Protein engineering has been a useful tool in the fight against human diseases. Human insulin was the first recombinant DNA-derived therapeutic protein (Humulin®) approved by the US FDA in 1982. However, many of the early protein drugs were only recombinant versions of natural proteins with no modification of their primary amino acid sequence and most of them did not make optimal drug products mainly due to their short half-life or suboptimal affinity, leading to poor therapeutic efficacy. The difficulty in the large-scale production of some therapeutic proteins was another important issue. In the past three decades, different protein engineering platforms have been developed to overcome the obstacles seen in the first generations of these treatments. With the help of these new techniques, proteins have been purposefully modified to improve their clinical potential. The focus of my dissertation is the engineering of potential protein drugs to make them therapeutically useful and more valuable. Previously, our research group has developed cocaine hydrolases (CocHs) from human butyrylcholinesterase (BChE) for treatment of cocaine addiction and prevention of acute cocaine intoxication. In the first project, CocHs were further engineered to improve their performance, e.g., Fc-fused CocHs with an extended serum half-life. Then, I investigated the potential application of a long-lasting CocH for protection against the acute toxic and stimulant effects of cocaine. In the second project, I investigated the potential inhibition of CocH-mediated cocaine hydrolysis by heroin (3,6-diacetylmorphine) or its initial host metabolite, 6-monoacetylmorphine (6-MAM). The investigation of this possible inhibition was important to determine the in vivo efficacy of CocHs, as heroin is one of the most commonly co-abused drugs by cocaine-dependent individuals, as well as a possible metabolite of CocHs. In the third project, I expressed and characterized the recombinant human UDP-glucuronosyltransferase 1A10 (UGT1A10) enzyme, which can inactivate many therapeutically valuable substances. In the fourth and final project, prostate apoptosis response-4 (Par-4), a tumor suppressor protein, was engineered to have a prolonged duration of action so that it may be more therapeutically valuable for cancer treatment.

Estrogens seem to play a role in the locomotor activating effects of cocaine. Japanese quail provide a good model for hormonal manipulation as alterations of their photoperiod controls hormone levels. The current study aims to examine the role of early life photoperiod manipulation in cocaine-induced behavioral sensitization in quail. It was expected that if quail were raised on a short photoperiod, they would have a reduction in gonadal hormones and this reduction in hormones would affect the acquisition of cocaine-induced behavioral sensitization. Quail were raised on an 8L:16D or a 16L:8D light cycle. Following 2 days of habituation, quail were administered saline, 5 mg/kg, or 10 mg/kg cocaine for 10 days. Restricted photoperiods in early life were correlated to lower gonadal hormone levels in females and males. Male quail raised on the short-light cycle developed a sensitized response to 10 mg/kg cocaine. Female quail raised on the short- or long-photoperiod developed behavioral sensitization to 5 mg/kg cocaine. Furthermore, early life reduction in estradiol in females modulated the amount of activity on day 10 of cocaine treatment. The current research extends previous research by finding a possible early life gonadal hormone control of behavioral sensitization in the quail.

Adolescent cannabis use has grown because of increased availability and higher societal acceptance. This increase in cannabis use is problematic as adolescents who experiment with cannabis are more likely to abuse cannabis and experiment with other illicit drugs such as cocaine. The reason for the greater susceptibility to drugs use is unclear and may be the result of altered drug sensitivity after cannabis exposure. Thus, the present investigation used the behavioral sensitization paradigm to examine the behavioral response of early adolescent rats to the cannabinoid agonist CP 55,940 (CP) or cocaine after repeated cannabinoid administration. It was hypothesized that: (1) CP would cause a sensitized response in both male and female adolescent rats, (2) female rats would have a greater behavioral response than male rats, (3) pretreatment with CP would induce cross-sensitization to cocaine, (4) pretreatment with cocaine would cause behavioral sensitization and conditioned activity in male and female adolescent rats. In the first experiment, 137 male and female Sprague-Dawley rats were given CP (4, 13.2, or 40 µg/kg, IP) or vehicle (50% DMSO/H2O) once daily for 5 consecutive days on postnatal day (PD) 30- PD 34. Distance traveled and stereotyped movement was assessed for 1 h after each drug injection. After a 48 h abstinence period (i.e., on PD 36), rats were given CP (4 or 13.2 µg/kg, IP) and distance traveled and stereotyped movement was monitored for 2 h. In the second experiment, 146 male and female rats were tested with the same protocol as in Experiment 1 except that rats were given CP (13.2 or 4 µg/kg), cocaine (20 mg/kg), or vehicle (saline or 50% DMSO/H2O) for five days and then tested with saline or cocaine (10 mg/kg) after 48 h. In the first experiment, no dose of CP altered distance traveled scores or stereotyped movement over the five pre-exposure days nor did CP cause behavioral sensitization on the test day. In the second experiment, pretreatment with cocaine led to enhanced distance traveled scores and stereotyped movement when challenged with cocaine (behavioral sensitization) or saline (conditioned activity) on test day. In contrast, CP-pretreated rats did not show greater activity when injected with cocaine or saline on test day. These data show that cannabinoids do not act like psychostimulant drugs, since CP did not cause the same changes in drug sensitivity as cocaine. The cocaine sensitization observed in adolescent rats indicates that this age group is particularly vulnerable to the rewarding effects of cocaine, and suggests that early cocaine exposure can augment drug seeking behavior. The failure to detect cannabinoid-induced sensitization, conditioned activity, or cocaine cross-sensitization during adolescence suggests that CP, when given at a consistent dose, does not increase the addictive properties of cannabinoids or cocaine. The results also indicate that cannabinoid use does not alter drug responsivity or lead to greater drug seeking and abuse in the adolescent population.

The neurochemical changes occurring between the preweanling period and adolescence could be crucial for understanding the role development plays in the manifestation of psychotic behaviors. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) fully attenuates the DA agonist-induced behaviors of adult rats, while potentiating the DA agonist-induced locomotor activity of preweanling rats. My specific hypotheses were as follows: (1) Systemically administered EEDQ would block the cocaine-induced locomotor activity of adult rats. (2) Systemically administered EEDQ would potentiate the cocaine-induced locomotion of preweanling rats. (3) EEDQ would increase the Emax values (a measure of D2 receptor sensitivity) of preweanling rats, but not adolescent or adult rats. And, (4) EEDQ would reduce dorsal striatal β-arrestin-2 (ARRB2) and GRK6 levels (measures of D2 receptor sensitivity) of preweanling rats. Behavioral results were as expected, because EEDQ attenuated the locomotion of adult and adolescent rats, while EEDQ potentiated locomotor activity of preweanling rats. EEDQ enhanced the GTPγS binding of preweanling rats, while depressing ARRB2 levels. These results are consistent with the overarching hypothesis that EEDQ causes DA supersensitivity in preweanling rats. Thus, it is here proposed that EEDQ inactivates a significant number of D2 receptors in preweanling rats, but that the remaining D2 receptors are supersensitive and capable of mediating a potentiated locomotor response.

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors.

This thesis explored the effect of cocaine use on treatment outcomes for heroin dependent individuals in Sydney, Australia. A naturalistic, longitudinal design was employed in order to examine the effects of cocaine on outcomes over a two year period. Study 1 assessed the prevalence and correlates of cocaine use among heroin dependent individuals. Cocaine use was found to be common among entrants to all three of the major treatment modalities in NSW. Heroin users who also used cocaine (CU) displayed a poorer clinical profile at baseline than non-cocaine users (NCU), reporting higher levels of drug use and dependence, and a greater prevalence of needle risk-taking and criminal behaviour. Study 2 examined outcomes three months post-study entry. CU and NCU were found to have been equally well retained in treatment. Despite significantly reduced levels of cocaine use amongst the cohort, however, CU continued to display the higher levels of drug-related harm that characterized them at baseline. In order to determine whether cocaine use itself was responsible for the greater levels of harm observed amongst CU, or whether instead CU were an inherently more dysfunctional group for whom cocaine use merely served as a marker, comparisons were made within groups on the basis of cocaine use patterns over the study period. The results of these analyses demonstrated that commencing cocaine use resulted in a clear decline in functioning, whereas cessation resulted in corresponding improvements. In Study 3 outcomes were examined twelve months post-study. Baseline cocaine use was again found to predict poorer outcome, despite a large scale reduction in cocaine use amongst the cohort. Importantly, CU were significantly less likely than NCU to be abstinent from heroin at twelve months and more likely to have been incarcerated since study entry. In addition, the effect of persistence of cocaine use was examined. Results indicated that the harms associated with cocaine use increased with increasing persistence of use. Outcomes at two years post-study entry were explored in Study 4. At this time CU and NCU recorded similar outcomes in most domains. Thus, it appeared that the harms caused by cocaine use may take a substantial period of time to diminish. Patterns of cocaine use and motivations for cessation and commencement were also examined. Responses suggested that cocaine use amongst the cohort was largely opportunistic, with participants ceasing use for a variety of reasons, including the financial and psychological problems caused by cocaine use. Past year prevalence of cocaine dependence was measured in this study, with the majority of those who had used cocaine in the past year meeting criteria for dependence. In Study 5, generalized estimating equations were used to measure the effect of baseline cocaine use on major outcome variables over the entire two year study period. Even after controlling for treatment variables, heroin use and other baseline polydrug use, the results of this study confirmed previous findings within the thesis by demonstrating the negative effect of baseline cocaine use on most outcome variables. Evidently, cocaine use among dependent heroin users has serious, long lasting, consequences. To date, however, there has been a stark lack of research examining the effect of cocaine use on treatment outcomes for heroin dependence. To that end, the results of this thesis are encouraging, suggesting that treatment for heroin dependence may also aid in reducing cocaine use among this group.

The current study examined the development of responding to joint attention (RJA), a prelinguistic skill, in a sample of children prenatally cocaine exposed. The sample used was part of a larger population of children randomly assigned to three levels of intervention. The growth of RJA in the current sample was best characterized by two linear growth groups determined by a semi-parametric growth modeling program. Each trajectory group was differentially associated with three language outcomes. Gender, treatment group, and birthweight were three risk factors that influenced the likelihood of belonging to either growth cluster. RJA?s predictive significance in terms of concurrent and subsequent language was also established, accounting for the variance associated with contemporaneous measures of cognition. The findings (regarding the relationship between RJA and language) were consistent with previous research examining joint attention behaviors in other types of samples. Additionally, this study contributed uniquely to the body of research on joint attention by exploring the growth of RJA, a precursor of language, in a sample of children at risk for language impairment.

In dieser Arbeit wird die Entwicklung eines bifunktionellen Biosensors nach dem Vorbild eines Baukastensystems beschrieben. Das Ziel wird durch die Kombination verschiedenster molekularer Erkennungselemente erreicht. Solche molekularen Erkennungselemente im verwendeten System sind: • Propidium und die periphere anionische Bindungsstelle der Acetylcholinesterase (AChE) • Organophosphate und das aktive Zentrum der AChE • ein an die AChE gekoppeltes Hapten und das Epitop eines Antikörpers • ein an die AChE gekoppeltes Hapten, das als Ligand ein weiteres Enzym bindet Neben dem molekularen Erkennungselement wird ein Biosensor ebenso durch die Art des Transducers charakterisiert. Hier werden Quarzplättchen mit Goldelektroden zur Signalumwandlung eingesetzt. Die Verwendung solcher Sensoren mit einem EQCM-Gerät (electrochemical quartz crystal microbalance) ermöglicht es zwei Messsignale gleichzeitig aufzunehmen: die piezoelektrische Bestimmung einer Massebeladung und die amperometrische Detektion von Enzymaktivität auf der Sensoroberfläche. Für die Analytik stehen somit zwei verschiedene Assay-Varianten zur Verfügung: die Bestimmung der Inhibition der ACHE-Aktivität und ein Bindungstest über das Hapten. Die Basis beider Tests ist die Modifizierung der piezoelektrischen Kristalle mit Propidium – einem reversiblen Inhibitor der Acetylcholinesterase. Dies ermöglicht die Beladung des Sensors mit AChE über die Wechselwirkung mit der peripheren anionischen Bindungsstelle des Enzyms. Die Aktivität der so immobilisierten AChE und die Inhibition durch Organophosphate (Pestizide) werden amperometrisch bestimmt. Durch die chemische Kopplung eines Hapten an die Cholinesterase wird ein weiteres Erkennungselement eingeführt. Das eröffnet die Möglichkeit, an die auf dem Propidium-modifizierten Sensor immobilisierte, haptenisierte Cholinesterase einen Antikörper zu binden. Als Voraussetzung für elektrochemische Bestimmung der AChE-Aktivität wurde zunächst die Optimierung der amperometrischen Messmethode vorgenommen. Die Oxidatationspotentiale für die Detektion von Thiocholin wurden im Bereich von 150 mV bis 300 mV variiert. Dabei wurde für die nachfolgenden Untersuchungen eine Arbeitspotential von 200 mV (vs. Ag/AgCl) festgelegt, da hier das beste Verhältnis von gemessenem Oxidationsstrom und Langzeitstabilität der Propidium-modifizierten Sensoren erzielt wurde. Dieses Potential war deutlich geringer als die bisher publizierten Mediator-freien AChE-Biosensoren. Es wurde ein Vergleich verschiedener Organophosphate über ihre Inhibitionskonstanten durchgeführt, um diejenigen herauszufinden, die möglichst schnell mit dem aktiven Zentrum der Acetylcholinesterase reagieren. Das verwendete Messsystem beruht nicht auf der Vorinkubation der AChE und damit einer Einstellung des Inhibitionsgleichgewichts. Stattdessen wurde die Inhibition der AChE direkt im Fließsystem verfolgt. Daher war eine schnelle Inhibitionskinetik für einen empfindlichen Organophosphat-Nachweis erforderlich. Da einige Inhibitoren nur als Phosphothionat vorlagen, wurde die Überführung dieser Substanzen in die entsprechenden Oxo-Formen mittels N-Bromsuccinimid untersucht. Die NBS-Aktivierung wurde erfolgreich durchgeführt, die erwartete Inhibitionsstärke konnte jedoch aufgrund hydrolytischer Vorgänge nicht erreicht werden. Untersuchungen mit Diisopropylfluorophosphat (DFP) und Chlorpyriphos-oxon (CPO) konnten die Voruntersuchungen über die Inhibitionskinetik in Bezug auf die erreichten Nachweisgrenzen von 2E-06 M für DFP und 5E-08 M für CPO bestätigen. Für die chemische Modifizierung der Acetylcholinesterase wurde zunächst 2,4-Dichlorphenoxyessigsäure (2,4-D) als Hapten ausgewählt. 2,4-D wird als Herbizid eingesetzt und in der EU über die Gewässerschutzrichtlinie reguliert. 2,4-D konnte in unterschiedlichen molaren Verhältnissen von 2,6 : 1 bis 260 : 1 (2,4-D : AChE) nach Aktivierung mit einem Norbornendicarboximido-Derivat an die AChE gekoppelt werden. Dabei konnte die spezifische Aktivität der Acetylcholinesterase erhalten und die Bindung eines anti-2,4-D-Antikörpers ermöglicht werden. Zur Verstärkung des piezolelektrischen Signals der Antikörperbindung wurden die Immunoglobuline zunächst an Goldnanopartikel gekoppelt. Damit konnte eine Verstärkung um den Faktor 10 erreicht werden. Allerdings waren die Antikörper-modifizierten Goldnanopartikel nicht langzeitstabil. Daher wurden auch Silica-Nanopartikel als Matrix für die Antikörperkopplung getestet. Mit diesem System konnte eine Verstärkung um den Faktor von 5 bis 13 je nach Grad der Beladung den Nanopartikel mit Antikörper bestimmt werden. Die hohe unspezifische Bindung der Antikörper-Nanopartikel-Konjugate an den Propidium-modifizierten QCM-Sensor konnte keinen empfindlichen 2,4-D-Nachweis ermöglichen. Als Alternative wurde Kokain (Benzoylecgonin, BZE) als Hapten an die Aceytlcholinesterase gekoppelt. Da Kokain selbst auch als Inhibitor im aktiven Zentrum der AChE binden kann, wurden zwei verschiedene Strategien zur Konjugatsynthese verfolgt. Durch Zugabe von Kokain während der Kopplung sollte die kovalente Fixierung des Kokain-Derivats BZE-DADOO im aktiven Zentrum verhindert werden (Konjugat B). In der Tat konnten mit dieser Synthesestrategie 67% der spezifischen Cholinesterase-Aktivität erhalten werden, während im Kokain-freien Ansatz (Konjugat A) nur 2% der Ausgangsaktivität wiedergefunden wurden. Das BZE-AChE-Konjugat ermöglichte auch die Untersuchung der Bindungskinetik der anti-BZE-Antikörper. Dabei konnte eine Assoziationsgeschwindigkeitskonstante ka von 12911 l/(mol•s) berechnet werden. Dieser Wert ist trotz der vergleichsweise geringen Oberflächenbeladung vergleichbar mit den in der Literatur angegebenen Werten. Die Dissoziationsgeschwindigkeitskonstante ist mit 2,89E−3 1/s um den Faktor 30 höher als der Literaturwert. Diese Abweichung ist auf Unterschiede im Bindungsmodell zurückzuführen. Mit beiden BZE-AChE-Konjugaten konnte ein kompetetiver Immunoassay mit Kokain im Fließsystem durchgeführt werden. Dabei zeigte sich für beide Konjugate ein ähnlicher Testmittelpunkt: IC50 = 4,40E−8 mol/l für Konjugat A bzw. IC50 = 1,77E−8 mol/l für Konjugat B. Diese Werte sind vergleichbar zu bereits publizierten Kokainassays im Fließsystem. Wie vorstehend beschrieben, bindet Kokain als Inhibitor auch im aktiven Zentrum von Cholinesterasen. Diese Eigenschaft wurde genutzt, um ein zweites Enzym – Butyrylcholinesterase (BChE) – an die BZE-AChE zu binden. Die Spezifität dieser Bindung konnte durch die Abwesenheit einer Affinität der BChE zum Propidium und durch die Blockierbarkeit der Bindung von BChE und BZE-AChE durch Kokain nachgewiesen werden. Damit konnte erfolgreich die Kombination mehrere molekularer Erkennungselemente demonstriert werden. Die Propidium-Plattform ermöglicht den Aufbau einer Architektur aus verschiedenen Cholinesterasen, die über unterschiedliche Bindungsstellen wechselwirken. Sowohl freie als auch BZE-modifizierte AChE können über die Affinität zum Propidium auf dem EQCM-Sensor immobilisiert werden. Mit Kokain als Substrat der Butyrylcholinesterase kann Benzoylecgonin nicht nur als Epitop für die Bindung eines Antikörpers, sondern auch als Erkennungselement für die BChE genutzt werden. Auf der anderen Seite erschwert die geringe Affinität der BChE im Gegensatz zum anti-BZE-Antikörper den Einsatz dieses Systems für analytische Zwecke. Durch die Verwendung anderer Ligand-Enzym-Kombinationen läßt sich das in dieser Arbeit vorgestellte Konzept noch weiter ausbauen und ermöglicht damit eine Entwicklung ausgehend von „einfachen“ molekularen Erkennungselementen (MRE) hin zu „multifunktionellen“ Erkennungselementsystemen. In dieser Arbeit konnte demonstriert werden, dass der Aufbau solch komplexe Systeme möglich ist, ohne Abstriche in Bezug auf die Empfindlichkeit der einzelnen Assays hinzunehmen. / This work describes the development of a bifunctional biosensor following a modular assembly approach. This aim is reached through the combination of various molecular recognition elements. The system presented herein uses the following recognition elements: • propidium and the peripheral anionic site of the acetylcholinesterase (AChE) • an organophosphate and the active site of the AChE • a hapten – covalently coupled to the AChE – and the epitope of an antibody • a hapten – covalently coupled to the AChE – binding as a ligand to another enzyme A biosensor is not only characterized by the molecular recognition element, but also by the type of signal transducer. This work is based on an electrochemical quartz crystal microbalance (EQCM) device that uses gold-plated quartz sensors for the signal transduction. This allows monitoring two distinct signals at the same time: the piezoelectric determination of a mass loading and the amperometrical detection of enzymatic activity on the sensor surface. Thus two different assay systems are provided: the determination of the inhibition of the AChE activity and ligand binding assay via the hapten. Both tests are based on the modification of the piezoelectric crystals with propidium – a reversible AChE inhibitor. This allows the deposition of AChE on the sensor surface via the interaction with the enzyme’s peripheral anionic site. The enzymatic activity of the in-situ immobilized AChE and the inhibition by organophosphates (pesticides) are measured amperometrically. Another recognition element is introduced by the chemical coupling of a hapten to the cholinesterase. This provides the opportunity bind an antibody to the haptenized cholinesterase that is immobilized on the propidium-modified sensor. Preliminary experiments were focussed on the improvement of the amperometric determination the AChE activity. The applied potential for the oxidation of thiocholine was changed over a range from 150 to 300 mV. The best results for the measured oxidation current and the long-term stability of the propidium-modified sensors were obtained at 200 mV (vs. Ag/AgCl). This potential was used throughout all subsequent experiments. This potential was also found to be lower as compared to mediator-free AChE-biosensors published hitherto. Different organophosphates were evaluated with regard to their inhibition constants to find those which react with active site of the acetylcholinesterase as fast as possible. The assay format used herein monitors the inhibition of the AChE directly in the flow-system. That is, it is not based preincubation of the enzyme with the inhibitor and therefore no inhibition equilibrium is reached. This approach requires fast inhibition kinetics in order to detect the organophosphates highly sensitively. Some of the inhibitors were only available in the phosphothionate form. Thus was necessary to convert these compounds to their respective oxon-forms by N-bromosuccinimide (NBS). The NBS-activation was performed successfully, though the expected inhibition potential could not be reached due to hydrolytic processes. Experiments with diisopropylfluorophosphate (DFP) und chlorpyriphos-oxon (CPO) could confirm the previous experiments on the inhibtion kinetics. Lower limits of detection of 2E-06 M for DFP and 5E-08 M for CPO could be reached with this approach. Initially 2,4-dichlorphenoxyacetic acid (2,4-D) was chosen as a hapten for the chemical modification of the acetylcholinesterase. The use of 2,4-D as a herbicide is regulated by the water protection directive of the European Union. 2,4-D was coupled to AChE in different molar ratios from 2,6 : 1 to 260 : 1 (2,4-D : AChE) after activation with a norbornendicarboximido derivative. The chosen coupling method allowed to recover the complete specific activity of the acetylcholinesterase and to bind a specific anti-2,4-D-antibody. Furthermore, the coupling of the immunoglobulins to gold nanoparticles was tested to enhance the piezoelectric signal of the antibody binding. An amplification factor of 10 was reached with this system. However the antibody-coated gold nanoparticles show a very poor long-term stability. Therefore also silica nanoparticles were tested as a matrix for the coupling of the antibodies. This approach yielded an amplification factor from 5 to 13 depending amount of antibodies bound to the nanoparticles. Unfortunately the high non-specific binding of the antibody nanoparticle conjugates did not allow a sensitive 2,4-D detection assay. Cocaine (benzoylecgonine, BZE) was coupled as a hapten to Acetylcholinesterase in an alternative approach. Two different strategies for the synthesis of the conjugate were pursued, since cocaine can bind also bind as an inhibitor for the AChE’s active site. The addition of excess cocaine during the coupling reaction should the covalent binding of the cocaine derivative BZE-DADOO at the active site (conjugate B). Indeed over two thirds of the original specific cholinesterase activity could be recovered with this strategy, while the cocaine-free batch (conjugate A) showed only 2% of the original activity. Furthermore the BZE-AChE conjugate allowed the evaluation of the binding kinetics of the anti-BZE-antibody. The association rate constant ka was calculated to 12911 l/(mol•s). Despite the low surface coverage this value is still comparable to other published results. The dissociation rate constant kd of 2,89E−3 1/s is thirty times higher than values found in the literature. This deviation is due to differences in the applied binding model. Both BZE-AChE conjugates could be applied in a competitive immunoassay for cocaine in the flow system. It was shown that for both conjugates a similar half maximal inhibitory concentration was reached: : IC50 = 4,40E−8 mol/l for conjugate A and IC50 = 1,77E−8 mol/l for conjugate B, respectively. These values are comparable to other published assay for cocaine in a flow system. As described earlier, cocaine is also able to bind to the active site of cholinesterases. This feature was used to examine the interaction of a second enzyme – butyrylcholinesterase (BChE) – with the BZE-AChE. Evidence for the specificity of this interaction was provided by two further experiments, i.e. BChE has no affinity towards Propidium and the binding of BChE towards BZE-AChE could be blocked by excess cocaine. Thus the successful integration of recognition elements on the molecular level could be demonstrated. The propidium-modifies sensor allowed the construction of a scaffold of cholinesterases that interact via different recognition sites. Unmodified and BZE-coupled AChE can be immobilized on the EQCM-sensor via the interaction with propidium. With cocaine being a substrate BChE this compound cannot only be used to capture anti-BZE-antibodies, but also as a recognition element for BChE. The affinity of the BChE towards is relatively low as compared to the antibody’s binding strength, thus making it difficult to employ this system for analytical purposes. Still the concept presented herein can be extended by other ligand-enzyme-combinations. On the basis of “simple” molecular recognition elements this enables the development of “multifunctional” recognition element systems. This work could show that the construction of such complex systems is possible without cutting back with regard to the sensitivity of the individual assays.

L'exposition répétée à la cocaine induit une plasticité cérébrale responsable de changements comportementaux de longue durée, dont les mécanismes de signalisation intracellulaire sont mal connus. Les neurones du striatum exprimant le recepteur à la dopamine D1 et ceux exprimant le recepteur à la dopamine D2 jouent un rôle important dans l'intégration de ces signaux. L'activation de ces récepteurs induit des cascades de signalisations opposées, il est donc primordial de pouvoir les étudier séparément. Afin d'analyser spécifiquement ces familles de neurones, nous avons adapté une méthode de tri et d'analyse des noyaux des neurones basée sur la cytométrie en flux. Notre étude a permis de quantifier les changements post-traductionels des histones, ainsi que les enzymes les controlant, specificiquement dans les noyaux des neurones D1 ou D2, suite à un traitement aigu ou chronique de cocaine. Avec cette approche, nous avons trouvé que les neurones D1 et D2 comportent des profils épigénétiques spécifiques, dynamiquement régulés par la cocaine. Plus particulièrement, nous avons trouvé que l'acétylation des histones H3K14, H4K5, H4K12 et la méthylation de H3K9 étaient régulées de manière opposée entre les deux types cellulaires, sous-tendant la disparité de leur réponse transcriptionelle à la drogue. Enfin, nous avons observé qu'il y avait une corrélation complexe entre les modifications post-traductionelles d'histones, spécifiques des neurones D1 ou D2, et qui est sensiblement altérée par la cocaine. Nous proposons une approche originale dans le domaine des neurosciences permettant l'étude des protéines nucléaires applicable potentiellement à tous les types neuronaux du cerveau

This thesis was undertaken to assess the characteristics of individuals who smoke crack cocaine and to examine the health-related risks and healthcare needs of this population. A literature review of 147 published articles was conducted to synthesize evidence regarding behaviours associated with crack use and to assess the risks of disease transmission through crack smoking behaviours. Qualitative interviews were subsequently conducted with thirty Ottawa residents who smoke crack to learn about their experiences with healthcare and harm reduction services. Results identified barriers related to accessing primary healthcare and drug treatment programming among people who smoke crack and gaps within existing harm reduction services. Individuals who smoke crack represent a marginalized population who are often missed through traditional health promotion and harm reduction programming. There is a need for increased coverage of current programming and a reduction of factors which currently hinder the delivery and effectiveness of crack-specific harm reduction programs.