A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.

Our understanding of subsurface flow depends on assumptions of how event characteristics and spatial scale affect the relationships between subsurface water velocity, discharge, water table dynamics, and runoff response. In this thesis, three chapters explore some of these patterns for a hillslope and small watershed in coastal British Columbia. In the first chapter, tracers were applied under natural and steady state conditions to determine the relationship between lateral tracer velocities and various hillslope and event characteristics; such as hillslope subsurface flow, rainfall intensity, water table level, hillslope length, and antecedent condition. The results showed that preferential flow made up a large percentage of the subsurface flow from the gauged hillslope. Flow velocities as measured by tracers were affected by slope length and boundary conditions. The flow velocity was most closely related to the rainfall intensity, and changes in flow velocity were large compared to the changes in the water table. In the second chapter, the preferential flow features that transmitted water during steady state were investigated by staining the soil with a food dye solution and excavating the soil. These data were used to explore the link between the topographical factors (slope and contributing area), the network of preferential features and soil properties. The contributing area appeared to be an indicator of the size of the preferential features and their connectivity. In the final manuscript chapter, water table level and stream discharge measurements were used to determine if areas within a watershed with runoff dominated by preferential flow could be grouped based on the observable physical information such as slope, contributing area, distance to stream, and vegetation. Preferential flow made the water table responses dynamic and thus, distinct zones could not be identified. Models of the water table – runoff were not able to predict the water table response for other sites with similar physical characteristics. Even though there was high variability in the results, the patterns and relationships revealed in this thesis conform to existing conceptual models of hillslope subsurface preferential flow. These patterns and relationships may be useful in developing or validating numerical models.

The aim of this study was to define and examin the capabilities of the low voltage transmission electron microscope LVEM 5 in the detection and identification of viruses, taking into consideration various contrasting agents, and a comparison to data from HV TEM.

Our understanding of subsurface flow depends on assumptions of how event characteristics and spatial scale affect the relationships between subsurface water velocity, discharge, water table dynamics, and runoff response. In this thesis, three chapters explore some of these patterns for a hillslope and small watershed in coastal British Columbia. In the first chapter, tracers were applied under natural and steady state conditions to determine the relationship between lateral tracer velocities and various hillslope and event characteristics; such as hillslope subsurface flow, rainfall intensity, water table level, hillslope length, and antecedent condition. The results showed that preferential flow made up a large percentage of the subsurface flow from the gauged hillslope. Flow velocities as measured by tracers were affected by slope length and boundary conditions. The flow velocity was most closely related to the rainfall intensity, and changes in flow velocity were large compared to the changes in the water table. In the second chapter, the preferential flow features that transmitted water during steady state were investigated by staining the soil with a food dye solution and excavating the soil. These data were used to explore the link between the topographical factors (slope and contributing area), the network of preferential features and soil properties. The contributing area appeared to be an indicator of the size of the preferential features and their connectivity. In the final manuscript chapter, water table level and stream discharge measurements were used to determine if areas within a watershed with runoff dominated by preferential flow could be grouped based on the observable physical information such as slope, contributing area, distance to stream, and vegetation. Preferential flow made the water table responses dynamic and thus, distinct zones could not be identified. Models of the water table – runoff were not able to predict the water table response for other sites with similar physical characteristics. Even though there was high variability in the results, the patterns and relationships revealed in this thesis conform to existing conceptual models of hillslope subsurface preferential flow. These patterns and relationships may be useful in developing or validating numerical models. / Forestry, Faculty of / Graduate

Background The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas. Principal Findings Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains. Significance Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

The methodology of visualization of soft tissue is in biology and medicine a topic for many years. During this period there were approving many techniques how to achieve accurate and authentic image of the researched object or structure. X-ray computed tomography is very helpful to get this goal but is necessary to improve contrasting techniques as well as the techniques of image post-processing. This thesis deals with imaging soft tissue. Specifically, it focuses on mouse liver contrasting with the artificial resin Microfil. Thesis also describes image processing technique (thresholding and region growing) for the data of the measurement with the goal of the visualization of the sample in 3D.

This diploma thesis deals with a segmentation of soft tissues in facial part of mouse embryos in Matlab. Segmentation of soft tissues of mouse embryos was not fully automated and every case needs a specific solution. Solving parts of this issues can provide valuable data for evolutionary biologists. Issues about staining and segmentation techniques are described. On the basis of accessible literature otsu thresholding, region growing, k-means clustering and segmentation with atlas were tested. In the end of this paper are those methods tested and evaluated on 3D microtomography data.