At foot of title page: Joint Coconut Research Scheme, Yandina, British Solomon Islands Includes bibliographical references. (p. 111-119)

<p>S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated.</p><p>Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.</p><p>Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate <i>in vitro</i> evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease.</p><p>Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.</p>

<p>Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. </p><p>A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest.</p><p>Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. </p><p>A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies.</p><p>Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients.</p><p>Successful <i>in vitro </i>transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.</p>

<p>Wilson’s disease is a rare inborn error of metabolism caused by a defect in ATP7B, a protein necessary for proper copper excretion into bile. It is characterised by copper accumulation with hepatic and central nervous system dysfunction.</p><p>We investigated 24 Swedish families with Wilson’s disease by sequencing the entire coding sequence using a new technique called manifold sequencing. Disease causing mutations were found in 44 out of 48 alleles.</p><p>From data obtained in the first study, the two most common mutations (C3207A and C2930T) were sought in 2640 anonymous DNA samples from a Swedish population, using a pooling strategy and solid-phase minisequencing. Four C3207A and one C2930T were found. From the number of C3207A, a prevalence of Wilson’s disease in Sweden of about 1 in 110,000 could be estimated.</p><p>Four groups with three patients each had four different genotypes concerning mutations in ATP7B. The patients’ psychopathological symptoms were investigated, using the Karolinska Scales of Personality rating (KSP) and Comprehensive Psychopathological Rating Scale (CPRS). A trend towards lower CPRS scores was seen in the groups with mutations known to render ATP7B completely without activity.</p><p>Using ⁶¹Cu liver PET in patients homozygous for mutations in ATP7B, heterozygotes, normal individuals and two patients with alcoholic liver cirrhosis, significantly slower uptake was seen in the homozygotes as compared to the heterozygotes and normal individuals. The patients with cirrhosis had values in between. This implies that ⁶¹Cu liver PET might be used as an additional rapid and little invasive diagnostic tool in Wilson’s disease.</p><p>In a retrospectively studied cohort consisting of 363 patients followed in Sweden and the UK, nine cases of aggressive intra-abdominal malignancies were seen, which is more than expected. Caution should be taken in the follow-up of Wilson’s disease patients.</p>

<p>Many Single Nucleotide Polymorphisms (SNPs) found in coding or regulatory regions within the human genome lead to phenotypic differences that make prediction of physical appearance, based on genetic analysis, potentially useful in forensic investigations. Complex traits such as pigmentation can be predicted from the genome sequence, provided that genes with strong effects on the trait exist and are known. Phenotypic traits may also be associated with variations in gene expression due to the presence of SNPs in promoter regions. In this project, the identification of genes associated with these physical traits of potential forensic relevance have been collated from the literature using a text mining platform and hand curation. The SNPs associated with these genes have been acquired from public SNP repositories such as the International HapMap project, dbSNP and Ensembl. Characterization of different population groups based on the SNPs has been performed and the results and data stored in a MySQL database. This database contains SNP genotyping data with respect to physical phenotypic differences of forensic interest. The potential forensicrelevance of the SNP information contained in this database has been verified through in silico SNP analysis aimed at establishing possible relationships between SNP occurrence and phenotype. The software used for this analysis is MATCH&trade / .</p>

S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated. Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated. Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate in vitro evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease. Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.

Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest. Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies. Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients. Successful in vitro transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.

Wilson’s disease is a rare inborn error of metabolism caused by a defect in ATP7B, a protein necessary for proper copper excretion into bile. It is characterised by copper accumulation with hepatic and central nervous system dysfunction. We investigated 24 Swedish families with Wilson’s disease by sequencing the entire coding sequence using a new technique called manifold sequencing. Disease causing mutations were found in 44 out of 48 alleles. From data obtained in the first study, the two most common mutations (C3207A and C2930T) were sought in 2640 anonymous DNA samples from a Swedish population, using a pooling strategy and solid-phase minisequencing. Four C3207A and one C2930T were found. From the number of C3207A, a prevalence of Wilson’s disease in Sweden of about 1 in 110,000 could be estimated. Four groups with three patients each had four different genotypes concerning mutations in ATP7B. The patients’ psychopathological symptoms were investigated, using the Karolinska Scales of Personality rating (KSP) and Comprehensive Psychopathological Rating Scale (CPRS). A trend towards lower CPRS scores was seen in the groups with mutations known to render ATP7B completely without activity. Using 61Cu liver PET in patients homozygous for mutations in ATP7B, heterozygotes, normal individuals and two patients with alcoholic liver cirrhosis, significantly slower uptake was seen in the homozygotes as compared to the heterozygotes and normal individuals. The patients with cirrhosis had values in between. This implies that 61Cu liver PET might be used as an additional rapid and little invasive diagnostic tool in Wilson’s disease. In a retrospectively studied cohort consisting of 363 patients followed in Sweden and the UK, nine cases of aggressive intra-abdominal malignancies were seen, which is more than expected. Caution should be taken in the follow-up of Wilson’s disease patients.

Single Nucleotide Polymorphisms (SNPs) are the most common DNA sequence variations where only a single nucleotide (A, T, C, G) in the human genome differs between individuals. Besides being the main genetic reason behind individual phenotypic differences, SNP variations have the potential to exploit the molecular basis of many complex diseases. Association of SNPs subset with diseases and analysis of the genotyping data with clinical findings will provide practical and affordable methodologies for the prediction of diseases in clinical settings. So, there is a need to determine the SNP subsets and patients&rsquo / clinical data which is informative for the prediction or the diagnosis of the particular diseases. So far, there is no established approach for selecting the representative SNP subset and patients&rsquo / clinical data, and data mining methodology that is based on finding hidden and key patterns over huge databases. This approach have the highest potential for extracting the knowledge from genomic datasets and to select the number of SNPs and most effective clinical features for diseases that are informative and relevant for clinical diagnosis. In this study we have applied one of the widely used data mining classification methodology: &ldquo / decision tree&rdquo / for associating the SNP Biomarkers and clinical data with the Alzheimer&rsquo / s disease (AD), which is the most common form of &ldquo / dementia&rdquo / . Different tree construction parameters have been compared for the optimization, and the most efficient and accurate tree for predicting the AD is presented.

Rice (Oryza sativa L.) supports half of the human population. However, predominant rice consumption leads to malnutrition due to mineral deficiencies. The research goal was to support identification of genes responsible for the uptake/accumulation of potassium (K), iron (Fe), zinc (Zn) and molybdenum (Mo), thus promoting the breeding for rice with high grain concentrations of these elements. Prior studies identified rice genotypes with high grain-K, -Fe, -Zn or -Mo concentrations that were hypothesized to be due to differences in root traits. The research objective was to identify root traits associated with these elements. These traits could be bases for identifying genes. The first study determined if these genotypes showed similar accumulation patterns in leaves as in grains, which would hint at influences of the roots and enable identifying distinct root traits and possible genes in vegetative growth stages. The second study determined if root traits of high grain-Mo genotypes displayed an acid-tolerance mechanism as these genotypes originated from Malaysia where acidic soils strongly adsorb Mo making it unavailable for plants. The third study identified root trait differences of high grain-K, -Fe, -Zn and -Mo genotypes in hydroponics media, while the fourth determined root trait differences in these genotypes in sand-culture media including a 1-Naphthalene Acetic Acid (NAA) seed treatment for perturbation. The first study identified several high grain-Mo genotypes with similar Mo accumulation patterns in V4 to V6 stage-leaves as in grains, suggestive of a root influence. The second study established that gross morphological and physiological root traits of a high grain-Mo genotype were not part of an acid-tolerance mechanism. Neither the third nor fourth study identified root traits related to shoot K, Fe, Zn or Mo concentration, however positive associations of seedling vigor traits with several beneficial elements, including K, and negative associations with numerous toxic elements were established. Lack of correlation with root traits suggests other mechanisms (e.g. active uptake transporters) instead control the observed grain accumulation differences. Based on the fourth study, either direct effects of NAA on element uptake/transfer or indirect effects on soil pH and redox potential altered tissue Fe and Zn levels.