The CsgD protein is the master regulator of a pathway leading to the formation of curli, in essence regulating the switch between a motile and a sessile lifestyle for bacteria. The 5’-UTR region of the csgD mRNA is a hotspot for multiple regulatory small RNAs (sRNA) involved in a complex regulatory network. Even though it is previously known how the interaction takes place it is unknown how sRNA binding affects the translational activity. In order to suggest a mode of regulation a mutational assay was performed by making changes in the csgD 5’-UTR and investigate what the translational effects were. Mutations in different regions are shown to affect the translation levels in various ways.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

The purpose of this investigation was to create and optimise a capture assay for the detectionof anti-drug antibodies (ADA) in human plasma, using Biacore. We also dealt with the nonspecificplasma binding to mouse-derived anti-biotin which may occur in the capture assay.By paying attention to these things we aimed at reaching as high sensitivity as possible for theADA detection. The capture assay also benefited and gained flexibility from using the same regenerationsolution irrespective of drug and from having a composition that minimises the risk ofdamaging drug epitopes.

Biofouling is a major issue in applying nanofiltration and reverse osmosis technologies for wastewater treatment. Biofilm formed on the surface of membranes will severely decline the flux and cause energy waste. In this study, a novel biofouling control method that applies D-amino acids to inhibit biofilm formation was investigated. The D-amino acids previously reported to inhibit biofilm formation and disrupt existing biofilm – D-tyrosine and the mixture of D-tyrosine, D-tryptophan, D-leucine and D-methionine were tested. Pseudomonas aeruginosa and Bacillus subtilis were used as model Gram-negative and Gram-positive bacteria, respectively. D-amino acids have little effect and some effect on inhibition of biofilm formation and disruption of exiting biofilm to Pseudomonas aeruginosa, but have good effect to Bacillus subtilis. A commonly used microtiter plate assay for quantitative biofilm measurement was systematically evaluated and optimized for screening biofilm control agents. The effect of D-tyrosine on inhibition of organic fouling and P. aeruginosa biofouling on NF90 membrane surface in bench scale dead end filtration experiment was examined, which shows that D-tyrosine can effectively inhibit organic fouling and P. aeruginosa biofouling on NF90 membrane surface.

The in situ proximity ligation assay (in situ PLA) is an immunoassay that enables directvisualisation of single protein targets or protein interactions in cell or tissue samples. This project revolves around designing and introducing several novel multiplexable components tobe used in conjunction with Olink Bioscience's Duolink product line. In this report, a novel in silico approach to DNA oligomer interaction design is presented. Using this in silico method, a multiplexed system of DNA oligomers has been designed andevaluated using in situ PLA and fluorescence microscopy.

The diagnosis of cancer today is obsolete, depending upon pattern recognition and non-quantifiable data. The time consuming diagnosis is often performed on biopsies, fixed using non standardised procedures, and leaves room for dubious results. The diagnosis is also invasive, exposing patients to risk of infections and discomfort due to the need of tissue samples. The knowledge about changes in protein expression levels related to cancer can instead be utilized to generate a new diagnostic tool. By adapting the in situ proximity ligation assay (in situ PLA) to cells in solution, it is possible to detect proteins, or protein interactions, within cells without the need for tissue samples. Since the method is both highly sensitive and specific, it delivers reliable results. In this report, the in situ PLA method for cells in solution is combined with flow cytometry readout. Hence, a new and less invasive diagnostic tool for cancer, delivering highly accurate high throughput single cell analysis, may be on the rise.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

Oxidative stress has been associated with many disease conditions in adults and neonates based on clinical and post mortem studies. Trauma is the commonest cause of oxidative stress. However a gap in knowledge of the natural history of oxidative stress in humans was identified as most studies have been post mortem or in animals. <p>The aim of this research is to understand treat and oxidative stress in trauma and surgical patients. The study involved three components including: the development and evaluation of the novel oxistress assay; study of clinical trauma and oxidative stress; and clinical trial of alanyl-glutamine supplementation following major surgery. The novel oxistress assay was used on urine samples in the normal population to determine reference values and subsequently on hospital patients to determine sensitivity and specificity. The study of clinical trauma and oxidative stress evaluated plasma antioxidants (FRAP assay), red cell glutathione (Asensis method), plasma and urine protein carbonyl (Levines method) and total oxidants in plasma and urine (oxistress assay) over 7 day period following trauma. The clinical trial was a double blind study of 69 major surgery patients evaluating biochemical and clinical parameters over 7 day period in comparison with pre-operative status. <p>The novel oxistress assay proves to be a sensitive and accurate bedside diagnostic tool for oxidative stress. It can also be used in the laboratory setting. Oxidative stress is associated with increased trauma severity resulting in antioxidant depletion, strong oxidant production and protein degradation. The presence of pre-morbid medical factors also increased oxidative stress in trauma patients. Oral alanyl-glutamine supplementation (0.3 g/kg) increased plasma glutamine and antioxidant levels while decreasing urine oxidant levels. It significantly reduced hospital stay in non-cancer and higher disease complexity patients. The intervention also reduced the resource intensity weighting (RIW) score. <p>Oxidative stress is a clinical problem in surgery and trauma patients that can now be easily diagnosed at the bedside using the novel oxistress assay. Treatment with alanyl-glutamine is effective in reducing oxidative stress and improving clinical outcome. It is highly recommended probably at a higher dose in order to achieve optimal results.