Bacterium Mycobacterium tuberculosis is an etiological agent of a deadly disease called tuberculosis that presents a global problem. According to The World Health Organization there are more than 2 billions people infected with latent tuberculosis all over the world. There is still need of specific, sensitive, quick and economic available method for identification of infected individuals. Currently in vitro blood tests are considered to be the best way of diagnosis. They are based on restimulation of specific T lymphocytes by mycobacterial antigens derived from virulent strains. There are several different approaches for enhancing of direct antigen delivery into antigen presenting cells and promising one is a genetically detoxified adenylate cyclase toxin (CyaA) of bacteria Bordetella pertussis. The main aim of the thesis includes construction and subsequent characterization of biological properties of CyaA protein carrying specific mycobacterial antigen TB7.7 in translocating domain. Here is shown that fusion protein CyaA-TB7.7 can form cation selective pores in target cell membranes and is able to deliver antigens into the cytosol of APC to be presented on surface with molecules MHC class II. Genetically detoxified CyaA- TB7.7 protein will be used to supplement current approaches such as also in vitro...

4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin

Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 67-89).

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [115]-133).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [78]-86).

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 154-163.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 78-93).

Bordetella pertussis produces many virulent factors including adenylate cyclase toxin (CyaA) This toxin preferentially invades cells of immune system with integrin receptor CD11b/CD18 and weakens the immune system of the host. CyaA affects invaded cells in two ways. First, CyaA creates a cation-selective pores in the membrane of invaded cell and causes colloidal osmotic lysis. Second, CyaA converts cytosolic ATP into signal molecule cAMP, which causes a loss of physiological function of invaded cell and also leads to cellular death. The aim of my thesis was to test a suitability of a new model system composed from synthetic lipids - diphytanoyls, for a characterization of pore-forming properties of adenylate cyclase toxin. In the past, asolectin model system comprising many different lipid was used for characterization but it was found to be too complex for defining the role of individual lipids in CyaA activity. Further the effect of cholesterol for activity of CyaA was studied in a new model system because it was found recently that translocation of adenylate cyclase domain takes place at lipids rafts with high concentration of cholesterol. The last aim of my thesis was to characterize a newly discovered type of channel with the two conductance levels. Key words: Bordetella pertussis, adenylate...