G-protein coupled receptors (GPCRs) implicated in disease are the predominant pharmaceutical targets. Growing evidence suggests that GPCRs form homo- and heteromeric complexes, resulting in allosteric functional changes. Ligands targeting one receptor can alter the function of the other receptor or receptors. Knowledge of these functional changes will provide unique opportunities to treat diseases. We examined two GPCR heteromers implicated in psychosis: mGlu2R-5HT2AR and D2R-5HT2AR. Using whole-cell patch clamp, we studied HEK-293 cells stably transfected with mGlu2R and 5HT2AR. Maximal heteromer formation allows inverse agonists to increase the G-protein activity of the opposite receptor, while sub-maximal heteromer formation does not. However, similar results are obtained in sub-maximal heteromer cells by applying a combination of a mGlu2R synthetic agonist with a 5HT2AR anti-psychotic drug. These results confirm our oocyte results, now in a mammalian cell line. Using two-electrode voltage clamp, we also investigated the allosteric changes upon heteromerization of D2R-5HT2AR in oocytes injected with appropriate cRNAs. Heteromer formation in the presence of dopamine or serotonin results in an increase in G-protein activity of each receptor while the simultaneous presence of both neurotransmitters further increases the G-protein activity. The addition of synthetic agonists or anti-psychotics decreases the G-protein activity of the opposite receptor while agonizing or antagonizing its target receptor, respectively. Maximal allosteric effects upon D2R-5HT2AR formation only occur at a specific cRNA injection ratio, but partial effects exist at other ratios. Our data suggest that allosteric functional changes upon heteromerization are physiologically relevant and are mostly different when comparing mGlu2R-5HT2AR to D2R-5HT2AR.

The ability to objectively index dopamine (DA) levels in the brain has the potential to revolutionize the field of neuropsychopharmacology, as having a peripheral biomarker of brain DA would enable the objective monitoring of the progression of Parkinson's disease (PD) and other DA-dependent psychiatric states. Of particular relevance to commercialization, it would provide an objective measure of treatment efficacy. We used a DA-depletion approach to determine if peripheral D2Rs are a biomarker for brain DA; mainly, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model and PD subjects, which are well-known models of DA depletion in the midbrain of rodents and humans, respectively. Dopamine depletion in the substantia nigra resulted a significant change to DA and norepinephrine (NE) levels in the plasma. Interestingly, these changes could be tracked as a time course from baseline to 15 days after injection with MPTP. Also, the proportion of D2R expressing leukocytes steadily increased (specifically B cells and T cells) during this same time of DA depletion. These results suggest that changes to the dopamine neuron population in the substantia nigra can be tracked with DA and NE level changes and D2R expression on B and T cells, providing a possible biomarker for nigral DA neuron loss to further investigate. In two cohorts of studies comparing subjects with PD vs controls, we found that Parkinson's subjects displayed significantly decreased D2R expression in all populations except for (natural killer) NK cells, CD16+ monocytes, and cytotoxic T cells. We also found that subjects with PD show increased levels in epinephrine (EPI) and DA as compared to control subjects. We did not, however, find any statistically significant correlations between the recorded leukocytic D2R downregulation in PD patients and their elevated DA and EPI plasma levels. Therefore, the results of this study did not provide a clear indication how brain DA levels are being represented in the periphery. Regardless, the modulation of peripheral D2Rs in PD and MPTP seen in this study do show that substantia nigra DA depletion in humans and rodents do manifest in the periphery. Although our study didn't result in a clear narrative of how nigral and peripheral DA system mirror each other, our result provide more evidence D2Rs may be both biomarkers and important substrates for treatment of DA-dependent disorders. Our results give a foundation from which future studies can investigate this connection further.

Recently, several studies have demonstrated that G protein coupled receptors exist as homo/heterodimers or oligomers. Adenosine A2A receptors and dopamine D2 receptors are present as both homo- and heterodimer. In the GABAergic striatopallidal neurons A2AR are co- localized with D2 receptors (D2R), and establish functional A2AR-D2R heteromers, which modulates dopaminergic activity. Due to be involved in physiological processes, these receptors bear critical roles. Dopamine receptors play critical role in dopaminergic pathways in regulation of memory, food intake and psychomotor activity, etc. On the other hand, adenosine A2A receptors are involved in the regulations of neurotransmission, immune response and cardiovascular systems. Dopamine D2R andadenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity The purpose of this study is to assess the functionality of EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) tagged adenosine A2A and dopamine D2 receptors and to detect homo/ hetero-dimerization of these receptors in live cells via Fluorescence Resonance Energy Transfer (FRET). Understanding the mechanisms of the interaction between adenosine and dopamine signaling will help us to figure out some molecular mechanism of neurophysiological disorders. Furthermore, the fluorescence based live cell model could be used to observe the effects of potential anti-psychotic drugs on the interaction of these two receptors.

Recent studies demonstrate that there are several G-protein coupled receptors (GPCRs) that dimerize with other GPCRs and form heterodimers. Adenosine A2A-Dopamine D2 receptor interaction is one of the examples for GPCR heterodimerization. Both receptors bear critical roles in physiological processes. Adenosine A2A receptor has functions in neurotransmission, cardiovascular system and immune response. On the other hand, dopamine receptors are the key point of dopaminergic system, which controls the regulation of memory, attention, food intake, endocrine regulation, psychomotor activity and positive reinforcement. Deregulation in dopamine signaling could cause neurological disorders such as Parkinson&rsquo / s disease and schizophrenia. Dopamine D2R and adenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity. The purpose of this study is to optimize Fluorescence Resonance Energy Transfer (FRET) method to detect dimerization of D2R and A2AR by tagging them with EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) in live N2a cell line using laser scanning confocal microscope. Establishing this model will pave the ways for understanding mechanisms of interaction between dopamine and adenosine signaling, thereby, contributing to the understanding molecular mechanisms of some neurophysiological events and disorders. Moreover, the fluorescence based live cell model will be used to detect effects of potential anti-psychotic drugs on the interaction of these two receptors. Indeed, follow-up studies are necessary to extend the limits of this project. Further imaging analyses and drug-receptor interaction studies can be readily applied to extract more information on dopamine-adenosine signaling by using the system developed with this thesis study.

During adolescent brain maturation, there are likely sensitive periods where environmental conditions, including drug exposure, may influence development by modifying neuronal connections. Altering neuronal function may produce different phenotypes than expected under normal conditions that may influence subsequent responding to drugs of abuse after the brain is fully mature. Experiment one investigated the relationship between novelty preference and cocaine place preference in adolescent and adult rats. High responding adolescent rats displaying greater free choice novelty exploration (but not forced novelty locomotion) expressed decreased cocaine place conditioning compared to low responding rats. No relationship was found in adult rats. Experiment two evaluated novelty-induced behaviors in adulthood after adolescent cocaine exposure. Repeated cocaine administration produced greater stress and anxiogenic behavioral responses to novelty in adult rats. Repeated alcohol administration produced less-inhibited novelty-induced behaviors in adulthood. Experiment three and four evaluated the consequence of developmental cocaine exposure on the rewarding efficacy of cocaine in adolescence and adulthood. Additionally, the interaction of D2 receptors and the rewarding efficacy of cocaine were investigated. After developmental cocaine exposure, adolescent and adult rats demonstrate decreased rewarding efficacy to cocaine. Importantly, blockade of the D2 receptor prevents cocaine-induced neurochemical changes, potentially regulating the behavioral and neurochemical alterations that occur after repeated drug use that increases the likelihood of dependence. Together, these data implicate both short and long-term behavioral adaptations that occur after developmental cocaine exposure that may result in a predisposition to develop adulthood drug dependence.

An emerging problem in organisations is that there exist a large number of tools storing data that communicate with each other too often, throughout the process of an application or product development. However, no means of communication without the intervention of a central entity (usually a server) or storing the schema at a central repository exist. Accessing data among tools and linking them is tough and resource intensive. As part of the thesis, we develop a software (also referred to as ‘adaptor’ in the thesis), which, when implemented in the lifecycle management systems, integrates data seamlessly. This will eliminate the need of storing database schemas at a central repository and make the process of accessing data within tools less resource intensive. The adaptor acts as a wrapper to the tools and allows them to directly communicate with each other and exchange data. When using the developed adaptor for communicating data between various tools, the data in relational databases is first converted into RDF format and is then sent or received. Hence, RDF forms the crucial underlying concept on which the software will be based. The Resource description framework (RDF) provides the functionality of data integration irrespective of underlying schemas by treating data as resource and representing it as URIs. The model of RDF is a data model that is used for exchange and communication of data on the Internet and can be used in solving other real world problems like tool integration and automation of communication in relational databases. However, developing this adaptor for every tool requires understanding the individual schemas and structure of each of the tools’ database. This again requires a lot of effort for the developer of the adaptor. So, the main aim of the thesis will be to automate the development of such adaptors. With this automation, the need for anyone to manually assess the database and then develop the adaptor specific to the database is eliminated. Such adaptors and concepts can be used to implement similar solutions in other organisations faced with similar problems. In the end, the output of the thesis is an approachwhich automates the process of generating these adaptors. / Resource Description Framework (RDF) ger funktionaliteten av dataintegration, oberoende av underliggande scheman genom att behandla uppgifter som resurs och representerar det som URI. Modellen för Resource Description Framework är en datamodell som används för utbyte och kommunikation av uppgifter om Internet och kan användas för att lösa andra verkliga problem som integrationsverktyg och automatisering av kommunikation i relationsdatabaser. Ett växande problem i organisationer är att det finns ett stort antal verktyg som lagrar data och som kommunicerar med varandra alltför ofta, under hela processen för ett program eller produktutveckling. Men inga kommunikationsmedel utan ingripande av en central enhet (oftast en server) finns. Åtkomst av data mellan verktyg och länkningar mellan dem är resurskrävande. Som en del av avhandlingen utvecklar vi en programvara (även hänvisad till som "adapter" i avhandlingen), som integrerar data utan större problem. Detta kommer att eliminera behovet av att lagra databasscheman på en central lagringsplats och göra processen för att hämta data inom verktyg mindre resurskrävande. Detta kommer att ske efter beslut om en särskild strategi för att uppnå kommunikation mellan olika verktyg som kan vara en sammanslagning av många relevanta begrepp, genom studier av nya och kommande metoder som kan hjälpa i nämnda scenarier. Med den utvecklade programvaran konverteras först datat i relationsdatabaserna till RDF form och skickas och tas sedan emot i RDF format. Således utgör RDF det viktiga underliggande konceptet för programvaran. Det främsta målet med avhandlingen är att automatisera utvecklingen av ett sådant verktyg (adapter). Med denna automatisering elimineras behovet att av någon manuellt behöver utvärdera databasen och sedan utveckla adaptern enligt databasen. Ett sådant verktyg kan användas för att implementera liknande lösningar i andra organisationer som har liknande problem. Således är resultatet av avhandlingen en algoritm eller ett tillvägagångssätt för att automatisera processen av att skapa adaptern.