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Dilated cardiomyopathy (DCM) is the third most common cause of heart failure, often associated with arrhythmias and sudden cardiac death if not controlled. Metabolic and/or environmental factors, such as alcohol abuse, obesity, diabetes and Chagas disease, alter glycoprotein glycosylation, can lead to DCM. Inherited genetic disease, such as the human congenital disorders of glycosylation (CDG), causes multi-system manifestations including DCM. Non-congenital changes in glycosylation are also occurred in humans with and in animal models of DCM and heart failure. However, mechanisms responsible for glyco-dependent DCM are not understood. Here we sought to investigate the impact of sialylation and N-glycosylation in cardiac function. Partial reduction of N-α2,3-sialylation achieved through ST3Gal4 deletion (ST3Gal4-/-) led to adult late-onset DCM. The DCM symptoms progressed gradually, developing thinner left ventricular walls and dilation of all four chambers by 18-month old, but with preserved systolic function. Transverse aortic constriction (TAC) was used as a chronic stressor on 16-20 week old mice to determine whether the ability of the ST3Gal4-/- heart to compensate against pathologic insult is compromised. TAC’d ST3Gal4-/- mice presented with insufficient hypertrophy and reduced systolic function that deteriorated into congestive HF within six weeks post-surgery, while constricted WT hearts remained well-adapted throughout (ejection fraction, ST3Gal4-/-=34±5.2%; WT =53.8±7.4%; p<0.05).Calcineurin expression was decreased in ST3Gal4-/- (compared to TAC’d WT), contributed to the maladaptation of TAC’d ST3Gal4-/-. In order to better understand the role of glycosylation on cardiac function, we generated a cardiomyocyte specific knockout (αMHC-Cre) of glycosyltransferase responsible for synthesizing complex and hybrid N-glycans, Mgat1, (Mgat1CKO). Similar to but much more severe than that observed in ST3Gal4-/-, Mgat1CKO developed early-onset of DCM, late adult mortality, severely impaired cardiac systolic and diastolic function and frequent arrhythmias. Marked sex-difference in cardiac phenotype was observed in this autosomal gene (Mgat1) deletion, with male Mgat1CKO more severely affected. Both ST3Gal4 and Mgat1 did not participate in murine cardiogenesis, evidenced by normal litter size, Mendelian distribution of genotypes, no septal defect or vessel deformation under autopsy or echocardiography. In conclusion, we provided here the first and direct evidence of desialylation-elicited idiopathic dilated cardiomyopathy (DCM), reporting the cardiac phenotype of ST3Gal4-/-and cardiac-specific knockout of Mgat1. Our data showed sialylation and complex N-glycosylation are essential for cardiac function, and reduced N-glycosylation or sialylation leads to DCM development, contractile dysfunction and arrhythmia.

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

CDG-Ia ist eine genetisch bedingte Stoffwechselstörung, die eine fehlerhafte Synthese von Glykoproteinen zur Folge hat. Ursache sind Mutationen in der genetischen Information für das Enzym Phosphomannomutase II (PMM2). Inhalt dieser Arbeit ist die Generierung von zwei Gene-Targeting-Vektoren (GTV) und den entsprechenden transgenen embryonalen Maus-Stammzellen, welche jeweils die beiden häufigsten Mutationen R141H und F119L (R137H und F115L im Maus-Genom) tragen. Ausgangsprodukt ist ein Teil des murinen Pmm2-Gens, welcher in einen pBlueScript-Vektor subkloniert wird. Mit gezielter in-vitro-Mutagenese erfolgt das Einbringen der jeweiligen Mutation. Als Selektionsmarker für beide GTV dient ein von zwei loxP- Sequenzen flankiertes Neomycin-Resistenz-Gen (Neo). Die beiden derart konstruierten GTV, welche jeweils die Mutation F115L bzw. R137H beinhalten, werden als linearisiertes Konstrukt durch Elektroporation in embryonale Mausstammzellen transferiert. Nach Überprüfung der erfolgreichen homologen Rekombination wird je ein Stammzell-Klon mit dem Genotyp F115L/WT und R137H/WT mit einem Cre-Rekombinase-Gen transfiziert und die erfolgreiche Entfernung des Neo-Gens überprüft. Mit diesen transgenen Stammzellen sollen hypomorphe Mausmodelle für CDG-Ia generiert werden.

The purpose of this study was to develop and describe a model for the enhancement of self-concept of people with oculocutaneous albinism based on the experience of the participants. The need to conduct the study was triggered by the researcher’s long-term interaction with people with oculocutaneous albinism, and observation that they are still stigmatised and marginalised by the community. A purposive sample of fifteen participants, consisting of twelve females and three males with oculocutaneous albinism, aged between 18 and 48, participated in the study. The study was conducted in urban and semi-urban areas around the Gauteng Province in South Africa. The study had two theory generative objectives, namely to identify the main concepts of the model from the findings, and to describe the model structure and process. A theory generative, qualitative, explorative, descriptive and contextual research design was used to achieve these objectives. Chinn and Kramer’s (1995:92) four steps of theory-generation were used, namely concept analysis, construction of theoretical relationships, description of the model, and development of guidelines to operationalise the model.Concept analysis involved identification, definition and classification of the concepts to develop the model. In-depth, phenomenological interviews were conducted to explore the participants’ experience. Data analysis and interpretation revealed three main themes from the findings: the participants’ perception of the self; experience with the external environment, and expression of need for development and growth. The main concepts deducted from the findings were “self-concept” and “enhancement” hence “A model for enhancement of self-concept of people with albinism”. Construction of theoretical relationships entailed using deductive reasoning to describe the interaction between the concepts of the model. Description of the model entailed visually portraying and describing the structure and process of the model, as well as evaluating it. Developing guidelines to operationalise the model entailed describing practical strategies to assist health professionals to enhance the self-concept of people with oculocutaneous albinism. / Health Studies / D. Litt. et Phil. (Health Studies)

The purpose of this study was to develop and describe a model for the enhancement of self-concept of people with oculocutaneous albinism based on the experience of the participants. The need to conduct the study was triggered by the researcher’s long-term interaction with people with oculocutaneous albinism, and observation that they are still stigmatised and marginalised by the community. A purposive sample of fifteen participants, consisting of twelve females and three males with oculocutaneous albinism, aged between 18 and 48, participated in the study. The study was conducted in urban and semi-urban areas around the Gauteng Province in South Africa. The study had two theory generative objectives, namely to identify the main concepts of the model from the findings, and to describe the model structure and process. A theory generative, qualitative, explorative, descriptive and contextual research design was used to achieve these objectives. Chinn and Kramer’s (1995:92) four steps of theory-generation were used, namely concept analysis, construction of theoretical relationships, description of the model, and development of guidelines to operationalise the model.Concept analysis involved identification, definition and classification of the concepts to develop the model. In-depth, phenomenological interviews were conducted to explore the participants’ experience. Data analysis and interpretation revealed three main themes from the findings: the participants’ perception of the self; experience with the external environment, and expression of need for development and growth. The main concepts deducted from the findings were “self-concept” and “enhancement” hence “A model for enhancement of self-concept of people with albinism”. Construction of theoretical relationships entailed using deductive reasoning to describe the interaction between the concepts of the model. Description of the model entailed visually portraying and describing the structure and process of the model, as well as evaluating it. Developing guidelines to operationalise the model entailed describing practical strategies to assist health professionals to enhance the self-concept of people with oculocutaneous albinism. / Health Studies / D. Litt. et Phil. (Health Studies)

South Africa has the highest prevalence of Foetal Alcohol Spectrum Disorders (FASD) worldwide. Despite the high prevalence of FASD in children and the resulting need for services, appropriate identification and management of most genetic and congenital disorders are critically lacking in South Africa. As a result of the established risk for communication delay from birth, young children with FASD need early communication intervention (ECI). There appears to be a dearth in research on the communication difficulties of local young children with FASD. Determining comprehensive communication profiles of young children may serve to emphasize their need for ECI. The main aim of the study was to present the distinctive communication profiles of a group of young children with FASD. The sub-aims entailed the description of their developmental histories, family/care-giving contexts, communication functioning and identifying developmental trends across the participants. A collective case study design following a quantitative approach was selected. The study was cross-sectional and descriptive statistics was used to describe the participants’ communication skills. A purposive sampling procedure was utilized as a limited number of participants could be found with a diagnosis of FASD. Five participants diagnosed with FASD, living in the Gauteng area, between the ages of 4 to 58 months took part in the study. The empirical study comprised of two phases. Phase 1 entailed the development of the Interview Schedule, the Assessment Protocol and the conduction of a pilot study. Phase 2 comprised the data collection, analysis and interpretation of results. The results of the study indicated that all five participants presented with incomplete case histories obtained from the foster parents and general developmental delays. According to the Four-Level Early Communication Assessment Framework the greatest delays included all aspects of communication skills. A developmental trend of the combined communication profiles of the participants indicated an increase in delay with an increase in chronological age. The participants presented with complex multiple neuro-developmental needs to be viewed within a developmental systems and ecological framework. Based on the results of this study the diverse roles of the speech-language therapist working with young children with FASD and their families were identified. A framework for interdisciplinary service delivery to this population was suggested. The study succeeded in the attempt to provide a comprehensive, in-depth description of a group of the five participants’ unique communication skills. In addition the study also highlighted the shortage of services, e.g. limited early identification and services dedicated to families affected by alcohol related problems. The study is of value to the speech-language therapist in the clinical and research domain. Future research is recommended to expand and support the findings. AFRIKAANS : Suid-Afrika vertoon die hoogste prevalensie van Fetale Alkohol Spektrumafwykings (FAS) wêreldwyd. Ten spyte van die hoë prevalensie van FAS in kinders en die voortspruitende behoefte aan dienste, is toepaslike identifikasie en hantering van die meeste genetiese en kongenitale afwykings in Suid-Afrika krities ontoereikend. As ‘n resultaat van die bevestigde risiko vir kommunikasie agterstand vanaf geboorte, benodig jong kinders met FAS vroëe kommunikasie intervensie (VKI). Daar blyk ‘n gaping in die navorsing aangaande die kommunikasie probleme van plaaslike jong kinders met FAS te wees. Omvattende kommunikasie profiele van jong kinders met FAS kan die behoefte aan VKI dienste beklemtoon. Die hoofdoel van die studie was om die kommunikasie profiele van ‘n groep jong kinders met FAS voor te lê. Die sub-doelwitte het die beskrywing van hulle ontwikkelings geskiedenis, gesin/sorggewer konteks, kommunikasie funksionering en die identifisering van ontwikkelings neigings in al die deelnemers behels. ‘n Saamgestelde gevallestudie is geselekteer volgens ‘n kwantitatiewe navorsings benadering. Die studie is ‘n kruisdeursnee ontwerp en beskrywende statistieke is gebruik om die deelnemers se kommunikasie vaardighede te beskryf. ‘n Doelgerigte steekproef prosedure is gebruik omdat ‘n beperkte aantal deelnemers met ‘n diagnose van FAS gevind kon word. Die vyf deelnemers wat geselekteer was vir die steekproef was gediagnoseer met FAS, woonagtig in die Gauteng area en het binne die 0 – 5 jaar ouderdomsgroep geval. Die empiriese studie het uit twee fases bestaan. Fase 1 het die ontwikkeling van die onderhoud skedule, die assesserings protokol en die uitvoering van ‘n loodsstudie ingesluit. Fase 2 het die data insameling, analise en interpretasie van resultate behels. Die resultate van die studie het aangedui dat al die deelnemers gepresenteer het met onvolledige gevalsgeskiedenisse wat van hul pleegouers verkry is en ‘n algemene ontwikkelings agterstand. Volgens die Vier-Vlak Vroëe Kommunikasie Assesserings Raamwerk het die grootste agterstande alle aspekte van kommunikasie vaardighede behels. ‘n Ontwikkelings neiging vanuit die gekombineerde kommunikasie profiele van die deelnemers het ‘n toename in agterstand met ‘n toename in kronologiese ouderdom aangedui. Die deelnemers presenteer met veelvuldige neuro-ontwikkelings behoeftes wat binne die ontwikkelings sisteem en ekologiese raamwerk beskou moet word. Gebasseer op die resultate is die diverse rolle van die spraak-taalterapeut wat met jong kinders met FAS en hulle families werk, geïdentifiseer. ‘n Raamwerk vir interdissiplinêre dienslewering aan hierdie populasie word voorgestel. Die studie het geslaag in die poging om ‘n omvattende, in-diepte beskrywing van die deelnemers se unieke kommunikasie vaardighede te verskaf. Addisioneel het die studie ook die tekort aan dienste, naamlik beperkte vroëe identifikasie en dienste toegewy aan families geaffekteer deur alkohol verwante probleme, uitgelig. Die studie is van waarde vir die spraak-taalterapeut in die kliniese en navorsings domein. Toekomstige navorsing vir die uitbreiding en ondersteuning van die studie se bevindinge word aanbeveel. / Dissertation (MCommunication Pathology)--University of Pretoria, 2011. / Speech-Language Pathology and Audiology / Unrestricted