Ph D.

<p>The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, <i>Hypocrea jecorina</i> and <i>Phanerochaete chrysosporium</i>, including both the action of the individual enzymes and their synergistic interplay. </p><p>The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases.</p><p>The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface.</p><p>A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases.</p><p>Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation.</p><p>Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.</p>

The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, Hypocrea jecorina and Phanerochaete chrysosporium, including both the action of the individual enzymes and their synergistic interplay. The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases. The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface. A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases. Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation. Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.

RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold. To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity. The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the iv collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues. In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n. Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.

Development of the central nervous system requires the precise coordination of intrinsic genetic programs to instruct cell fate, synaptic connectivity and function. The MEF2 family of transcription factors (TFs) plays many essential roles in neural development; however, the mechanisms of gene regulation by MEF2 in neurons remain unclear. This dissertation focuses on the molecular mechanisms by which MEF2 binds to the genome, activates enhancers, and regulates gene expression within the developing nervous system. We find that one MEF2 family member in particular, MEF2D, is an essential regulator of the development and function of retinal photoreceptors, the primary sensory neurons responsible for vision. Despite being expressed broadly across many tissues, in the retina MEF2D binds to retina-specific enhancers and regulates photoreceptor-specific transcripts, including critical retinal disease genes. Functional genome-wide analyses demonstrate that MEF2D achieves tissue-specific binding and action through cooperation with a retina-specific TF, CRX. CRX recruits MEF2D away from canonical MEF2 binding sites by promoting MEF2D binding to retina-specific enhancers that lack a strong consensus MEF2 binding sequence. MEF2D and CRX then synergistically co-activate these enhancers to regulate a cohort of genes critical for normal photoreceptor development. These findings demonstrate that MEF2D, a broadly expressed TF, contributes to retina-specific gene expression in photoreceptor development by binding to and activating tissue-specific enhancers cooperatively with CRX, a tissue-specific co-factor. A major unresolved feature of MEF2D function in the retina is that the number of MEF2D binding sites significantly exceeds the number of genes that are dependent on MEF2D for expression. We investigated causes of this discrepancy in an unbiased manner by characterizing the activity of MEF2D-bound enhancers genome-wide. We find that many MEF2D-bound enhancers are inactive. Furthermore, less than half of active MEF2D-bound enhancers require MEF2D for activity, suggesting that significant redundancies exist for TF function within enhancers. These findings demonstrate that observed TF binding significantly overestimates direct TF regulation of gene expression. Taken together, our results suggest that the broadly expressed TF MEF2D achieves tissue specificity through competitive recruitment to enhancers by tissue-specific TFs and activates a small subset of enhancers to regulate genes.

A novel approach of using near infrared region (NIR) dyes is applied to elucidate the binding interaction between human serum albumin (HSA) and mangiferin (MGF). HSA is a blood carrier protein used for drug delivery, while mangiferin is a natural polyphenol found in mangoes that possesses numerous beneficial health properties. The NIR dyes are used as a probe to investigate MGF binding interaction with HSA via monitoring fluorescence of Trp-214 residue. Molecular modeling is used for docking and semi-empirical analysis. The investigation of the binding interaction between Trp-214 and MGF is significant, for it may offer broader pharmacological insight and applications for the polyphenol. Mangiferin in proposed to bind with a 2:1 stoichiometric ratio with HSA to the Trp-214 residue in subdomain IIA and another possible binding site to be determined in future studies. Spectral changes suggest a stabilized protein conformation upon mangiferin binding with the addition of NIR dye E-06 and MHI-06.

RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold. To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity. The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the iv collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues. In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n. Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.

No description available.

Endothelial cells separate the bloodstream from the underlying tissue and play a crucial role in vascular homeostasis. They also form an important barrier for vascular drug delivery. This thesis contains preliminary studies targeted at understanding the mechanisms of binding and transport across endothelial cells cultured in vitro. Specifically, the first study investigates how the recombinant source of Factor IX (FIX), a blood coagulant protein used in the treatment of Hemophilia B, impacts surface ligand binding (FIX to its specific receptors) to bovine aortic endothelial cells (BAECs). Competitive binding experiments between 125I-FIX and FIX were undertaken to quantify the interaction of recombinant and transgenic FIX with BAECs and human collagen IV and determine if there was a measurable difference in binding affinity. Results indicate limited specific binding of 125I-FIX to BAECs and no binding to human collagen IV. Concrete conclusions were not drawn from this data due to technical issues during the experimental process. The second study investigates insulin-like growth factor-I (IGF-I) transport across both BAEC and MAC-T cells, a mammary epithelial cell line, cultured on tissue culture inserts. IGF-I is a circulatory growth factor implicated in the regulation of cell division and tissue proliferation. Competitive binding experiments between 125I-IGF-I and unlabeled protein (IGF-I, Y60L-IGF-I, a mutant of IGF-I, and IGF Binding Protein-3 (IGFBP-3)) were undertaken to quantify the binding and transport of IGF-I under various experimental conditions. Results confirmed earlier work from the Williams' laboratory indicating that 125I-IGF-I transport was enhanced by incubation with its non-receptor-binding analog, Y60L-IGF-I, but cell surface associated 125I-IGF-I was decreased by its presence. Other studies were undertaken but conclusive results could not be drawn. / Master of Science

<p>Parkinson’s disease is a neurodegenerative disorder characterized by a loss of dopaminergic neurons, where mitochondrial dysfunction and neuroinflammation are implicated in this process. However, the exact mechanisms of mitochondrial dysfunction, oxidative stress and neuroinflammation leading to the onset and development of Parkinson’s disease are not well understood. There is a lack of tools necessary to dissect these mechanisms, therefore we engineered genetically encoded fluorescent biosensors to monitor redox status and an inflammatory signal peptide with high spatiotemporal resolution. To measure intracellular redox dynamics, we developed red-shifted redox sensors and demonstrated their application in dual compartment imaging to study cross compartmental redox dynamics in live cells. To monitor extracellular inflammatory events, we developed a family of spectrally diverse genetically encoded fluorescent biosensors for the inflammatory mediator peptide, bradykinin. At the organismal level, we characterized the locomotor effects of mitochondrial toxicant-induced dopaminergic disruption in a zebrafish animal model and evaluated a behavioral assay as a method to screen for dopaminergic dysfunction. Pairing our intracellular redox sensors and our extracellular bradykinin sensors in a Parkinson’s disease animal model, such as a zebrafish toxicant-induced model will prove useful for dissecting the role of mitochondrial dysfunction and inflammation in Parkinson’s disease. </p>