No description available.

Three residues, Phe190, Leu193 and Val302, which have been proposed to define the S₁ site of prolidase of <i>Lactococcus lactis</i> NRRL B-1821 (<i>L. lactis</i> prolidase), may limit the size and polarity of specific substrates accepted by this enzyme (Yang, S. I., and Tanaka, T. 2008. Characterization of recombinant prolidase from <i>L. lactis</i> changes in substrate specificity by metal cations, and allosteric behavior of the peptidase. FEBS J. 275, 271-280). These residues form a hydrophobic pocket to determine the substrate specificity of <i>L. lactis</i> prolidase towards hydrophobic peptides, such as Leu-Pro and Phe-Pro, while little activity was observed for anionic Asp-Pro and Glu-Pro. It is hypothesized that the substrate specificity of <i>L. lactis</i> prolidase would be changed if these residues are substituted with hydrophilic amino acid residues individually or in combinations by site-directed mutagenesis (SDM). In addition to the changes in substrate specificity, other characteristics of wild type prolidase, such as allosteric behaviour and substrate inhibition may receive influences by the mutations (Yang & Tanaka, 2008). To test this hypothesis, mutations were conducted on these three residues at the S₁ site. Mutated <i>L. lactis</i> prolidases were subsequently analyzed in order to examine the roles of these residues in the substrate specificity, allosteric behaviour, pH dependency, thermal dependency and metal dependency of prolidase. The results showed the significant changes in these kinetic characteristics of single mutants, such as L193E, L193R, V302D and V302K and double mutants, L193E/V302D and L193R/V302D. Leu193 was suggested to be a key residue for substrate binding. The mutants L193R, V302D, L193R/V302D and L193E/V302D lost their allosteric behaviour, and the substrate inhibition of the wild type was no longer observed in V302D and L193E/V302D. The results indicated Val302 to be more important for these properties than other S₁ site residues. Moreover, together with the observations in molecular modelling of the mutants, it was proposed that interactions of Asp302 with Arg293 and His296 caused the loss of allosteric behaviour and substrate inhibition in the V302D mutant. The investigations on the pH dependency suggested that His296 acted as proton acceptor in <i>L. lactis</i> prolidase's catalysis. It was expected that the electrostatic microenvironment surrounding His296 was influenced by the charged mutated residues and side chains of dipeptide substrates, thus the protonation of His296 was affected. It was suggested that the introduced positive charge would stabilize the deprotonated form of His296 thus to maintain the activities of the mutants in more acidic condition compared to wild type prolidase. The study of thermal dependency revealed that all non-allosteric prolidases had higher optimum temperatures, suggesting that the loss of allosteric behaviour resulted in more rigid structures in these prolidases.

Three residues, Phe190, Leu193 and Val302, which have been proposed to define the S₁ site of prolidase of <i>Lactococcus lactis</i> NRRL B-1821 (<i>L. lactis</i> prolidase), may limit the size and polarity of specific substrates accepted by this enzyme (Yang, S. I., and Tanaka, T. 2008. Characterization of recombinant prolidase from <i>L. lactis</i> changes in substrate specificity by metal cations, and allosteric behavior of the peptidase. FEBS J. 275, 271-280). These residues form a hydrophobic pocket to determine the substrate specificity of <i>L. lactis</i> prolidase towards hydrophobic peptides, such as Leu-Pro and Phe-Pro, while little activity was observed for anionic Asp-Pro and Glu-Pro. It is hypothesized that the substrate specificity of <i>L. lactis</i> prolidase would be changed if these residues are substituted with hydrophilic amino acid residues individually or in combinations by site-directed mutagenesis (SDM). In addition to the changes in substrate specificity, other characteristics of wild type prolidase, such as allosteric behaviour and substrate inhibition may receive influences by the mutations (Yang & Tanaka, 2008). To test this hypothesis, mutations were conducted on these three residues at the S₁ site. Mutated <i>L. lactis</i> prolidases were subsequently analyzed in order to examine the roles of these residues in the substrate specificity, allosteric behaviour, pH dependency, thermal dependency and metal dependency of prolidase. The results showed the significant changes in these kinetic characteristics of single mutants, such as L193E, L193R, V302D and V302K and double mutants, L193E/V302D and L193R/V302D. Leu193 was suggested to be a key residue for substrate binding. The mutants L193R, V302D, L193R/V302D and L193E/V302D lost their allosteric behaviour, and the substrate inhibition of the wild type was no longer observed in V302D and L193E/V302D. The results indicated Val302 to be more important for these properties than other S₁ site residues. Moreover, together with the observations in molecular modelling of the mutants, it was proposed that interactions of Asp302 with Arg293 and His296 caused the loss of allosteric behaviour and substrate inhibition in the V302D mutant. The investigations on the pH dependency suggested that His296 acted as proton acceptor in <i>L. lactis</i> prolidase's catalysis. It was expected that the electrostatic microenvironment surrounding His296 was influenced by the charged mutated residues and side chains of dipeptide substrates, thus the protonation of His296 was affected. It was suggested that the introduced positive charge would stabilize the deprotonated form of His296 thus to maintain the activities of the mutants in more acidic condition compared to wild type prolidase. The study of thermal dependency revealed that all non-allosteric prolidases had higher optimum temperatures, suggesting that the loss of allosteric behaviour resulted in more rigid structures in these prolidases.

No description available.

The field of tissue engineering has grown immensely since its inception in the late 1980s. However, currently commercialized tissue engineered products are simple in structure. This is due to a pre-clinical bottleneck in which complex tissues are unable to be fabricated. 3D bioprinting has become a versatile tool in engineering complex tissues and offers a solution to this bottleneck. Characterizing the mechanical properties of engineered tissue constructs provides powerful insight into the viability of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogel materials used in tissue engineering destroy the sample and ignore the effect of 3D bioprinting on the overall mechanical properties of a construct. Herein, this work reports on the novel use of a non-destructive method of viscoelastic analysis to demonstrate the influence of 3D bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. 3D bioprinting is demonstrated as a versatile tool with the ability to control mechanical and physical properties. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include effective real-time monitoring of crosslinking, and mechanical characterization of multi-material scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering. / Graduate

1 ABSTRACT In this Thesis, we studied properties of μ-, δ-, and κ-opioid receptors in lymphocytes isolated from rat spleen. This splenocytes were exposed to mitogen concanavalin A or opiate morphine and cultivated for 48 hours. Under physiological conditions, level of opioid receptors in immune cells is very low. Due to various factors such as presence of opioids, mitogens, long-term exposition to stress, expression of these receptors can be amplified. In this study we demonstrated, that concanavalin A causes up-regulation of μ-, δ- and κ-opioid receptors in lymphocytes isolated from rat spleen. In control cells no significant signal of μ- or δ-receptors was observed. In contrast, κ-opioid receptors were detected already in control cells. Concanavalin A stimulation caused a 2.4 - fold increase of these receptors. In lymphocytes treated with morphine only μ-opioid receptors were up-regulated, whereas in control cells, there was no signal for these receptor type. δ-opioid receptors were not detected in control or morphine treated cells. κ-opioid receptors were determined in control and also in morphine affected lymphocytes but the amount of these receptors wasn't changed by morphine. Detection of μ-, δ- and κ-opioid receptors using Western blot technique in lymphocytes isolated from rat spleen, that were...

Les mécanismes adhésifs jouent un rôle crucial en biologie. Les cadhérines classiques constituent une des principales familles d'adhésion cellulaire dépendante du calcium. Ces glycoprotéines transmembranaires sont impliquées dans des interactions principalement homophiles. Ces interactions régulent des voies de signalisation impliquées dans de nombreux phénomènes biologiques. Cette thèse porte sur l'étude comparative des dynamiques d'interactions des cadhérines E- et -11, prototypes respectivement des cadhérines classiques de type I et II. Le ciblage d'acides aminés particuliers de l'interface adhésive nous a permis de montrer que pour les cadhérines de type I, l'échange de brin avec le Trp2 ont un rôle clé ; pour les types II un mécanisme différent intervient. Nous avons aussi développé une chimie innovante pour contrôler l'immobilisation orientée et covalente de protéines. Enfin une revue décrit une étude de l'activation de voies de signalisation par engagement des cadhérines. / Cell adhesion receptors of the classical cadherin family are involved in Ca2+-dependent homophilic interactions. In order to dissect the molecular mechanisms of cadherin-based cellcell adhesion, this Ph.D. thesis describes a comparative dynamic study of interactions between cadherins E- & -11, chosen as classical type I and II cadherins prototypes respectively. Modifications of particular residues in the E-cadherin adhesive interface showed that the ?-strand exchange with its Trp2 had a prominent feature; for type II cadherins, a different mechanism was described involving a larger domain swapping. We then developed a new protocol for immobilizing proteins in an orientated and covalent manner on surfaces. These interactions regulate signalization pathways in various biological processes. Studies describing Stat3 activation through direct cadherin engagement are reviewed.

Das Ziel der kognitiven Neurowissenschaften ist es, Struktur-Funktions-Beziehungen im Gehirn aufzudecken. Durch Fortschritte in der funktionellen Bildgebung und der Graphentheorie konnten zentrale Knotenpunkte im Gehirn identifiziert werden, welche neuronale Informationen integrieren und dementielle Prozesse erklären können. Datenbankgetriebene metaanalytische Methoden wurden genutzt, um tausende Bildgebungsstudien auszuwerten und das funktionelle Profil von Hirnregionen zu dekodieren. Das Systems-Level Decoding zielt darauf ab, eine Seed-Region und mit ihr verbundene kortikale Regionen funktionell zu dekodieren. In Studie 1 wurden thalamische Subregionen und in Studie 2 drei Subregionen des Intraparietalen Sulcus (hIP) als Seed-Regionen verwendet. Studie 3 untersuchte Unterschiede in der Rich-Club-Struktur zwischen Alzheimer-Patienten, Patienten mit milder kognitiver Beeinträchtigung und gesunden Probanden. Es wurden zwei große, Thalamus-zentrierte Systeme identifiziert, die mit autobiographischem Gedächtnis und Nozizeption assoziiert sind; für den hIP wurden neun Systeme, die mit Prozessen wie Arbeitsgedächtnis und numerischem Denken zusammenhängen, gefunden. Studie 3 fand Hinweise, dass periphere Regionen bei Patienten im Vergleich zu gesunden Kontrollen stärker aktiviert sind als Rich-Club-Regionen. Das Systems-Level Decoding lieferte neue Erkenntnisse über die Einbettung des Thalamus und des hIP in kortikale funktionelle Systeme. Allerdings waren die Ergebnisse hinsichtlich der funktionellen Charakterisierung thalamischer Kerne und der Aktivitätsunterschiede in Rich-Club- und peripheren Regionen zwischen Patienten und gesunden Kontrollpersonen begrenzt. Diese Ergebnisse lassen sich möglicherweise durch Limitationen des metaanalytischen Ansatzes erklären. Das Systems-Level Decoding ist insgesamt ein vielversprechender Ansatz für die Formulierung von Hypothesen über Struktur-Funktionsbeziehungen innerhalb der Netzwerkarchitektur des menschlichen Gehirns. / In cognitive neuroscience, there is great interest in unraveling structure-function relationships in the human brain. Advances in functional neuroimaging and graph theory methods have identified key brain nodes relevant for neuronal information integration and functional deficits in degenerative diseases like Alzheimer's. Database-driven meta-analytical methods have also evaluated knowledge from thousands of neuroimaging studies, to decode the functional profile of brain regions. The systems-level decoding aims to identify brain systems that provide insight into the functional characteristics of a seed region and its connected cortical regions. In Study 1, thalamic subregions were used as seed regions. Study 2 applied systems-level decoding to three distinct regions in the intraparietal sulcus (hIP). In Study 3, we attempted to substantiate activation differences in the Rich Club structure between Alzheimer's patients, patients with mild cognitive impairment, and healthy subjects. Two major, thalamus-centered systems associated with autobiographical memory and nociception were identified. Additionally, nine large systems associated with processes such as working memory, numerical cognition, and recognition memory were uncovered for the hIP. Finally, evidence showed that peripheral regions in patients are more activated than central Rich Club regions compared to healthy controls. Systems-level decoding provided significant new insights into the embedding of the thalamus and intraparietal sulcus in functional systems. However, results were limited in the functional characterization of thalamic nuclei and activity differences in Rich Club and peripheral regions between patients and healthy controls. Limitations of the meta-analytical approach may explain these findings. The systems-level decoding represents a promising approach for formulating hypotheses about brain structure-function relationships within the functional network architecture.

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.