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Physiological and pathophysiological role of GCPII in the body
Sedlák, František ; Konvalinka, Jan (advisor) ; Klener, Pavel (referee) ; Smetana, Karel (referee)
Glutamate carboxypeptidase II (GCPII) is a metalloprotease responsible for cleaving the neurotransmitter N-acetyl-aspartyl-glutamate in the central nervous system to N-acetyl aspartate and glutamate. At the same time, in the human small intestine, it facilitates folate absorption by cleaving γ-linked glutamate from folyl-poly-γ-glutamate. In humans, GCPII is also expressed in a number of other organs (e.g., kidney and prostate) and tumors, where its physiological function is unknown. In an attempt to characterize the physiological function of the enzyme, we first characterized the commercially available monoclonal antibodies against GCPII. Further, we developed a fully synthetic replacement based on a hydrophilic polymer with bound GCPII inhibitors. We evaluated the suitability of using a murine biomodel to study GCPII function in vivo. We found the difference in GCPII expression profile in mouse and human. We did not observe GCPII in either the mouse prostate or small intestine. To assess physiological and pathophysiological functions of the enzyme we analyzed a GCPII-deficient mouse model. Apart from the observation of enlarged seminal vesicles in older males, we did not detect any other obvious phenotype. Similarly, we confirmed that GCPII cannot cleave amyloid peptides (Aβ1-40 and Aβ1-42)....
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Analysis of antibody response during BK virus infection
Tomanová, Tereza ; Španielová, Hana (advisor) ; Saláková, Martina (referee)
BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...
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Characterization of viral nanoparticles derived from mouse papillomavirus
Vomáčka, Petr ; Španielová, Hana (advisor) ; Šmahelová, Jana (referee)
The L1 and L2 capsid proteins of papillomaviruses are characterized by the ability to self- assemble into viral capsids, which can be divided into pseudovirions (PsVs) and virus-like particles (VLPs) by inner content. In addition to the fact that such particles can serve as "nano-containers" for diagnostic and therapeutic agents, it has also been shown that papillomaviruses, whether wild, PsVs or VLPs have a higher affinity for tumor tissue than non-tumor tissue. This thesis deals with relatively newly discovered (2011) mouse papillomavirus (MusPV) and nanoparticles derived from this virus. This papillomavirus has been chosen for its positives, including easy preparation of VLPs and PsVs, as well as an available model organism for possible testing. Furthermore, MusPV has the potential for use in gene therapy and cancer diagnosis, because there is no immune response in the human population. The aim of this diploma thesis is to prepare an expression system for the production of PsVs and VLPs. In additional it will also look at the quality and quantity of PsVs and VLPs, characterization of these particles and verification of existing postulates regarding higher affinity of papillomaviruses for tumor cells. Finally, it will also to verify whether the same effect is observed in MusPV. In the results of...
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Analysis of antibody response during BK virus infection
Tomanová, Tereza ; Španielová, Hana (advisor) ; Saláková, Martina (referee)
BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...
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Influence of size and geometry of nanoparticles on cellular internalization pathways
Číhařová, Barbora ; Španielová, Hana (advisor) ; Lišková, Petra (referee)
Nanoparticles can be used in biomedical disciplines as carriers for transport of diagnostic as well as therapeutic substances into cells. Variety of different shapes, sizes and different compositions are used experimentally. Despite the discoveries already made in this area, the exact nature of the interaction between a nanoparticle and a cell has not been fully understood yet. The objective of this thesis is to provide the knowledge about possibilities of utilisation and aspects influencing the interaction between the cell membrane and several types of nanoparticles: liposomes, gold nanoparticles and virus-like nanoparticles. The comparison shows that generalisation of the mechanism of nanoparticle entry into the cell is problematic, although it seems that the spherical nanoparticles with the diameter of 50 nm provide the most efficient entry.
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