Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-Oglucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3-O-GT in particular.

Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases are the enzymes that mediate glucosylation by transferring glucose from a high energy sugar donor to the acceptor substrates. Our study focuses on the structural and functional analysis of a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) clone from Citrus paradisi that has been characterized previously in our lab. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-OGT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at 7-OH position forming prunin. Product identification with other substrates is in progress. Results are being used to revisit and refine the structure model. Structural and functional analysis of flavonoid GTs may contribute to custom design of GTs for the synthesis of novel glucosides by changing glucosylation patterns.

La lipase B de Candida antarctica (CALB) est un enzyme présentant des propriétés énantiosélectives très intéressantes pour l'obtention de molécules énantio pures par dédoublement cinétique de mélanges racémiques,molécules utilisées comme synthons dans l'industrie pharmaceutique. En effet, le principe actif de nombreux médicaments est efficace sous une forme énantio pure, l'autre forme chirale pouvant se révéler délétère pour l'organisme.Les travaux de la thèse s'intéressent à mieux comprendre l'origine de l'énantiosélectivité de la lipase B de Candida antarctica, en particulier pour la résolution d'alcools secondaires par des réactions de transestérification.Nous utilisons pour la première fois la méthode de la perturbation de l'énergie libre pour estimer la différence d'énergie libre entre les intermédiaires tétraédriques obtenus avec les formes R et S d'alcools énantiomères pour une série d'alcools secondaires, dans le but de prédire in silico l'énantiosélectivité de la CALB. Les paramètres cinétiques apparents d'une réaction avec deux alcools substrats énantiopurs sont expérimentalement déterminés et permettent de définir la contribution respective du Km et du kcat de chaque énantiomère pour la définition de l'énantiosélectivité. L'étude expérimentale de l'effet d'empreinte par des molécules co-substrats est réalisée,ainsi qu'une étude par modélisation moléculaire de l'effet d'empreinte par le premier ester substrat de la réaction qui pourrait modifier la conformation du site actif de la CALB. La troisième partie porte sur l'étude de la CALB et de trois variants (T42V, S47A et T42V/S47A) chez lesquels les acides aminés dans la poche stéréospécifiques ont mutés. T42V et S47A permettent d'obtenir une augmentation de l'énantiosélectivité. L'étude propose une étude détaillée de la conformation du site actif à partir de simulations de trajectoires de dynamique moléculaire

Much experimental evidence shows that the cytoskeleton is a downstream target and effector during cell death in numerous neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's diseases. However, recent evidence indicates that cytoskeletal dysfunction can also trigger neuronal death, by mechanisms as yet poorly understood. We studied a mathematical model of cytoskeleton-induced neuron death in which assembly control of the neuronal cytoskeleton interacts with both cellular stress levels and cytosolic free radical concentrations to trigger neurodegeneration. This trigger mechanism is further modulated by the presence of cell interactions in the form of a diffusible toxic factor released by dying neurons. We found that, consistent with empirical observations, the model produces one-hit exponential and sigmoid patterns of cell dropout. In all cases, cell dropout is exponential-tailed and described accurately by a gamma distribution. The transition between exponential and sigmoidal is gradual, and determined by a synergetic interaction between the magnitude of fluctuations in cytoskeleton assembly control and by the degree of cell coupling. We concluded that a single mechanism involving neuron interactions and fluctuations in cytoskeleton assembly control is compatible with the experimentally observed range of neuronal attrition kinetics. We also studied the transit of neurons through states intermediate between initial viability and cell death. We found that the stochastic flow of neuron fate, from viability to cell death, self-organizes into two distinct temporal phases. There is a rapid relaxation of the initial neuron population to a more disordered phase that is long-lived, or metastable, with respect to the time scales of change in single cells. Strikingly, cellular egress from this metastable phase follows the one-hit kinetic pattern of exponential decline now established as a principal hallmark of cell death in neurodegenerative disorders. Intermediate state metastability may therefore be an important element in the systems biology of one-hit neurodegeneration. Further, we studied the full spatiotemporal dynamics of death factor pulses released from dying neurons to emphasize the effects of the cell-to-cell coupling strength on neuron death rates. The rate of neuron cell loss monotonically increased with increased diffusion-dependent intercellular communication. Death factor diffusion effects may therefore be important moderators of one-hit neurodegeneration.

<i>Staphylococcus aureus</i> is a ubiquitous organism causing world-wide morbidity and mortality. This species readily develops resistance to antimicrobial agents. Current dosing strategies are based, in part, on minimum inhibitory concentrations (MICs). This susceptibility test fails to detect the presence of first-step resistant mutants often present in large heterogeneous populations of infecting bacteria. Dosing strategies based on MIC results may, in fact, allow for the selective proliferation of resistant subpopulations. The mutant-prevention concentration (MPC) is the drug concentration at which all first-step resistant mutants will be eradicated along with the susceptible cells. Determination of the mutant-selection window (MSW) is possible using MIC and MPC data. When considered together with achievable drug concentrations in human bodily sites, the MSW helps determine which antimicrobials are likely to select for resistance. MIC and MPC testing on clinical isolates of methicillin-susceptible (MSSA) and -resistant (MRSA) S. aureus was performed. Characterization via the polymerase chain reaction, sequencing, and electron microscopy (EM) was done on selected organisms recovered from MPC studies (MPC-recovered). MIC and MPC testing was performed on organisms isolated sequentially from patients with recurring S. aureus infections. Pulsed field gel electrophoresis was performed on these sequential isolates. Based on the MIC and the MPC values, the most potent agents for systemic MSSA and MRSA infections are gemifloxacin and vancomycin, respectively. Re-testing MPC-recovered populations by the MIC showed increased MIC results compared to the parent populations. Macrolide-resistance genes were discovered in S. aureus MPC-recovered populations; in contrast, parental isolates lacked these resistance determinants. EM revealed an increase in cell wall thickness of a vancomycin MPC-recovered population compared to its parental population. Moxifloxacin and vancomycin had the lowest and narrowest MSWs for systemic MSSA and MRSA infections, respectively, compared to the other agents tested. Sequential isolates showed no change in MIC and MPC values. The data presented provides evidence for the application of the MPC test to S. aureus organisms. The MPC data is significant when determining appropriate dosing strategies aimed at preventing resistance.

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) cause the neurodevelopmental disorder Rett syndrome, a common genetic cause of mental retardation in females. Although alterations in performance of MeCP2-deficient mice in specific behavioural tasks have been documented, it remains unclear if, and to what degree, MeCP2 dysfunction affects patterns of periodic behavioural and electroencephalographic activity. To address this, we monitored daily rhythmic patterns of core body temperature, gross motor activity, and cortical delta power from MeCP2-deficient mice and correlated it against regional MeCP2 expression levels. Our results show that normal daily rhythmic behavioural patterning of delta wave activity, body temperature and mobility are disrupted in these mice. Moreover, MeCP2-deficient mice displayed lower average core body temperature and significantly greater body temperature fluctuation than wild-type female mice. Finally, we also found that epileptiform discharge activity in MeCP2-deficient mice is more predominant during times of behavioural activity compared to inactivity.

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) cause the neurodevelopmental disorder Rett syndrome, a common genetic cause of mental retardation in females. Although alterations in performance of MeCP2-deficient mice in specific behavioural tasks have been documented, it remains unclear if, and to what degree, MeCP2 dysfunction affects patterns of periodic behavioural and electroencephalographic activity. To address this, we monitored daily rhythmic patterns of core body temperature, gross motor activity, and cortical delta power from MeCP2-deficient mice and correlated it against regional MeCP2 expression levels. Our results show that normal daily rhythmic behavioural patterning of delta wave activity, body temperature and mobility are disrupted in these mice. Moreover, MeCP2-deficient mice displayed lower average core body temperature and significantly greater body temperature fluctuation than wild-type female mice. Finally, we also found that epileptiform discharge activity in MeCP2-deficient mice is more predominant during times of behavioural activity compared to inactivity.

Much experimental evidence shows that the cytoskeleton is a downstream target and effector during cell death in numerous neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's diseases. However, recent evidence indicates that cytoskeletal dysfunction can also trigger neuronal death, by mechanisms as yet poorly understood. We studied a mathematical model of cytoskeleton-induced neuron death in which assembly control of the neuronal cytoskeleton interacts with both cellular stress levels and cytosolic free radical concentrations to trigger neurodegeneration. This trigger mechanism is further modulated by the presence of cell interactions in the form of a diffusible toxic factor released by dying neurons. We found that, consistent with empirical observations, the model produces one-hit exponential and sigmoid patterns of cell dropout. In all cases, cell dropout is exponential-tailed and described accurately by a gamma distribution. The transition between exponential and sigmoidal is gradual, and determined by a synergetic interaction between the magnitude of fluctuations in cytoskeleton assembly control and by the degree of cell coupling. We concluded that a single mechanism involving neuron interactions and fluctuations in cytoskeleton assembly control is compatible with the experimentally observed range of neuronal attrition kinetics. We also studied the transit of neurons through states intermediate between initial viability and cell death. We found that the stochastic flow of neuron fate, from viability to cell death, self-organizes into two distinct temporal phases. There is a rapid relaxation of the initial neuron population to a more disordered phase that is long-lived, or metastable, with respect to the time scales of change in single cells. Strikingly, cellular egress from this metastable phase follows the one-hit kinetic pattern of exponential decline now established as a principal hallmark of cell death in neurodegenerative disorders. Intermediate state metastability may therefore be an important element in the systems biology of one-hit neurodegeneration. Further, we studied the full spatiotemporal dynamics of death factor pulses released from dying neurons to emphasize the effects of the cell-to-cell coupling strength on neuron death rates. The rate of neuron cell loss monotonically increased with increased diffusion-dependent intercellular communication. Death factor diffusion effects may therefore be important moderators of one-hit neurodegeneration.

The structure of 11S cruciferin has been solved; however, how the individual subunits contribute to its physico-chemical and functional properties are not well known. The cruciferin isoforms in Arabidopsis thaliana, CRUA, CRUB, and CRUC, were investigated with respect to their molecular structures and the relationship of structural features to the physico-chemical and functional properties of cruciferin using homology modeling and various analytical techniques. Comparison of these models revealed that hydrophobicity and electrostatic potential distribution on the surface of the CRUC homotrimer had more favorable interfacial, solubility, and thermal properties than those of CRUA or CRUB. Flavor binding and pepsin digestion were associated with hypervariable regions (HVRs) and center core regions, respectively, moreso for CRUA and CRUB homotrimers than for CRUC. Chemical imaging of a single cell area in wild type (WT) and double-knockout seeds (CRUAbc, CRUaBc, and CRUabC) using synchrotron FT-IR microscopy (amide I band, 1650 cm-1, νC=O) showed that seed storage proteins were concentrated in the cell center and protein storage vacuoles, whereas lipids were closer to the cell wall. Secondary structure components of proteins of double-knockout lines did not show major differences. Changes in protein secondary structure components of pepsin-treated CRUabC (CRUC) mutant were minimal, indicating low enzyme accessibility. A three-step chromatographic procedure allowed isolation of the hexameric form of cruciferin with high purity (>95%). Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic analysis of the secondary structure of these proteins revealed cruciferins were folded into higher order secondary structures; 44−50% β-sheets and 7−9% α-helices. The relative subunit ratio was approximately 1:3:6 (CRUA:CRUB:CRUC) in the WT cruciferin. The Tm values of purified cruciferin at pH 7.4 (μ = 0.0) were in the order of WT = CRUA = CRUB < CRUC. The order of surface hydrophobicity as determined by ANS (1-anilinonaphthalene-8-sulfonate) probe binding was CRUA > CRUB = WT >> CRUC. Intrinsic fluorescence studies revealed a compact molecular structure for the CRUC homohexamer compared to the CRUA and CRUB homohexamers. The order of emulsion forming abilities was CRUA = CRUB > WT > CRUC (no emulsion formation) and the order of heat-induced network structure strength was WT > CRUA = CRUB > CRUC (no gel formation). The inability of CRUC to form gels or emulsions may be attributed to its low surface hydrophobicity and molecular compactness. At pH 2.0, CRUC hexamers dissociated into trimers which allowed the formation of an O/W emulsion and heat-induced network structures. Solubility of cruciferin as a function of pH at low ionic strength gave two minima around pH 4 and 7.4 yielding a “W” shape solubility profile deviating from the typical “U” or “V” shape solubility profile of other 11S globulins. The high ionic strength (μ = 0.5) was not favorable for emulsification, heat-induced gel formation, or solubilization for all cruciferins. Furthermore, the CRUA and CRUB homohexamers exhibited rapid pepsinolysis, while the CRUC homohexamer and WT heterohexamer were digested more slowly. Although fairly well conserved regions were found in the primary structure of these three cruciferin subunits, differences were found in the hypervariable regions and extended loop regions resulting in slight differences in 3D structures and interactions that occur during association to form superstructures, such as hexamers. These differences were reflected in the physico-chemical and techno-functional properties of hexamers and trimers composed of each subunit. In silico predictions for certain functionalities were highly correlated with empirical data from laboratory experiments.

Rollspel är en form av sällskapsspel som går ut på att deltagarna tillsammans berättar en historia, som ofta utspelar sig i fiktiva världar och utgår från perspektivet av den enskilda spelarens skapade rollperson. En av deltagarna är spelledare och är den som skildrar spelets miljöer och karaktärer. I studien diskuteras rollspelens identitetsskapande och sociala funktion samt rollspel som kunskaps- och färdighetsfrämjande, med utgångspunkt i tanken att rollspel kan skapa ett intresse för och främja bearbetandet av existentiella frågor. En enkät utdelad till trettio rollspelare samt fem mer djupgående intervjuer av erfarna spelare visar att rollspelsdeltagare genom spelet utforskar och behandlar existentiella frågor, samt att spelare själva rapporterar om rollspelandets positiva inflytande på det individuella och sociala livet samt spelandets kunskaps-, färdighets- och intressefrämjande förmåga.