Apple proliferation (AP) is one of the most serious diseases of apple trees in Europe. It is caused by a phytoplasma, Candidatus Phytoplasma mali. The goal of the present study was to analyze transcriptional profiles of Malus pumila during infection by Ca. P. mali using cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique in order to gain insight into molecular and physiological changes in diseased plants. We used a rootstock of apple (MM106) susceptible to Ca. P. mali to maximise the range of the potential host responses, and two strains (AP and AT) of the pathogen. Gene expression comparisons were studied in 3 categories of plant materials: healthy sample versus infected samples, symptomatic versus non-symptomatic sample, and AP-infected sample versus AT-infected sample. Forty-five genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were isolated and identified. Of 45 partial cDNA sequences, twenty-seven showed similarity to international DNA or protein data bases. Of these, 18 were previously characterized in plants (the rest was related to unknown or hypothetical proteins). Eighteen out of 45 did not show any similarity with sequences in data bases, and so may be present novel genes. The majority of fragments were differently expressed between healthy sample and infected samples (fewer differences between symptomatic and non-symptomatic samples, or between the samples infected by different strains of phytoplasma). Quantitative Real-time RT-PCR (qRT-PCR) was used to confirm differential expression of sequences isolated by cDNA-AFLP. We chose the most stable reference housekeeping genes (GAPDH and actin) for normalisation of our data. The gene expression ratios were calculated by means of ΔΔCt method. Consequently, the second methodology (qRT-PCR) showed the similar profile expression as primary elucidation technique (cDNA-AFLP) for 11 known genes (between 18) and 13 unknown, hypothetical or novel genes (between 27). Changes in gene expression involved a wide spectrum of biological functions, including processes of metabolism, cell defence, senescence, photosynthesis, transport, transcription, signal transduction and protein synthesis. The possible effect of phytoplasma infection on these processes and their relationships with disease development, symptom appearance and probably plant defence system is discussed. A model is proposed to explain the mode of action of the Ca. P. mali in its host plant, apple tree. This is the first study of global gene profiling in plants in response to phytoplasma infections using cDNA-AFLP.

Chitin, a homopolymer of β (1-4)-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature. It is widely distributed in the exoskeleton of crustaceans and insects, in the cell walls of most fungi and some algae. Chitin is an extremely insoluble material with limited industrial applicability. The deacetylated derivative of chitin, chitosan, is a water soluble cationic biopolymer having a broad range of applications (Hirano, 1999). Chitosan is naturally found in the cell wall of Zygomycetes, in the ascospore of Saccharomyces cerevisiae (Briza et al., 1988) and in the cyst wall of Entamoeba invadens (Das et al., 2006). Chitosan biosynthesis requires the coordinated action of chitin synthase (E.C.2.4.1.16) and chitin deacetylase (E.C.3.5.1.41) (Davis & Bartnicki, 1984). Chitin synthase polymerizes N-acetyl glucosamine precursor molecules into chitin and chitin deacetylase catalyzes the deacetylation of the nascent chitin chains. The chitin deacetylase enzymes are members of the family 4 of carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/~cazy/CAZY] (Couthino et al., 1999), which includes several members sharing a conserved region in the primary structure assigned as the NodB homology domain(Caufrier et al., 2003) or polysaccharide deacetylase domain. Chitin deacetylase was first identified and partially purified from extracts of the fungus Mucor rouxii. Since then, chitin deacetylase has been purified from several fungi and chitin deacetylase open reading frames have been cloned from a few microorganisms including M. rouxii (Kafetzopoulos et al., 1993), Colletotrichum lindemuthianum (Tokuyasu et al., 1999; Shresta et al., 2004), Phycomyces blakesleeanus (GenBank AB046690), Schizophillum commune (GenBank AF271216), Blumeria graminis (GenBank AAK84438), Saccharomyces cerevisiae (Christodoulidou et al., 1999) and Schizosaccharomyces pombe (Matsuo et al., 2005). The structure and the catalytic mechanism of chitin deacetylase from C. lindemuthianum were recently studied (Blair et al., 2006). Chitin deacetylase plays a role in the cell wall biosynthesis in M. rouxii and Absidia coerulea (Gao et al., 1995). In C. lindemuthianum and Aspergillus nidulans, it was suggested that chitin deacetylase participates in plant-pathogen interactions to promote plant invasion (Tsigos et al., 2000). In S. cerevisiae, chitin deacetylase is essential for the ascospore cell wall rigidity and the resistance against lytic enzymes (Christodoulidou et al., 1996). The use of chitin deacetylase enzyme for the industrial deacetylation of chitin awaked a great interest. Different fungal strains were screened and compared for their ability to produce a chitin deacetylase secreted, active on insoluble substrates and showing low inhibition with acetate, a product of reaction. Rhizopus circinans proved to be a good chitin deacetylase producer with the targeted characteristics. The second part of the work was to isolate the cDNA encoding for the chitin deacetylase of R. circinans. The native enzyme was purified to homogeneity for sequencing the N-Terminal extremity. The enzyme was purified in only two steps from the culture supernatant of R. circinans. Then, the purified enzyme was sequenced and the first nine amino acids were identified. In the same way, a R. circinans cDNA library was also constructed. The cDNA library was screened using two approaches: on the one hand with radiolabeled homologous probe and on the other hand by PCR with primers designed for the 5 extremity, on the basis of the deduced sequence of the N-Terminal extremity of the native enzyme and for the 3 extremity, from the deduced R. oryzae chitin deacetylase. Two cDNA sequences (D2 and I3/2) with homology to fungal chitin deacetylase genes were isolated with the radiolabeled probe and one sequence (RC+) by PCR approach. The sequences were analyzed and characterized. The three sequences possessed several characteristics of chitin deacetylase sequence: homology with known chitin deacetylase cDNA, the presence of the deacetylase polysaccharide domain, and the same potential glycosylation sites than M. rouxii chitin deacetylase. The cDNA D2, I3/2 and RC (RC sequence is the mature protein sequence of RC+ sequence) were expressed in the yeast Pichia pastoris to confirm their potential chitin deacetylase activity. Numerous constructions were tested. A poly-histidine tag was cloned to facilitate the further purification of the recombinant enzyme. Only the RC sequence showed a high chitin deacetylase activity. Several hypotheses were emitted to explain the low chitin deacetylase activity level measured with the inserts D2 and I3/2. The recombinant RC protein was purified to homogeneity in one step, and partially characterized.

Research in positive psychology continues to contribute to the understanding of the significance of human virtues as well as the value that experiencing positive emotions has on individual well-being, including building strengths, broadening resources, and increasing mental health. The benefits that the creative arts have on increasing psychological health and creating positive emotions are also an important but understudied area of research. The purpose of the current study was to examine, in a manner similar to the written disclosure paradigm, how the creation of mandalas while reflecting on the positive emotions of love and joy related to increased psychological well-being, and continued positive affect in a college sample. It was also hypothesized that the mere act of expressing personally felt emotions, regardless of positive or negative, while creating mandalas would reveal a significant increase in psychological and physical health relative to the control condition. Benefits to participants were measured in terms of changes in the variables of post-traumatic stress severity symptoms, depressive symptoms, anxiety, spiritual meaning, the frequency of physical symptoms and illness, as well as positive and negative affect. A series of one-way analyses of covariance (ANCOVA) comparing the experimental and control groups were conducted for all outcome measures at Time 2 and at 1-month follow-up. Results revealed no significant differences between the groups on any of the health measures. Next, a series of ANCOVA were also conducted comparing the experimental and control groups for general positive and negative affect and basic positive and negative emotion before and after each drawing session at Time 1, 2 and 3 as well as at the 1-month follow-up. Although sustained positive emotion was not supported between Time 3 and the 1-month follow-up, participants felt higher general positive affect and basic positive emotion after each drawing session focusing on love and joy. Implications of these results and further research will be discussed.

Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.

Gene expression, which connects genomic information to functional units in living cells, has received substantial attention since the completion of The Human Genome Project. Quantitative characterization of gene expression will provide valuable information for understanding the behavior of living cells, and possibilities of building synthetic gene circuits to control or modify the behavior of naturally occurring cells. Many aspects of quantitative gene expression have been studied, including gene expression dynamics and noise in E. coli. The gene expression process itself is stochastic, and modelling approaches have been broadly used to study gene expression noise; however, stochastic gene expression models are usually large and time intensive to simulate. To speed up simulations, we have developed a systematic method to simplify gene expression models with fast and slow dynamics, and investigated when we can ignore the gene expression from the background genome when modelling the gene expression from plasmids. When modelling the noise in gene expression, one usually neglected aspect is the slow maturation process of fluorescent proteins, necessary for the protein to give out fluorescence after it is produced. By modelling, we show that the maturation steps can bring large changes to both the mean protein number and the noise in the model. An unstudied aspect of gene expression dynamics is the time dependent gene expression behavior in E. coli batch culture. Contrary to the usual assumption, we have found, in E. coli batch culture gene expression, that there is no steady state in terms of both the mean number of proteins and the noise. Negative feedback is thought to be able to reduce the noise in a system, and experiments have shown that negative feedback indeed suppresses the noise in gene expression, but the modelling shows that negative feedback will increase the noise. We have found that the increase of noise by feedback is due to the exclusion of extrinsic noise from the model, and that negative feedback will suppress the extrinsic noise while increasing the intrinsic noise. Living cells are crowded with macromolecules, which will, predicted by modelling, make the reaction constant time dependent. Our experimental observation has confirmed this prediction.

There is an inherent conflict over land use between humans and wildlife. Human activities can alter habitat, creating pressure on North American large carnivore populations. Traditional wildlife techniques can be slow to show population declines, especially in long lived species with slow reproduction rates and high mortality of young, such as grizzly bears (Ursus arctos), which leads to delayed information for land managers trying to find the balance between human use of land and preservation of wildlife. Concern about population health of grizzlies in Western Alberta, Canada has lead to investigation of the impacts of current land use within grizzly bear habitat. The objective of this work was to develop a protein microarray that could detect patterns of physiological stress in a rapid manner with small samples of grizzly bear tissue. Sampling from four regions in the foothills of the Rocky Mountains in Alberta resulted in the capture of 133 bears. During the developmental phase, proteins involved with mitochondrial function were found, using two dimensional gel electrophoresis, to be altered in situations of increased stress. Limited cross-reactivity was found when evaluating grizzly bear stress protein expression using commercially available protein microarrays. The protein microarray developed in this thesis consists of 31commercial antibodies validated for grizzly bears. These antibodies recognize proteins associated with different aspects of the stress response, including the hypothalamic-pituitary-adrenal axis, apoptosis/cell cycle, cellular stress, and oxidative stress and inflammation. Skin was selected as the tissue for evaluation of protein expression. Strong correlations were found between many of the proteins within functional categories. Model selection for the protein categories revealed variation that corresponded with region, serum markers of stress (total cortisol and hsp60), growth, the density of roads in the habitat and the amount of anthropogenic change in the bears home range. Regional trends of expression found bears in Swan Hills and bears from North highway 16 having elevated expression of the proteins measured by the microarray. The protein microarray was thus able to detect expression patterns reflecting physiological and environmental markers. The array shows great promise for future use in detection of potential distress in wildlife populations due to alterations of their habitat.

This study examined the differential gene expression patterns between endothelial cells (EC) from 2D monolayer and EC from angiogenic capillary-like network in 3D matrices. Our microarray analysis comparing 3D to 2D EC cultures detected upregulation of 854 protein-coding genes and downregulation of 863 genes. We show that Notch signaling pathway is highly regulated in angiogenesis, induced by change in ECM dimension. Notch target genes Hey1, HeyL, Hes1 and Hes4 transcription factors were upregulated in 3D angiogenic EC, which were confirmed with qRT-PCR. Moreover, we are the first to report enrichment of FoxS1 transcription factor mRNA during angiogenesis in 3D ECM. Next, we asked whether epigenetic mechanisms partly mediate cis-trans response in angiogenesis. Our sodium bisulfite sequencing analyses did not indicate a role for DNA methylation in the expression of key Notch signaling components. However, our pilot studies indicate a potential role for lncRNAs in controlling EC phenotype in angiogenic response.

This study examined the differential gene expression patterns between endothelial cells (EC) from 2D monolayer and EC from angiogenic capillary-like network in 3D matrices. Our microarray analysis comparing 3D to 2D EC cultures detected upregulation of 854 protein-coding genes and downregulation of 863 genes. We show that Notch signaling pathway is highly regulated in angiogenesis, induced by change in ECM dimension. Notch target genes Hey1, HeyL, Hes1 and Hes4 transcription factors were upregulated in 3D angiogenic EC, which were confirmed with qRT-PCR. Moreover, we are the first to report enrichment of FoxS1 transcription factor mRNA during angiogenesis in 3D ECM. Next, we asked whether epigenetic mechanisms partly mediate cis-trans response in angiogenesis. Our sodium bisulfite sequencing analyses did not indicate a role for DNA methylation in the expression of key Notch signaling components. However, our pilot studies indicate a potential role for lncRNAs in controlling EC phenotype in angiogenic response.

Gene expression, which connects genomic information to functional units in living cells, has received substantial attention since the completion of The Human Genome Project. Quantitative characterization of gene expression will provide valuable information for understanding the behavior of living cells, and possibilities of building synthetic gene circuits to control or modify the behavior of naturally occurring cells. Many aspects of quantitative gene expression have been studied, including gene expression dynamics and noise in E. coli. The gene expression process itself is stochastic, and modelling approaches have been broadly used to study gene expression noise; however, stochastic gene expression models are usually large and time intensive to simulate. To speed up simulations, we have developed a systematic method to simplify gene expression models with fast and slow dynamics, and investigated when we can ignore the gene expression from the background genome when modelling the gene expression from plasmids. When modelling the noise in gene expression, one usually neglected aspect is the slow maturation process of fluorescent proteins, necessary for the protein to give out fluorescence after it is produced. By modelling, we show that the maturation steps can bring large changes to both the mean protein number and the noise in the model. An unstudied aspect of gene expression dynamics is the time dependent gene expression behavior in E. coli batch culture. Contrary to the usual assumption, we have found, in E. coli batch culture gene expression, that there is no steady state in terms of both the mean number of proteins and the noise. Negative feedback is thought to be able to reduce the noise in a system, and experiments have shown that negative feedback indeed suppresses the noise in gene expression, but the modelling shows that negative feedback will increase the noise. We have found that the increase of noise by feedback is due to the exclusion of extrinsic noise from the model, and that negative feedback will suppress the extrinsic noise while increasing the intrinsic noise. Living cells are crowded with macromolecules, which will, predicted by modelling, make the reaction constant time dependent. Our experimental observation has confirmed this prediction.

Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.