Background: Lichen planus is a chronic inflammation of skin and mucosa with unknown cause. Oral Lichen Planus, OLP, affects around 2% of the population. Autoimmunity has been suggested as a possible cause as the disease has autoimmune features such as female predominance, cyclic nature and cytotoxic T-cell infiltrate. It has been suggested that the intense inflammatory response seen in OLP is caused by factors on the keratinocyte surface triggering the immune system. Chronic inflammation is one of the hallmarks of oral lichen planus and chronic inflammation is connected to increased risk of tumor development. WHO classifies OLP as a potentially malignant condition with increased risk of developing Squamous cell carcinoma of head and neck, SCCHN, but malignant transformation of OLP is a matter of controversy. The aim of these studies was to further elucidate the autoimmune and premalignant character of OLP. Factors involved in malignant transformation, autoimmunity and inflammation were analyzed in normal oral mucosa, OLP and SCCHN. Factors studied were the signal transducers of Transforming growth factor-β the Smad proteins, microRNAs, COX-2, the receptor CXCR-3 and its ligands CXCL-10 and -11 and ELF-3. Material and methods: In the study on Smad protein expression formalin fixed and paraffin embedded biopsies from normal oral mucosa, OLP and SCCHN was used. For the remaining studies fresh frozen biopsies from OLP and normal controls was used. All of the fresh frozen OLP samples and their controls were micro dissected to be able to analyze the epithelial part only as well as sections of the whole biopsy. Methods used are immunohistochemistry, qRT-PCR and Western blot. Results: Analyses of smad proteins expression showed a clear increase of smad3 and smad7 in OLP compared to normal oral mucosa. The expressions of smad proteins in the tumors were more heterogeneous. Some of the SCCHN samples showed a similar expression as OLP while others did not. Micro RNA analyzes showed that miR-21 and miR-203 was significantly increased in OLP epithelium compared to normal oral epithelium while the expression of miR-125b and their potential targets p53 and p63 was decreased in OLP. The presence of COX-2 was significantly higher in OLP than normal controls. At the same time the expression of miR-26b, a suggested repressor of COX-2 was decreased in OLP compared to normal mucosa. The receptor CXCR-3 and its ligands CXCL-10 and -11 were increased in OLP. Expressions of the differentiation involved factor ELF-3 mRNA as well as protein were decreased in OLP. Conclusion: The factors studied are involved in differentiation, malignant transformation and inflammation. Some of the results in these studies indicate a similar expression pattern for OLP and SCCHN. Several of the factors studied are involved in differentiation and their deregulation suggests a disturbed differentiation pattern and this could indicate a premalignant character of OLP but malignant transformation of OLP lesions are relative rare. A lot of these factors are also involved in inflammatory processes and connected to autoimmune diseases and their deregulation in OLP could also support an autoimmune cause of the disease. Based on our studies a suggestion is that the disturbed differentiation pattern triggers the intense immune response directed against the epithelial cells seen in OLP.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

Primary cultures of stromal-vascular (S-V) cells from adipose tissue were used to investigate the regulation of preadipocyte development. Differentiation of S-V cells was found to be under hormonal control. Insulin and glucocorticoids are essential for S-V cell differentiation in culture. S-V cells from both newborn and mature pig adipose tissue and sera from both ages were used to examine the effect of age on preadipocyte development. S-V cells from newborn pigs replicated faster and appeared more responsive to serum borne factors influencing S-V cell growth and development in culture. Serum source (newborn vs mature) did not affect differentiation of S-V cells from newborn or mature pig adipose tissue. When sera from fed or fasted pigs were used to culture newborn pig S-V cells, fasted pig sera stimulated greater differentiation and decreased cell replication as indicated by DNA content of rat S-V cell culture. Lean pig serum compared to obese pig serum, increased differentiation activity in culture of S-V cells an effect which may be influenced by sex. When sera from rat and pig were subjected to gel filtration fractionation on Sephacryl S-200 column, the elution profiles of both sera were similar. Rat serum contained six additional peaks (280 nm) not present in pig serum. Rat serum fraction two (apparent molecular size 67-150 kD) promoted greater differentiation of S-V cells than other rat serum fractions or pig serum fraction two. Fraction three (apparent molecular size 17-43 kD) of both sera inhibited differentiation and lipid filling in cultures of S-V cells but only rat fraction three promoted cell proliferation. Rat and pig S-V cells have different morphology when differentiated. Differentiated rat S-V cells appeared as individual cells when cultured in serum free or serum supplemented medium while differentiated pig S-V cells appeared as individual cells in serum free medium and as a tight cluster of cells in serum supplemented medium. Both cells responded differently to sera obtained from pigs of differing ages and development of rat S-V cells was influenced by anatomic site. / Graduation date: 1992

The fungus <i>Colletotrichum truncatum</i> is a potential biocontrol agent (BA) against the noxious weed scentless chamomile (<i>Metricaria perforata</i> Mérat; syn.: <i>Tripleurospermum perforatum</i> (Mérat) Lainz) in western Canada. This potential BA, however, is taxonomically related to the anthracnose pathogen on lentil, raising questions about crop safety. Ribosomal DNA (rDNA) internal transcribed space (ITS) regions of <i>C. truncatum</i> isolates collected from different plant hosts were examined, and compared with additional Colletotrichum species. Sequences were amplified with the universal primers its4 and its5, and <i>C. truncatum</i> isolates from scentless chamomile and selected legume crops were differentiated consistently. All scentless chamomile isolates fell within a single cluster in phylogenetic trees, regardless of their geographic origins. These isolates were more closely related to lentil isolates of <i>C. truncatum</i> than to isolates from the other host species. Soybean isolates, with more falcate and slender conidia and slightly bigger appressoria, were distinguishable from other <i>C. truncatum</i> isolates, while the isolates from scentless chamomile, lentil and pea were morphologically more similar. Based on sequence information, strain-specific PCR primers were designed for <i>C. truncatum</i> isolates from these hosts and used to amplify specific DNA bands (markers) from isolates of <i>C. truncatum</i>. This technique may be used for rapid detection and differentiation of <i>C. truncatum,</i> from scentless chamomile and designated legume species, as well as for tracking the BA after release. Inoculation trials were conducted using detached leaves and whole plants to determine potential cross infection of these <i>C. truncatum</i> isolates. Isolates from scentless chamomile caused disease only on their original host, but not on lentil, pea, soybean or alfalfa. In contrast, lentil isolates caused severe disease on lentil and pea, light symptoms on alfalfa, but no disease on the other hosts tested. Potential penetration of lentil leaves by scentless chamomile isolates was tested, with 2-23% incidence of the fungus from inoculated detached, senescence leaves but disease symptoms were not observed on either detached leaves or whole plants. Examination of the infection process revealed that scentless chamomile and lentil isolates had a similar pattern of infection and disease development on their respective hosts; infection vesicles were produced 24 h after inoculation, both primary and secondary infection hyphae were present, and the onset of disease symptoms tended to coincide with the development of secondary hyphae. The current study provided molecular and pathological evidence that differentiates the potential BA of scentless chamomile from <i>C. truncatum</i> isolates from lentil, pea and soybean.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

Recent advancements in mass spectrometry have facilitated new analytical approaches capable of comprehensively characterizing metabolites in biological samples. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) combines excellent mass accuracy (ppm<1) and ultra-high resolution, which enables the separation and identification of individual components within complex mixtures, and the determination of elemental composition for each detected mass. FTICR-MS is an ideal method for non-targeted metabolomics as the majority of small molecular compounds (100-1000 Da) in a biological sample can be detected. The objective of this research was to investigate metabolomic alterations associated with key cellular processes deemed fundamental to cancer development and progression. Differentiating U937 cells, fibroblasts synchronously progressing through the cell cycle and a transformed cell line containing a temperature sensitive oncogene were collected and subject to FTICR-MS analysis for non-targeted comprehensive metabolomics. Putative metabolite identifications were confirmed with targeted metabolite analysis using multiple reaction monitoring triple quadrupole mass spectrometry. Analysis of the resulting metabolic profiles revealed robust metabolic alterations associated with fundamental cellular processes. Changes in glycerolipid content were observed in all cellular processes studied. During cell cycle progression, elevated levels of triacylglycerols and vinyl acylglycerols were detected as cells approached mitosis; increased levels of plasmalogens were detected during the induced differentiation of human leukemic cells and activation of the oncogene p130gag-fps in fibroblasts resulted in increased levels of phospholipids, including plasmalogens. When de novo fatty acid synthesis was inhibited in the differentiation cell model, the cells were not able to complete the differentiation process. Removal of the inhibitor resulted in increased lipid content, particularly plasmalogens, and the continuation of differentiation, suggesting a requirement for the de novo synthesis of lipids during this cellular process. This work demonstrates the advantages of non-targeted metabolic profiling for identifying non-intuitive metabolic associations with specific cellular processes. Collectively, the results of this thesis have implicated glycerolipids, in particular phospholipids, in the processes of cell cycle progression, differentiation and tumourigenic transformation. A broadened understanding of the role of global lipid metabolism during fundamental cellular processes may one day lead to new approaches for their modulation, and potentially new therapeutic strategies.

Major depressive disorder is a common severe psychiatric disorder with unknown etiology. Recent studies show that the loss and malfunction of oligodendrocytes are closely related to the neuropathological changes in depression, which can be reversed by antidepressant treatment. St. Johns wort is an effective and safe herbal treatment for depression in several clinical trials. However, the underlying mechanism of its therapeutic effects is unclear. In this study, we evaluated the effects of hyperforin, a major active component of this herb, on the proliferation, mitochondrial function and development of oligodendrocytes. We have demonstrated that hyperforin increases mitochondrial function and prevents mitochondrial toxin-induced cytotoxicity in oligodendrocyte lineage cells. Hyperforin promotes the maturation of oligodendrocytes but does not increase the proliferation of oligodendrocyte progenitor cell line and neural stem/progenitor cells. Our findings suggest that chronic hyperforin treatment may stimulate the development and function of oligodendrocytes. These results suggest a new mechanism of hyperforin in depression treatment. Future in vitro and in vivo studies are required to further characterize the mechanisms of hyperforin.

Recent advancements in mass spectrometry have facilitated new analytical approaches capable of comprehensively characterizing metabolites in biological samples. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) combines excellent mass accuracy (ppm<1) and ultra-high resolution, which enables the separation and identification of individual components within complex mixtures, and the determination of elemental composition for each detected mass. FTICR-MS is an ideal method for non-targeted metabolomics as the majority of small molecular compounds (100-1000 Da) in a biological sample can be detected. The objective of this research was to investigate metabolomic alterations associated with key cellular processes deemed fundamental to cancer development and progression. Differentiating U937 cells, fibroblasts synchronously progressing through the cell cycle and a transformed cell line containing a temperature sensitive oncogene were collected and subject to FTICR-MS analysis for non-targeted comprehensive metabolomics. Putative metabolite identifications were confirmed with targeted metabolite analysis using multiple reaction monitoring triple quadrupole mass spectrometry. Analysis of the resulting metabolic profiles revealed robust metabolic alterations associated with fundamental cellular processes. Changes in glycerolipid content were observed in all cellular processes studied. During cell cycle progression, elevated levels of triacylglycerols and vinyl acylglycerols were detected as cells approached mitosis; increased levels of plasmalogens were detected during the induced differentiation of human leukemic cells and activation of the oncogene p130gag-fps in fibroblasts resulted in increased levels of phospholipids, including plasmalogens. When de novo fatty acid synthesis was inhibited in the differentiation cell model, the cells were not able to complete the differentiation process. Removal of the inhibitor resulted in increased lipid content, particularly plasmalogens, and the continuation of differentiation, suggesting a requirement for the de novo synthesis of lipids during this cellular process. This work demonstrates the advantages of non-targeted metabolic profiling for identifying non-intuitive metabolic associations with specific cellular processes. Collectively, the results of this thesis have implicated glycerolipids, in particular phospholipids, in the processes of cell cycle progression, differentiation and tumourigenic transformation. A broadened understanding of the role of global lipid metabolism during fundamental cellular processes may one day lead to new approaches for their modulation, and potentially new therapeutic strategies.

Members of the Mef2 transcription factor family are extensively studied within the muscle field for their ability to cooperate with the myogenic regulatory factors MyoD and myogenin during muscle differentiation. Although it is known that Mef2 pre-mRNAs undergo alternative splicing, the different splice forms have not been functionally annotated. In this thesis, my studies aimed to characterize three Mef2D splice forms: MEF2Dα'β, MEF2Dαβ, MEF2Dαø. Our results show that MEF2D splice forms can be differentially phosphorylated by p38 MAPK and PKA in vitro. Gene expression analysis using cell lines over-expressing each Mef2D splice form suggests that they can differentially activate desmin, myosin heavy chain and myogenin expression. Mass spectrometry analyses from our pull-down assays reveal known and novel MEF2D binding partners. Our work suggests that Mef2D splice forms have overlapping but distinct roles and provides new insight into the importance of Mef2D alternative splicing during skeletal myogenesis.