Washington University Record, October 17, 1996

https://digitalcommons.wustl.edu/record/1736/thumbnail.jp

Photochemical and photophysical reaction dynamics of chemical and biological systems

Texto en inglés, y resumen y conclusiones en inglés y españolEl proyecto realizado en esta Tesis consiste en el desarrollo y aplicación de metodologías teóricas y computaciones, usadas en la descripción estática y dinámica de procesos fotofísicos y fotoquímicos de compuestos químicos y de interés biológico. Estas metodologías computacionales fueron implementadas aplicando técnicas punteras usadas en el campo de la ciencia de la computación. La presente Tesis se compone de 4 bloques principales. El primero de estos bloques estudia el proceso de transferencia de energía intermolecular, especialmente transferencia de energía triplete. Por su parte, el segundo bloque examina los mecanismos y comportamiento dinámico de dos procesos biológicos fotoinducidos de intereses tecnológico. Mientras el tercer bloque, consiste en el estudio del efecto de fuerzas externas sobre las propiedades espectroscópicas de los sistemas moleculares. Finalmente, el último bloque considera el diseño de dispositivos moleculares que usan cambios conformacionales fotoinducidos en la generación de movimiento controlado. En la sección de transferencia de energía ha sido estudiado el problema de encontrar las principales coordenadas moleculares que modulan de forma eficiente el proceso de transferencia de energía triplete. Así mismo, se llevó a cabo una aproximación dinámica al proceso de transferencia energía triplete a temperatura constante, que completa el estudio estático llevado a cabo en la primera parte de la sección. En la primer parte del segundo bloque, se lleva a cabo la caracterización estática y dinámica de modelos moleculares en el estudio de los fenómenos de quimioluminiscencia y bioluminiscencia. Donde se analiza en detalle el mecanismo de descomposición concertado de la familia de 1,2-dioxetanes. Por su parte, en la segunda sección de este bloque es analizado el efecto del ambiente proteico en la emisión de fluorescencia de la proteína fluorescente IrisFP. En el tercer bloque de la presente Tesis ha sido explorado la respuesta fotodinámica de sistemas moleculares al efecto de una fuerza externa. Discutiendo en detalle el efecto sobre el cambio de la reactividad química a causa de la disrupción del sistema molecular por parte de la fuerza externa. Simultáneamente, se muestran los resultados obtenidos con respecto al cambio en las propiedades espectroscópicas debidos a la fuerza externa y se plantea su posible aprovechamiento en aplicaciones tecnológicas Finalmente en el último bloque del presente trabajo, se expone el diseño y operación de dispositivos moleculares como motores e interruptores controlados mediante ciclos fotoinducidos, controlado la rotación unidireccional en el caso de los motores moleculares a través de puentes de hidrógenos

Molecular Aspects of Hepatitis C Virus Infection and Associated Mitochondrial DNA Damage

Hepatitis C virus (HCV) is the main cause of viral hepatitis in the UK and leads to chronic liver disease in many infected individuals. There is a substantial burden on diagnostic laboratories to provide rapid, cost-effective tests for monitoring HCV infection. Commercial assays are expensive and so the development of validated in-house methods is beneficial. This thesis describes the development and implementation of rapid and inexpensive real-time PCR assays for HCV quantitation and genotyping to support clinical practice. Additionally, the development of methods for defining HCV isolates at the subtype level, important in epidemiological and transmission studies, is described. These assays were utilised in a study on spontaneous HCV clearance, the results of which suggest that HCV type 1 infection and younger age at infection are factors which are associated with spontaneous viral clearance. Chronic HCV infection is linked to oxidative stress with numerous deleterious cellular effects. Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA making it an ideal marker to assess the overall level of cellular DNA damage - including deletions and mutations. This thesis illustrates the development of real-time PCR assays to detect and quantify two major mtDNA deletions. The D-loop region of mtDNA is particularly prone to damage - with two well recognised 'hotspots of mutation'. The creation of an RSCA (heteroduplex-based) method using capillary electrophoresis, to detect and quantify damage in this region, is described. To evaluate the clinical utility of these assays, a study of mtDNA damage in patients with liver disease was undertaken. The aim of this study was to identify whether chronic HCV infection results in increased levels of mtDNA damage compared to other liver pathologies. Low levels of mtDNA deletions were detected in the majority of liver biopsy specimens and there was no correlation between liver aetiology and quantity of deletions. The RSCA method identified numerous D-loop mtDNA species within the liver tissue of several individuals. There was no correlation between liver aetiology and the presence of multiple D-loop species

The Prostaglandin E2 Receptor Subtype 3E and its involvement in tauopathies

Neuroinflammation is becoming increasingly recognised as key to the pathogenesis of Alzheimer’s disease and tauopthies. Epidemiological studies report a delay in the onset of Alzheimer’s in subjects using nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit enzymes in the cyclooxygenase-2 (COX-2) pathway which play a key role in synthesising prostaglandin E2 (PGE2) from arachidonic acid. PGE2 has been implicated in preclinical stages of Alzheimer’s disease, where elevated levels of PGE2 in the cerebrospinal fluid can be found, as well as aberrant amyloid processing in experimental models of disease. PGE2 signals via 4 E-prostanoid (EP) receptors, EP1-EP4, all G-protein coupled receptors (GPCR). The EP3 receptor, the most abundant PGE2 receptor in the brain, is unique in that it is alternatively spliced giving rise to species specific isoforms. One of the EP3 receptor isoforms, EP3Re, is human specific and an incidental finding within a project to investigate its distribution in brain, suggested that it could be associated with tau tangles. The aim of this project was to further investigate the unknown distribution of EP3Re in human brain, to determine its signalling mechanism and explore whether any meaningful interaction between EP3Re, tau and its pathology exists. We use immunohistochemistry, proximity ligation assays and electron microscopy to map out the distribution of EP3Re in the human brain and explore the interaction between EP3Re and tau. We also use gene reporter and second messenger assays to characterise EP3Re signalling and what role if any this may be playing in tauopathies. We show that EP3Re is expressed throughout the brain, with strong expression in brain stem nuclei, and signals predominantly through a Gi coupling pathway. Moreover, using a combination of human tissue, primary cell lines and neurons derived from induced pluripotent stem cells, we show that EP3Re appears to be associated with tau neurofibrillary tangles in disease. We also show, using the EP3 agonist sulprostone, that signalling through the receptor increases tau phosphorylation in our cellular systems. Further work will be required to fully clarify the specificity of the interaction and understand the mechanism behind this and if targeting inflammatory EP3Re signalling has the potential to affect tau pathology in disease.John Van Geest studentship; Alzheimer's Research UK (ARUK