This thesis is focused on immunochemical determination of concentration of active and inactive form of cathepsin B in patients with bladder cancer in order to compare diagnostic efficiency of methods for their possible use for routine diagnosis. Cathepsin B and procathepsin B were measured in serum and urine in 82 patients with bladder cancer (47 men and 35 women), with the average age of 66.5 year. The control group contain of 72 healthy subjects (31 men and 41 women), with the average age of 58.5 year. The concentration of cathepsin B and procathepsin B in the urine were corrected to creatinine, which was determined by the enzymatic creatinase method. The concentrations of cathepsin B in urine were singnificantly elevated in patients than in control group (median = 3.5 µg/l vs. 0.9 µg/l, P = 0.01), similarly the results of the cathepsin B/creatinine ratio were elevated (median = 0.4 µg/mmol vs. 0.1 µg/mmol, P = 0.01). There were no significant difference in concentration in serum between patients and control group (median = 4.8 µg/l vs. 4.2 µg/l, P = 0.8). The concentration values of procathepsin B were significantly higher in patients compare to control group both in urine (median = 3.9 µg/l vs. 1.4 µg/l, P < 0.0001), in serum (median = 73.3 µg/l vs. 58.7 µg/l, P = 0.0005) and similarly in...

Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...

Cell death is a fundamental aspect of development and homeostasis; its dysregulation is commonly associated with disease. Historically, apoptosis has been the most heavily studied type of cell death, but there are many other non-apoptotic forms of cell death. The Drosophila ovary provides a powerful in vivo model to study non-apoptotic cell death. Each egg chamber in the ovary contains 15 nurse cells that support an oocyte throughout development, and at the end of oogenesis the nurse cells are surrounded by stretch follicle cells and undergo non-apoptotic cell death. The work in this dissertation investigated the role of stretch follicle cells in nurse cell death. Genetic ablation of the stretch follicle cells revealed that they are required for multiple nurse cell death events including the transport of cytoplasm to the oocyte and DNA fragmentation. We found that phagocytic machinery is required in the stretch follicle cells for the acidification and elimination of nurse cells, suggesting nurse cells die by phagoptosis. Furthermore, live imaging and a transgenic engulfment detector demonstrated that nurse cells are not engulfed piece-wise despite the requirement of phagocytosis machinery, but are instead surrounded and acidified extracellularly. To determine the mechanism driving nurse cell acidification, we performed a targeted RNAi screen against lysosome-associated genes. Using tissue-specific RNAi, we demonstrated that the V-ATPase proton pump is required in the stretch follicle cells for nurse cell acidification. GFP fusion proteins and antibody staining revealed that V-ATPases become enriched and localize to the stretch follicle cell plasma membranes to acidify the nurse cells that they surround. Following acidification, the stretch follicle cells were found to release cathepsins, lysosomal proteases, to break down and degrade the nurse cell. To uncover novel pro-death proteins that mediate signaling between the stretch follicle cells and nurse cells, we utilized proximity-dependent protein labeling and identified proteins enriched in the stretch follicle cells. Altogether this work uncovers a new role for lysosomal machinery acting at the plasma membrane of stretch follicle cells to drive nurse cell death, and identifies pro-death proteins in the stretch follicle cells that promote nurse cell death.

An affinity column for cathepsin D was prepared making use of the strong affinity of pepstatin for cathepsin D. Pepstatin is an N-acylated pentapeptide from Actinomycetes with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy- 6-methyl heptanoic acid. A relatively rapid and efficient method for cathepsin D purification has been developed; Steps include homogenization, ammonium sulfate fractionation, and chromatography on pepstatin-Sepharose column. The final preparation has a specific activity of 38 units/mg. and shows a single protein band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate corresponding to a subunit molecular weight of 42,000. Polyacrylamide gel electrophoresis studies did not reveal any impurities. The proteolytic activity of isolated cathepsin D on bovine myofibrils and myosin was examined at pH 3.80, 37 °C. The heavy chains of myosin, as well as other smaller regulatory proteins of the myofibrils were degraded. Actin was degraded less rapidly than myosin heavy chain. Degradation became more extensive when the substrate-enzyme incubation time was increased.

Trichomonas vaginalis is a sexually-transmitted parasite that is the causative agent in the disease trichomoniasis. Resistance to the only FDA-approved medication to this disease, metronidazole, has been on the increase giving rise to the need for finding targets for new inhibitors to exploit. New inhibitors can target enzymes such as 4-coumarate:CoA ligase and S-adenosylhomocysteine hydrolase. Another potential target is a cathepsin D-like protease found in T. vaginalis . This aspartic protease in humans is responsible for degrading proteins in the lysosome, and degrading hemoglobin in P. falciparum as the homologue plasmepsin. Searching the gene database, only one cathepsin-D like protease was discovered throughout the organism's genome. Utilizing RT-PCR, this gene is found to be expressed in two different strains of the organism. Transfection of an epitope-tagged version of this cathepsin D-like protease into T. vaginalis was accomplished, and subsequent immunofluorescence of this tagged version shows it to be localized in intracellular compartments, which can be colocalized using the SNARE and VAMP proteins found in T. vaginalis .

The objective of this research was to develop and validate new macromolecular imaging agents to detect and characterize malignant tumours. Using well-defined, highly branched macromolecules called dendrimers as the structural scaffold, efficient functionalization of the periphery was demonstrated using “click” chemistry in order to prepare multivalent imaging probes. Furthermore, a transmetalation was demonstrated to displace chelated copper with technetium, enabling “click” reactions to be performed in the presence of the dipicolylamine (DPA), a ligand known to chelate many metals. The dendritic scaffold was functionalized with either hydrophobic or hydrophilic targeting vectors. The hydrophobic ligand, an acyloxymethyl ketone targeting the overexpression of cathepsin B exhibited poor in vitro affinity when coupled to either G1 or G2 dendrimers, despite the use of various linkers. A glu-urea-lys dipeptide, representing a hydrophilic prostate specific membrane antigen targeting vector, demonstrated excellent affinity in vitro. The lead compound, a G2 dendrimer bearing four PSMA targeting vectors attached via an alkyl spacer was further investigated in vitro and in vivo. Unfortunately, poor tumor uptake was observed and the compound was hypothesized to hydrolyze readily (<15min), based on the in vitro plasma stability data. To rectify the aforementioned problem, non neo-pentyl esters were replaced with either carbamate or ether linkages. In vitro plasma stability analysis of the analogous compounds demonstrated increased stability. In particular, the ether analogue was found to be most stable, with minimal degradation observed after 4 hours. / Thesis / Doctor of Philosophy (PhD)

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Cysteinproteaset cathepsin B har en trolig roll i vävnadsnedbrytningen vid kronisk parodontit då det visats kunna bryta ner extracellulära komponenter. Cystatin C är en fysiologisk inhibitor av cathepsin B och båda har kopplats till parodontal sjukdom. Syftet med studien var att undersöka närvaro av samt lokalisera cathepsin B och cystatin C i gingivala biopsier från patienter med kronisk parodontit. Närvaro och lokalisation av cathepsin Bs och cystatin Cs demonstrerades genom immunohistokemisk metod av kryostatsnittad vävnad. Polyklonal kanin anti-human primär antikropp för både enzym och inhibitor användes. För identifiering av primära antikroppar användes Dako REAL™ EnVision™ Detection System. Granskning skedde i ljusmikroskop. Studien visar på en närvaro av både enzym och inhibitor i epitel och bindväv i parodontalt sjuk vävnad. Cathepsin B lokaliserades i stor utsträckning till bindväven, främst perivaskulärt. I epitelet lokaliserades endast ett fåtal infärgade celler. Cystatin C hade i bindväven en diffus utbredning vilket gjorde lokalisation till specifika cellpopulationer svår. I epitelet sågs en tydlig infärgning kring/i celler med dendritiskt utseende. Dessa celler identifierades som sannolika langerhanska celler. / The cysteine protease cathepsin B has been shown to have a possible role in tissue destruction in chronic periodontitis, as it has been shown to degrade extracellular components. Cystatin C is a physiological inhibitor of cathepsin B and both are associated with periodontal disease. The aim with this study was to investigate the presence and localization of cathepsin B and cystatin C in gingival biopsies from patients with chronic periodontitis. Cathepsin B's and cystatin C’s presence and localization was demonstrated by immunohistochemical method of cryostat sectioned tissue from patients with chronic periodontitis. Polyclonal rabbit anti-human primary antibody for both the enzyme and the inhibitor were used. Identification of the primary antibodies were confirmed with Dako REAL ™ EnVision ™ Detection System. The sectioned tissue was analysed by using light microscopy. Our study indicates a presence of both enzyme and inhibitor in epithelial and connective tissue from patients with periodontal disease. Cathepsin B was localized mainly to the connective tissue, primarily to the perivascular space. In the epithelium only a few stained cells were identified. In the connective tissue, cystatin C had a diffuse distribution. Therefor it was difficult to distinguish the specific cell populations associated with the inhibitor. The epithelium showed a clear staining around/in dendritic cells. These cells were identified as probable Langerhans cells.