Deriving structural information from non-coding ribonucleic acids by mass spectrometry

The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.

Associations of interleukin-1 (IL-1) gene variation and IL-1 receptor antagonist phenotypes with immunological responses, metabolic dysregulation and type 2 diabetes

The upstream proinflammatory interleukin-1 (IL-1) cytokines, together with a naturally occurring IL-1 receptor antagonist (IL-1Ra), play a significant role in several diseases and physiologic conditions. The IL-1 proteins affect glucose homeostasis at multiple levels contributing to vascular injuries and metabolic dysregulations that precede diabetes. An association between IL-1 gene variations and IL-1Ra levels has been suggested, and genetic studies have reported associations with metabolic dysregulation and altered inflammatory responses. The principal aims of this study were to: 1) examine the associations of IL-1 gene variation and IL-1Ra expression in the development and persistence of thyroid antibodies in subacute thyroiditis; 2) investigate the associations of common variants in the IL-1 gene family with plasma glucose and insulin concentrations, glucose homeostasis measures and prevalent diabetes in a representative population sample; 3) investigate genetic and non-genetic determinants of IL-1Ra phenotypes in a cross-sectional setting in three independent study populations; 4) investigate in a prospective setting (a) whether variants of the IL-1 gene family are predictors for clinically incident diabetes in two population-based observational cohort studies; and (b) whether the IL-1Ra levels predict the progression of metabolic syndrome to overt diabetes during the median follow-up of 10.8 and 7.1 years. Results from on patients with subacte thyroiditis showed that the systemic IL-1Ra levels are elevated during a specific proinflammatory response and they correlated with C-reactive protein (CRP) levels. Genetic variation in the IL-1 family seemed to have an association with the appearance of thyroid peroxidase antibodies and persisting local autoimmune responses during the follow-up. Analysis of patients suffering from diabetes and metabolic traits suggested that genetic IL-1 variation and IL-1Ra play a role in glucose homeostasis and in the development of type 2 diabetes. The coding IL-1 beta SNP rs1143634 was associated with traits related to insulin resistance in cross-sectional analyses. Two haplotype variants of the IL-1 beta gene were associated with prevalent diabetes or incident diabetes in a prospective setting and both of these haplotypes were tagged by rs1143634. Three variants of the IL-1Ra gene and one of the IL-1 beta gene were consistently identified as significant, independent determinants of the IL-1Ra phenotype in two or three populations. The proportion of the phenotypic variation explained by the genetic factors was modest however, while obesity and other metabolic traits explained a larger part. Body mass index was the strongest predictor of systemic IL-1Ra concentration overall. Furthermore, the age-adjusted IL-1Ra concentrations were elevated in individuals with metabolic syndrome or diabetes when compared to those free of metabolic dysregulation. In prospective analyses the systemic IL-1Ra levels were found as independent predictors for the development of diabetes in people with metabolic syndrome even after adjustment for multiple other factors, including plasma glucose and CRP levels. The predictive power of IL-1Ra was better than that of CRP. The prospective results also provided some evidence for a role of common IL-1 alpha promoter SNP rs1800587 in the development of type 2 diabetes among men and suggested that the role may be gender specific. Likewise, common variations in the IL-1 beta coding region may have a gender specific association with diabetes development. Further research on the potential benefits of IL-1Ra measurements in identifying individuals at high risk for diabetes, who then could be targeted for specific treatment interventions, is warranted. It has been reported in the recent literature that IL-1Ra secreted from adipose tissue has beneficial effects on glucose homeostasis. Furthermore, treatment with recombinant human IL-1Ra has been shown to have a substantial therapeutic potential. The genetic results from the prospective analyses performed in this study remain inconclusive, but together with the cross-sectional analyses they suggest gender-specific effects of the IL-1 variants on the risk of diabetes. Larger studies with more extensive genotyping and resequencing may help to pinpoint the exact variants responsible and to further elucidate the biological mechanisms for the observed associations. This would improve our understanding of the pathways linking inflammation and obesity with glucose and insulin metabolism.

Genetic alterations in microsatellite-unstable colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.

Molecular genetics of Meckel syndrome : Ciliary genes are defective in MKS

Meckel syndrome (MKS, MIM 249000) is a severe developmental disorder that leads to death already in utero or shortly after birth. MKS diagnosis can be established by a careful ultrasound examination already at 11-14 weeks of gestation. The main features of MKS are occipital meningoencephalocele, cystic kidney dysplasia and fibrotic changes of the liver. In addition, polydactyly is frequently reported in the cases. The aim of the study was to characterize the molecular and functional defects in MKS. In this study we were able to identify two major MKS mutations in Finnish population, which cover over 90% of the cases. The first mutation is a 29 bp intronic deletion in the MKS1 gene (c.1483-7_35del) that is found in 70% of the families and the second is a C>T substitution in the coding region of CC2D2A (c.1762C>T), that is found in 20% of the MKS families. Both of these mutations result in abnormal splicing. The discovery of the disease genes has revealed that MKS is caused by primary cilia dysfunction. MKS1 gene has a conserved B9 domain, and it is found in the predicted ciliary proteome. CC2D2A protein is also found in the predicted ciliary proteome and it has a Ca2+ binding domain. The number of genes behind MKS has increased rapidly in the past years and to date, mutations have been identified in five genes (MKS1, TMEM67/MKS3, CEP290/MKS4, RPGRIP1L/MKS5 and CC2D2A/MKS6). Identification of the disease genes mutations has also revealed that MKS is an allelic disorder with other syndromes with overlapping phenotypes. Disorders that are caused by primary cilia dysfunction are collectively known as ciliopathies. Sequence analysis of all the known MKS genes in Finnish and non-Finnish families available to us, where the mutation was still unknown, revealed mutations in 14 out of the 30 families included in the study. When we collected all the reported mutations in MKS genes in different syndromes we could see that there was clearly a genotype-syndrome correlation between the mutations and the syndromes, since the same pair of mutations has never been reported in different syndromes. The basic molecular events behind MKS will not only give us information of this syndrome, but also significant novel information on early fetal development in general.

ATM, RAD50 and p53 in breast cancer

Breast cancer is the most commonly occurring cancer among women, and its incidence is increasing worldwide. Positive family history is a well established risk factor for breast cancer, and it is suggested that the proportion of breast cancer that can be attributed to genetic factors may be as high as 30%. However, all the currently known breast cancer susceptibility genes are estimated to account for 20-30% of familial breast cancer, and only 5% of the total breast cancer incidence. It is thus likely that there are still other breast cancer susceptibility genes to be found. Cellular responses to DNA damage are crucial for maintaining genomic integrity and preventing the development of cancer. The genes operating in DNA damage response signaling network are thus good candidates for breast cancer susceptibility genes. The aim of this study was to evaluate the role of three DNA damage response associated genes, ATM, RAD50, and p53, in breast cancer. ATM, a gene causative for ataxia telangiectasia (A-T), has long been a strong candidate for a breast cancer susceptibility gene because of its function as a key DNA damage signal transducer. We analyzed the prevalence of known Finnish A-T related ATM mutations in large series of familial and unselected breast cancer cases from different geographical regions in Finland. Of the seven A-T related mutations, two were observed in the studied familial breast cancer patients. Additionally, a third mutation previously associated with breast cancer susceptibility was also detected. These founder mutations may be responsible for excess familial breast cancer regionally in Northern and Central Finland, but in Southern Finland our results suggest only a minor effect, if any, of any ATM genetic variants on familial breast cancer. We also screened the entire coding region of the ATM gene in 47 familial breast cancer patients from Southern Finland, and evaluated the identified variants in additional cases and controls. All the identified variants were too rare to significantly contribute to breast cancer susceptibility. However, the role of ATM in cancer development and progression was supported by the results of the immunohistochemical studies of ATM expression, as reduced ATM expression in breast carcinomas was found to correlate with tumor differentiation and hormone receptor status. Aberrant ATM expression was also a feature shared by the BRCA1/2 and the difficult-to-treat ER/PR/ERBB2-triple-negative breast carcinomas. From the clinical point of view, identification of phenotypic and genetic similarities between the BRCA1/2 and the triple-negative breast tumors could have an implication in designing novel targeted therapies to which both of these classes of breast cancer might be exceptionally sensitive. Mutations of another plausible breast cancer susceptibility gene, RAD50, were found to be very rare, and RAD50 can only be making a minor contribution to familial breast cancer predisposition in UK and Southern Finland. The Finnish founder mutation RAD50 687delT seems to be a null allele and may carry a small increased risk of breast cancer. RAD50 is not acting as a classical tumor suppressor gene, but it is possible that RAD50 haploinsufficiency is contributing to cancer. In addition to relatively rare breast cancer susceptibility alleles, common polymorphisms may also be associated with increased breast cancer risk. Furthermore, these polymorphisms may have an impact on the progression and outcome of the disease. Our results suggest no effect of the common p53 R72P polymorphism on familial breast cancer risk or breast cancer risk in the population, but R72P seems to be associated with histopathologic features of the tumors and survival of the patients; 72P homozygous genotype was an independent prognostic factor among the unselected breast cancer patients, with a two-fold increased risk of death. These results present important novel findings also with clinical significance, as codon 72 genotype could be a useful additional prognostic marker in breast cancer, especially among the subgroup of patients with wild-type p53 in their tumors.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.

Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.

Molecular Background of Common Dyslipidemias

The leading cause of death in the Western world continues to be coronary heart disease (CHD). At the root of the disease process is dyslipidemia an aberration in the relevant amounts of circulating blood lipids. Cholesterol builds up in the arterial wall and following rupture of these plaques, myocardial infarction or stroke can occur. Heart disease runs in families and a number of hereditary forms are known. The leading cause of adult dyslipidemia presently however is overweight and obesity. This thesis work presents an investigation of the molecular genetics of common, hereditary dyslipidemia and the tightly related condition of obesity. Familial combined hyperlipidemia (FCHL) is the most common hereditary dyslipidemia in man with an estimated population prevalence of 1-6%. This complex disease is characterized by elevated levels of serum total cholesterol, triglycerides or both and is observed in about 20% of individuals with premature CHD. Our group identified the disease to be associated with genetic variation in the USF1 transcription factor gene. USF1 has a key role in regulating other genes that control lipid and glucose metabolism as well as the inflammatory response all central processes in the progression of atherosclerosis and CHD. The first two works of this thesis aimed at understanding how these USF1 variants result in increased disease risk. Among the many, non-coding single-nucleotide polymorphisms (SNPs) that associated with the disease, one was found to have a functional effect. The risk-enhancing allele of this SNP seems to eradicate the ability of the important hormone insulin to induce the expression of USF1 in peripheral tissues. The resultant changes in the expression of numerous USF1 target genes over time probably enhance and accelerate the atherogenic processes. Dyslipidemias often represent an outcome of obesity and in the final work of this thesis we wanted to address the metabolic pathways related to acquired obesity. It is recognized that active processes in adipose tissue play an important role in the development of dyslipidemia, insulin resistance and other pathological conditions associated with obesity. To minimize the confounding effects of genetic differences present in most human studies, we investigated a rare collection of identical twins that differed significantly in the amount of body fat. In the obese, but otherwise healthy young adults, several notable changes were observed. In addition to chronic inflammation, the adipose tissue of the obese co-twins was characterized by a marked (47%) decrease in amount of mitochondrial DNA (mtDNA) a change associated with mitochondrial dysfunction. The catabolism of branched chain amino acids (BCAAs) was identified as the most down-regulated process in the obese co-twins. A concordant increase in the serum level of these insulin secretagogues was identified. This hyperaminoacidemia may provide the feed-back signal from insulin resistant adipose tissue to the pancreas to ensure an appropriately augmented secretory response. The down regulation of BCAA catabolism correlated closely with liver fat accumulation and insulin. The single most up-regulated gene (5.9 fold) in the obese co-twins was osteopontin (SPP1) a cytokine involved in macrophage recruitment to adipose tissue. SPP1 is here implicated as an important player in the development of insulin resistance. These studies of exceptional study samples provide better understanding of the underlying pathology in common dyslipidemias and other obesity associated diseases important for future improvement of intervention strategies and treatments to combat atherosclerosis and coronary heart disease.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

Molecular epidemiology of human rhinoviruses

The first part of this work investigates the molecular epidemiology of a human enterovirus (HEV), echovirus 30 (E-30). This project is part of a series of studies performed in our research team analyzing the molecular epidemiology of HEV-B viruses. A total of 129 virus strains had been isolated in different parts of Europe. The sequence analysis was performed in three different genomic regions: 420 nucleotides (nt) in the VP4/VP2 capsid protein coding region, the entire VP1 capsid protein coding gene of 876 nt, and 150 nt in the VP1/2A junction region. The analysis revealed a succession of dominant sublineages within a major genotype. The temporally earlier genotypes had been replaced by a genetically homogenous lineage that has been circulating in Europe since the late 1970s. The same genotype was found by other research groups in North America and Australia. Globally, other cocirculating genetic lineages also exist. The prevalence of a dominant genotype makes E-30 different from other previously studied HEVs, such as polioviruses and coxsackieviruses B4 and B5, for which several coexisting genetic lineages have been reported. The second part of this work deals with molecular epidemiology of human rhinoviruses (HRVs). A total of 61 field isolates were studied in the 420-nt stretch in the capsid coding region of VP4/VP2. The isolates were collected from children under two years of age in Tampere, Finland. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades. Seasonal clustering was found. Also, several distinct serotype-like clusters were found to co-circulate during the same epidemic season. Reappearance of a cluster after disappearing for a season was observed. The molecular epidemiology of the analyzed strains turned out to be complex, and we decided to continue our studies of HRV. Only five previously published complete genome sequences of HRV prototype strains were available for analysis. Therefore, all designated HRV prototype strains (n=102) were sequenced in the VP4/VP2 region, and the possibility of genetic typing of HRV was evaluated. Seventy-six of the 102 prototype strains clustered in HRV genetic group A (HRV-A) and 25 in group B (HRV-B). Serotype 87 clustered separately from other HRVs with HEV species D. The field strains of HRV represented as many as 19 different genotypes, as judged with an approximate demarcation of a 20% nt difference in the VP4/VP2 region. The interserotypic differences of HRV were generally similar to those reported between different HEV serotypes (i.e. about 20%), but smaller differences, less than 10%, were also observed. Because some HRV serotypes are genetically so closely related, we suggest that the genetic typing be performed using the criterion "the closest prototype strain". This study is the first systematic genetic characterization of all known HRV prototype strains, providing a further taxonomic proposal for classification of HRV. We proposed to divide the genus Human rhinoviruses into HRV-A and HRV-B. The final part of the work comprises a phylogenetic analysis of a subset (48) of HRV prototype strains and field isolates (12) in the nonstructural part of the genome coding for the RNA-dependent RNA polymerase (3D). The proposed division of the HRV strains in the species HRV-A and HRV-B was also supported by 3D region. HRV-B clustered closer to HEV species B, C, and also to polioviruses than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D coding region than in the VP4/VP2 coding region, in contrast to HEV. Moreover, the diversity of HRV in 3D exceeded that of HEV. One group of HRV-A, designated HRV-A', formed a separate cluster outside other HRV-A in the 3D region. It formed a cluster also in the capsid region, but located within HRV-A. This may reflect a different evolutionary history of distinct genomic regions among HRV-A. Furthermore, the tree topology within HRV-A in the 3D region differed from that in the VP4/VP2, suggesting possible recombination events in the evolution of the strains. No conflicting phylogenies were observed in any of the 12 field isolates. Possible recombination was further studied using the Similarity and Bootscanning analyses of the complete genome sequences of HRV available in public databases. Evidence for recombination among HRV-A was found, as HRV2 and HRV39 showed higher similarity in the nonstructural part of the genome. Whether HRV2 and HRV39 strains - and perhaps also some other HRV-A strains not yet completely sequenced - are recombinants remains to be determined.

Adenoviral gene therapy for non-small cell lung cancer

Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities.

Non-enzymatic Degradation of (1→3)(1→4)-β-D-glucan in Aqueous Processing of Oats

Cereal water-soluble β-glucan [(1→3)(1→4)-β-D-glucan] has well-evidenced health benefits and it contributes to the texture properties of foods. These functions are characteristically dependent on the excellent viscosity forming ability of this cell wall polysaccharide. The viscosity is affected by the molar mass, solubility and conformation of β-glucan molecule, which are further known to be altered during food processing. This study focused on demonstrating the degradation of β-glucan in water solutions following the addition of ascorbic acid, during heat treatments or high pressure homogenisation. Furthermore, the motivation of this study was in the non-enzymatic degradation mechanisms, particularly in oxidative cleavage via hydroxyl radicals. The addition of ascorbic acid at food-related concentrations (2-50 mM), autoclaving (120°C) treatments, and high pressure homogenisation (300-1000 bar) considerably cleaved the β-glucan chains, determined as a steep decrease in the viscosity of β-glucan solutions and decrease in the molar mass of β-glucan. The cleavage was more intense in a solution of native β-glucan with co-extracted compounds than in a solution of highly purified β-glucan. Despite the clear and immediate process-related degradation, β-glucan was less sensitive to these treatments compared to other water-soluble polysaccharides previously reported in the literature. In particular, the highly purified β-glucan was relatively resistant to the autoclaving treatments without the addition of ferrous ions. The formation of highly oxidative free radicals was detected at the elevated temperatures, and the formation was considerably accelerated by added ferrous ions. Also ascorbic acid pronounced the formation of these oxidative radicals, and oxygen was simultaneously consumed by ascorbic acid addition and by heating the β-glucan solutions. These results demonstrated the occurrence of oxidative reactions, most likely the metal catalysed Fenton-like reactions, in the β-glucan solutions during these processes. Furthermore, oxidized functional groups (carbonyls) were formed along the β-glucan chain by the treatments, including high pressure homogenisation, evidencing the oxidation of β-glucan by these treatments. The degradative forces acting on the particles in the high pressure homogenisation are generally considered to be the mechanical shear, but as shown here, carbohydrates are also easily degraded during the process, and oxidation may have a role in the modification of polysaccharides by this technique. In the present study, oat β-glucan was demonstrated to be susceptible to degradation during aqueous processing by non-enzymatic degradation mechanisms. Oxidation was for the first time shown to be a highly relevant degradation mechanism of β-glucan in food processing.

Carotid Surgery : A posse ad esse non valet consequentia

According to a large body of evidence, carotid endarterectomy (CEA) can prevent strokes, provided that appropriate inclusion criteria and high-quality perioperative treatment methods are utilised with low complication rates. From the patient s perspective, it is of paramount importance that the operation is as safe and effective as possible. From the community s point of view, it is important that CEA provision prevents as many strokes as possible. In order to define the stroke preventing potential of CEA in different communities, a comparison between eight European countries and Australia was performed including 53 077 carotid interventions. A more detailed evaluation was performed in Finland, the United Kingdom and Egypt. It could be estimated that many potentially preventable strokes occur due to insufficient diagnostics and CEA provision. The number of CEAs should be at least doubled in the Helsinki region. The theoretical power of CEA provision in stroke prevention varied significantly between the countries. Delay from symptom to surgery has been identified as one of the most important factors influencing the effectiveness of CEA. In 2008 only 11% of CEAs in Helsinki university central hospital (HUCH) were performed within the recommended14 days. Registered data of 673 CEAs in HUCH during 2000-2005 was analyzed. There was no systematic error that would have changed the outcome analysis. However it is important that registers are audited regularly and cross matching of different registries is possible. A previously unpublished method of combining medial mandibulotomy, neck incision and carotid artery interposition was carried out as a collaboration of maxillofacial, ear, nose and throat and vascular surgeons. Five patients were operated on with a technique that was feasible and possible to perform with little morbidity, but due to the significant risks involved, this technique should be reserved for carefully selected cases. In stroke prevention, organisational decisions seem far more important than details in interventional procedures when CEA is performed with low complication rates, as was the case in the present study. A TIA clinic approach with close co-operation between the on-call vascular surgeons, neurologists and radiologists should be available at all centres treating these patients. Patients should have a direct and fast admission to the hospital performing CEA.