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Monolayer or suspension cell cultures are of only limited value as experimental models for human cancer. Therefore, more sophisticated, three-dimensional culture systems like spheroid cultures or histocultures are used, which more closely mimic the tumor in individual patients compared to monolayer or suspension cultures. As tissue culture or tissue engineering requires more sophisticated culture, specialized in vitro techniques may also improve experimental tumor models. In the present work, a new miniaturized hollow-fiber bioreactor system for mammalian cell culture in small volumes (up to 3 ml) is characterized with regard to transport characteristics and growth of leukemic cell lines (chapter 2). Cell and medium compartment are separated by dialysis membranes and oxygenation is accomplished using oxygenation membranes. Due to a transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60 and REH were cultivated up to densities of 3.5 x 107/ml without medium change or manipulation of the cells. Growth and viability of the cells in the bioreactor were the same or better, and the viable cell count was always higher compared to culture in Transwellâ plates. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with the flow rate. Influx of low molecular weight substances in the cell compartment could be regulated by variation of the concentration in the medium compartment. Thus, time dependent concentration profiles (e.g. pharmacokinetic profiles of drugs) can be realized as illustrated using glucose as a model compound. Depending on the molecular size cut-off of the membranes used, added growth factors like GM-CSF and IL-3 as well as factors secreted from the cells are retained in the cell compartment for up to one week. Second, a method for monitoring cell proliferation the hollow-fiber bioreactor by use of the Alamar BlueTM dye was developed (chapter 3). Alamar BlueTM is a non-fluorescent compound which yields a fluorescent product after reduction e.g. by living cells. In contrast to the MTT-assay, the Alamar BlueTM-assay does not lead to cell death. However, when not removed from the cells, the Alamar BlueTM dye shows a reversible, time- and concentration-dependent growth inhibition as observed for leukemic cell lines. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows to discriminate between cell densities of 105, 106 and 107 of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 106 cells/ml, in particular. In conclusion, the Alamar BlueTM-assay combined with the hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system. In chapter 4 the use of the hollow-fiber bioreactor as a tool for toxicity testing was investigated, as current models for toxicity as well as efficacy testing of drugs in vitro allow only limited conclusions with regard to the in vivo situation. Examples of the drawbacks of current test systems are the lack of realistic in vitro tumor models and difficulties to model drug pharmacokinetics. The bioreactor proved to be pyrogen free and is steam-sterilizable. Leukemic cell lines like HL-60 and primary cells such as PHA-stimulated lymphocytes can be grown up to high densities of 1-3 x 107 and analyzed during growth in the bioreactor by light-microscopy. The cytostatic drug Ara-C shows a dose-dependent growth inhibition of HL-60 cells and a dose-response curve similar to controls in culture plates. The bioreactor system is highly flexible since several systems can be run in parallel, soluble drugs can be delivered continuously via a perfusion membrane and gaseous compounds via an oxygenation membrane which also allows to control pO2 and pH (via pCO2) during culture in the cell compartment. The modular concept of the bioreactor system allows realization of a variety of different design properties, which may lead to an improved in vitro system for toxicity testing by more closely resembling the in vivo situation. Whereas several distinct advantages of the new system have been demonstrated, more work has to be done to promote in vitro systems in toxicity testing and drug development further and to reduce the need for animal tests.
This study investigates mechanisms and consequences of sexual selection in a polygynous population of dusky warblers Phylloscopus fuscatus, breeding near Magadan in the Russian Far East. In particular, the study focuses on individual variation in the reproductive behaviours of both females and males. The mating system of this population is characterised by facultative polygyny (17 per cent of the males mated with more than one female), and by an outstandingly high rate of extra-pair paternity (45 per cent of the offspring was not sired by the social partner of the female). The occurrence of polygyny is best explained by the ‘polygyny-threshold model’ (PTM). A novel finding of this study is that female mating decisions follow a conditional strategy. First-year females that have no prior breeding experience prefer monogamy over territory quality, while older females more often mate polygynously. I argue that the costs of receiving no male help may be higher for inexperienced females, while the benefits of having a free choice between territories may be higher for individuals that know which territories had the highest breeding success in previous years. Furthermore, I find support for the existence of two female mating strategies. The ‘emancipated’ female which is not dependent on male help, is free to choose the best territory and the best copulation partners. The ‘help-dependent’ female, in contrast, is bound to find a partner who is willing to assist her with brood care, thus she will have to accept territories and genetic fathers of lesser quality. The most unexpected finding on female mating behaviours is that this dichotomy between emancipated and help-dependent females is accompanied by morphological specialisation, which indicates that there is genetic variation underlying these female mating strategies. Male mating behaviours are characterised by competition for ownership of the best territories and by advertisement of male quality to females, as these are the factors which largely determine male reproductive success. Male success in obtaining copulations depended on the quality of their song, a fact that explains why males spend most of the daytime singing during the period when females are fertile. Individuals that were able to maintain a relatively high sound amplitude during rapid frequency modulations were consistently preferred by females as copulation partners. Studies of physiological limitations on sound production suggest that such subtle differences in male singing performance can provide an honest reflection of male quality. The present study is the first to indicate that females may judge the quality of a male’s song by his performance in sound production. Quality of song was also related to winter survival, which suggests that females can enhance the viability of their offspring by seeking extra-pair fertilisations from good singers (good-genes hypothesis). In general, the present study demonstrates that a complete understanding of avian mating systems is not possible without a detailed analysis of alternative behavioural strategies and of how individuals adjust their reproductive tactics according to their individual needs and abilities.
T cell activation is supposed to require two signals via engagement of the TCR and a costimulatory molecule. However, the signaling cascade of costimulatory molecules has remained elusive. Here, I provide evidence that CD44 supports proliferation as well as apoptosis mainly, if not exclusively, by enhancing signal transduction via the TCR/CD3 complex. Blockade of CD44 interferes with mounting of an immune response. This has been demonstrated by the significantly decreased IL-2 production of a T helper line, when stimulated in the presence of a competing CD44 receptor globulin. To evaluate the underlying mechanism, CD44 was cross-linked by an immobilized antibody (IM7). Cross-linking of CD44 induces proliferation of peripheral T cells and apoptosis of thymocytes and a T helper line in the presence of subthreshold levels of anti-CD3. CD44-induced proliferation was accompanied by an upregulation of the activation markers CD25 and CD69 and an increased cytokine production. TCR-mediated apoptosis was accompanied by an upregulation of CD95 ligand and CD95 receptor, which could be greatly enhanced by costimulation via CD44. On the level of signal transduction, coligation of CD44 with CD3 resulted in a strong and sustained increase of early tyrosine phosphorylation events and upregulated downstream signal transduction pathways, such as the ras/ERK and the JNK signaling cascades. These pleiotropic effects of CD44 are due to its involvement in the most proximal events in TCR signaling, as demonstrated by a strong increase in the phosphorylation of the TCR z-chain and ZAP-70. Notably, cross-linking of CD44 was binding-site dependent and was only effective when supporting colocalization of the TCR/CD3 complex and CD44. Cross-linking of CD44 via immobilized IM7 also induced profound changes in cell morphology, characterized by strong adhesion, spreading and development of surface extensions, which were dependent on a functional tubulin and actin cytoskeleton. These cytoskeletal rearrangements were mediated by rac1, a small GTPase of the rho subfamily, and src-family kinases, two of which, fyn and lck, were found to be associated with CD44. By cross-linkage of CD44 these kinases were redistributed into so called lipid rafts. It is supposed that for T cell activation a relocation of the TCR/CD3 complex into the same membrane microdomains is required. The data are interpreted in the sense that the costimulatory function of CD44 relies on its cooperativity with the TCR. Most likely by recruitment of phosphokinases CD44 significantly lowers the threshold for the initiation of signaling via the TCR. The requirement for immobilized anti-CD44, the necessity for neighbouring anti-CD3 and the dependence on the binding site of CD44 strongly suggest that the costimulatory mechanism involves cytoskeletal rearrangements, which facilitate recruitment and redirection of src-family protein kinases in glycolipid enriched membrane microdomains.
The present thesis reports on four years of field research on stingless bee ecology in Sabah, Malaysia. Hereby, it was the main focus to evaluate the effect of selective logging for timber extraction on communities of bees, and to elucidate causative relationships involved in regulating bee populations. Included were background studies on resource use (3.1, 3.2, 3.3) and nesting biology (3.4) as well as comparative studies on stingless bee diversity and abundance in logged and unlogged lowland rainforest sites (4.1, 4.2). Stingless bees proved to be generalist foragers that used a large range of plant species as pollen sources. Nevertheless, different species of bees had rather distinct pollen diets, a findind that was independent of fluctuations in flowering activity in the habitat. At one particular point in time colonies of one species (Trigona collina)collected mold spores (Rhizopus sp.) as a pollen surrogate. In order to obtain low-effort estimates of meliponine pollen sources a new method was developed: Trapping of bee garbage (with funnel traps) and the quantitative analysis of pollen in garbage samples. Pollen in bee garbage reflected pollen import with a certain time lag and could therefore be used for an assessment of long-term pollen foraging (see below). The majority of stingless bee nests (275 nests of 12 species) were found in cavities in trunks or under the bases of large, living canopy trees. Nest trees mostly belonged to commercial species and were of the correct size and (partly) timber quality to warrant harvesting. It was estimated that roughly one third of stingless bee nests in an given forest area would be killed during a selective logging operation. Besides causing direct mortality, logging may also indirectly affect bee populations by reducing the availability of potential nest sites (trees). However, in a comparison of primary and differentially logged forest sites (10 to 30 years after logging) no effect of the degree of disturbance on meliponine nest density was found. Instead, the variation in nest density (0 to 16.2 nest/ha) was best explained by differences in the available floral resources (assessed by analysis of pollen in bee garbage). Bee populations in forest edge situations were favored: there was a positive correlation between nest density and the proportion of external non-forest pollen (e.g. from crop plants, road edge vegetation, mangroves) in the bees’ diet. The highest nest density was found in a site bordering the mangroves in Sandakan Bay. Here, the mangrove tree Rhizophora apiculata represented a extraordinary large fraction of the pollen volume. Presumably, external pollen sources effectively supplement bee diets at times when little flowering occurs inside the forest, thus increasing overall bee carrying-capacity. The idea of differential pollen limitation was strengthened by direct measurements of pollen import and foraging activity over a period of five months. Both were elevated in colonies in a site with high bee density. It is concluded that the abundance of stingless bees in forests in Sabah is chiefly dependent on the local availability of food resources. Hereby, bee populations strongly benefit from edge effects and increased habitat diversity. Although direct negative effects of selective logging are strongly indicated by a close association of bee nests with commercial trees, no clear effects were detected in regenerating forests ten to 30 years after logging.
The MEK5/ ERK5 kinase module is a relatively new discovered mitogen-activated protein kinase (MAPK) signalling pathway with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle a function of the cascade during muscle differentiation was examined. ERK5 becomes activated upon induction of differentiation in mouse myoblasts. The selective activation of the pathway results in promoter activation of differentiation-specific genes, such as the cdk-inhibitor p21 gene, the myosin light chain (MLC1A) gene, or an E-box containing promoter element, where myogenic basic-helix-loop-helix proteins such as MyoD or myogenin bind. Moreover, myogenic differentiation is completely blocked, when ERK5 expression is inhibited by antisense RNA. The effect can be detected also on the expression level of myogenic determination and differentiation markers such as p21, MyoD and myogenin. Another new finding is that stable expression of ERK5 in C2C12 leads to differentiation like phenotype and to increased p21 expression levels under growth conditions. These results provide first evidence that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.
Food borne pathogens that cause systemic disease must cross the intestinal barrier. Many of these pathogens, eg Salmonella typhimurium and Shigella flexneri, use M cells, found only within the follicle associated epithelium (FAE) that overlies Peyer’s patches and other lymphoid follicles, to enter the host. This study is primarily an investigation into the interaction of S. typhimurium and Listeria monocytogenes with the intestinal epithelium, representing the early stage of an infection.
The mode of action of phloretin and its analogs on the permeability of natural membranes for neutral and charged molecules, such as urea, glucose and chloride has been characterized 25 years ago. In contrast to signal molecules with primary effects on transport systems of natural membranes, phloretin also affects model membranes, i.e., artificial membranes, which do not contain proteins. Since the dipole potential reducing effect of phloretin on mono- and bilayers has been found, it became clear that its primary effect must be a biophysical one: phloretin adsorbs to lipid layers and changes biophysical parameters of these layers. The aim of this work was the characterization of the interaction between the surface-active molecule phloretin and artificial lipid layers. We were able to describe structural and functional parameters of the model systems mono- and bilayer as functions of one or few variables. One of these parameters, the dipole potential, measured as a function of the aqueous phloretin concentration, allowed a critical examination of the Langmuir adsorption model that has been postulated for the interaction between phloretin and lipid layers. Surface pressure versus area per lipid molecule isotherms and surface (dipole) potential change versus area per lipid molecule isotherms, measured at lipid monolayers, allowed a structural description of the phloretin-lipid interaction: phloretin integrates into monolayers dependent on the surface pressure and the phase state of the lipid. Calorimetric measurements confirmed the integration of phloretin into membranes because of the strong decrease of the phase transition temperature, but they also showed that the cooperativity of phase transition is hardly affected, even at very high amounts of phloretin in the membrane. Obviously the interaction between phloretin and lipids is restricted to the head groups, an integration into the hydrocarbon layer is unlikely. 2H NMR measurements with spherical unilamellar vesicles of headgroup-deuterated lipid showed changed quadrupolar splittings indicating the interaction between phloretin and headgroups of the lipids.
Large parts of the tropical lowland rain forests of Sabah (Malaysia) were transformed into secondary forests due to heavy logging. Additionally the remaining forest remnants are isolated from each other by large scale oil palm plantations. Biodiversity patterns and responses of the community of leaf litter ants were studied in anthropogenically disturbed habitats and primary forests of different size. In logged over forests, only 70 per cent of the species of a primary forest were present even 25 years after timber extraction. The ant communities were thinned and could be described by a lower species density producing lower species numbers and a different community composition. The similarity in species number and community composition between logged over forests of different degrees of disturbance was explained by source-sink dynamics within a heterogeneous forest matrix. Rain forest fragments displayed even higher reductions in species density, numbers and diversity due to a more pronounced thinning effect. Even forest isolates exceeding 4 000 ha in size did not support more than 50 per cent of the species of the leaf litter ant community of a contiguous primary rain forest. Additionally, an increase in tramp species was recorded with decreasing size of the forest fragments, leading to a very different community composition. Regarding the leaf litter ant community, the remaining rain forest fragments of Sabah are effectively isolated by a barrier of oil palm plantation, now stretching all over the lowlands of the east coast. Only 13 species, which belonged to the forest ant community in highly disturbed areas were collected in these plantations. Some of the 10 other species of the highly reduced ground-dwelling ant community in the plantations are known as invasive tramp species, forming large exclusive territories. Correlative evidence and a field experiment implied, that leaf litter humidity, volume and temperature affect the distribution and community composition of forest leaf litter ant species. The smaller primary forests and the most disturbed logged over forests in this study revealed higher temperatures and lower humidity levels and a reduction in leaf litter volume compared to a large primary forest or forests affected by a lower impact of timber harvesting. If the pattern for leaf litter ants is confirmed for other taxa, the implications for any efficient management design aiming to preserve the majority of the biodiversity of the country are tremendous and current concepts need rethinking.
Most natural learning situations are of a complex nature and consist of a tight conjunction of the animal's behavior (B) with the perceived stimuli. According to the behavior of the animal in response to these stimuli, they are classified as being either biologically neutral (conditioned stimuli, CS) or important (unconditioned stimuli, US or reinforcer). A typical learning situation is thus identified by a three term contingency of B, CS and US. A functional characterization of the single associations during conditioning in such a three term contingency has so far hardly been possible. Therefore, the operational distinction between classical conditioning as a behavior-independent learning process (CS-US associations) and operant conditioning as essentially behavior-dependent learning (B-US associations) has proven very valuable. However, most learning experiments described so far have not been successful in fully separating operant from classical conditioning into single-association tasks. The Drosophila flight simulator in which the relevant behavior is a single motor variable (yaw torque), allows for the first time to completely separate the operant (B-US, B-CS) and the classical (CS-US) components of a complex learning situation and to examine their interactions. In this thesis the contributions of the single associations (CS-US, B-US and B-CS) to memory formation are studied. Moreover, for the first time a particularly prominent single association (CS-US) is characterized extensively in a three term contingency. A yoked control shows that classical (CS-US) pattern learning requires more training than operant pattern learning. Additionally, it can be demonstrated that an operantly trained stimulus can be successfully transferred from the behavior used during training to a new behavior in a subsequent test phase. This result shows unambiguously that during operant conditioning classical (CS-US) associations can be formed. In an extension to this insight, it emerges that such a classical association blocks the formation of an operant association, which would have been formed without the operant control of the learned stimuli. Instead the operant component seems to develop less markedly and is probably merged into a complex three-way association. This three-way association could either be implemented as a sequential B-CS-US or as a hierarchical (B-CS)-US association. The comparison of a simple classical (CS-US) with a composite operant (B, CS and US) learning situation and of a simple operant (B-US) with another composite operant (B, CS and US) learning situation, suggests a hierarchy of predictors of reinforcement. Operant behavior occurring during composite operant conditioning is hardly conditioned at all. The associability of classical stimuli that bear no relation to the behavior of the animal is of an intermediate value, as is operant behavior alone. Stimuli that are controlled by operant behavior accrue associative strength most easily. If several stimuli are available as potential predictors, again the question arises which CS-US associations are formed? A number of different studies in vertebrates yielded amazingly congruent results. These results inspired to examine and compare the properties of the CS-US association in a complex learning situation at the flight simulator with these vertebrate results. It is shown for the first time that Drosophila can learn compound stimuli and recall the individual components independently and in similar proportions. The attempt to obtain second-order conditioning with these stimuli, yielded a relatively small effect. In comparison with vertebrate data, blocking and sensory preconditioning experiments produced conforming as well as dissenting results. While no blocking could be found, a sound sensory preconditioning effect was obtained. Possible reasons for the failure to find blocking are discussed and further experiments are suggested. The sensory preconditioning effect found in this study is revealed using simultaneous stimulus presentation and depends on the amount of preconditioning. It is argued that this effect is a case of 'incidental learning', where two stimuli are associated without the need of reinforcement. Finally, the implications of the results obtained in this study for the general understanding of memory formation in complex learning situations are discussed.
The proventriculus regulates the food passage from crop to midgut. As the haemolymph provides a constantly updated indication of an insect’s nutritional state, it is assumed that the factor controlling the proventri-culus activity is to be found in the haemolymph. The purpose of this doctoral thesis was to investigate how output (metabolic rate), input (food quality and food quantity) and internal state variables (haemolymph osmolarity and haemolymph sugar titer) affect each other and which of these factors controls the activity of the proventriculus in the honeybee. Therefore free-flying foragers were trained to collect con-trolled amounts of different sugar solutions. Immediately after feeding, metabolic rates were measured over different periods of time, then crop-emptying rates and haemolymph sugar titers were measured for the same individual bees. Under all investigated conditions, both the sugar transport rates through the proventriculus and the haemolyph sugar titers depended mainly on the metabolism. For bees collecting controlled amounts of 15 per cent, 30 per cent or 50 per cent sucrose solution haemolymph trehalose, glucose and fructose titers were constant for metabolic rates from 0 to 4.5 mlCO2/h. At higher metabolic rates, trehalose concentration decreased while that of glucose and fructose increased with the exception of bees fed 15 per cent sucrose solution. As the supply of sugar from the crop via the proventriculus was sufficient to support even the highest metabolic rates, the observed pattern must result from an upper limit in the capacity of the fat body to synthesise trehalose. The maximal rate of conversion of glucose to trehalose in the fat body was therefore calculated to average 92.4 µg glucose/min. However, for bees fed 15 per cent sucrose solution both the rate of conversion of glucose to trehalose and the rate of sugar transport from the crop to the midgut were limited, causing an overall decrease in total haemolymph sugar titers for metabolic rates higher than 5 mlCO2/h. Haemolymph sucrose titers were generally low but increased with increasing metabolic rates, even though sucrose was not always detected in bees with high metabolic rates. Though foragers were able to adjust their sugar transport rates precisely to their metabolic rates, a fixed surplus of sugars was transported through the proventriculus under specific feed-ing conditions. This fixed amount of sugars increased with increasing concentration and in-creasing quantity of fed sugar solution, but decreased with progressing time after feeding. This fixed amount of sugars was independent of the metabolic rates of the bees and of the molarity and viscosity of the fed sugar solution. As long as the bees did not exhaust their crop content, the haemolymph sugar titers were unaffected by the sugar surplus, by the time after feeding, by the concentration and by the viscosity of fed sugar solution. When bees were fed pure glucose (or fructose) solutions, un-usually little fructose (or glucose) was found in the haemolymph, leading to lower total haemolymph sugar titers, while the trehalose titer remained unaffected. In order to investigate the mechanisms underlying the regulation of the honeybee proven-triculus, foraging bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable sugars (sorbose). Bees reacted to injections of metabolisable sugars with reduced crop-emptying rates, but injection of non-metabolisable sugars had no influence on crop emptying. Therefore it is concluded that the proventriculus regulation is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemo-lymph osmolarity. A period of 10min was enough to observe reduced crop emptying rates after injections. It is suggested that glucose and fructose have an effect on the proventriculus activity only via their transformation to trehalose. However, when the bees were already in-jected 5min after feeding, no response was detectable. In addition it was investigated whether the overregulation is the result of feed-forward regulation for the imminent take-off and flight. In a first experiment, we investigated whether the bees release an extra amount of sugar solution very shortly before leaving for the hive. In a second experiment, it was tested whether the distance covered by the bees might have an influence on the surplus amount released prior to the take-off. In a third experiment, it was investigated if walking bees fail to release this extra amount of sugars, as they do not have to fly. Though we were not able to demonstrate that the overregulation is the result of feed-forward regulation for the imminent take-off and flight, it is conceivable that this phenome-non is a fixed reaction in foragers that can not be modulated. To investigate whether regulated haemolymph sugar titers are also observed in honeybee foragers returning from natural food sources, their crop contents and haemolymph sugar titers were investigated. While the quantity of the collected nectar was without influence on the haemolymph sugar titers, foragers showed increasing haemolymph sugar titers of glucose, fructose and sucrose with increasing sugar concentration of the carried nectar. In contrast no relationship between crop nectar concentrations and haemolymph trehalose titers was observed. We are sure that the regulation of food passage from crop to midgut is controlled by the trehalose titer. However, under some conditions the balance between consumption and income is not numerically exact. This imprecision depends on the factors which have an impact on the foraging energetics of the bees but are independent of those without influence on the foraging energetics. Therefore we would assume that the proventriculus activity is modulated by the motivational state of the bees.
Different transgenes that can be expressed in neurons to kill or block them were compared. Tetanus neurotoxin blocked chemical synapses very efficiently. Synapses consisting of a chemical and an electrical component were blocked more reliably by expressing a human inwardly rectifying potassium channel. To gain temporal control over neuronal function, three genetic tools have been investigated. None of the systems is without drawbacks, however, the recombination induced tetanus neurotoxin expression is a promising approach. The knowledge gained from the comparative methodological study was used to investigate the role of neurons in sensory systems in processing different sensory informations. Receptor neurons sensitive for chemical or mechanical stimuli were correlated to specific olfactory behaviors or locomotor tasks. The main topic of this thesis is the much discussed question of which neurons are involved in motion processing in the visual system of flies. Neither L2 nor L4 neurons in the first visual neuropil are essential for motion-detection. The results indicate that maybe motion is detected by the network of amacrine cells (a). The vertical motion-sensitive VS cells in the lobula plate are not necessary for behavioral responses to vertical motion. This finding implies that the lack of VS cells in the structural mutant optomotor blind is not causally related to the altered responses to motion stimuli. Other abnormalities in optomotor blind are responsible for this behavioral phenotype. This work shows the potential of the described methods in studying information processing in the Drosophila brain. Groups of neurons were correlated to complex behavioral responses and theories about information processing were tested by behavioral experiments with transgenic flies. The refinement of the genetic tools to interfere with neuronal function will make the Drosophila brain an even better model to study information processing in nervous systems.
Recent advances in the development of immunoassays and nucleic acid assays have improved the performance and increased the sensitivity of sensors that are based on biochemical recognition. The new approaches taken by researchers include detecting pathogens by detecting their nucleic acids, using new nontoxic reporter entities for generating signals, and downscaling and miniaturizing sensors to micromigration and microfluidic formats. This dissertation connects some of these successful approaches, thereby leading to the development of novel nucleic acid sensors for rapid and easy detection of pathogens. The author's goal was to develop diagnostic tools that enable investigators to detect pathogens rapidly and on site. While the sensors can be used to detect any pathogen, the author first customized them for detecting particularly Cryptosporidium parvum, a pathogen whose detection is important, yet presents many challenges. Chapter 2 of this thesis presents a novel test-strip for the detection of C. parvum. The test-strip is designed to detect nucleic acids rather than proteins or other epitopes. While test strips are commonly used for sensors based on immunological recognition, this format is very new in applications in which nucleic acids are detected. Further, to indicate the presence or absence of a specific target on the test strip, dye-entrapped, oligonucleotide-tagged liposomes are employed. Using liposomes as reporter particles has advantages over using other reporter labels, because the cavity that the phospholipidic membranes of the liposomes form can be filled with up to 106 dye molecules. By using heterobifunctional linkers liposomes can be tagged with oligonucleotides, thereby enabling their use in nucleic acid hybridization assays. The developed test-strip provides an internal control. The limit of detection is 2.7 fmol/mL with a sample volume of 30 mL. In chapter 3 the detection of nucleic acids by means of oligonucleotide-tagged liposomes is scaled down to a microfluidic assay format. Because the application of biosensors to microfluidic formats is very new in the field of analytical chemistry, the first part of this chapter is devoted to developing the design and the method to fabricate the microchip devices. The performance of the microchips is then optimized by investigating the interactions of nucleic acids and liposomes with the material the chips consist of and by passivating the surface of the chips with blocking reagents. The developed microfluidic chip enabled us to reduce the sample volume needed for one assay to 12.5 mL. The limit of detection of this assay was determined to be 0.4 fmol/mL. Chapters 4 and 5 expand on the development of the microfluidic assay. A prototype microfluidic array that is able to detect multiple analytes in a single sample simultaneously is developed. Using such an array will enable investigators to detect pathogens that occur in the same environment, for example, C. parvum and Giardia duodenalis by conducting a single test. The array's ability to perform multiple sample analysis is shown by detecting different concentrations of target nucleic acids. Further, the author developed a microfluidic chip in which interdigitated microelectrode arrays (IDAs) that consist of closely spaced microelectrodes are integrated. The IDAs facilitate electrochemical detection of cryptosporidial RNA. Electrochemical detection schemes offer benefits of technical simplicity, speed, and sensitivity. In this project liposomes are filled with electrochemically active molecules and are then utilized to generate electrochemical signals. Chapter 6 explores the feasibility of liposomes for enhancing signals derived from nucleic acid hybridization in surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy offers advantages because nucleic acid hybridization can be monitored in real time and under homogeneous conditions because no washing steps are required. SPR spectroscopy is very sensitive and it can be expected that, in the future, SPR will be integrated into microfluidic nucleic acid sensors.