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Selected proteolytic aspects as targets to combat ticks and tick borne pathogens
HARTMANN, David
Ticks and tick-borne diseases (TBD) represent a growing global burden for both human and animal health. Tick-host-pathogen interactions have evolved through dynamic processes that accommodated the genetic traits of the hosts, pathogens transmitted, and the vector tick species that mediate their development and survival. As in other parasites, proteases and proteolysis have been found as one of the key factors in this interaction triangle. This thesis is focused on selected proteolytic aspects of tick and tick-borne diseases: (i) processing of host blood as a source of nutrients and energy (hematophagy) as a continuum of the long-term goal of the Laboratory of Vector Immunology, that established the currently accepted model of multienzyme degradation of host blood proteins by ticks (ii) proteases in innate immunity (iii) validation of Babesia proteasome as a potential therapeutic target against the tick transmitted apicomplexan parasites.
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Cathepsins L of Diplostomum pseudospathaceum cercariae
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Hartmann, David (referee)
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...
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Cathepsins L of Diplostomum pseudospathaceum cercariae
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Hartmann, David (referee)
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...
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Characterization and function of Factor C from the tick \kur{Ixodes ricinus}
HARTMANN, David
Factor C is a multi-domain serine protease which recognizes Gram-negative bacteria via binding to lipopolysaccharides and triggers hemolymph clotting cascade in the horseshoe crab. A closely related molecule was also found to be present in the genome of the tick Ixodes scapularis. In this work, the full sequence of Factor C ortholog from Ixodes ricinus (IrFC) was determined. IrFC is mainly expressed in tick hemocytes and the heavy chain of the activated molecules is present in tick hemolymph as confirmed by Western blotting with antibodies raised against recombinant fragments of IrFC. The function of the IrFC in tick innate immunity was assessed using its silencing by RNA interference.
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Genetic screening for the dominant suppressors of adgf-a mutant phenotype in Drosophila
HARTMANN, David
Extracellular adenosine is an important signaling molecule in various physiological processes. When not regulated properly it can cause various pathologies such as Severe Combined Immunodeficiency. To study the effects of increased extracellular adenosine level a Drosophila model has been established. This model is based on mutation in the main adenosine deaminase gene in Drosophila (ADGF-A, Adenosine Deaminase-related Growth Factor A). This work is a consequence of large screening for the suppressors of adgf-a mutant phenotype and presents a more detailed study of 3 previously identified regions containing dominant suppressors.
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