Dyskeratosis congenita (DC) is a multi system disorder that exhibits considerable clinical and genetic heterogeneity. It is characterised by mucocutaneous features, bone marrow failure and a predisposition to cancer. Research has identified mutations affecting several telomerase components and patients often have short telomeres, implicating defective telomere maintenance in this disease. Affected components include dyskerin, NOP10 and NHP2, which together with GAR1 form a protein core common to telomerase and all other H/ACA ribonucleoprotein complexes (H/ACA RNPs). Initially characterised as H/ACA RNP components important for pseudouridylation and rRNA processing, their role in the functionally distinct telomerase complex and telomere maintenance is less defined. In order to better understand their implications in DC, this study investigated the importance of these core proteins for the integrity and function of telomerase in human cells. RNAi knockdown studies demonstrated that dyskerin, NOP10 and NHP2 are necessary for the accumulation of TERC (telomerase RNA component); dyskerin and NOP10 for telomerase activity. Moreover, dyskerin was found to be important for maintaining telomere length over time. The impact of NOP10 and NHP2 missense mutations was also analysed in vitro, which indicated that they impair TERC accumulation. The potential effect on pseudouridylation was also considered in this study; the analysis of other H/ACA RNA levels in these knockdown experiments and in a cohort of patients with DKC1 mutations revealed an irregular and inconsistent impact compared to that observed on TERC. Finally, defective telomere maintenance is heavily implicated as the primary cause of DC and very short telomeres have been proposed as a diagnostic marker. This study investigated telomere length in a patient cohort of unprecedented size. It demonstrated the prevalence of the telomere length defect, but telomere length was not found to correlate with either genetic subtype or disease severity, implicating the rate of telomere shortening as the correlating factor instead.

Background: In recent years, there has been an increase in the use of automated healthcare databases for drug safety in pregnancy evaluation; their suitability for this purpose needs to be evaluated. Aim: To evaluate the utility of the United Kingdom’s General Practice Research Database (GPRD) to act as an alternative to pregnancy registries, using anticonvulsants as a case study. Methods: Pregnancies in women with epilepsy were identified and first trimester anticonvulsant exposure was determined. Major congenital malformations in the offspring were identified and verified. The risk of major congenital malformations following exposure to a range of anticonvulsants was calculated and compared to those reported by the UK Epilepsy and Pregnancy Register. The ability to identify a known teratogenic association using GPRD data was also assessed. An algorithm was created to identify and classify different types of pregnancy loss in an automated manner. Results: The risks of a pregnancy outcome with a major congenital malformation following first trimester anticonvulsant exposures, were found to be similar in the GPRD to those of the UK Register. The number of exposures to individual products in the GPRD was often small and therefore lacked statistical power. It was, however, possible to identify a known teratogenic association using data from the GPRD. Verification of the algorithm developed to classify pregnancy losses demonstrated that, although not perfect, it would be a beneficial tool when using the GPRD for drug safety in pregnancy research. Conclusion: It is unlikely a single data source or study design will be sufficient for monitoring all aspects of the safety of medicine use during pregnancy. The GPRD has the potential to make a valuable contribution to this field of research and could play an important role in complementing the work of other surveillance systems.

This thesis is about partnerships involving congenitally deafblind people journeying towards language. The focus will be on the first steps of that journey: how partnerships make initial moves away from the here-and-now. In order to understand how this happens in the tactile medium, this thesis will draw on Reddy’s model (2003 and 2008) of the expanding awareness of the objects of the other’s attention to analyse how both partners are able to share attention to self, what self does, what self perceives and finally what self remembers. Demonstrating that both partners can operate at each of these four stages in the tactile medium then allows me to focus particularly on the final stage, what self remembers, and ask: what happens within partnerships if either partner brings movements, gestures or signs that refer to people, objects, places or events not present? Do both partners come to comprehend and produce such referential movements, gestures and signs in forms perceivable by both? Such questions will be considered against the backdrop of the dialogical framework, since in any exploration of human interaction it makes no sense simply to consider it from one perspective. At all times throughout this thesis, the focus will be on partnership. This thesis raises a number of practical recommendations about approaches and attitudes to be adopted by non-deafblind partners if language is going to be an outcome for their partnerships with congenitally deafblind people. But it will also conclude with a number of theoretical questions about how we define language in the first place.

Severe congenital neutropenia (SCN) is a rare blood disorder characterized by abnormally low levels of circulating neutrophils. Mutations in multiple genes like neutrophil elastase gene (ELANE) and granulocyte colony stimulating factor receptor (CSF3R) may cause SCN. The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF) which elevates the neutrophil count and hence improves the survival and quality of life. Long term survivorship on G-CSF is however linked to development of MDS (myelodysplastic syndrome)/AML (acute myeloid leukemia). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R. In this project, we hypothesized that this coding region of CSF3R constitutes a hotspot, vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to study the effect of induced oxidative or ER stress on the mutation rate in our hypothesized hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and a leukemia-associated gene Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a Green Fluorescent Protein (GFP) tag was subjected to cellular stress inducing mutagen treatment for a prolonged period of time (30 days). The amplicon based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all genes, there were more mutations in the GFP region as compared to the GC-rich partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Thus, we conclude that there are other mechanisms to acquired mutations of CSF3R that help drive the evolution of SCN to MDS/AML. To test this hypothesis, further experiments using unique barcoding system are in progress to characterize the clonal competition between different mutant CSF3R and ELANE expressing cell lines. This study will shed further light on the selection advantage that is provided to cells because cooperativity between mutations in different genes.

<p> </p><p><em><strong>Bakgrund:</strong> Varje år föds ett av hundra barn med hjärtsjukdom i Sverige. Att få ett hjärtsjukt barn medför ofta sorg och besvikelse, men också stora påfrestningar. Barn som behandlats för hjärtfel kan tvingas leva med olika besvär och behåller ofta kontakten med vårdpersonal långt upp i åren <strong><em>Syftet</em>: </strong>Syftet med detta arbete var att belysa föräldrars erfarenheter av att få ett hjärtsjukt barn, med tonvikt på föräldrars informationsbehov, känsloupplevelser och coping. <em><strong>Metod:</strong> </em>Studien utfördes som en litteraturöversikt där fem kvalitativa och fem kvantitativa artiklar valdes ut, granskades och kvalitetsbedömdes. Artiklarna analyserades med hjälp av innehållsanalys enligt Graneheim och Lundman (2003). Analysmetoden identifierar skillnader och likheter i ett textinnehåll. <em><strong>Resultat:</strong> </em>Föräldrar till hjärtsjuka barn hade svårt att acceptera barnets sjukdom beroende på att barnet ser friskt ut. Föräldrarna upplevde en enorm stress och andra mentala hälsoproblem. Att som förälder känna sig förstådd och trygg med den vård som ges till deras barn, kan minimera risken för krisreaktioner längre fram. <em><strong>Diskussion:</strong> </em>Det är av betydelse att ge föräldrar till hjärtsjuka barn individuellt anpassad information, vilket gör att de känner trygghet i vården. Dessutom underlättar denna information det kliniska arbetet. </em></p><p> </p>

Bakgrund: Varje år föds ett av hundra barn med hjärtsjukdom i Sverige. Att få ett hjärtsjukt barn medför ofta sorg och besvikelse, men också stora påfrestningar. Barn som behandlats för hjärtfel kan tvingas leva med olika besvär och behåller ofta kontakten med vårdpersonal långt upp i åren Syftet: Syftet med detta arbete var att belysa föräldrars erfarenheter av att få ett hjärtsjukt barn, med tonvikt på föräldrars informationsbehov, känsloupplevelser och coping. Metod: Studien utfördes som en litteraturöversikt där fem kvalitativa och fem kvantitativa artiklar valdes ut, granskades och kvalitetsbedömdes. Artiklarna analyserades med hjälp av innehållsanalys enligt Graneheim och Lundman (2003). Analysmetoden identifierar skillnader och likheter i ett textinnehåll. Resultat: Föräldrar till hjärtsjuka barn hade svårt att acceptera barnets sjukdom beroende på att barnet ser friskt ut. Föräldrarna upplevde en enorm stress och andra mentala hälsoproblem. Att som förälder känna sig förstådd och trygg med den vård som ges till deras barn, kan minimera risken för krisreaktioner längre fram. Diskussion: Det är av betydelse att ge föräldrar till hjärtsjuka barn individuellt anpassad information, vilket gör att de känner trygghet i vården. Dessutom underlättar denna information det kliniska arbetet.

Congenital hypothyroidism (CH) is a pediatric endocrine disorder caused by an insufficiency of thyroid hormone. Despite treatment following newborn screening, CH is associated with persisting memory weaknesses. Given animal research has shown thyroid hormone plays a crucial role in the development of the hippocampus, a brain structure required for normal declarative memory, it is possible that altered hippocampally-dependent processes underlie the memory weakness associated with CH. Previous studies of individuals with CH have found reduced memory abilities and left hippocampal volumes but no study has thoroughly assessed memory abilities or how the hippocampus functions to support memory. Thus, the present study compared individuals with CH and typically developing adolescents using clinical memory tests and two associative memory tasks shown in adults to activate the hippocampus during functional magnetic resonance imaging (fMRI). Results indicated groups did not differ in memory accuracy on clinical measures or either fMRI task. However, fMRI revealed hippocampal activation differed between the groups when performing the associative memory tasks. The first task utilized a visuospatial paired associates novelty detection paradigm to show the CH group increased activation relative to controls in left hippocampus and recruited right hippocampus when controls did not. Since previous research suggested the left hippocampus and verbal memory were more vulnerable to the effects of CH, the second task utilized a verbal paired associates paradigm to demonstrate that when making old and new judgments about associations versus items, the CH group increased activation relative to controls in left hippocampus. Further investigation revealed that when recognizing old associations versus items, the CH group had increased bilateral posterior hippocampal activation whereas controls showed increased activation in right anterior hippocampus, a distinction noted in previous research with this paradigm which suggests individuals with CH may retrieve associations in a less flexible manner than controls. In addition, worse memory performance and increased hippocampal activation, particularly on the left, was predicted by severity of hypothyroidism experienced early in life. In conclusion, these studies demonstrate that early thyroid hormone insufficiency associated with CH alters the functioning of the hippocampus and engenders use of compensatory mechanisms to support associative memory functions.

Congenital hypothyroidism (CH) is a pediatric endocrine disorder caused by an insufficiency of thyroid hormone. Despite treatment following newborn screening, CH is associated with persisting memory weaknesses. Given animal research has shown thyroid hormone plays a crucial role in the development of the hippocampus, a brain structure required for normal declarative memory, it is possible that altered hippocampally-dependent processes underlie the memory weakness associated with CH. Previous studies of individuals with CH have found reduced memory abilities and left hippocampal volumes but no study has thoroughly assessed memory abilities or how the hippocampus functions to support memory. Thus, the present study compared individuals with CH and typically developing adolescents using clinical memory tests and two associative memory tasks shown in adults to activate the hippocampus during functional magnetic resonance imaging (fMRI). Results indicated groups did not differ in memory accuracy on clinical measures or either fMRI task. However, fMRI revealed hippocampal activation differed between the groups when performing the associative memory tasks. The first task utilized a visuospatial paired associates novelty detection paradigm to show the CH group increased activation relative to controls in left hippocampus and recruited right hippocampus when controls did not. Since previous research suggested the left hippocampus and verbal memory were more vulnerable to the effects of CH, the second task utilized a verbal paired associates paradigm to demonstrate that when making old and new judgments about associations versus items, the CH group increased activation relative to controls in left hippocampus. Further investigation revealed that when recognizing old associations versus items, the CH group had increased bilateral posterior hippocampal activation whereas controls showed increased activation in right anterior hippocampus, a distinction noted in previous research with this paradigm which suggests individuals with CH may retrieve associations in a less flexible manner than controls. In addition, worse memory performance and increased hippocampal activation, particularly on the left, was predicted by severity of hypothyroidism experienced early in life. In conclusion, these studies demonstrate that early thyroid hormone insufficiency associated with CH alters the functioning of the hippocampus and engenders use of compensatory mechanisms to support associative memory functions.

Children with Down Syndrome (DS) experience cognitive delays with language being one of the most impaired domains. Exploring the effects of congenital heart defects (CHD), hospitalization, hearing impairment, and parental concern can provide a more precise view of factors affecting language development. Participants were 49 children with DS, 22 to 54 months of age. Expressive and receptive vocabulary size was obtained using a word count with the MacArthur Communication Development Inventory (MCDI). Medical information was obtained from the child’s medical file. Results showed expressive vocabulary was marginally significantly different between children with DS and no CHD, a CHD that did not require surgery, and a CHD that did require surgery, such that children with a CHD requiring surgery had the smallest vocabulary. Children had significantly more health problems when they had a CHD that required surgery. Expressive and receptive vocabularies were significantly smaller for children with hearing impairment.

To identify genes related to the heart developments, a total of 92 CHD families from Kaohsiung Veteran General Hospital, including 290 individuals with 95 affected, were genotyped in this study. Among these CHDs families, 54 were diagnosed as VSDs, accounted for 56.8% of all CHDs. Ten highly polymorphic markers, D22S420, D22S427, D22S1638, D22S941, D22S1648, D22S944, D22S1623, D22S264, D22S303 and D22S315, from centromere to the HSA22q telomere, covering HSA22q11 were genotyped by using a semi-quantitative fluorescent PCR. LOH at loci on 22q11 have been identified in 31 VSDs affected individuals. Candidate genes in HSA22q11 region was identified by bioinformatic methods firstly based on Ensembl (EMBL-EBI and the Sanger Institute), Genome browser (UCSU) and Map viewer (NCBI), and then FatiGO Data mining with Gene Ontology and Swiss-Prot annotations. In order to narrow down more specific cardiac development-related candidate genes within HSA22q11, TUBA8, DGCR2, DGCR14, CLTCL1, HIRA, TBX1 and GNB1L, from the centromere to telomere, were further subjected to dosage analyses by quantitative PCR. Results indicated the most frequent LOH region was localized on D22S1648. There are 48.3% and 38.7% of 31 VSDs patients with one copy deletion in TUBA8 and HIRA, respectively. Two VSDs patients were deleted in all seven candidate genes. Furthermore, there are one CAVC and one VSD patient were deleted in five consecutive genes, TUBA8, DGCR2, DGCR14, CLTCL1 and HIRA.