Content is focused on fluorescence lifetime imaging techniques. Fluorescence lifetime is computed from data acquired with using of Leica TCS SP8X confocal microscope sequential scanning. Algorithms and software for the computation, imaging and analysis of fluorescence lifetime is presented. Software is allowing both 2D and 3D imaging of fluorescence lifetime. Techniques are used for fluorescence lifetime imaging of mesenchymal cells and fibroblasts tainted with SPIO-Rhodamin complex.

This work focuses on examination of hyaluronan-sulfactant aggregates in term of determination of aggregate number. The value of critical micellar concentration (CMC) cetyltrimethylammonium bromide (CTAB) in three different solvents (water, phosphate buffer and physiological solution). Next the effect of the native hyaluronan supplement on the value of CMC was examined. It wasfound out that the solvent has the biggest effect on the value of CMC whilst the hyaluronan supplement affects CMC only a little. The aggregate number (Nagg) CTAB and the effect of the native hyaluronan supplement were determined out by means of fluorescence quenching. Pyrene was used as a fluorescence probe. Iodine and cetylpyridinium chloride (CPC) were used as quenchers. Sulfactant was dissolved in three different solvents (water, phosphate buffer and physiological solution). Not only the hyaluronan supplement but also the used solvent has the strong effect on the value of the aggregate number. When using 10mM CTAB dissolved in physiological solution the value of Nagg was 119 ± 4 while the value was half in buffer. Then we found out that in most cases the hyaluronan supplement reduces the value of the aggregate number.

Call number: LD2668 .T4 1979 H83 / Master of Science

No description available.

La cytogénétique est une branche de la génétique qui étudie les chromosomes et leur intégrité ainsi que les conséquences de leur transmission et de leur expression. Plusieurs syndromes et maladies ont pu être expliqués par cette discipline. Certaines anomalies chromosomiques de structure ont d'ailleurs contribué à identifier des gènes, des fonctions de gènes ou à caractériser des structures chromosomiques. Dans cet ordre d'idées, nous avons utilisé deux isodicentriques du bras long du chromosome Y (idic(Y)(p11.3)) pour étudier la fonction d'une protéine du kinétochore, CENP-B, dans le mécanisme d'inactivation de kinétochore. Pour ce faire, nous avons premièrement fait une caractérisation cytogénétique des deux idic(Y)(p11.3) à l'aide des techniques de cytogénétique classique et de cytogénétique moléculaire. Nous avons ainsi déterminé approximativement le point de cassure des deux isodicentriques, soit en Yp11.3. Comme les deux chromosomes avaient une structure très semblable, mais que les patients présentaient un phénotype clinique très différent, nous avons investigué les niveaux de mosaïcisme dans différents tissus chez les deux patients. Il est connu qu'un chromosome possédant deux centromères capables de former le kinétochore peut être très instable lors des divisions cellulaires. Ainsi, la cellule a mis au point un mécanisme permettant d'inactiver un des kinétochores du chromosome dicentrique. Une récente revue a proposé que la protéine CENP-B jouerait un rôle dans ce mécanisme. Cependant, comme le chromosome Y ne possède pas la séquence d'ADN liant cette protéine, il était intéressant de vérifier si une inactivation des kinétochores avait eu lieu dans nos idic(Y)(p11.3). À l'aide d'un anticorps dirigé contre la protéine CENP-C, connue comme un marqueur de kinétochore actif, nous avons montré que plus de 40% des chromosomes dicentriques avaient subi une inactivation d'un de leur kinétochore. Enfin, la présence de la protéine CENP-B dans ces kinétochores a été étudiée. Nous avons montré que la protéine CENP-B était présente à tous les autres centromères, sauf ceux de l'idic(Y)(p11.3). Ainsi, nous proposons que la protéine CENP-B n'est pas impliquée directement dans le mécanisme d'inactivation de kinétochore du chromosome Y. Par contre, nous ne pouvons pas exclure qu'elle joue un rôle indirect, soit par une interaction protéine/protéine, soit à une étape en amont dans le mécanisme d'inactivation.

Submitted in complete fulfilment for Masters Degree in Technology: Biotechnology, Durban University of Technology, 2013. / A comparative thermostability analysis of different partially purified xylanases from Rhodothermus marinus, Bacillus halodurans, Thermomyces lanuginosus and Pulpzyme HC was studied using differential scanning fluorometry (DSF), fluorescence spectroscopy and circular dichroism (CD). The R. marinus xylanase was found to have an optimum temperature and pH of 90oC and 6 respectively while the B. halodurans xylanase was optimally active at 70oC and a broad range of alkaline pH of 8 - 10. The commercially available xylanase from T. lanuginosus showed optimal activity at 50oC and pH 7 while the Novozyme xylanase Pulpzyme HC showed optimal activity at 60oC and pH 7. Fluorescence spectroscopy monitored the microenvironment and fluorescence emission of Trp residues. In their native folded state, Trp are generally located in the core of the protein but during unfolding they become exposed. The fluorescence changes as the enzyme undergoes denaturation due to conformational changes and exposure of Trp residues. Differential scanning fluorometry (DSF) monitors thermal unfolding of proteins in the presence of a fluorescent dye such as Spyro Orange. A wide range of buffers were tested for their ability to increase the xylanase stability. T. lanuginosus had the greatest increase in melting temperature with 0.73M Bis Tris pH 6.5 and peaked highest at 78°C. The B. halodurans xylanase exhibited high pH stability (pH 4-10) and exhibited very little change in melting temperature, from 74°C-77°C over the twenty four different conditions. The R. marinus xylanase had no increase in melting temperature showing a maximum melting temperature of 90oC. Circular dichroism (CD) measures unequal absorption of right- and left-handed circularly polarized light by the molecule. The xylanase from R. marinus exhibited the lowest ΔG of 34.71kJ at 90°C as was expected. The B. halodurans xylanase showed a much higher ΔG of -52.71 at its optimum temperature of 70°C when compared with the xylanases from R. marinus and T. lanuginosus. When comparing the three xylanases activities at 70°C, it can be seen that the B. halodurans xylanase exhibited a lower relative activity then both R. marinus and T. lanuginosus xylanases. All three techniques offered different information on the structure and function relationship. Fluorescence spectroscopy, the change in conformation due to fluorescence emission as a result of increased temperature and salt concentrations. DSF, optimal conditions for increased stability and activity at higher temperatures and CD, conformational changes, the fraction of folded protein and change in Gibbs free energy over a range of temperature. / National Research Foundation

This thesis presents improvements to a method for measuring the production of biogenic silica (bSiO2) by diatoms, a group of microscopic algae with siliceous cell walls (frustules) that dominate the marine cycling of silicon (Si) and account for a significant proportion of global marine primary productivity. Using the fluorescent dye PDMPO, diatom bSiO2 can be labeled as it is produced and then quantified using fluorometry to determine community-wide bSiO2 production. A distinct advantage of PDMPO over more traditional tracers of bSiO2 production is that the combination of measurements of PDMPO by fluorometry and by fluorescence microscopy allows for the quantification of cell (and thus taxa) specific bSiO2 production within a mixed community. However, the robustness of PDMPO as a quantitative tracer of diatom bSiO2 production has not been sufficiently investigated. To address this, experiments were conducted both in the lab, and at two field locations where diatoms are known to be abundant, namely the continental shelf off the west coast of Vancouver Island, and Saanich Inlet, a highly productive fjord located on southern Vancouver Island. Laboratory culture experiments demonstrated that concentrations of PDMPO >500 nmol L-1 reduced growth rate in the diatom Thalassiosira pseudonana, and affected the Si:PDMPO ratio of incorporation. The relationship between SiO2 and PDMPO incorporation was significantly affected by diatom species, though this effect was small (8%) when cells were lysed. From these experiments, a Si:PDMPO incorporation ratio of 4200 ± 380:1 was determined, which predicted 30% more bSiO2 production for PDMPO incorporation than previous studies, and better agreed with bSiO2 production rates determined using established methods in Saanich Inlet. However, bSiO2 production rates were over-estimated by the PDMPO method when rates were less than 1 µmol L-1 d-1. In a few cases, this occurred when dinoflagellates were numerically dominant, but for the majority of samples, dinoflagellates were low in abundance, and over-estimation by PDMPO may be related to low dissolved Si(OH)4 concentration. Protocols for quantifying PDMPO fluorescence by microscopy were optimized by using a low numerical aperture microscope objective. Additionally, measurements of fluorescence intensity were calibrated using a fluorescent microscope slide as a standard, which served to correct for unevenness of illumination across the field of view. With these protocol modifications, quantification of PDMPO by microscopy agreed with PDMPO measured by fluorometry. When PDMPO was measured by microscopy in the field, the contribution of diatom taxa to PDMPO fluorescence differed from their contribution to cell numbers. In many cases this was due to large diatom taxa producing more bSiO2 per cell than smaller taxa. However, much of the difference between cell numbers and PDMPO fluorescence was not explained by differences in cell size. This suggests that the diatom taxa had different specific bSiO2 production rates, which could be estimated using PDMPO. This thesis highlights the strength of the PDMPO tracer for understanding diatom community dynamics. The use of PDMPO should allow the relationship between diatom community composition, growth and productivity to be better illuminated in the oceans. / Graduate / 0416 / jelong@uvic.ca

In the field of cell biology, there is an increasing use of time-lapse data to understand cellular function. Using automated microscopes, large numbers of images can be acquired, delivering videos of cell samples over time. Analysing the images manually is extremely time consuming as there are typically thousands of individual images in any given sequence. Additionally, decisions made by those analysing the images, e.g. labelling a mitotic phase (one of a set of distinct sequential stages of cell division) can be subjective, especially around transition boundaries between phases, leading to inconsistencies in the annotation. There is therefore a need for tools which facilitate automated high-throughput analysis. In this thesis we develop systems to automatically detect, track and analyse sub-cellular structures in image sequences to address biological research needs in three areas: (i) Mitotic phase labelling, (ii) Mitotic defect detection, and (iii) Cell volume estimation. We begin by presenting a system for automated segmentation and mitotic phase labelling using temporal models. This work takes the novel approach of using temporal features evaluated over the whole of the mitotic phases rather than over single frames, thereby capturing the distinctive behaviour over the phases. We compare and contrast three different temporal models: Dynamic Time Warping, Hidden Markov Models, and Semi Markov Models. A new loss function is proposed for the Semi Markov model to make it more robust to inconsistencies in data annotation near transition boundaries. We then present an approach for detecting subtle chromosome segregation errors in mitosis in embryonic stem cells, targeting two cases: misaligned chromosomes in a metaphase cell, and lagging chromosomes between anaphase cells. We additionally explore an unsupervised approach to detect unusual mitotic occurrences and test its applicability to detecting misaligned metaphase chromosomes. Finally, we describe a fully automated method, suited to high-throughput analysis, for estimating the volume of spherical mitotic cells based on a learned membrane classifier and a circular Hough transform. We also describe how it is being used further in biological research.

Rotaviruses are the single most common cause of fatal and severe childhood diarrhoeal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialise in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organisation. To test these opposing models, a novel single-molecule assay was developed for the capture and identification (CID) of newly synthesised specific RNA transcripts. CID combines the hybridisation of transcripts with biotinylated and FRET compatible labelled ssDNAs with the implementation of recent developments in single molecule fluorescence such as alternating laser excitation (ALEX) and total internal reflection fluorescence (TIRF) microscopy. CID identifies and quantifies specific transcripts of rotavirus based on a FRET/Stoichiometry (E*/S) value of the hybridised labelled probes. I used CID to pull down the capsid on the surface slide and identify partially extruded transcripts of three different segments 2, 6 and 11. The findings presented in this thesis support a model in which each channel specialises in extruding transcripts of a specific segment, that in turn is linked to a single transcription complex. The method can be extended to study other transcription systems including E.coli, and can be further developed as a potential diagnostic tool.

The extensive use of nanoparticles (NPs) has started receiving increased attention because of the knowledge gaps regarding their fate in the environment and the possible impact on the environment and human health. The production of silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (TiO2-NPs) is increasing and it is expected that, due to their great number of applications, their concentration in waste streams will increase in the future. The presence of NPs in waste streams may affect the treatment process (e.g., composting) and, if they are not successfully removed from the waste streams, their presence in the treated waste (e.g., compost) may present an environmental risk. Composting of the biodegradable fractions of municipal solid waste (MSW) is a widely used waste management practice, mainly because it is a cost-effective treatment technology and the final product (i.e., compost) presents several benefits to the environment, particularly as a soil conditioner. The overall aim of this thesis is to assess the effect of Ag-TiO2NPs and AgNPs that may be present in the biodegradable fractions of municipal solid waste on composting and subsequent soil application of compost. For that purpose in-vessel composting of artificial municipal solid waste contaminated with commercial nanoparticles was investigated at laboratory scale, simulating a range of relevant concentration levels. Subsequently, the fate of NPs present in mature compost use as a top-layer soil conditioner was investigated using a column approach at laboratory scale. The toxicity effect of NPs present mature compost on plant growth was further investigated. The impact of NPs during composting was assessed by monitoring the temporal dynamics of organic matter (OM) using Excitation Emission Matrix (EEM) fluorescence spectroscopy. The fate of NPs following application of contaminated mature compost as a top-soil conditioner and potential release to groundwater was investigated using a column leaching experiment while the phytotoxicity of mature compost contaminated with NPs was assessed using a seed germination bioassay. Finally, to investigate further possible environmental impacts due to the application of mature compost contaminated with NPs to soils, a Life Cycle Assessment (LCA) was conducted. The impact of commercial Ag-TiO2 NPs and AgNPs on the in-vessel composting of biodegradable municipal solid waste was investigated over 21 days, using initial concentrations of 0, 5, 10, 20 and 50 mg Ag / kg of OM. Microbial activity was inhibited in the biodegradable waste reactors using 2% NaN3 to evaluate abiotic losses. Physicochemical parameters such as pH, ash content, weight loss, and the formation of humic substances (HS) were determined after 0, 4, 7, 14 and 21 days of composting and after a maturation phase. The results indicated that the presence of 2% NaN3 in biodegradable MSW inhibited effectively the microbial activity during the first week of composting. The microbial population was activated during the second week of composting but the decomposition rate was so low that did not result in the formation of humic substances (HS) following 21 days of composting when 2% NaN3 was used. Both treatments, using Ag-TiO2-NPs and AgNPs, did not show any inhibition of the decomposition process for all the tested concentrations and EEM peaks shifted towards the HS region during in-vessel composting. Higher inorganic carbon removal resulted from NP-contaminated compost with higher NP concentrations. This may indicate that the formation of humins was higher for non-contaminated compost and decreased as the NP concentration in waste increased. The shift of the peaks towards the HS region during composting for all the treatments suggested that NPs did not have an effect on humification and therefore on compost stability. The leaching properties of the NP-contaminated compost were investigated using a column leaching test. Five samples of leachate, of 50 mL each, were collected. The highest concentrations of HS were observed in the first two leaching samples. The leaching results suggested that only a low percentage of the total NPs (in weight) in compost, up to ca. 5% for Ag and up to ca. 15% for Ti, leached out from the columns, which was assumed the amount that potentially could leach to the environment. These results suggested that NPs will mainly accumulate in soils’ top layers following application of compost contaminated with NP. The phytotoxicity of NP-contaminated compost was assessed using a seed germination bioassay and the germination index was then calculated. The results indicated that the NP-contaminated compost did not present any toxic effects to cress germination. The possible environmental impacts due to the NP-contaminated compost application to soils were investigated by conducting a comparative LCA study. The LCA study indicated that the effects of NP-contaminated compost to human health and ecosystems endpoint categories increased due to the presence of NPs. The risks are associated with terrestrial ecotoxicity and human toxicity midpoint categories and are mainly attributed to the accumulation of Ag to soils.