Les cellules de mélanome humain expriment différents antigènes tumoraux qui sont reconnus par les lymphocytes T cytotoxiques CD8 + (CTL) induisant des réponses spécifiques de la tumeur in vivo. Cependant, chez les patients atteints de mélanome l'efficacité de la réponse naturelle des CTL ou stimulée par thérapie est limitée. Les mécanismes sous-jacents de l'échec de la phase effectrice des CTL contre les mélanomes sont encore largement méconnus. Notre hypothèse est que l'efficacité limitée des CTL dans leur combat contre les tumeurs est le résultat d'une balance défavorable entre la capacité des CTL à tuer les tumeurs et une résistance tumorale intrinsèque à l'activité cytolytique des CTL. Au cours de ma thèse je me suis concentrée sur la dynamique moléculaire qui se produit à la synapse lytique afin de pouvoir identifier un mécanisme précoce mis en place par les cellules de mélanome face à l'attaque des CTL. En combinant l'utilisation d'approches de microscopie de pointe et des outils moléculaires, j'ai pu montrer que, lors de l'interaction avec les CTL, les cellules de mélanome humain subissent une activation de leur trafic vésiculaire endosomal et lysosomal, lequel est intensifié à la synapse lytique et corrèle avec la dégradation par la cathepsine de la perforine et un défaut de pénétration d'entrée du granzyme B. De plus, j'ai démontré que le blocage du trafic lysosomal dépendant de SNAP23, la modification du pH (intra-vésiculaire) et l'inhibition de l'activité lysosomale protéotlytique des cellules de mélanome permet de restaurer leur sensibilité à l'attaque des CTL. Nos résultats révèlent une stratégie sans précédent d' " auto-défense " des cellules de mélanome à la synapse immunologique basée sur une sécrétion lysosomale massive et sur la dégradation de la perforine sécrétée par les CTL. Ainsi pouvoir interférer avec cette stratégie synaptique d'auto-défense des cellules de mélanome pourrait contribuer à potentialiser les réponses des CTL et les immunothérapies chez les patients atteints de mélanome. / Human melanoma cells express various tumor antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL) and elicit tumor-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying the failure of CTL effector phase against melanomas are still largely elusive. Our hypothesis is that the limited efficacy of CTL in their fight against tumors is the result of an unfavorable balance between CTL ability to kill tumors and an intrinsic tumor resistance to CTL cytolytic activity. During my thesis I focused on the molecular dynamics occurring at the lytic synapse in order to identify possible "early response-mechanism" of melanoma cells to CTL attack. Using a combination of cutting edge microscopy approaches and molecular tools, I showed that upon conjugation with CTL, human melanoma cells undergo an exacerbated late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Our results reveal an unprecedented strategy of melanoma cell "self-defense" at the immunologic synapse based on a lysosome secretory burst and perforin degradation at the lytic synapse. Interfering with this synaptic self-defense strategy might be instrumental to potentiate CTL-mediated therapies in melanoma patients.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Kyeong-Ok Chang / Caliciviruses are important pathogens of humans and animals. Noroviruses are major causes of foodborne gastroenteritis cases, but their research is hindered due to the inability to grow human noroviruses in cell culture. Detailed studies on entry events of caliciviruses are lacking and may be crucial for development of cell culture models. We examined the entry events of caliciviruses using porcine enteric calicivirus (PEC), feline calicivirus (FCV) and murine norovirus-1 (MNV-1). PEC replication in LLC-PK cells requires bile acid in the medium, but the mechanism is not well understood. Our studies showed that bile acids are required in the early stage of virus replication, and while internalization of PEC is not dependent of them, they are required for endosomal escape and successful replication. Further examination on virus entry, we demonstrated that endosomal acidification and cathepsin L activity are essential in the replication of PEC, FCV and MNV-1. The results showed that inhibition of endosomal acidification or cathepsin L activity led to retention of viruses in the endosomes. Also we demonstrated that recombinant cathepsin L cleaved structural protein of PEC, FCV or MNV-1, which suggests that the enzyme may facilitate uncoating viruses in endosomes. In addition to bile acids, we found that a cold shock treatment during virus entry supported PEC replication by facilitating the endosomal escape. While PEC alone did not induce ceramide formation, bile acids or cold shock treatment induce ceramide formation on endosomes through activation acid sphingomyelinase (ASM), and this event was crucial for virus replication because inhibition of ASM blocked ceramide formation and significantly reduced PEC replication. Incubation of FCV or MNV-1 with cells led to ceramide formation during virus entry, and inhibition of ASM also significantly reduced their replication. Inhibition of ASM led to endosomal retention of PEC, FCV or MNV-1 during virus entry, which may be the reason for the reduction of viral replication. These studies revealed the important and common events during calicivirus entry for successful replication, including virus endosomal escape, cathepsin L activity and ASM/ceramide formation. This detailed information may provide clues for understanding the replication of fastidious caliciviruses and for potential therapeutic targets.

Cathepsin S is a protease important in major histocompatibility complex (MHC) class II antigen presentation and also in degrading the extracellular matrix. Studies, most of them experimental, have shown that cathepsin S is involved in different pathological conditions such as obesity, inflammation, atherosclerosis, diabetes, and cancer.    The overall hypothesis of this report is that high levels of circulating cathepsin S, is a biomarker that reflects pathology induced by inflammation and obesity. The overall aim of this report was to investigate possible associations between circulating cathepsin S, inflammation, glucometabolic disturbance, and its associated diseases in the community. As cathepsin S appears to be a novel risk marker for several pathological conditions, we also wanted to examine the effect of dietary intervention on circulating cathepsin S concentrations.    This thesis is based on data from three community-based cohorts, the Uppsala longitudinal study of adult men (ULSAM), the prospective investigation of the vasculature in Uppsala seniors (PIVUS), and a post-hoc study from the randomized controlled NORDIET trial.    In the first study, we identified a cross-sectional positive association between serum cathepsin S and two markers of cytokine-mediated inflammation, CRP and IL-6. These associations were similar in non-obese individuals. In longitudinal analyses, higher cathepsin S at baseline was associated with higher CRP and IL-6 levels after six years of follow-up. In the second study, we identified a cross-sectional association between increased serum levels of cathepsin S and reduced insulin sensitivity. These associations were similar in non-obese individuals. No significant association was observed between cathepsin S and insulin secretion. In longitudinal analysis, higher cathepsin S levels were associated with an increased risk of developing diabetes during the six-year follow-up. In the third study, we found that higher serum levels of cathepsin S were associated with increased mortality risk. Moreover, in the ULSAM cohort, serum cathepsin S was independently associated with cause-specific mortality from cardiovascular disease and cancer. In the fourth study, we identified that adherence to an ad libitum healthy Nordic diet for 6 weeks slightly decreased the levels of plasma cathepsin S in normal or marginally overweight individuals, relative to the control group. Changes in circulating cathepsin S concentrations were correlated with changes in body weight, LDL-C, and total cholesterol.    Conclusion: This thesis shows that circulating cathepsin S is a biomarker that independently reflects inflammation, insulin resistance, the risk of developing diabetes, and mortality risk. Furthermore, a Nordic diet moderately reduced cathepsin S levels in normal-weight and overweight men and women. This effect may be partially mediated by diet-induced weight loss and possibly by reduced LDL-C concentrations.

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A characteristic feature of chronic wounds is a prolonged inflammatory response as well as susceptibility to infection. Studies have shown that during the inflammatory response, there is a significant increase in the levels of neutrophil-derived enzymes. The purpose of this work was to determine whether the anionic macromolecule polystyrene sulfonate (PSS) and five of its salt forms, namely PSS-calcium, PSS-chlorhexidine, PSS-doxycycline, PSS-glutathione and PSS-silver are able to inhibit the activity of three of the enzymes whose levels are elevated in chronic wounds: elastase, cathepsin G and myeloperoxidase. In addition to the enzyme inhibition study, the various formulations’ antimicrobial properties were analyzed by evaluating their ability to inhibit the growth of three common clinical isolates: Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumanii. It is worthy to note that the structure of PSS makes it a very flexible platform to which other molecules can be added in order to address a variety of “targets” as well as tailor quantitative strength. The results from this project showed that purified PSS and the various salt derivatives were able to inhibit elastase and cathepsin G activity. In addition, three of the therapeutic cations attached to PSS: silver, doxycycline and chlorhexidine retained their intrinsic antimicrobial properties without having an adverse effect on healthy tissue. In summary, this study demonstrated that PSS possessed an intrinsic ability to inhibit a number of proteases and that it could also be used as a delivery vehicle for other compounds with potential therapeutic value.

Colorado potato beetle (Leptinotarsa decemlineata) is an economically important herbivorous pest. Cathepsin D-like aspartic peptidase (LdCD) plays an important role during protein degradation in the midgut of Colorado potato beetle. This work describes the preparation of two expression systems, namely in Escherichia coli and Pichia pastoris, for the production of recombinant LdCD. The protocol for refolding of denatured LdCD was designed and optimized. Activation of the inactive LdCD zymogen and cleavage of the propetide (activation peptide) were investigated. This process proceeds autocatalytically at acidic pH or with the assistance of the cysteine peptidase legumain. The proteolytic activity of LdCD was characterized using fluorogenic peptidic substrate and protein substrates, and kinetic parameters and pH optimum were determined. The inhibition specificity of LdCD was analyzed using a panel of peptidase inhibitors. LdCD was significantly inhibited by PDI (potato cathepsin D inhibitor), a protein inhibitor produced in potato leaves. This suggests that PDI is a natural defense protein, which is directed against LdCD in the midgut of Colorado potato beetle in order to block the digestion. The potential application of PDI in the construction of transgenic crops resistant against insects is discussed.

The blood fluke Schistosoma mansoni and the hard tick Ixodes ricinus produce an aspartic protease cathepsin D which initiates degradation of hemoglobin, their key nutrient. First, in the presented work, the protocol for refolding and activation of the zymogen of cathepsin D from I. ricinus (IrCatD) was developed and optimized. In acidic pH the propeptide of IrCatD zymogen was removed by an auto-activation mechanism. Further, a kinetic assay with fluorogenic substrates was employed to study functional properties of IrCatD including pH optimum, substrate and inhibition specificities. Second, two isoforms of cathepsin D from S. mansoni (SmCatD) were produced using recombinant expression in E. coli. These recombinant proteases were isolated from inclusion bodies using affinity chromatography under denaturating conditions, and protocol for their refolding was developed and optimized. The studied aspartic proteases are pharmacological targets: inhibitors of SmCatD represent potential chemotherapeutics for the treatment of schistosomiasis, and IrCatD is a candidate antigen for the development of novel anti-tick vaccines.

Our experimental organism Fascioloides magna is a digenetic liver fluke from Fasciolidae family which parasitizes in domestic and free-living ruminants of North America and Central Europe including Czech Republic. In Czech Republic this highly pathogenic worm causes a severe liver damage to cervids and bovids and the prevalence locally reaches up to 95%. The biology of F. magna including e.g. the characteristics of host-parasite molecular interaction and the functions of particular molecules produced by the parasite are not fully understood. According to results of our previous research the excretory-secretory products of F. magna adults contain number of molecules which play the crucial role in host tissue invasion, digestion and evasion of the host immune response. One of the most abundant is cysteine peptidase cathepsin L (FmCL). FmCL is supposed to play various key roles in biological processes of all stages during a life cycle and therefore we can suppose its different expression level in particular life stages. In order to define the expression level of FmCL we performed the pilot study with miracidia and adults where the qPCR method was applied. The results of this experiment revealed much higher expression level of FmCL1 in adults than in miracidia. The attempt to in situ localize the mRNA...

This study is focused on cercarial cysteine peptidases of the trematode Diplostomum pseudospathaceum. It follows previous research which confirmed the presence of a 24kDa cysteine peptidase in cercariae biochemically and by mass spectrometry. It was postulated, that the function of this peptidase is histolytic, when cercariae penetrate the tissues. During an attempt to purify this peptidase and characterize its peptidolytic activity, it was found out that the cercarial homogenate containsmore different peptidases varying in their pI. Tests of peptidolytic activity and inhibition have shown that these peptidases are cathepsin L-like. They are active over a broad spectrum of pH with optima of activities in weakly acidicor neutral pH. Using degenerate primers based on conserved motifs of cysteine pepridases, partial sequences of three genes for cathepsin L of D. pseudospataceum (DpCL1, 2 a 3) were obtained. Then the complete sequences of DpCL2 and 3 genes and partial sequence (without 5'end) of DpCL1 were obtained by RACE PCR. To confirm function of these peptidases we tried to immunolocalize them. We assumed that they are localized in penetration glands. Preliminary results suggested that some of the cathepsins could be also localized in the gut of cercariae. For more detailed biochemical...

The blood processing mechanisms in monogeneans of the subclass Polyopisthocotylea are known from ultrastructural and histochemical analyses only. In contrast to other blood- feeding parasites, just few biochemical and molecular analyses have been done on digestive enzymes in monogeneans. Therefore, we focused on the biochemical and molecular characterization of hydrolytic enzymes (peptidases) in the hematophagous species Paradiplozoon bliccae and Eudiplozoon nipponicum. The presence of the cysteine class peptidases, mainly cathepsin L, in excretory- secretory products and soluble protein extracts of P. bliccae and E. nipponicum we found. Detection was carried out using fluorogenic substrates, specific inhibitors and the labelled probe DCG-04. On the gels / membranes after electrophoresis / blotting we detected bands of approximately size of 35 kDa in the case of both species and 24 kDa for E. nipponicum. Soluble protein extracts of worms were separated by 2D gel electrophoresis and relevant spots around 35 kDa (P. bliccae) and around 25 ˗ 35 kDa (E. nipponicum) were confirmed by mass spectrometry as cathepsins L. Using degenerate primers based on the conserved motifs of cysteine class peptidases, a partial sequence of cathepsin L gene from E. nipponicum was obtained. Furthermore, 3'RACE PCR method...