Vortrag ueber das Warum und Wie der Kryptografie mit oeffentlichen Schluesseln am Beispiel von GNU Privacy Guard

The ABC protein P-glycoprotein (Pgp, ABCB1) transports many structurally diverse substrates from the lipid bilayer. Previous studies demonstrated the importance of the membrane environment, but few have quantified these effects. In the present work, purified Pgp reconstituted into defined lipid systems was employed. Drug binding affinities were determined using Trp quenching, and drug-lipid partitioning by equilibrium dialysis. Pgp bound substrates from the bilayer with affinities in the millimolar range; both drug-Pgp and drug-lipid interactions were important. The kinetics of Pgp-mediated drug transport were sensitive to drug structure and lipid environment. The rate of transport is proposed to depend on the affinity of Pgp for substrate and conformational changes. The lipid bilayer affected the stability of Pgp catalytic activity which provided evidence for distinct basal and drug-stimulated ATPase cycles. Overall, the lipid environment had pronounced effects on Pgp-mediated drug binding, transport and catalytic functions. / Canadian Cancer Society

The purpose of this project was to determine how Dlc-2 and Rho signaling modulate the ceramide induction of PGP synthase. This induction was studied at the transcriptional, post-transcriptional, and post-translational levels using cell culture, Real-Time RT-PCR, protein purification, phage display, and western blotting techniques. We have demonstrated that the PGP synthase gene is not controlled at the transcriptional level by ceramide and Rho, nor is the mRNA stability of PGP synthase affected. However, ceramide and Rho do seem to exhibit translational or post-translational control over the PGP synthase protein. The relationships between Dlc-2 (and Rho), ceramide, and PGP synthase (and CL) are important to understand. All three are involved in cancer and apoptotic responses. The knowledge gained by the experiments discussed in this thesis will contribute to an understanding of how these proteins and lipids interact. This knowledge may then be used in the future to develop cancer treatments.

The purpose of this project was to determine how Dlc-2 and Rho signaling modulate the ceramide induction of PGP synthase. This induction was studied at the transcriptional, post-transcriptional, and post-translational levels using cell culture, Real-Time RT-PCR, protein purification, phage display, and western blotting techniques. We have demonstrated that the PGP synthase gene is not controlled at the transcriptional level by ceramide and Rho, nor is the mRNA stability of PGP synthase affected. However, ceramide and Rho do seem to exhibit translational or post-translational control over the PGP synthase protein. The relationships between Dlc-2 (and Rho), ceramide, and PGP synthase (and CL) are important to understand. All three are involved in cancer and apoptotic responses. The knowledge gained by the experiments discussed in this thesis will contribute to an understanding of how these proteins and lipids interact. This knowledge may then be used in the future to develop cancer treatments.

Huvudfrågan i arbetet är hur produktivitet och säkerhet kombineras i de säkerhetslösningar som existerar idag samt huruvida högre säkerhet leder till ökad komplexitet. Det tillvägagångssätt som använts består av en lastmätning av Secure Socket Layer (SSL), och Pretty Good Privacy (PGP), samt en teoretisk jämförelse av säkerhetslösningarna PGP, SSL och Secure Electronic Transaction (SET). Detta innebär att PGP som har jämförelsevis hög säkerhet när det kommer till kryptering även är den säkerhetslösning är mest resurskrävande. SSL däremot är mindre säkert med betydligt lägre resursanvändning. Generellt kan man med facit i hand säga att den lösning som är säkrast även är den mest komplexa. Den säkerhetsaspekt lastmätningen bygger på är främst krypteringsalgoritm och nyckellängd. Även om dessa aspekter är allmänt vedertagna i säkerhetstänkande måste även hänsyn tas till säkerhetstänkandet som helhet. SET visar ett sätt att kringgå den klassiska motsättningen mellan säkerhet och produktivitet genom att begränsa användningsområdet, men detta på bekostnad av flexibilitet och ger vidare potential för interoperabilitet.

Im Vortrag werden die Funktionsprinzipien von PGP sowie verwendete Verfahren dargelegt. Ziel ist die Schaffung einer geeigneten Technologie im Universitaetsrechenzentrum zur Sicherung des Mailbetriebs.

The rates of opioid prescription and use have continued to increase over the last few decades. In turn, a greater number of patients suffer from opioid tolerance. Treatment of acute pain is a clinical challenge for these patients. Acute pain can arise from common occurrences like surgical pain and pain resulting from the injury. P-glycoprotein (p-gp) is a transporter at the blood-brain barrier (BBB) associated with a decrease in the analgesic efficacy of morphine. Peripheral inflammatory pain (PIP) is a pain state known to cause a change in p-gp trafficking at the BBB. P-gp traffics from the nucleus to the luminal surface of endothelial cells making up the BBB. This surface where circulating blood interfaces with the endothelial cell is where p-gp will efflux morphine back into circulation. Osmotic minipumps were used as a long-term delivery method in this model of opioid tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase when animals were exposed to opioids for 6 days. This observation presents a possible relationship between p-gp trafficking and the challenges of treating post-surgical pain in opioid tolerant patients. This could reveal potential strategies for improving pain management in these patients.

Primary brain tumours, and especially the most common form malignant gliomas, usually display a pronounced resistance to other treatment modalities when surgery fails to cure. Growth factors, such as EGF and its receptor, frequently amplified and overexpressed in malignant gliomas, and factors associated with multidrug resistance have been suggested to at least partially explain the poor outcome. The aim of this thesis was to characterise factors in primary brain tumours associated with the development of resistance with focus on the epidermal growth factor receptor (ErbB) family, and multidrug resistance (MDR). Influences of irradiation on the expression and activity of P-glycoprotein (Pgp) in malignant gliomas was evaluated. The effects showed that irradiation increased the efflux activity of Pgp in rat brain vascular endothelial cells, but not in glioma cells. In the intracranial BT4C glioma model, Pgp was detected in the capillary endothelium in the tumour tissue but not in glioma cells. Expression of several factors coupled to MDR (Pgp, MRP1, LRP, and MGMT) in primary brain tumours were analysed and correlated to clinical data. In gliomas, Pgp and MRP1 were predominantly observed in capillary endothelium and in scattered tumour cells, whereas LRP occurred only in tumour cells. In meningiomas, expression of the analysed markers was demonstrated in the capillary endothelium, with a higher expression of Pgp and MRP1 in transitional compared to meningothelial meningiomas. A pronounced expression of MGMT was found independently of the histopathological grade or tumour type. Survival analysis indicated a shorter overall survival for patients suffering from low-grade gliomas with high expression of Pgp. To explore the importance of the epidermal growth factor receptor (EGFR), expression levels of the family members (EGFR, ErbB2-4) were analysed and their relations to various clinical parameters were evaluated in gliomas and meningiomas. In gliomas, the highest EGFR expression was observed in high-grade tumours, while ErbB4 expression was most pronounced in low-grade tumours. In meningiomas, expression of EGFR, ErbB2, and ErbB4 was observed in the majority of the tumours. An intriguing observation in low-grade gliomas was a significantly decreased overall survival for patients with high EGFR protein expression. The effects of different time schedules for administration of the selective EGFR inhibitor ZD1839 in relation to irradiation of glioma cells were analysed. The analyses showed a heterogeneity in the cytotoxic effects of ZD1839 between cell lines, and it was obvious that some of the cell lines showed sensitivity to ZD1839 despite no or low expression of EGFR. The study also demonstrated the importance of timing of ZD1839 administration when this agent is combined with irradiation. In conclusion, in order to enhance the efficacy of radiotherapy by various drugs in malignant gliomas it may be essential to inhibit drug efflux activity in endothelial cells and to deliver drugs in an optimal timing in relation to radiotherapy. The heterogeneity in expression of drug resistance markers, as well as the ErbB family reflects the complexity in classification of primary brain tumours, and indicates that subgroups of patients with low-grade gliomas expressing Pgp and EGFR might benefit from more aggressive and individualised treatment.

P-glycoprotein (Pgp, ABCB1) is a polyspecific efflux transporter implicated in multidrug resistance in human cancers. In this study, tetramethylrhodamine-5-carbonyl azide (AzTMR) was covalently crosslinked to the Pgp drug binding pocket with a stoichiometry of 1. The Pgp-AzTMR adduct was functionally equivalent to unlabelled Pgp and retained its ability to transport Hoechst 33342. The binding site of AzTMR in Pgp was nonpolar, with a similar environment to that of propanol. Pgp-AzTMR could bind a second drug molecule, with a higher affinity for H-site drugs and lower affinity for other R-site drugs. Unlabelled Pgp interacted with dimeric versions of known Pgp modulators, binding them with higher affinity than the monomer. These compounds were also found to either stimulate or inhibit Pgp ATPase activity depending on the concentration. Pgp-AzTMR was able to bind dimeric drugs, indicating that 3 substrate moieties can fit into the binding pocket. / The Canadian Cancer Society

Central mechanisms involving aldosterone - mineralocorticoid receptor (MR) activation mediate the increase in sympathetic tone after myocardial infarction (MI). We hypothesized that an increase in cardiac sympathetic activity (CSA) post MI facilitates cardiac sympathetic axonal sprouting, and that central MR blockade attenuates CSA and reduces cardiac sympathetic hyperinnervation post MI. Western blotting and qRT-PCR were used to assess protein and gene expression, and fluorescent immunohistochemistry was used to study changes in sympathetic innervation. Tyrosine hydroxylase (TH) and Norepinephrine transporter protein content in the non-infarcted base of the heart remained unaltered. In contrast, protein gene product (PGP 9.5) protein was significantly increased 2 fold in the base of the heart, and 6 fold in the peri-infarct area at 1 wk post MI, and associated with increased ubiquitin expression. Cardiac myocytes rather than sympathetic axons were identified as the main source of elevated PGP 9.5 expression. At the infarct border sympathetic hyperinnervation was observed with a 4 fold increase in growth associated protein 43 (GAP 43), a 2 fold increase in TH and a 50% increase in PGP 9.5 positive fibers when compared to the epicardial side of the left ventricle in sham rats. Central infusion of the MR blocker eplerenone at 5 ug/day for 9 days post MI markedly attenuated the increase in TH, GAP 43 and PGP 9.5 nerve densities at the infarct border. Central MR blockade may attenuate sympathetic hyperinnervation by several mechanisms, including decreasing CSA post MI, or affecting expression or function of nerve growth factor protein. Marked PGP 9.5 expression occurs in cardiomyocytes early post MI, which may contribute to the increase in ubiquitin and the early cardiac remodeling post MI.