously. This transducer is instrumental to the motivation for controlling an advanced prosthesis. As well, specialized hardware is designed for the cadaver-based studies. Overall, this thesis successfully demonstrated the proposed tracking algorithms and newly designed hardware, for tracking the displacement of single and multiple flexor tendons. It has provided several important contributions to the field. / Graduate / 0548 / 0986 / 0760