Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the 'clearing-up' process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies.

Alkaline glutaraldehyde (GTA) has been used as a high level chemical disinfectant and sterilant for many years and is known to kill a broad spectrum of organisms ranging from vegetative eukaryotes to bacterial endospores. Although the mechanism of sporicidal action has been studied on numerous occasions, GTA's exact mechanism(s) of action are still debated. In addition to the uncertainty of GTA's mechanism(s) of action, GTA has also shown significant variability in the time required to kill endospores and naked viruses. A better knowledge of the lethal mechanism(s) of GTA is needed to understand this discrepancy in kill times for GTA against spores of different species. Similar trends have been observed in GTA's activity against non-enveloped viruses. Based on previous work, one proposed major mechanism of GTA's sporicidal activity is related to the number of available primary amines located on the surface of microbes. In this study, we have compared the efficacy of GTA on spores from 5 Bacillus species. We have also developed a method for staining these spores with amine reactive dyes to create fluorescent profiles correlating to the abundance of free amino groups on each spore type. We also describe a method for staining non-enveloped viruses to identify exposed primary amino groups on capsid proteins that may act as targets for GTA, using amine reactive Gold nanoparticles. We found that GTA 6-Log10 reduction times for various spore types varied at both the batch and species level. Spore coat thickness and fluorescence were useful tools in predicting the susceptibility of spores to GTA. Amine reactive gold particles (AuNPs) also proved useful in identifying virus susceptibility to GTA. Ultimately, more reliable disinfection testing methods are needed, and caution should be used when trying to extrapolate data generated from surrogate organisms to other species.

Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the `clearing-up¿ process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies. / Covance Laboratories Ltd.

Poor water quality is a global health concern affecting one billion people around the world. It is important to monitor water sources in order to maintain the quality of our drinking water and to avoid disease outbreaks. Targeting Escherichia coli as a faecal indicator is a widely used procedure, but the current methods are time consuming and not adequate to prevent spreading of faecal influence.   This Master thesis demonstrates the development of a near infrared fluorescence flow cytometer sensor system targeting Escherichia coli, using fluorescently labeled chicken IgY antibodies. The near infrared light was chosen to avoid fluorescence from blue-green algae that are present in the water source.   The hardware was developed with a 785  nm laser line to detect Alexa Fluor 790 labeled antibodies, using a photomultiplier tube or two different CMOS cameras. The antibodies were labeled using a commercial labeling kit, and evaluated using antibody binding assays and the developed hardware.   The IgY antibodies were successfully labeled with Alexa Fluor 790 and the function was maintained after the labeling process. The result demonstrates the principles of the sensor system and how it solved to the problem with fluorescence from blue-green algae. An aperture was used to overcome the suboptimal laser and filter setup, and to increase the sensitivity of the system. However, only a small fraction of the cells could be detected, due to challenges with the focal depth and loss of sensitivity in the photomultiplier tube at near infrared wavelengths. Further development is required to create a working product.

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.