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Human serum transferrin (hTF) is a bilobal glycoprotein that plays a central role in iron metabolism. Each lobe of hTF (N- and C-lobe) can reversibly bind a single ferric iron. Iron binds to hTF at neutral pH in the plasma; diferric hTF binds to specific hTF receptors (TFR) on the cell surface and the complex undergoes receptor mediated endocytosis. The pH within the endosome is lowered to ~5.6 and iron is released from hTF. Apo hTF remains bound to the TFR and recycles back to the cell surface. Upon fusion with the plasma membrane, apo hTF dissociates from the TFR and is free to bind more iron and continue the cycle. The iron release process is complicated by various factors which include pH, anions, a chelator, lobe-lobe cooperativity and interaction with the TFR. All of these influence iron release in a complex manner. Because they are intricately linked, it is difficult to determine the effect of any single parameter. We have utilized stopped-flow and steady-state fluorescence and urea gel electrophoresis to dissect the iron release process as a function of lobe-lobe interactions, the presence of the TFR, and changes in pH and salt concentration. Application of recombinant protein production and site-directed mutagenesis has allowed us to generate a variety of hTF constructs in which the iron status of each lobe is completely controlled. Thus, we have created authentic monoferric hTFs unable to bind iron in one lobe, diferric hTFs with iron locked in one lobe and diferric hTF in which iron can be removed from both lobes. Importantly, we have produced the soluble portion of the TFR (sTFR) to analyze interactions between hTF and the sTFR and to monitor iron release from hTF/sTFR complexes. Together, we are able to provide a more precise picture of iron release from the two lobes of hTF in the presence and absence of the TFR. Steady-state fluorescence emission scans and urea gel electrophoresis provide a qualitative evaluation of the iron status of each construct after a predetermined incubation in iron removal buffer (i.e. an endpoint). However, these techniques do not provide information regarding the kinetic pathway to reach that endpoint. Combined with stopped-flow fluorescence time-based kinetics, a more precise assessment of the iron release process has been obtained. We have determined that changes in pH and salt affect endpoint iron release from the C-lobe, but not the N-lobe, however, the kinetics of iron release from both lobes are highly sensitive to pH and salt. Kinetic analysis in the absence and presence of the sTFR reveals the complexity of the iron release process. In the absence of the sTFR, the kinetics of iron release are insensitive to the iron status of the opposite lobe. However, in the presence of the sTFR, the kinetics of iron release from both lobes are affected by the iron status of the opposite lobe. Determination of conformational changes induced by anion binding, lobe-lobe communication and sTFR interactions have now been confidently assigned. We have created kinetic models of iron release from diferric hTF ± the sTFR and incorporated specific events pertaining to anion binding, lobe-lobe communication and conformational changes associated with sTFR interactions. We provide irrefutable evidence that a critical role of the sTFR is to accelerate the rate of iron release from the C-lobe, while decreasing the rate of iron release from the N-lobe such that the two lobes effectively release iron on a time scale relevant to one cycle of endocytosis.

The thesis deals with the issues of pre-trial detention in the criminal procedure of the Czech Republic. It comprises and compares opinions voiced by various scholars, high courts and myself. The thesis also includes a section about the rules of pre-trial detention in the American federal criminal procedure and compares it with the Czech system. The first part of the thesis is the Introduction, in which the goals of the thesis are set out. The second part is the main part of the thesis and it is divided into five chapters. The first chapter consists of a general description of pre-trial detention in the Czech Republic and the principles that govern it. The second part is called Formal Rules of Pre-Trial Detention, and it deals with the issues of competence, court procedure and decision-making. The third chapter is where the focus point of the thesis lies, as it investigates the conditions of ordering a pre-trial detention of a person and adresses the numerous problems that arise in this area. The fourth chapter describes the conditions for pre-trial release of an accused person, including the legislative changes recetly made in this area. The fifth chapter explores the American federal criminal procedure and compares it with the Czech criminal procedure. It also includes American scholars'...