Microtubules (MTs) are one of the essential cell structure that participate in a number of key events in the plant cells and their properties and functions are influenced and modified by many other proteins. These proteins belong to a group of microtubule- associated proteins (MAPs, microtubule-associated proteins). One of the MAPs, the molecular chaperone Hsp90, examines and fulfills a large number of different functions in the cell. Its colocalization with MTs has been demonstrated previously by Freudenreich and Nick (1998) and Petrášek et al. (1998). However, direct interaction with MTs was described only recently using cosedimentation assay. The specific cytosolic isoform of tobacco Hsp90 bound to MTs was called Hsp90_MT due to its ability to bind MTs. It has been also found that the binding to MTs is independent on the activity of ATP (Krtková et al., 2012). The authors also described a positive effect of Hsp90_MT on MT recovery after their exposure to cold stress. Although MT cytoskeleton dynamics is influenced by a large number of MAPs, it is surprising that the molecular mechanism of MAPs interaction with MTs and their MT-binding domains have not been described yet. Therefore, we decided to determine the tobacco Hsp90_MT MT-binding domain by production of a set of recombinant proteins...

NK cells are a component of innate immunity system, which is derived from lymphoid progenitor. By a sophisticated receptor repertoire, which is expressed on their surface, they provide a surveillance against pathogenic, virus infected or tumour cells. Simultaneously they produce cytokines, thereby are involved in adaptive immune response. This work is focused on the study of structure of mice soluble mNkr-p1a isoform. Recently this short isoform was identified at the transcriptional level by a member of our laboratory and it is designated as isoform 2. The aim was to produce mNkr-p1a iso2 protein in the prokaryotic expression system and to perform its renaturation and purification in vitro. In the next phase of work, the obtained product was analyzed by the mass spectrometry methods. Recieved results made us think about that our protein is in unfolded state. This assumption was refuted by following biophysical methods, nuclear magnetic resonance, circular dichroism and dynamic light scattering measurement. Keywords: NK cells Receptor mNkr - p1a Short isoform mNkr - p1a iso2 Alternative splicing Protein biosynthesis Recombinant protein production Protein purification Mass spectrometry Disulfide bond Chemical cross-linking NMR, CD, DLS 5

11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...

Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...

Natural killer (NK) cells represent indispensable part of the innate immunity as they are capable of promptly identifying virally infected or tumor cells and participating in the regulation of adaptive immune responses. These functions are ensured by the interplay between NK receptors, creating a complex regulatory system. Solving the receptors' structure may contribute to an overall understanding of NK cell biology. Presented thesis describes an elucidation of the structure of the inhibitory C-type lectin-like receptor (CTLR) Nkrp1b with an emphasis toward structural features (stalk, loop and oligomerization state) which might affect conformation or interactions of this receptor. The interaction of Nkrp1b with its ligand, Clr-b protein, is immunologically significant as it regulates NK cells' activity independently and monitors changes that are not visible to cytotoxic T lymphocytes. To study individual structural aspects of Nkrp1b, two protein variants were recombinantly prepared in bacterial expression system: entire ectodomain and ligand-binding domain lacking the stalk. Using a range of mass spectrometric techniques in combination with homology modeling and molecular dynamics, we proposed the Nkrp1b structure including its monomeric and dimeric arrangements. In addition, the oligomerization...

The existing seasonal influenza vaccine does not provide broad long-term protection against seasonal influenza and must be remanufactured yearly due to frequens mutations and reassortment of theinfluenza genes. A universal influenza vaccine with the ability to raise long lasting immunity is the focus of several studies, including the Edufluvac project. Edufluvac is based on virus-likeparticles, a modern recombinant platform wellsuited for vaccinatin applications. Redbiotec's rePAX® technology allows the generation of multivalent recombinant baculovirus which generatesvirus-like particles presenting multiple proteins on the surface in insect cell culture. Any effects oninsect cell culture protein expression brought on by the regulatory elements controlling each gene in the baculovirus, or by the genome position of the baculovirus genes, could affect the composition of the virus-like  particles. The ai of this thesis was to elicit a better understanding of the protein expression by analysing multi-protein influenza virus-like particles and virus-like particles encoding a reporter gene regulated by different promoter and terminator combinations. Different bivalent and tetravalent influenza gene bacmids were cloned as well as seven bacmids encoding a YFP gene regulated by different promoter and terminator combinations. Spodopera frugiperda cells weretransfected with the bacmids and harvested recombinant baculovirus was used to perform testexpressions in High-Five™ cells. The resulting protein expression levels from the bivalent andtetravalent recombinant baculovirus were analyzed and compared by Western blots and ELISA assays. The expression of YFP in infected Spodoptera and High-Five™ cells was monitored byfluorescence microscopy and measured with FACS to quantify protein expression differencesbetween the seven promoter and terminator combinations. Analysis of the bivalent constructs indicated that the order of the genes in a recombinant baculovirus does not affect the protein expression in High-Five™ cells. The analysis of the tetravalent constructs revelaed positionalvariations in expressin of the H1 and M1 genes, but the number of test expressions and recombinant baculovirus construct clones included in the analysis were not hogh enough to allow a definitive conclusion. Of the different promoter and terminator constructs highest mean fluorescence intensity was reached with the reference combination. The early promoter yielded mean fluorescent intensitites that were close to the values of the negative control in both cell lines.

No description available.

Malaria is a fatal tropical disease affecting billions of people in impoverished countries world-wide. An alarming fact is that a child in Africa dies of malaria every 30 seconds that amounts to 2500 children per day (www.who.int/features/factfiles). Malaria is caused by the intraerythrocytic forms of Plasmodium species, notably P. falciparum, P. vivax, P. ovale and P. malariae (Hyde 2007). The spread of drug-resistant strains, failure of vector control programs, rapid growth rate of the parasite, and lack of a vaccine have further exacerbated the effects of malaria on economic development and human health. It is therefore imperative that novel drug targets are developed or current antimalarial drugs optimized (Foley and Tilley 1998). One such target is folate biosynthesis, given that folates and their derivatives are required for the survival of organisms (Muller et al. 2009). DHFR and DHPS are currently the only folate targets exploited however, their antifolate drugs are almost useless against parasite resistant strains. As such, guanosine-5’triphosphate cyclohydrolase I (GTPCHl) among other antifolate candidates are considered for intervention (Lee et al. 2001). Knock-out studies (of P. falciparum gtpchI) resulted in the suppression of DHPS activity (Nzila et al. 2005). Additionally, gtpchI amplified 11-fold in P. falciparum strains resistant to antifolates due to mutations in dhps and dhfr and this may be a mechanism for the compensation of reduced flux of folate intermediates (Kidgell et al. 2006; Nair et al. 2008). Over-expression of P. falciparum proteins in E. coli remains a challenge mainly due to the A+T rich Plasmodium genome resulting in a codon bias. This results in the expression of recombinant proteins as insoluble proteins sequestered in inclusion bodies (Carrio and Villaverde 2002; Mehlin et al. 2006; Birkholtz et al. 2008a). Comparative expression studies were conducted of native GTPCHI (nGTPCHI), codon optimized GTPCHI (oGTPCHI) and codon harmonized (hGTPCHI) in various E. coli cell lines, using alternative media compositions and co-expression with Pfhsp70. The nGTPCHI protein did not express because the gene consisted of codons rarely used by E. coli (codon bias). The expression levels of purified hGTPCHI were a greater in comparison to oGTPCHI using the different expression conditions. This is because codon-harmonization involves substituting codons to replicate the codon frequency preference of the target gene in P. falciparum, as such the translation machinery matches that of Plasmodium (Angov et al. 2008). Furthermore, greater expression levels of GTPCHI were achieved in the absence of Pfhsp70 due to expression of a possible Nterminal deletion product or E. coli protein. Purification conditions could be improved to obtain homogenous GTPCHI and further analysis (mass spectrometry and enzyme activity assays) would be required to determine the nature of soluble GTPCHI obtained. To improve the expression of soluble proteins the wheat germ expression system was used as an alternate host. However, GTPCHI expression was not effective, possibly due to degradation of mRNA template or the absence of translation enhancer elements. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Intracellular calcium is a major determinant of a wide variety of cell functions and thus of organ function. In order to get a clear picture of the intracellular calcium status it is preferable to assess the content of the various intracellular calcium pools as well as the characteristics of the transmembrane calcium movements, Le., the magnitude of the transmembrane Ca2+ flux upon stimulation and the rate of the subsequent return to baseline levels. The first aim of this study was to establish and evaluate the methods in the laboratory. The methods investigated include atomic absorption spectrometry, graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry for the determination of the total cell calcium content, fluorescence spectrophotometry for the determinations of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The methods eventually identified as feasible included fluorescence spectrophotometry for the determination of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The newly developed fluorescent calcium indicator, fura-PE3, was presently shown to be the most reliable fluorescent indicator for the intracellular free Ca2+ determinations. The best method for the calcium localisation by transmission electron microscopy was an adaptation of the antimonate precipitation technique. The following objectives were set in order to contribute to the knowledge in chronic renal failure; examination of the intracellular free Ca2+ content in the neutrophils of end stage renal failure patients on maintenance haemodialysis treatment, as the result of renal failure, dialysis treatment and medication combined; examination of the characteristics of the transmembrane Ca2+ movements; investigation of the intracellular calcium distribution in the neutrophils; exploration of a possible link between the alterations in intracellular calcium status and factors known to influence the calcium status, including the lipid composition of the membrane, the oxidative status as reflected by anti-oxidant vitamin levels, as well as the levels of parathyroid hormone, and ionised serum calcium. This study involved 14 chronic renal failure patients on maintenance haemodialysis. An increase in intracellular free Ca2+, the magnitude of the transmembrane Ca2+ flux upon fMLP stimulation and an increase in the rate of the subsequent decrease in intracellular free calcium were found. In separating the patients into those receiving rHuEPO and those not receiving rHuEPO, it was seen that the significance in the increase in intracellular free Ca2+ could be ascribed to the values obtained in those patients receiving rHuEPO - despite the fact that they were the only patients receiving calcium channel blockers. No overt indications of oxidative stress could be detected by anti-oxidant vitamin levels. Nevertheless, a decrease in the content of specific membrane fatty acids occurred, supporting the previous suggestions of the presence of a mild chronic inflammatory condition in the chronic renal failure patient on maintenance haemodialysis treatment. These results suggest that factors other than those associated with uraemia, such as rHuEPO administration, might result in an increase in intracellular free Ca2+ in cells of CRF/MHT patients. The magnitude of the rHuEPD-induced increase in intracellular free Ca2+ and the effects of the various calcium channel blockers need urgent further investigation as ineffective counteraction of the rHuEPO effect, as indicated by the relative ineffectivity of Norvasc, may have serious side-effects. / Dissertation (MSc)--University of Pretoria, 2000. / Physiology / unrestricted

>Magister Scientiae - MSc / This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.