Candida parapsilosis is an emerging human opportunistic pathogen causing a wide spectrum of potentially life-threatening infections in immunocompromised hosts. One of the most important virulence factors of Candida spp. is a production of secreted aspartic proteinases (Saps). Presented thesis is mainly focused on the study of secreted aspartic proteinase 1 (Sapp1p) of C. parapsilosis, its processing and secretion under variable conditions and by use of various experimental models. Sapp1p is secreted by C. parapsilosis cells into the extracellular space as a completely processed and fully active enzyme. Experiments studying the C. parapsilosis cell wall (CW) confirmed the prolonged presence of completely processed Sapp1p on the cell surface (CW- Sapp1p). Proteolytic activity assay performed with the intact cells showed that CW-Sapp1p is proteolytically active prior to its release into the extracellular space and is capable of substrate cleavage. Biotinylation experiments with consecutive MS analysis revealed that CW-Sapp1p biotinylation is incomplete but saturable process, leaving partially unlabelled molecules. The accessibility of individual lysine residues in the Sapp1p molecule varied, with exception of four residues that were labelled in all of our experiments performed. The final step of...

Today, the demand for speed in drug discovery is constantly increasing, particularly in the iterative processes of hit validation and expansion and lead optimization. Irradiation with microwaves (MWs) has been applied in the area of organic synthesis to accelerate chemical reactions and to facilitate the generation of new chemical entities since 1986. In the work presented in this thesis, the use of MW-mediated heating has been expanded to address three fields of drug discovery, namely hit expansion, chemical library generation and genomics. In the first project, potential inhibitors of malaria aspartic proteases were designed and synthesized, partly by MW-assisted organic chemistry, and evaluated with regard to their inhibitory efficacy on five malaria aspartic proteases and their selectivity over two human aspartic proteases. The synthetic work included the development of fast and convenient methods of MW-assisted formation of thiazolidines and epoxy esters. Some of the resulting structures proved to be efficacious inhibitors of the aspartic protease that degrades haemoglobin in all four malaria parasites infecting man. No inhibitor affected the human aspartic proteases. Expedient, two-step, single-operation synthetic routes to heterocycles of medicinal interest were developed in the second and third projects. In the former, the use of a versatile synthon, Ph3PCCO, provided α,β-unsaturated lactones, lactams and amides within 5–10 minutes. In the latter project, saturated lactams were formed from amines and lactones in 35 minutes, in the absence of strong additives. These two MW-mediated protocols allowed the reduction of the reaction time from several hours or days to minutes. In the fourth project, a fully automated MW-assisted protocol for the important enzyme-catalysed polymerase chain reaction (PCR) was established. In addition, the PCR reaction could be performed in unusually large volumes, 2.5 mL and 15 mL, with yields corresponding to those from conventional PCR. Good amplification rates suggested that the thermophilic enzyme, Taq polymerase, was not affected by the MW radiation.

Enolate formation by proton abstraction from an sp3-hybridized carbon atom situated next to a carbonyl or carboxylate group is an abundant process in nature. Since the corresponding nonenzymatic process in water is slow and unfavorable due to high intrinsic free energy barriers and high substrate pKa s, enzymes catalyzing such reaction steps must overcome both kinetic and thermodynamic obstacles. Computer simulations were used to study enolate formation catalyzed by glyoxalase I (GlxI) and 3-oxo-Δ5-steroid isomerase (KSI). The results, which reproduce experimental kinetic data, indicate that for both enzymes the free energy barrier reduction originates mainly from the balancing of substrate and catalytic base pKas. This was found to be accomplished primarily by electrostatic interactions. The results also suggest that the remaining barrier reduction can be explained by the lower reorganization energy in the preorganized enzyme compared to the solution reaction. Moreover, it seems that quantum effects, arising from zero-point vibrations and proton tunnelling, do not contribute significantly to the barrier reduction in GlxI. For KSI, the formation of a low-barrier hydrogen bond between the enzyme and the enolate, which is suggested to stabilize the enolate, was investigated and found unlikely. The low pKa of the catalytic base in the nonpolar active site of KSI may possibly be explained by the presence of a water molecule not detected by experiments. The hemoglobin-degrading aspartic proteases plasmepsinI and plasmepsin II from Plasmodium falciparum have emerged as putative drug targets against malaria. A series of C2- symmetric compounds with a 1,2-dihydroxyethylene scaffold were investigated for plasmepsin affinity, using computer simulations and enzyme inhibition assays. The calculations correctly predicted the stereochemical preferences of the scaffold and the effect of chemical modifications. Calculated absolute binding free energies reproduced experimental data well. As these inhibitors have down to subnanomolar inhibition constants of the plasmepsins and no measurable affinity to human cathepsin D, they constitute promising lead compounds for further drug development.

Natural plant growth regulators (phytohormones) are a group of organic compounds which, in very small amounts, act as regulators of physiological processes in plants.Methods were developed for the analysis of phytohormones in samples from Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris (L.) Karst»). Identification of abscisic acid, 3-indoleacetic acid, gibbe-rellin Ag and the conjugate N-(3-indoleacetyl)aspartic acid was performed by GC-MS as their methyl esters. A quantitative determination of abscisic acid was made by GC-ECD and this method was also applied to anther samples of Anemone canadensis. 3-Indole-acetic acid and N-(3-indoleacetyl)aspartic acid were quantified by reversed-phase HPLC and spectrofluorimetric detection. Dichlorophene, used as a growth regulator in containerized seedlings of pine and spruce, was analysed by GC-MID in peat and paper. / digitalisering@umu

Στην παρούσα διατριβή παρουσιάζεται μια νέα μεθοδολογία σύνθεσης μη-πρωτεϊνικών αμινοξέων (ΜΠΑ) κατάλληλων για εφαρμογές στην πεπτιδική σύνθεση με χρήση του ασπαραγινικού οξέος ως χειρόμορφου εκμαγείου. Η μεθοδολογία αυτή βασίζεται στην επιτυχή και σε καλές αποδόσεις σύνθεση της β-αλδεΰδης του Ν-τριτυλοασπαραγινικού τριτ-βουτυλεστέρα, η οποία αποτελεί την ένωση «κλειδί». Αυτή στη συνέχεια έδωσε μια ποικιλία νέων ΜΠΑ μέσω αντιδράσεων Wittig, Horner-Emmons ή με επίδραση οργανοψευδραργυρικών αντιδραστηρίων. Ανάλογες μελέτες με σκοπό την σύνθεση των αντίστοιχων ομολόγων τους με έναν άνθρακα λιγότερο ή περισσότερο στην πλευρική αλυσίδα, ξεκινώντας από σερίνη ή γλουταμινικό οξύ δεν απέδωσαν τα αναμενόμενα αποτελέσματα ή ήταν ανεπιτυχείς. / In the present dissertation a new methodology is reported for the synthesis of novel non-proteinogenic amino acids (NPAAs) suitable for applications in solid phase peptide synthesis. This methodology involves aspartic acid as chiral template and relies on the successful and in good yields pepraration of the key N-tritylaspartic tert-butyl ester’s β-aldehyde, which when followed by Wittig, Horner-Emmons or Grignard type reactions may result in a variety of new NPAAs. Furthermore unsuccessful attempts towards the application of the present methodology for the synthesis of NPAA homologues bearing one carbon atom less or more in the side chain by using as chiral templates serine or glutamic acid, respectively, are also reported.

L’Opacification Capsulaire Postérieure (OCP) est la fibrose de la capsule développée sur la lentille intraoculaire implantée (LIO) suite à la dé-différenciation de cellules épithéliales cristalliniennes (LECs) subissant une transition épithélio-mésenchymateuse (EMT). La littérature a montré que l'incidence de l’OCP est multifactorielle, dont l'âge ou la maladie du patient, la technique de chirurgie, le design et le matériau de la LIO. La comparaison des LIOs en acryliques hydrophiles et hydrophobes montre que les premières ont une OCP plus sévère, médiée par la transition EMT. En outre, il est également démontré que l'adhérence des LECs est favorisée sur des matériaux hydrophobes par rapport à ceux hydrophiles. Une stratégie biomimétique destinée à promouvoir l’adhérence des LECs sans dé-différenciation en vue de réduire le risque de développement de l’OCP est proposée. Dans cette étude, les peptides RGD, ainsi que les méthodes de greffage et de quantification sur un polymère acrylique hydrophile ont été étudiés. La surface fonctionnalisée des LIOs favorisant l'adhérence des LECs via les récepteurs de type intégrine peut être utilisée pour reconstituer la structure capsule-LEC-LIO en sandwich, ce qui est considéré dans la littérature comme un moyen de limiter la formation de l‘OCP. Les résultats montrent que le biomatériau innovant améliore l'adhérence des LEC, et présente également les propriétés optiques (transmission de la lumière , banc optique) similaires et mécaniques (force haptique de compression, force d'injection de la LIO) comparables à la matière de départ. En outre, par rapport au matériau hydrophobe IOL, ce biomatériau bioactif présente des capacités similaires vis à vis de l’adhérence des LECs, le maintien de la morphologie, et l'expression de biomarqueurs de l’EMT. Les essais in vitro suggèrent que ce biomatériau a le potentiel de réduire certains facteurs de risque de développement de l’OCP. / Posterior Capsular Opacification (PCO) is the capsule fibrosis developed onto the implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LEC) undergoing Epithelial-Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including patient’s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs show the former has more severe PCO after EMT transition. Additionally, the LEC adhesion is favored onto the hydrophobic materials compared to the hydrophilic ones. A biomimetic strategy to promote LEC adhesion without de-differentiation to reduce PCO development risk is proposed. RGD peptides, as well as their grafting and quantification methods on a hydrophilic acrylic polymer were investigated. The surface functionalized IOL promoting LEC adhesion via integrin receptors can be used to reconstitute the capsule-LEC-IOL sandwich structure, which is considered to prevent PCO formation in literature. The results show the innovative biomaterial improves LEC adhesion, and also exhibits similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties comparing to the starting material. In addition, comparing to the hydrophobic IOL material, this bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression. The in vitro assays suggest this biomaterial has the potential to reduce some risk factors of PCO development.

No description available.

PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...

Applications in paleontological research are far from being limited to taxonomic collection and identification. Nor is such research limited to working solely on fossil data. Actualistic paleontology is the study of modern or recent organisms and processes to better understand those of the past. The bulk of this body of research falls under the category of actualistic paleontology, and examines geochronological methods and error biases in dating biological specimens ranging in age from modern to thousands of years old. Although such methods are arguably not perfect, error rates of ± a few hundred to few thousand years can be extremely important when considering ecological relationships among both Holocene taxa and time-averaged paleocommunities, but quite diminished when considering implications on more traditional dating techniques for ancient strata. Regardless, understanding implications of time resolution is important in analyses of and comparisons between any biological dataset. The following chapters are united by quantitative and statistical management of data with varying levels of temporal resolution, and represent four manuscripts that either are in press or soon to be submitted for publication. / Ph. D.

Die vorliegende Arbeit beschäftigt sich mit der Synthese und den Eigenschaften von doppelthydrophilen Blockcopolymeren und ihrer Anwendung in einem biomimetischen Mineralisationsprozeß von Calciumcarbonat und Bariumsulfat. Doppelthydrophile Blockcopolymere bestehen aus einem hydrophilen Block, der nicht mit Mineralien wechselwirkt und einem zweiten Polyelektrolyt-Block, der stark mit Mineraloberflächen wechselwirkt. Diese Blockcopolymere wurden durch ringöffnende Polymerisation von N-carboxyanhydriden (NCA′s) und a-methoxy-ω-amino[poly(ethylene glycol)] PEG-NH2 als Initiator hergestellt.<br /> Die hergestellten Blockcopolymere wurden als effektive Wachstumsmodifikatoren für die Kristallisation von Calciumcarbonat und Bariumsulfat Mineralien eingesetzt. Die so erhaltenen Mineralpartikel (Kugeln, Hantel, eiförmige Partikel) wurden durch Lichtmikroskopie in Lösung, SEM und TEM charakterisiert. Röntgenweitwinkelstreuung (WAXS) wurde verwendet, um die Modifikation von Calciumcarbonat zu ermitteln und die Größe der Calciumcarbonat- und Bariumsulfat-Nanopartikel zu ermitteln. / This work describes the synthesis and characterization of double hydrophilic block copolymers and their use in a biomimetic mineralization process of Calcium Carbonate and Barium Sulfate.<br /> Double hydrophilic block copolymers consist of a hydrophilic block that does not interact with minerals and another hydrophilic polyelectrolyte block that strongly interacts with mineral surfaces. These polymers were synthesised via ring opening polymerisation of N-carboxyanhydride (NCA), and the first hydrophilic block a-methoxy-ω-amino[poly(ethylene glycol)] PEG-NH2 was used as an initiator.<br /> The prepared block copolymers were used as effective crystal growth modifiers to control the crystallization of Calcium Carbonate and Barium Sulfate minerals. The resulting mineral particles (spheres, dumbbells, egg-like particles) were characterised by light microscopy in solution, by SEM, and by TEM. X-Ray scattering measurements (WAXS) were used to prove the modification of Calcium Carbonate particles and to calculate the size of Calcium Carbonate and Barium Sulfate nanoparticles.