No description available.

Thesis (doctoral)--Albert-Ludwigs-Universität Freiburg im Breisgau, 1999.

EN The growing emergence of bacteria resistant to conventional antibiotics is very alarming. This has prompted an intensive search for alternative antimicrobial agents which kill bacteria with different modes of action than do traditional antibiotics and do not develop drug resistance. Among these, antimicrobial peptides (AMPs) are considered as promising compounds against resistant pathogens. These positively charged peptides permeabilize or disrupt bacterial cell envelope which leads to leakage of cytoplasmic components and cell death. The aim of my dissertation thesis was the study of the action mechanism of novel antimicrobial peptides which I have isolated from the venom of different wild bees. I identified six novel AMPs which were named panurgines (PNG), codesane (COD) and antapines (ANTPs). These peptides were isolated from the venom of three different bee species (Panurgus calcaratus, Collete daviesanus and Anthophora plumipes). I was also involved in the structural studies of lasiocepsin (Las), the antimicrobial peptide identified in the venom earlier in our laboratory. All studied peptides possess activity against various strains of bacteria and low or moderate hemolytic activity. We prepared series of PNG, COD and ANTP analogs in order to study the effect of physicochemical properties...

Research in the field of molecular neuroscience contributes a better perception of the events that trigger neurodegeneration. At the forefront of this work is the study of intracellular calcium as a consequence of mitochondrial dumping and NMDA receptor activation by glutamate. Increased intracellular calcium presages excitotoxicity with ultimate apoptosis of the cell. Among the many disorders involving this sequence is depression, a disorder that in and of itself is a risk factor for neurodegenerative disorders such as Alzheimer disease. The successful use of anti-depressants to alleviate the depressive state leads to the question about whether these pharmacological agents, as part of their effect to ameliorate depression, might have an effect on intracellular calcium. Until now, this has not been explored directly but such exploration was initiated with this thesis. As a model for astrocytes and neurons, two cell lines, C6 and SH-SY5Y were used. These were differentiated with all-trans retinoic acid into astrocyte-like and neuron-like cells. Graphic imaging of intracellular calcium by ratiometrics is not new, but what is new is using this technique to evaluate the effect of the antidepressants mirtazapine and citalopram on intracellular calcium fluxes induced by glutamate. Furthermore, comparing the ratiometric intracellular calcium flux in the presence of mirtazapine and citalopram to that of known NMDA blockers was also done for the first time. Also studied were the acute and chronic effects of mirtazapine and citalopram on cell viability. The antidepressant agents, mirtazapine and citalopram, were chosen for this study. Mirtazapine blocks the adrenergic and serotonergic inhibitory autoreceptors which results in the increased release of these neurotransmitters and increases their concentration in the synapse. And it also has been shown to have an anti-oxidant and a calcium modulatory effect. Citalopram has the highest degree of serotonin reuptake selectivity of all the selective serotonin reuptake inhibitors. The ratiometric studies found that mirtazapine and citalopram reduce the effect of glutamate-induced increase in relative [Ca2+]i by either a direct or indirect action on NMDA receptors. This effect is not similar to the NMDA blockers memantine and AP5. The supporting evidence is that CCCP, which normally releases calcium from mitochondria, has no effect in cells treated with acute mirtazapine or citalopram. This indicates that no calcium entered the cell – and subsequently none was taken up by mitochondria – in response to glutamate. However, these observations were with a limited number of cells and, therefore, these results will have to be verified by different techniques by different laboratories. In microplate studies, all drugs studied reduced cell viability but the mechanism behind this reduced viability remains to be determined. This may be due to mutations in enzymatic expression, uptake of drug through the cell membrane, or other perturbations. The reduction in cell viability induced by acute glutamate was attenuated by pretreatment with mirtazapine or citalopram. Moreover, chronic treatment of the cells with mirtazapine or citalopram for 10 weeks before acute treatment with glutamate either attenuated the effect on viability or reversed it. Based on this present study, mirtazapine and citalopram may be useful as neuroprotective agents to alleviate not only depression but also to reduce cell death in neurodegenerative diseases, trauma and stroke.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

No description available.

La résistance pléiotropique (MDR) est une propriété de certaines cellules relative à la capacité de rejeter ou d’évacuer une très large variété de substances potentiellement toxiques. Les pompes à l’origine de ce rejet sont des protéines membranaires appartenant à la superfamille ABC (ATP-Binding Cassette). Deux membres de cette famille ABC confèrent la propriété de résistance pléiotropique, P-gp (P-glycoprotein) et MRP1 (Multidrug Resistance-associated Protein). Nous avons mené une étude fonctionnelle sur l’activité de ces deux transporteurs dans la muqueuse olfactive à la fois chez le rat et la souris. Nous avons employé le test fluorométrique à la calcéine-AM sur des tranches coronales de la muqueuse olfactive incubées en présence d’inhibiteurs spécifiques des transporteurs de la résistance pléiotropique, vérapamil et cyclosporine A comme inhibiteurs de Pgp ainsi que probénécide et MK571 comme inhibiteurs de MRP1. Chacun de ces quatre inhibiteurs provoque une augmentation significative de l’intensité de la fluorescence.Afin de savoir si les transporteurs de la résistance pléiotropique peuvent être impliqués dans la réponse olfactive nous avons examiné les réponses évoquées par des odorants seuls ou mélangés à l’aide d’enregistrements d’électro-olfactogrammes (EOG). En présence des deux inhibiteurs de MRP1, l’amplitude maximale des EOG est significativement réduite pour chaque stimulus odorant testé, tandis que les inhibiteurs de Pgp n’ont qu’un effet modéré ou nul. L’expression des gènes codant pour Pgp et MRP1 dans l’épithélium olfactif ont ensuite été confirmées par RT-PCR. L’ensemble de ces résultats suggère que les transporteurs MRP1 et Pgp sont présents et fonctionnels dans l’épithélium olfactif principal des rongeurs et sont impliqués dans la réponse aux odorants. Leur fonction précise dans l’olfaction reste à élucider / Multidrug resistance (MDR) is a property of various cells associated with the capacity to reject or efflux a wide range of potentially harmful substances out of the cell. Pumps that effect such efflux are membrane proteins and belong to the ATP- binding cassette (ABC) superfamily. Among the members of the ABC family two are conferring MDR, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP1). In this study we investigated the functional activity of MDR transporters in olfactory mucosa of two species, rat and mouse. We used the fluorometric calcein-AM uptake assay on olfactory mucosal slices incubated with specific inhibitors of the MDR-transporters, verapamil and cyclosporin A as Pgp-inhibitors, and probenecid and MK571 as MRP-inhibitors. All four inhibitors caused significant increases in fluorescence intensities. To test if MDR transporters may be involved in the olfactory response we examined odorant evoked responses to single and mixed odorants by means of electro-olfactograms recordings (EOG). In the presence of the two MRP inhibitors, maximum EOG amplitudes were significantly reduced for all odorants tested, while Pgp inhibitors had only a moderate or no effect. Expression of Pgp and MRP1 encoding genes in the olfactory epithelium was further confirmed by RT-PCR. The results together suggest that MRP and Pgp transporters are present and functional in the main olfactory epithelium of rodents and are implicated in the olfactory response. The precise functional role in olfaction remains to be elucidated.

People receiving chemotherapy not only suffer from side effects of therapeutics but also must buy expensive drugs. Targeted drug and gene delivery directed to specific tumor-cells is one way to reduce the side effect of drugs and use less amount of therapeutics. In this research, two novel liposomal nanocarriers were developed. This nanocarrier, called an eLiposome, is basically one or more emulsion droplets inside a liposome. Emulsion droplets are made of perfluorocarbons which usually have a high vapor pressure. Calcein (as a model drug) and Paclitaxel were used to demonstrate drug delivery, and plasmids and siRNA were used to exemplify gene delivery. Drugs or genes were encapsulated inside the interior of the liposomes along with emulsion droplets; targeting moieties were attached to the outside of the phospholipid bilayer. Ultrasound was used to break open the bilayer by changing the liquid emulsion droplets to gas, which released the content of the eLiposomes. Transmission electron microscopy (TEM) was used to prove the formation of eLiposomes and confocal microscopy showed the uptake of drugs and genes in vitro. Cell viability was measured to show the effect of uptake in cancer cells. Results indicate that eLiposomes were successfully made and that they were endocytosed into the cell. It was observed that the emulsion and the targeting moiety in combination with ultrasound are the essential elements required to produce release from eLiposomes.

Hypertension is the chronic elevation in blood pressure that is regulated in part through the retention and regulation of sodium retention and excretion in the kidneys. Hence the kidney has been considered the organ that regulates blood pressure. There are a cohort of patients that suffer with high blood pressure due to lack of 11β-hydroxysteroid dehydrogenase-type 2 (11β-HSD2) expression (which inactivates glucocorticoids (GCs), allowing selective activation of mineralocorticoid receptors (MR) by aldosterone) that results in hypertensive and increased salt appetite phenotypes - a condition known as syndrome of apparent mineralocorticoid excess (SAME). This disorder can be recapitulated in the mouse through the global deletion of 11β-HSD2, which results in over activation of the MR driving an elevation in blood pressure. However, the distinction between blood pressure elevation because of kidney dysfunction with loss of 11β-HSD2 or increased salt appetite due to loss of brain 11β-HSD2 expression is not clear from the global 11β-HSD2 knockout model. Salt appetite is regulated by regions of the brain out-with the blood-brain barrier, known as circumventricular organs. In the mouse, salt appetite is controlled by aldosterone-sensitive cells in the nucleus of the solitary tract (NTS) in the brain stem, where 11β-HSD2 is expressed to provide mineralocorticoid selectivity. However, in the fetal brain, 11β-HSD2 is widely expressed, protecting against adverse GC action that alters brain development and increases susceptibility to psychiatric disorders as adults. 11β-HSD2 deletion solely in the brain from embryonic day 12 resulting in GC fetal programming (HSD2BKO) causes effects on both behaviour and salt appetite. To determine the role of developmental versus adult expression of brain 11β- HSD2, mice with deletion of brain 11β-HSD2 from mid gestation (HSD2BKO) and mice with adult deletion of 11β-HSD2 in the NTS using lentivirus (HSD2.v- BKD) were compared. The phenotypes (salt appetite, blood pressure (BP), baroreceptor response (BRR) and cognition), can be categorised as either due to GC fetal programming (as indicated by HSD2BKO groups), or increased activation of MR in adult 11β-HSD2 expressing neurons (recapitulated in the HSD2.v-Cre groups). Salt appetite increased in both HSD2BKO and HSD2.v-BKD cohorts (mean percentage increase 65% n=8 and 46% n=6, compared to their respective controls), leading to an increased BP in both groups (+12% and +8%, respectively) as well as an impaired BRR, indicating all phenotypes are mediated by adult NTS neurons. However, spatial recognition memory (Object-in-Place task) is abolished in HSD2BKO mice, whereas, HSD2.v-BKD mice still retain short-term memory. Our data suggest that neural 11β-HSD2 protects against inappropriate activation of MR by corticosterone to regulate salt appetite and salt-induced rises in blood pressure. However, spatial recognition memory is not influenced by deletion of 11β-HSD2 in the adult brain, confirmation that this phenotype is underpinned by developmental programming by GCs, which is observed in the 11β-HSD2 brain KO. Salt appetite has been shown to be centrally regulated through the adult deletion of 11β-HSD2. From this, our data suggest that an increased salt appetite is due to adult loss of function of 11β-HSD2 rather than GC programming during development. Highlighting the NTS as a region for drug delivery to try and control salt appetite in salt sensitive individuals who struggle with administering a recommended change in diet. To develop this further, minimally invasive modes of delivery of viruses and drugs into the brain were investigated. In so doing, a non-invasive and reversible method to temporarily disrupt the blood brain barrier (BBB) was optimised. The technique required acoustic insonation of ultrasonic contrast agents (CAs) (gas microbubbles) adjacent to the BBB. These microbubbles (SonoVueTM, Bracco) were delivered via tail vein injection into the vasculature. To target the BBB, an ultrasonic transducer was suspended and focused through coupling gel onto the area of interest in the brain with skull the intact. The optimisation of this technique required determination of the focal position of the 3.5MHz transducer that was utilised, in addition to optimisation of the pulse length, pulse repetition frequency and power output of the ultrasound beam to enable the BBB to be disrupted. In addition, measurement of the attenuation of the ultrasound beam through ex vivo mouse skulls were measured. These results showed a 50% reduction in pressure amplitude from the baseline of 335.2mV (Baseline mean = 100% +/-SEM 0 n=3 (No skull), five regions across the skull averaged 47.79% +/-SEM 1.913 n=25 (using 5 different animals). In in vivo mice, after co-injection of the microbubbles with Evans Blue and insonation of the brain, disruption of the BBB was confirmed by the presence of Evans Blue dye in the brain, with no measurable damage occurring in the brain. This was confirmed by cell and nuclear morphology with no red blood cell extravasation into the surrounding tissue. The parameters used to open the BBB used a peak negative pressure of 2.1MPa (single pulse), transducer frequency 3.5MHz, 35,000 cycles over a 10ms burst at a pulse repetition frequency of 10Hz. The technique when applied in vivo in recovery animals is speculated to work by the focused ultrasound causing the microbubbles to oscillate within the vasculature adjacent to the BBB, resulting in high-shear stresses being generated on the tight junctions within the BBB. The resultant gaps in the BBB allow free circulating compounds (e.g. large dye molecules (Evans Blue - 960.8g/mol molecular weight) and adeno-associated-viruses (25nm with a packing capacity of 4.5kb) within the blood to pass into the brain, but there is no penetration of red blood cells (7μm). Longitudinal mouse experiments demonstrated that within 12-hours these gaps close with no long-term damage observed. Currently, utilising this technique, successful passage of an adeno-associated virus expressing GFP (as a marker) has been shown to pass into the brain (n=6 for each cohort including control) - indicating that the virus requires the ultrasound and microbubbles to facilitate its movement into the brain. Further technique optimisation is being explored looking at the role of CAs used in the opening and disruption of the BBB, comparing composition and size of the CAs. Microbubbles (2-3μm) and nanobubbles (200nm) were compared as well as lipid and non-ionic surfactant surface compositions, using volume of drug delivery and degree of disruption as outputs. Using this technique, the hydrophilic drug mimic calcein was delivered into the brain (n=5 non-ionic surfactant nanobubble, n=5 lipid nanobubble). Results have indicated that the delivery of calcein is most efficient when using non-ionic surfactant nanobubbles as opposed to lipid nanobubbles - with a greater volume of the drug being delivered into the cerebral tissue. Furthermore, the concentration and surface composition of the nanobubble have an effect as to the size and potential damage to the brain when opening the BBB. In conclusion, it has been shown that it is possible to non-invasively open the BBB and deliver viruses and dye into the brain. In addition, this thesis has investigated the use of nanobubbles as both facilitators to opening the BBB and delivery vectors for potentially therapeutic drugs. Finally, a non-invasive opening of the BBB has been achieved using focused ultrasound. Ultimately this non-invasive opening of the BBB can be used to achieve delivery of larger molecules (such as antibodies and viruses) into the brain to target treatments. Focused ultrasound brain targeting can be applied to the potential treatment of salt appetite regulation in the NTS. For the individuals who suffer from salt sensitive hypertension, the NTS can be targeted to reduce the drive to ingest high salt diets. Furthermore, the continuation of research into the central control of BP, salt appetite and baroreceptor reflex control can become better understood, using less invasive delivery techniques to the brain.