Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon. In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.

The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4′-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4′,7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 μmol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (ΔΨm) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Complexity lies halfway between stochasticity and determinism, suggesting that brain activity is neither fully random nor fully predictable but lives by the rules of nonlinear high- and low-complexity dynamics. One important aspect of brain function is noise-like activity (NLA), defined as background, electrical potential fluctuations in the nervous system distinct from spiking rhythms in the foreground. The objective of this thesis was to investigate the neurodynamical complexity of NLA recorded at the cellular and local network scales in in vitro preparations of mouse and human hippocampal tissue, under healthy and epileptiform conditions. In particular, it was found that neuronal NLA arises out of the physiological contributions of gap junctions and chemical synaptic channels and is characterized by a spectrum of complexity, ranging from high- to low-complexity, that was measured using methods from nonlinear dynamical systems theory. Importantly, the complexity of background, neuronal NLA was shown to depend on the degree of cellular interconnectivity to the surrounding local network. In addition, the complexity and multifractality of NLA was further studied at the cellular and local network scales in epileptiform transitions to seizure-like events, identifying emergent low-complexity and reduced multifractality (bordering on monofractal-type dynamics) in the pathological ictal state. Finally, dual intracellular recordings of hippocampal epileptiform activity were analyzed to measure NLA synchronicity, showing evidence for increased same- and cross-frequency correlations and increased phase synchronization in the pathological ictal state. Convergence towards increased phase synchrony manifested in lower frequency regions including theta (4-10 Hz) and beta (12-30 Hz), but also in higher frequency bands (gamma, 30-80 Hz). In summary, there is evidence to suggest that background NLA captures important neurodynamical information pertinent to the classification and identification of brain state transitions in healthy and epileptiform hippocampal dynamics, using sophisticated neuroengineering analyses of these physiological signals.

Sur l’épiderme des feuilles des végétaux supérieurs, se trouvent des pores appelés stomates et composés de deux cellules spécialisées, les cellules de garde. La régulation des mouvements stomatiques en réponse aux signaux de l’environnement permet le contrôle des échanges gazeux de la plante avec l’atmosphère. Ces mouvements sont dus à des variations rapides de la turgescence des cellules de garde. Les H+-ATPases sont des pompes à protons situées sur la membrane plasmique des cellules des végétaux. En excrétant les ions H+ dans l’apoplasme, ces protéines génèrent une force protomotrice capable de fournir l’énergie nécessaire à l’activation de canaux et transporteurs ioniques membranaires. Ce processus permet de contrôler la turgescence des cellules de garde. Des analyses d’expression nous ont permis d’identifier les trois isoformes d’H+-ATPases, AHA1, AHA2 et AHA5, exprimées dans la cellule de garde. Des études in silico nous ont révélé qu’en dépit d’une forte proximité génétique, ces trois protéines semblent être impliquées dans des voies de signalisation différentes. La caractérisation phénotypique d’une collection de mutants affectés dans l’expression de chacune des isoformes nous a permis de mettre en évidence qu’AHA1 est l’H+-ATPase spécifique de la réponse stomatique à la lumière bleue et que la perte d’expression de cette isoforme confère à la plante une croissance plus faible et une meilleure tolérance à la sécheresse que le sauvage. Enfin, nous avons confirmé par des études d’électrophysiologie et par l’élaboration de sondes fluorescentes pH-sensibles l’implication directe d’AHA1 dans la voie de signalisation de la lumière bleue dans la cellule de garde. / Stomata are pores laocated on high plant leaf epidermis and formed by two specialized guard cells. Stomatal movement regulation in response to environment allows the control plant-atmosphere gas exchanges. Stomatal movements are ruled by rapid turgor changes in guard cells. H+-ATPases are proton pomps expressed on plant cell plasma membrane. By extruding H+ in the apoplast, these proteins generate a protomotive force able to activate secondary ionic transporters and channels. This process leads to changes in ion homeostasy and in fine to turgor changes in the guard cell. Expression analyses allowed us to identify the three AHAs isoforms expressed in the guard cell : AHA1, AHA2 and AHA5. In silico study indicates that despite the high identity percentage between the three proteins, they seem involved in differents signalisation pathways. Phenotypic characterization of mutants impaired in the expression of each AHAs allowed us to conclude that AHA1 is specific for the blue light stomatal response. Furthermore, the lack of AHA1 leads to a lower growth and a better tolerance to drought stress of aha1 mutant compare to the wild type. Sugar metabolism studies in aha1 mutant gave us informations on the compensatory mecanism for stomatal opening in this mutant. Finally, we confirmed by electrophisiologcal studies and by fluorescent pH sensitive probes elaboration, the specific involvement of AHA1 in blue light signalisation pathway in the guard cell.

>Magister Scientiae - MSc / Cissampelos capensis, is commonly known by the Afrikaans name ‟dawidjies” or ‟dawidjieswortel”. C. capensis is the most important and best known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis rhizome extracts on sperm function.

Les neurones interagissent à travers leur potentiel de membrane qui a en général une évolution temporelle complexe due aux nombreuses entrées synaptiques irrégulières reçues. Cette évolution est mieux décrite en termes probabilistes, en raison de ces entrées irrégulières ou «bruit synaptique». L'évolution temporelle du potentiel de membrane est stochastique mais aussi déterministe: stochastique, car conduite par des entrées synaptiques qui arrivent de façon aléatoire dans le temps, et déterministe, car un neurone biologique a une évolution temporelle très similaire quand soumis à une même séquence d'entrées synaptiques. Nous étudions les propriétés statistiques d'un modèle simplifié de neurone soumis à des entrées à taux variable d'où en résulte l'évolution non-stationnaire du potentiel de membrane. Nous considérons un modèle passif de membrane neuronale, sans mécanisme de décharge neuronale, soumis à des entrées à courant ou à conductance sous la forme d'un processus de «shot noise». Les fluctuations du potentiel de membrane sont aussi modélisées par un processus stochastique similaire, de «shot noise» filtré. Nous avons analysé les propriétés statistiques de ces processus dans le cadre des transformations de processus ponctuels de Poisson. Des propriétés de ces transformations sont dérivées les statistiques non-stationnaires du processus. Nous obtenons ainsi des expressions analytiques exactes pour les moments et cumulants du processus filtré dans le cas général des taux d'entrée variables. Ce travail ouvre de nombreuses perspectives pour l'analyse de neurones dans les conditions in vivo, en présence d'entrées synaptiques intenses et bruitées. / Neurons interact through their membrane potential that generally has a complex time evolution due to numerous irregular synaptic inputs received. This complex time evolution is best described in probabilistic terms due to this irregular or "noisy" activity. The time evolution of the membrane potential is therefore both stochastic and deterministic: it is stochastic since it is driven by random input arrival times, but also deterministic, since subjecting a biological neuron to the same sequence of input arrival times often results in very similar membrane potential traces. In this thesis, we investigated key statistical properties of a simplified neuron model under nonstationary input from other neurons that results in nonstationary evolution of membrane potential statistics. We considered a passive neuron model without spiking mechanism that is driven by input currents or conductances in the form of shot noise processes. Under such input, membrane potential fluctuations can be modeled as filtered shot noise currents or conductances. We analyzed the statistical properties of these filtered processes in the framework of Poisson Point Processes transformations. The key idea is to express filtered shot noise as a transformation of random input arrival times and to apply the properties of these transformations to derive its nonstationary statistics. Using this formalism we derive exact analytical expressions, and useful approximations, for the mean and joint cumulants of the filtered process in the general case of variable input rate. This work opens many perspectives for analyzing neurons under in vivo conditions, in the presence of intense and noisy synaptic inputs.

Parkinson's disease (PD) is the second most common chronic neurodegenerative disease characterized by dopamine decrease in the substantia nigra. Currently, there is no promising cure for PD and this has resulted in extensive research into alternative medicines. The aim of this study was to investigate the effect of methanol and ethyl acetate extracts of Lannea schweinfurthii (Engl. Engl) (Anacardiaceae), Zanthoxylum capense (Thunb. Harv) (Rutaceae), Scadoxus puniceus ((L.) Friis & Nordal) (Amaryllidaceae) and Crinum bulbispermum (Burm. f.) Milne-Redh. & Schweick) (Amaryllidaceae) on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells. The latter which mimics PD symptoms in vitro. Cytotoxicity of the plant extracts was assessed using sulforhodamine B (SRB) assay. Intracellular reactive oxygen species (ROS) were measured fluorometrically with the use of the fluorescent dye 2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA). Intracellular glutathione content was measured fluorometrically after staining with monochlorobimane (MCB). Fluorescent dye 5,5‟ ,6,6‟ -tetrachloro-1,1‟ ,3,3‟ -tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to assess the mitochondrial membrane potential (MMP) status of cells. Apoptosis was assessed by determining caspase-3 activity through detection of 7-amino-4-methylcoumarin (AMC) which is a product of caspace-3 substrate, acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Ac-DEVD-AMC), cleaved by the caspase-3 enzyme. Rotenone was used as an in vitro model to induce PD-like symptoms. Cytotoxicity studies for methanol extract of Zanthoxylum capense revealed the highest IC50 value of 121.3 μg/mL, indicating low toxicity. The ethyl acetate extract of Crinum bulbispermum was observed to have no effect on the normal proliferation of the SH-SY5Y cells and produced an IC50 value >100 μg/mL. The calculated IC50 value obtained from rotenone cytotoxicity studies was 112 iv nM. Zanthoxylum capense and Scadoxus puniceus plant extracts were observed to be neuroprotective against rotenone-induced toxicity. A decrease in intracellular glutathione content as well as MMP was also observed in cells exposed to rotenone alone (50 nM). There was no intracellular ROS generation observed in cells exposed to rotenone alone (50 nM) after 24 h and 72 h. However, apoptotic cell death was observed in cells treated with rotenone (50 nM). Intracellular ROS production was observed to be elevated by methanol and ethyl acetate extracts of C. bulbispermum. Methanol extracts of Z. capense was observed to increase intracellular glutathione content. MMP was increased effectively following treatment with ethyl acetate extract of C. bulbispermum. Moreover, both methanol and ethyl acetate plant extracts were found to decrease caspase-3 activity significantly (p<0.05), in cells exposed to 50 nM rotenone. Z. capense methanol extract reduced caspase-3 activity the most effectively. Treatment with plant extracts was protective and decreased cell death. Furthermore, L. schweinfurthii, Z. capense, S. puniceus and C. bulbispermum, demonstrated strong antioxidant and anti-apoptotic effects against rotenone-toxicity, making them potential agents in developing therapies for treating PD. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

The Na/K pumps are essential for living system and widely expressed in all eukaryotic cell membranes. By actively transporting sodium ions out of and potassium ions into the plasma membrane, Na/K pumps creates both an electrical and a chemical gradient across the plasma membrane, which are crucial for maintaining membrane potential, cell volume, and secondary active transporting of other solutes, etc. Previously, oscillating electric field with a frequency close to the mean physiological turnover rate was used to synchronize and modulate the Na/K pump molecules. Results showed that the turnover rate of Na/K pumps can be accelerated by folds. However, this what we called first generation synchronization modulation (SM) technique can only synchronize sodium and potassium translocations into their corresponding half cycles. The detailed location of each sodium extrusion and potassium intrusion can not be determined. As a result, the synchronized pumps were uniformly distributed, generating steady-state macroscopic currents. Based on these studies, Dr.Chen developed a new generation synchronization modulation technique. The waveform of original SM by adding an overshoot pulse at the end of each half cycle. This overshoot pulse has a function of energy barrier which will force all of the Na/K pumps into the same state in the pumping cycle until the membrane polarity change. As a result, Na/K pump molecules are not only synchronized into half cycles of oscillating electric field, but individual steps of the pumping cycle. Accordingly, transient pump currents or so called 'pre-steady state' pump currents are generated, from which some detailed information abut the mechanism of Na/K pumps can be dissected. In this dissertation, we firstly characterized the synchronized pump currents by modified SM. The results showed that transient currents were induced at the beginning of each half cycle as expected. The ratio between positive and negative transient currents was close to 3:2, stoichiometric number of Na/K pump. Moreover, the transient currents were significantly reduced in the presence of ouabain in a time dependent manner. In addition, by gradually increasing the frequency of SM electric field in a step-wise fashion, the synchronized pump current can be modulated to the corresponding level. Next,we utilized this technique to study some detailed mechanisms of Na/K pump, including single channel configuration in transmembrane domain and extracellular D2O effect on the turnover rate. Lastly, we extended our study to applications of this new technique and found that the modified Synchronization Modulation technique can significantly hyperpolarize the membrane potential of skeletal muscle fiber in both physiological and high potasssium conditions. During intensive exercise, the interstitial potassium ions are accumulated and temporarily reach a high level, which will attenuate the contraction force and induce muscle fatigue. Na/K pumps are crucial in the maintenance of skeletal muscle excitability and contractility by restoring the Na and K concentration gradients. By accelerating the turnover rate of Na/K pumps, SM can efficiently re-establish the membrane potential and enhance skeletal muscle contractivity, which unleashes its potential in improving certain pathological conditions, such as exercise-induced hyperkalemia.

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.

Aging is a multifaceted process of accumulation of damage and waste in cells and tissues; age-related changes in mitochondria and in respiratory metabolism have the focus of aging research for decades. Studies of aging in nematodes, flies and mammals all revealed age-related decline in respiratory functions, with somewhat controversial causative role. Here we investigated age-related changes in respiration rates, lactate/pyruvate ratio, a commonly used proxy for NADH/NAD+ balance, and mitochondrial membrane potential in 4 genotypes of an emerging model organism for aging research, a cyclic parthenogen Daphnia magna. We show that total body weight-adjusted respiration rate decreased with age, although this decrease was small in magnitude and could be fully accounted for by the decrease in locomotion and feeding activity. Neither total respiration normalized by protein content, nor basal respiration rate measured in anaesthetized animals decreased with age. Lactate/pyruvate ratio and mitochondrial membrane potential (∆Ψ) showed no age-related changes, with possible exceptions of ∆Ψ in epipodites (excretory and gas exchange organs) in which ∆Ψ decreased with age and in the optical lobe of the brain, in which ∆Ψ showed a maximum at middle age. We conclude that actuarial senescence in Daphnia is not caused by a decline in respiratory metabolism and discuss possible mechanisms of maintaining mitochondrial healthspan throughout the lifespan.