Immobilization is an effective method to increase ethanol production, as proven by previous research. Results almost exclusively demonstrate an increase in ethanol production by and decrease in reproduction rate of immobilized Saccharomyces cerevisiae cells. Recently, research has been conducted to determine the cause of this change. The extreme variance in results due to lack of technology makes it difficult to determine the cellular changes induced by immobilization. With the advent of new technology, specifically gene expression analysis, the RNA content of cells can be easily and rapidly analyzed. S. cerevisiae cells were immobilized in 3% (w/v) calcium alginate beads and grown inside of a packed bed reactor for comparison to planktonic cells growing in batch and chemostat cultures. Temperature inside of the reactor was maintained at 33 C with a pH of 5.5. Cell concentration inside of the beads was monitored periodically in order to create growth curves. Bud scar numbers of immobilized cells were also counted and compared to suspended cells. Scanning electron microscopy images of the alginate beads were taken to determine cell growth inside of the beads. Affymetrix Yeast 2.0 gene chips were used, and the data retrieved was analyzed with GeneSpring software using the Bioconductor packages. Results indicated changes in expression of 3,559 genes with significant difference among treatments by a factor of 2-fold or greater. One-way ANOVA of the filtered data yielded 380 highly significantly different genes between immobilized and suspended cells. Many of the genes pertaining to glycolysis exhibited increased expression levels. Several genes necessary for reproduction were expressed at lower levels in the immobilized cells than in their planktonic counterparts. Many different gene ontologies are discussed, and the expressed genes are mapped onto biochemical pathways.

Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations. Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15. These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment. / QC 20110121

Winter wheat (<i>Triticum aestivum</i> L.), seeded in the fall, cold acclimates when exposed to low fall temperatures. Growth resumes in spring, culminating in early summer harvest. Winter wheat yield is generally 20-25% higher than spring wheat. However, winter damage/kill can reduce its yield. A better understanding of the cold acclimation/tolerance process could help in the development of improved breeding strategies for winter wheat hardiness. Transcriptional activators and specific cold regulated (COR) genes are induced as a result of exposure to low temperatures. Thus, the objective of this study was to determine the quantitative expression of three COR genes (Wcs120, Wcor410 and Wcor14b) and one transcriptional activator (WCBF1) in field-grown wheat using real-time PCR and to establish any association with LT50 (temperature at which 50% of plants are killed). Winter Norstar (vrn-A1/vrn-A1), spring Manitou (Vrn-A1/Vrn-A1) and two near-isogenic lines (Spring Norstar (Vrn-A1/vrn-A1) and Winter Manitou (vrn-A1/vrn-A1), respectively) were used in these studies. Plants were sampled on three dates (Sept. 29, Oct. 12 and Oct. 26) in the fall of 2004. Accumulation of WCBF1 transcripts was highest in Norstar, but in all four genotypes there was an increase in transcripts by the second sampling date, followed by a decline on the third sampling date. Wcs120 transcripts increased from the first to the third sampling date in Norstar, Spring Norstar and Winter Manitou, but increased to the second sampling date and decreased by the third in Manitou. For Wcor14b, generally there was an increase to the second sampling date, followed by a decrease or steady levels on the third. Wcor410 showed a similar pattern, except for Spring Norstar wherein transcript levels increased by the third sampling date. With the exception of Wcor410 in Manitou, the Vrn-A1 locus affected gene expression in all genotypes. However, only Wcs120 expression followed the low-temperature tolerance pattern in these genotypes.

Winter wheat (<i>Triticum aestivum</i> L.), seeded in the fall, cold acclimates when exposed to low fall temperatures. Growth resumes in spring, culminating in early summer harvest. Winter wheat yield is generally 20-25% higher than spring wheat. However, winter damage/kill can reduce its yield. A better understanding of the cold acclimation/tolerance process could help in the development of improved breeding strategies for winter wheat hardiness. Transcriptional activators and specific cold regulated (COR) genes are induced as a result of exposure to low temperatures. Thus, the objective of this study was to determine the quantitative expression of three COR genes (Wcs120, Wcor410 and Wcor14b) and one transcriptional activator (WCBF1) in field-grown wheat using real-time PCR and to establish any association with LT50 (temperature at which 50% of plants are killed). Winter Norstar (vrn-A1/vrn-A1), spring Manitou (Vrn-A1/Vrn-A1) and two near-isogenic lines (Spring Norstar (Vrn-A1/vrn-A1) and Winter Manitou (vrn-A1/vrn-A1), respectively) were used in these studies. Plants were sampled on three dates (Sept. 29, Oct. 12 and Oct. 26) in the fall of 2004. Accumulation of WCBF1 transcripts was highest in Norstar, but in all four genotypes there was an increase in transcripts by the second sampling date, followed by a decline on the third sampling date. Wcs120 transcripts increased from the first to the third sampling date in Norstar, Spring Norstar and Winter Manitou, but increased to the second sampling date and decreased by the third in Manitou. For Wcor14b, generally there was an increase to the second sampling date, followed by a decrease or steady levels on the third. Wcor410 showed a similar pattern, except for Spring Norstar wherein transcript levels increased by the third sampling date. With the exception of Wcor410 in Manitou, the Vrn-A1 locus affected gene expression in all genotypes. However, only Wcs120 expression followed the low-temperature tolerance pattern in these genotypes.

Gene expression analyses using native populations can provide information on the genetic and molecular mechanisms that determine intraspecific variation and contribute to the understanding of plant development and adaptation in multiple ways. Using quantitative real time – polymerase chain reaction (qRT-PCR), we analyzed the expression of 111 genes with probable roles in wood development in 400 loblolly pine individuals belonging to a population covering much of the natural range. Association mapping techniques are increasingly being used in plants to dissect complex genetic traits and identify genes responsible for the quantitative variation of these traits. We used candidate-gene based association studies to associate single nucleotide polymorphisms (SNPs) in candidate genes with the variation in gene expression. The specific objectives established for this study were to study natural variation in expression of xylem development genes in loblolly pine (Pinus taeda L.) using qRT-PCR, to associate SNPs in candidate genes with the variation in gene expression using candidate-gene based association analyses and to detect loblolly pine promoter polymorphisms and study their effect on gene expression. Out of the 111 genes analyzed using qRT-PCR, there were significant differences in expression among clones for 106 genes. Candidate-gene based association studies were performed between 3937 single nucleotide polymorphisms (SNPs) and gene expression to associate SNPs in candidate genes with the variation in gene expression. To the best of our knowledge, this is the first association genetic study where expression of a large number of genes, analyzed in a natural population, has been the phenotypic trait of interest. We cloned and sequenced promoters of 19 genes, 16 of which are transcription factors involved in wood development and drought response. SNP discovery was done in 13 of these promoters using a panel of 24 loblolly pine clones (unique genotypes). SNP genotyping is underway in the entire association population and association analyses will be done to study the effects of promoter SNPs on gene expression. The results from this project are promising and once these associations have been tested and proved, we believe that they will help in our understanding of the genetics of complex traits.

RNA-binding proteins (RBP) shuttle between cellular compartments either constitutively or in response to stress and regulate localization, translation and turn over of mRNAs. In our laboratory, cytosolic proteome map of Phanerochaete chrysosporium was established and upon Pb exposure, the changes in cytosolic protein expressions were determined. The identified RBPs were a newly induced polyadenylate-binding protein (RRM superfamily) as well as two up-regulated proteins, namely splicing factor RNPS1 and ATP-dependent RNA helicase, all being very important candidates of post-transcriptional control in response to stress. This finding inspired us to conduct Real Time PCR studies in order to have a better understanding of the changes in the expression of corresponding genes at mRNA level in response to Pb exposure, thus the present study aims at examining the effect of lead exposure on the transcript levels of the genes coding for ATP-dependent RNA helicase, splicing factor RNPS1 and polyadenylate binding protein. As shown via expression analysis based on Real Time PCR, the mRNA level of splicing factor RNPS1 showed 2.68, 2.62 and 4.86 fold increases in a dose-dependent manner when the cells were grown for 40 h in the presence of 25, 50 and 100 &micro / M Pb, repectively. ATP-dependent RNA helicase mRNA level showed no significant increase in response to 25 &micro / M Pb exposure while increased 2 and 1.84 fold in response to 50 and 100 &micro / M Pb, respectively. Polyadenylate binding protein mRNA levels revealed no significant increase when exposed to 25, 50 and 100 &micro / M Pb. As to the mRNA dynamics as a function of duration of lead exposure, the mRNA level of this protein showed 2.54-fold increase upon 1 h exposure to 100 &micro / M Pb. Splicing factor RNPS1 mRNA level showed a significant increase of 19.22 fold at 2nd h of 50 &micro / M Pb exposure. Expression level of ATP-dependent RNA helicase was not affected by the time of exposure to Pb.

<p>The central question investigated in this project was whether clustering of gene expression patterns could be done more biologically accurate by providing the clustering technique with additional information about the genes as input besides the expression levels. With the term biologically accurate we mean that the genes should not only be clustered together according to their similarities in expression profiles, but also according to their functional similarity in terms of functional annotation and metabolic pathway. The data was collected at AstraZeneca R&D Mölndal Sweden and the applied computational technique was self-organising maps. In our experiments we used the combination of expression profiles together with enzyme classification annotation as input for the self-organising maps instead of just the expression profiles. The results were evaluated both statistically and biologically. The statistical evaluation showed that our method resulted in a small decrease in terms of compactness and isolation. The biological evaluation showed that our method resulted in clusters with greater functional homogeneity with respect to enzyme classification, functional hierarchy and metabolic pathway annotation.</p>

<p>Recent methods to infer genetic networks are based on identifying gene interactions by similarities in expression profiles. These methods are founded on the assumption that interacting genes share higher similarities in their expression profiles than non-interacting genes. In this dissertation this assumption is validated when using mutual information as a similarity measure. Three algorithms that calculate mutual information between expression data are developed: 1) a basic approach implemented with the histogram technique; 2) an extension of the basic approach that takes into consideration time delay between expression profiles; 3) an extension of the basic approach that takes into consideration that genes are regulated in a complex manner by multiple genes. In our experiments we compare the mutual information distributions for profiles of interacting and non-interacting genes. The results show that interacting genes do not share higher mutual information in their expression profiles than non-interacting genes, thus contradicting the basic assumption that similarity measures need to fulfil. This indicates that mutual information is not appropriate as similarity measure, which contradicts earlier proposals.</p>

<p>The metabolic syndrome is a cluster of metabolic risk factors such as diabetes type II, dyslipidemia, hypertension, obesity, microalbuminurea and insulin resistance, which in the recent years has increased greatly in many parts of the world. In this thesis decision trees were applied to the BioExpress database, including both clinical data about donors and gene expression data, to investigate nuclear receptors ability to serve as markers for the metabolic syndrome. Decision trees were created and the classification performance for each individual risk factor were then analysed. The rules generated from the risk factor trees were compared in order to search for similarities and dissimilarities. The comparisons of rules were performed in pairs of risk factors, in groups of three and on all risk factors and they resulted in the discovery of a set of genes where the most interesting were the Peroxisome Proliferator Activated Receptor - Alpha, the Peroxisome Proliferator Activated Receptor - Gamma and the Glucocorticoid Receptor. These genes existed in pathways associated with the metabolic syndrome and in the recent scientific literature.</p>

<p>Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations.</p><p>Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes <i>BRAF</i> and <i>NRAS</i>, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with <i>BRAF</i> mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene <i>MGMT</i> was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15.</p><p>These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment.</p>