Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 59-63). Also available on the World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 152-176).

abstract: This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics. Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination. Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Barbora Hadámková Determination the mechanism of entry F. tularensis into B lymphocytes Diploma thesis Charles University, Faculty Of Pharmacy in Hradec Králové Study program: Pharmacy Background: Besides processing the research with basics knowledge of the problem, the main aim of the study was the analysis of mechanism of entrance of intracellular bacteria Francisella tularensis into B cells. Methods: The B cells, which we obtained through peritoneal lavage from mice Balb/c, we blocked using antibodies individual complement receptors, B cell receptor and Fcƴ receptor. The population of the cells was infected by bacteria F. tularensis LVS/GFP opsonized by complement and/or by antibodies. Using flow cytometry we measured the percentage of infection of individual subpopulations of B cell B1a, B1b and B2 and we evaluated the influence of blocking and opsonization on the infection. Results: From the measured data, we can say that the percentage of infected B cells after infection by F. tularensis opsonized by complement is increased. This increase was more distinct in subtype of B cells B1b and B2. On the other hand, the opsonization F. tularensis by antibodies did not affect the infection. We also found out, that blocking of Fcƴ receptor has decrease the infection, if we used for infection of B cells...

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