A genome sequence can be conceptualized as a 'book' written with four nucleotide 'letters' in oligonucleotide (oligo) 'words'. These words can be used in genomics, bioinformatics and the development of molecular markers. The whole-genome sequence for rice (Oryza sativa L.) is almost finished and has been assembled into pseudomolecules. For barley ( Hordeum vulgare L.) expressed sequence tags (ESTs) have been assembled into 21,981 tentative consensus sequences (TCs). The availability of such sequence information provides opportunities to investigate oligo usage within and between genomes. For the first of three studies reported in this thesis, a C++ program was written to automatically design oligos that are conserved between two sets of sequence information. In silico mapping between rice coding sequences (CDS) and barley TCs indicated that oligos between 18 and 24 bp provide good specificity and sensitivity (83% and 86%, respectively, for 20mers). Conserved oligos used as PCR primers had a high (91%) success rate on barley lines. Sequencing of PCR products revealed conservation in exon sequence, size and order between barley and rice. Introns were not conserved in sequence but were relatively stable in size. Map locations of eight new markers in barley revealed both genome colinearity and rearrangements between barley and rice. The second study reported in this thesis examined word frequency within the rice genome. A non-random landscape composed of high-frequency and low-frequency zones was observed. Interestingly, high-frequency words seemed to be rice specific while single-copy words were gene specific and conserved across species. As in the first study, oligos of 12 bp or less were not specific, and 18 bp seemed to be a critical length for the specificity of oligos. The third study reported in this thesis involved the development of molecular markers for known genes using public sequence information. Six new polymorphic markers were d

Stachybotrys elegans (Pidopl.) W. Gams is a destructive mycoparasite of the soilborne plant pathogen Rhizoctonia solani. It colonizes effectively all types of cells of R. solani, and is considered as an effective biological control agent (BCA). Monitoring the presence of this mycoparasite in the field trials requires the development of a reliable and sensitive diagnostic assay that is able to detect and differentiate the BCA from their target host. To achieve this, designed SCAR (sequenced characterized amplified regions) primers designated as SE-13F and SE-13R were generated from informative RAPD markers. They were tested in conventional PCR assays alone or in conjunction with the recently developed SCAR primers (SBU-177/336) designed for Rhizoctonia solani (Kuhn) on several types of DNA. These included DNA extracted from pure cultures, co-cultures of the BCA and the pathogen, plant tissue and several types of soils inoculated with both the BCA and the pathogen. Irrespective of the type of the biological samples from which the DNA was extracted, the primers SE-13F/SE-13R successfully amplified only S. elegans. No cross-reaction was observed when the primers were used to amplify DNA of other fungi, bacteria and plant tissues. Likewise, the primer pair SBU-177/336 detected only its target organism, i.e., R. solani. The detection limit using these primers on amplified DNA was as little as 1 pg DNA extracted from pure cultures of S. elegans, 100 pg DNA extracted from greenhouse soil and 33 pg DNA extracted from natural soil. This work is the first report on the development of SCAR markers for the BCA, S. elegans. These molecular markers offer not only an alternative diagnostic assay to conventional detection methods, but also the possibility of being used in ecological studies.

Two studies were conducted on gene mapping analysis. For the first study, genetic simulation experiments were conducted to address the effects of marker density, method of mapping analysis, and gaps in a marker map on the efficiency of QTL detection and the accuracy of QTL parameter estimation. The simulated genome consisted of seven chromosomes with seven or eight segregating QTL affecting the simulated quantitative trait. A set of six randomly segregating QTL outside the test region was consistently used to represent 40% of phenotypic variation. An individual QTL or a linkage block of two QTL on a target chromosome contributed 10% of phenotypic variation. The marker map was either dense (with markers every 4 cM) or sparse (with markers every 20 cM). The gap in the marker map was either 32 cM or 56 cM. Interval mapping and composite interval mapping were used to map QTL on the target chromosome. A dense map provided more power of QTL detection, better accuracy of QTL parameter estimation, and higher false-positive error rates for the target chromosome than a sparse map. Composite interval mapping provided more power of QTL detection, better accuracy of QTL parameter estimation, and lower false-positive error rates than interval mapping. Presence of a large gap in a marker map affected QTL detection and QTL parameter estimation for a QTL inside or near the gap. The use of a dense map with composite interval mapping was the most efficient combination tested in this study. For the second study, a mixed factorial regression model for interval mapping was developed for conducting QTL-by-environment interaction analysis and for providing inferences about QTL that are applicable beyond the environments used in the experiments. Genetic simulation was used to test the model for the power of detecting QTL-by-environment interaction and identifying the types of such interaction as crossover or non-crossover, and for the accuracy of estimating QTL parameters. The model prov

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Background: Patients with heart failure (HF) are characterized by exercise intolerance, breathlessness, fatigue and excessive neurohormonal activation associated with premature mortality. Recently, inflammatory activation has been described as an important factor in the progression of HF. Increased levels of certain pro-inflammatory cytokines (e.g., TNF-ɑ, IL-6) have been related to increased severity of left ventricular dysfunction, the activation of the sympathetic and renin-angiotensin systems and the catabolism of skeletal muscle. Although exercise training is important in the management of HF, acute bouts of exercise may lead to increases in proinflammatory cytokines. It is believed that the skeletal muscle abnormalities associated with HF may increase the risk of damage to skeletal muscle, (i.e., exercise-induced muscle injury (EIMI) with associated inflammatory activation) especially following unaccustomed exercise training. Recently, several training methods have been proposed for patients with HF that challenge the traditional “steady-state” (SS) training model, including interval training (IT). Interval training methods employ greater muscular loading than SS and therefore may increase the risk of inflammatory system activation EIMI, and/or reduced muscle function. There is no study that has examined the effects of IT on EIMI, muscle function and/or inflammatory markers. Material and Methods: Fourteen male participants with HF (mean age: 59 +/- 7.8 yrs; mean VO2 peak: 13.64 +/- 4.5 ml/kg/m-1; EF < 45%) were matched (for body mass and aerobic fitness) and randomized into SS or IT for 20 minutes. The IT involved 2 minute work:recovery phases of 90% and 40% of heart rate reserve, respectively. The SS involved continuous exercise at 65% of heart rate reserve. Biochemical markers of muscle damage and acute inflammation, concentric and eccentric isokinetic muscle torque, and subjective indicators of delayed onset muscle soreness (DOMS) and lower extremity function were evaluated at baseline, and then immediately following the training bout, and at 6, 24, and 48 hours post. Results: There were no significant differences between the IT and the SS training group for markers of skeletal muscle injury or inflammatory activation. Conclusions: The findings from the present study suggest that IT or SS do not result in excessive inflammatory system activation or skeletal muscle injury. These results have important implications for clinicians prescribing exercise regimes for HF patients who may be starting back into activity after a prolonged sedentary period. Additionally, results from this study indicate that there is a need for future research looking at the actual and perceived effect of even a single about of exercise on lower extremity function.

My thesis research has examined the fatty acid composition of serum triacylglycerol, phospholipid and cholesterol ester in 2793 participants who took part in the 1997 National Nutrition Survey - a national population-based survey of New Zealand adolescents and adults aged or [greater than or equal to]̲15 y. Differences in serum fatty acids by sex, age, ethnicity, body mass index and smoking - independent of dietary fat intake - were determined. Serum fatty acids were used as biomarkers of saturated and polyunsaturated fat intake to predict population serum total cholesterol concentrations. The association between n-3 long-chain polyunsaturated fatty acids in serum phospholipid and mental and physical wellbeing, as assessed by the short form-36, was determined. Serum fatty acids have been used as biological markers of fat intake and to predict the risk of disease. The fatty acid composition of serum triacylglycerol, phospholipid and cholesterol ester is subject to alteration by dietary fat but overall, is largely controlled by metabolic enzymes. Non-dietary variables - sex, age, body mass index or cigarette smoking - may influence the activity of these enzymes, which will subsequently alter the fatty acid composition but the extent to which these affect serum fatty acid composition in the general population is poorly understood. Our results showed that the proportion of docosahexaenoic acid in serum phospholipid and cholesterol ester was significantly greater in women by 0.15 and 0.02 mol%, respectively in comparison to men whereas, the proportion of eicosapentaenoic acid was significantly greater in men by 0.08 and 0.1 mol%, respectively, after adjusting for age, ethnicity, body mass index and smoking. A number of differences in the proportion of palmitoleic acid in serum triacylglycerol, phospholipid and cholesterol ester were detected; palmitoleic acid increased across the age categories in women (15-24, 25-44, 45-64 65+ y), was higher in women compared to men, New Zealand Europeans compared to New Zealand Maori and Pacific People, those with a body mass index or [greater than or equal to] 30 kg/m� compared to those with a body mass index < 25 kg/m� and in current smokers in comparison to non-smokers. In women, there was an inverse trend in the proportion of linoleic acid in serum phospholipid and cholesterol ester across the age categories. The proportion of linoleic acid in serum triacylglycerol, phospholipid and cholesterol ester was lower in smokers by 2.19, 1.04 and 0.75 mol%, respectively in comparison to non-smokers. None of these differences could be explained by a difference in dietary fat intake. Consequently, sex appears to affect the metabolism of n-3 long-chain polyunsaturated fatty acids independent of dietary fat intake and metabolic differences associated with age, body mass index and smoking may be at play for a number of other serum fatty acids notably, palmitoleic and linoleic acids. Evidence for a role of dietary fat as a predictor of serum cholesterol concentrations in the general population is conflicting. On one hand, results from cholesterol-lowering dietary intervention trials show unequivocally that decreasing saturated fat intake produces a meaningful reduction in serum cholesterol concentrations. On the other hand, the results of large observational studies show little association between saturated fat intake and cholesterol concentrations. The lack of association in the latter studies may result from errors in dietary assessment and therefore, using serum fatty acids as biomarkers of fat intake may overcome the limitations associated with typical dietary assessment techniques. Participants were divided into quintiles of increasing proportion of serum fatty acids. Each one SD increase in the myristic acid composition of serum cholesterol ester, triacylglycerol and phospholipid was associated with an increase in cholesterol of 0.19, 0.10 and 0.13 mmol/L, respectively after adjusting for confounding variables. The difference in cholesterol concentrations between those categorised into the highest and lowest quintiles of serum cholesterol ester myristate was 0.48 mmol/L. A one SD increase in the linoleic acid composition of serum cholesterol ester, triacylglycerol and phospholipid corresponded to a decrease in cholesterol of 0.07, 0.05 and 0.07 mmol/L, respectively. The difference in cholesterol concentrations between the 1st and 5th quintiles of serum cholesterol linoleate was 0.18 mmol/L. Intake of saturated and polyunsaturated fats, as measured using serum fatty acids, are important determinants of cholesterol concentrations in New Zealanders. It has been hypothesised that a lower intake of n-3 long-chain polyunsaturated fatty acids, largely of marine origin, is implicated in the aetiology of depressive disorder. Results from the majority of observational studies have shown that depressed participants have a lower proportion of eicosapentaenoic or docosahexaenoic acid in phospholipids compared to controls but evidence for an improvement in depressive symptoms after supplementation with n-3 long-chain polyunsaturated fatty acids is conflicting. There is little known about the role that n-3 long-chain polyunsaturated fatty acids may have as predictors of mental wellbeing in the general population. Participants were categorised into quintiles of increasing n-3 long-chain polyunsaturated fatty acids in serum phospholipid. There was no significant trend in self-reported mental wellbeing - the mental component score - across the quintiles of eicosapentaenoic, docosapentaenoic and docosahexaenoic acids or the sum of these three fatty acids after adjusting for confounding variables. There was a significant trend in the mental component score across the quintiles of the ratio of eicosapentaenoic/arachidonic acid; the difference between the highest and the lowest quintile was 6.6 points. There were significant positive trends in self-reported physical health - the physical component score - across the quintiles of eicosapentaenoic and docosapentaenoic acids as well as the ratio of eicosapentaenoic/arachidonic acid ratio; the difference between the 1st and 5th quintiles were 8.6, 6.0 and 8.9 points, respectively. Overall, there appears to be little association between the n-3 long-chain polyunsaturated fatty acid composition of serum phospholipid and self-reported mental health in a population of low fish consumers; however, the proportion of n-3 long-chain polyunsaturated fatty acids may be an important predictor of physical wellbeing in New Zealanders.

Colorectal cancer (CRC) is the fourth most common malignancy in the developed Western countries and represents the second leading cause of death from cancer after lung cancer. Despite better diagnostic tools and improvement of surgical standards, the prognosis of this disease is still unsatisfactory with a mean 5-year cancer-specific survival of approximately 50%, mainly due to our inability to predict liver failure secondary to hepatic involvement and still poorly effective palliative treatments. The metastatic cascade is a complex, multistep process driven by progressive accumulation of genetic alterations which result in proto-oncogene activation and inactivation of tumour suppressor genes. Among the additional pathways involved in this process, secretion of angiogenesis factors, proteolysis of the extra-cellular matrix (ECM) molecules, and probably inhibition of apoptosis are also known to facilitate tumour progression. We undertook a retrospective study in a series of paraffin-embedded human colonic tissues to investigate the prognostic significance of new tumour markers as predictors of liver metastasis and survival in sporadic CRC. This research was conducted in three parts: first, immunohistochemical studies of protein markers and development of a new quantitative method of measurement in a subset of the immunostained sections using a colour video image analysis (VIA) procedure; second, concomitant determination of the expression of the bcl-2 gene at the messenger RNA (mRNA) level, using a more specific methodology of in situ hybridisation (ISH) and investigation of the relationship between bcl-2 mRNA and its encoded protein expression; and third, investigation of microsatellite alterations at four loci using a fluorescent microsatelllite polymerase chain reaction (PCR) assay coupled with an automated DNA sequencer. In our initial immunohistochemical experiment, we found a good correlation between colour VIA and semiquantitative evaluation of nm23 immunoreactivity (IR) confirming the validity of such quantitative analysis (Pearson's correlation coefficient r=0.88; P<0.001). Furthermore, overexpression of nm23 was associated with an increased risk of developing liver metastases (logrank test for trend, P<0.001) and cancer-related death (P=0.002). We used the same quantitative method to determine the expression of urokinase-type plasminogen activator (u-PA) and c-erbB-2 (HER2/neu) proteins and found that neither predicted patient outcome. However, CRC showing overexpression of u-PA (above 85 pixels) had an increased risk of liver metastasis (P=0.013). Since this was a post hoc analysis we can not be confident that this represents a real effect. There were significant positive correlations among expression of all three markers, u-PA, c-erbB-2 and nm23 proteins (u-PA vs c-erbB-2, Spearman rank correlation coefficient, P=0.003; u-PA vs nm23, P<0.001; c-erbB-2 vs nm23, P=0.001) suggesting that, in vivo, all proteins interact or are similarly regulated. Semiquantitative analysis of the vascular endothelial growth factor (VEGF) protein showed that expression of VEGF was significantly reduced in the metastatic liver tumours compared to their matched primary ones (Wilcoxon test, P=0.002), suggesting VEGF activity to be secondarily down-regulated once the tumour cells reach the hepatic parenchyma. There was no strong evidence from our data that the level of VEGF in the primary tumour could predict risk of liver metastasis or survival duration. Finally, when semiquantitatively assessing five protein markers (nm23, p53, c-erbB- 2, u-PA, and VEGF) individually or in combination, we found that only nm23 protein expression was positively related to the risk of liver metastasis (logrank test, P<0.001); p53 protein expression was only marginally associated (P=0.091). Furthermore, patients with Dukes' stage B tumours showing positive expression of nm23 protein also demonstrated an increased risk of liver metastasis (P=0.001). Although the risk of developing liver secondaries was statistically correlated with the number of positive markers (NPM) and the cumulative intensity score (CIS) (logrank test for trend, P=0.004 and P=0.001 respectively), these two parameters did not improve the predictive value over and above that of nm23 protein alone. These results suggest that the only marker of the five we studied that provides prognostic information about the risk of developing liver metastasis in CRC is nm23. Its evaluation may be particularly useful in selecting high risk Dukes' stage B patients who should be considered for adjuvant chemotherapy. In the second part of this study, immunohistochemical analysis using a monoclonal mouse antibody to the bcl-2 protein and in situ hybridisation using digoxigenin-labelled bcl-2 cRNA probes were carried out on the same paraffin-embedded specimens and semiquantitatively assessed. These specimens also included adenomas with various degrees of dysplasia. The expression of bcl-2 protein was gradually and significantly lost during the progression from moderately dysplastic adenoma to primary CRC [moderate/severe dysplasia: Mann-Whitney U-test, P=0.0001; severe dysplasia/primary CRC: P=0.027], whereas the cellular expression of bcl-2 mRNA was progressively increased during the dysplasia/adenoma-carcinoma neoplastic sequence. Our observation suggests that in a proportion of CRCs the bcl-2 proto-oncogene expression may be down-regulated at a post-transcriptional level. In the final section of this study, we investigated the possible relationship between loss of heterozygosity (LOH) and microsatellite instability (MIN) at four microsatellite loci spanning the 17q21-23 region that includes the nm23-H1 and nm23- H2 genes, to the risk of liver metastasis and nm23 protein expression. Genomic DNA was extracted from the same series of paraffin-embedded colorectal specimens and a fluorescent PCR coupled with DNA fragment analysis in an automated DNA sequencer was applied. In 45% and 48% of the primary and secondary lesions, LOH was present in at least one locus. We found a positive correlation when looking for a trend comparing the fraction of sites with LOH at these loci to risk of liver recurrence (logrank test for trend, P=0.005). This remained an independent predictor after adjusting to T-stage (Multivariate Cox regression, P=0.022), N-stage (P=0.007), or Dukes' stage (P=0.012). MIN was present in 2 loci in 41% and 30% of the primary and secondary tumours respectively. Considering only the Dukes' B tumours, we found that an increasing number of sites showing MIN was associated with a reduced risk of liver recurrence (logrank test for trend, P=0.032). When comparing LOH and MIN status of the primary and matched liver secondary tumours with their corresponding normal tissue samples, we found concordant genomic alterations in 72% (NME1 locus) to 43% (D17S579). Finally, we observed a trend in association between the proportion of loci with LOH and nm23 positivity ( ?? 2 test for trend, P=0.024). These findings suggest that, genomic alterations in the 17q21-23 region may affect prognosis of CRC as well as regulation of the nm23 protein expression via a stillmechanism.

The physiologically important free radicals, nitrogen monoxide and superoxide, can combine to form the reactive intermediate peroxynitrite. Peroxynitrite can react with proteins and their constituent amino acids, such as tyrosine, resulting in protein peroxidation, oxidation and nitration. The nitration of proteins, assessed by the analysis of 3-nitrotyrosine, is a proposed index of pathophysiological activity of peroxynitrite. The aim of the work was to investigate the reaction products between peroxynitrite and protein, develop an assay for 3-nitrotyrosine and measure its levels in biological samples. To study the amino acid products arising from the reaction of peroxynitrite and protein, both liquid chromatography (LC) and gas chromatography (GC) combined with mass spectrometry (MS) were adopted. Approaches to 3-nitrotyrosine assay development were first, to take advantage of the intrinsic sensitivity of electron capture negative ionization GC-MS. Secondly, to avoid possible artefactual problems associated with the derivatisation step in GC-MS, an assay for 3-nitrotyrosine based on combined LC-MS-MS was developed. When a selection of peptides was exposed to peroxynitrite under physiological conditions in vitro, the hydrolysis products showed that 3-nitrotyrosine was the major product. Detectable minor products were 3,5-dinitrotyrosine and DOPA. The GC-MS assay was found to be fraught with difficulty due to artefactual formation of 3-nitrotyrosine. In order to quantify and correct for artefact formation, this complication was approached by incorporating a second isotopomer. This method, however, was confounded by large errors that reduced the overall sensitivity. Either negative or zero levels of endogenous 3-nitrotyrosine were found in tested samples after correction for artefact formation. The LC-MS-MS assay was then used to analyse 3-nitrotyrosine levels in a range of biological samples, including human plasma from healthy volunteers, synovial fluid samples from arthritis patients and tissue extracts from a mouse model of amyotropic lateral sclerosis. In contrast to published data, 3-nitrotyrosine levels were found to be below the limit of detection (1 pg/????L, 10 pg o/c) for all samples - a result somewhat consistent with the negative GC-MS data. It is suggested that the high 3-nitrotyrosine levels previously reported in the literature might reflect artefactual generation of 3-nitrotyrosine and that other approaches to assessing pathophysiological nitration should be sought in future.

Personality is a key component of characters that inhabit immersive virtual environments, such as games and virtual agent applications. In order to be distinguishable from other characters in the environment, each character should have its own personality in the form of different observable behaviour, not solely in its physical appearance or animation. Previous work in this field has mostly relied on time-consuming, handcrafted characters and static, trait-based approaches to personality. Our goal is a method to develop complex, individual personalities without handcrafting every behaviour. Unlike most implemented versions of personality theories, cognitive-social theories of personality address how personality is developed and adapts throughout childhood and over our lifetimes. Cognitive-social theories also emphasise the importance of situations in determining how we behave. From this basis, we believe that personality should be individual, adaptive, and based on context. Characters in current state-of-the-art games and virtual environments do not demonstrate all of these features without extensive handcrafting. We propose a model where personality influences both decision-making and evaluation of reward. Characters use their past experiences in the form of simple somatic markers, or gut-instinct, to make decisions; and determine rewards based on their own personal goals, rather than via external feedback. We evaluated the model by implementation of a simple game and tested it using quantitative criteria, including a purpose-designed individuality measure. Results indicate that, although characters are given the same initial personality template, it is possible to develop different personalities (in the form of behaviour) based on their unique experiences in the environment and relationships with other characters. This work shows a way forward for more automated development of personalities that are individual, context-aware and adapt to users and the environment.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.