No description available.

Lead (Pb²⁺) is an environmental toxin that is known to cause lasting cognitive deficits following early life exposure. Previously, our laboratory demonstrated increased sensitivity to the psychostimulant effects of cocaine in animals with elevated blood Pb²⁺ levels (BLL). This effect was abolished following introduction of dopamine (DA) receptor antagonists, indicating that the dopaminergic (DAergic) system may be a target of Pb²⁺’s toxic effects. However, the biological mechanisms through which Pb²⁺ increased sensitization to cocaine’s psychostimulant effects have not been fully elucidated. There is some disagreement regarding the magnitude and direction of Pb²⁺’s effects on the DAergic system. Furthermore, many studies to date have measured the effects of Pb²⁺ in only one sex (usually male), one exposure, and one or two time-points, making it difficult to determine any potential sex-, age-, and exposure-dependent effects. In the present study, we used a well-validated animal model and Pb²⁺ exposure paradigm that uses chronic dietary exposure to 180ppm and 1500ppm Pb²⁺ acetate (PbAC) in the diet. These levels of Pb²+ in the diet resulted in low and moderate levels of BLLs that on average approximated 4.5 and 22.0µg/dl in young adult rats. These levels of Pb²⁺ exposure are relevant to contemporary levels of BLL in intoxicated children in many cities in the United States and in many parts of the world where Pb²⁺ exposure continues to be a major public health concern. It should be noted that at the low level of Pb²⁺ exposure, the resulting BLL of 4.5µg/dl is just below the current CDC level of action. Using this well-defined rat model of chronic Pb²⁺ exposure, in Aim 1, we measured DA concentration and turnover in the dorsal striatum (STR) of juvenile (PN14), adolescent (PN28), and young adult (PN50) male and female rats. Tyrosine hydroxylase (TH) protein, the rate-limiting step in the synthesis of DA, and phosphorylation of TH at serine 40 (pser40TH) were assessed as an indirect measure of TH activity. Thus, we measured the ratio of pser40TH to total TH protein. We also measured vesicular monoamine transporter-type 2 (VMAT2) levels in the STR, nucleus accumbens (NAC), and olfactory tubercle (OT) since this protein is critical for the sequestration of DA in presynaptic vesicles and has been used as a biomarker for DA terminal integrity. In Aim 2, we examine the effect of chronic Pb²⁺ exposure on D1 and D2 dopamine receptor (D1R and D2R) in the OT, NAC, and STR. Analysis of D1R and D2R is important since the downstream effects of DA are dependent on the DA receptor subtype it activates. In Aim 1, we observed significant increases in DA and its metabolites homovanillic acid (HVA) and 3,4-Dihydroxyphenylacetic acid (DOPAC) in the STR of adolescent and young adult male rats with BLL as low as 4.5µg/dl in the absence of phosphorylation at the serine 40 residue of TH or altered VMAT2 levels. In Aim 2, a significant increase in D2R was detected in the juvenile male rat STR. We also observed increases in D1R expression in adolescent male rats in the NAC, OT, STR, and in the OT of adolescent female rats. Together, these results demonstrate that chronic Pb²⁺ exposure alters DA receptor levels in a manner characteristic of a hyperactive DAergic state. The observations presented in this work suggest that a hyperactive DAergic system underlies the heightened sensitization to cocaine we previously observed in Pb²⁺-exposed animals. This work builds upon the current understanding of how Pb²⁺ modulates the DAergic system and provides some elucidation of the mechanisms underlying increased drug sensitization our laboratory has previously observed in rats exposed to Pb²⁺.

Introdução: A doença de Cushing apresenta elevada morbimortalidade. Seu tratamento de escolha é a cirurgia transesfenoidal que possui resultados satisfatórios em cerca de 70% dos casos. Na doença persistente ou recorrente, reabordagem cirúrgica, radioterapia e adrenalectomia bilateral podem ser realizadas, porém, essas opções apresentam como desvantagens o desenvolvimento de hipopituitarismo e a dependência de terapia de reposição. Até o momento, nenhuma droga tem se mostrado eficaz no tratamento do corticotrofinoma. Os esquemas terapêuticos mais eficazes são os inibidores da esteroidogênese que não atuam no tumor hipofisário. Objetivos: avaliar o efeito do octreotide e da cabergolina administrados isoladamente e em associação nas concentrações urinárias de cortisol e plasmáticas de ACTH em pacientes com corticotrofinomas; correlacionar esse efeito com a expressão tumoral dos receptores SSTR2, SSTR5 e DRD2; correlacionar a expressão tumoral desses receptores através de RT-PCR quantitativa e imunohistoquímica; avaliar se o uso prévio dessas drogas altera a expressão desses receptores. Casuística e Métodos: grupo controle composto por 11 pacientes (10 mulheres e 1 homem) entre 21 e 43 anos sem tratamento prévio à neurocirurgia e um grupo tratado formado por 11 pacientes (2 homens e 9 mulheres) entre 22 e 53 anos que receberam o seguinte tratamento antes da cirurgia: coleta de três amostras de cortisol urinário e ACTH plasmático, seguida da introdução de octreotide 100 g, subcutâneo, 8/8h durante 30 dias e nova coleta de três amostras de cortisol urinário e ACTH plasmático. Em seguida, iniciou-se a cabergolina 0,5 mg via oral 3 vezes na semana durante 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. A seguir, o octreotide era associado por mais 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. Resultados: Os valores de cortisol urinário apresentaram queda significante após o uso de cabergolina isolada (P = 0,016) e em associação ao octreotide (P = 0,012). A eficácia do tratamento combinado não foi maior que a da cabergolina isolada. Os valores de ACTH plasmático não revelaram diferença significante durante o tratamento e não se correlacionaram com os valores de cortisol. A média de expressão do mRNA do gene DRD2 foi maior no grupo tratado (1,170 ± 0,417) quando comparada ao grupo controle (0,776 ± 0,252) (P = 0,036). Houve dissociação entre os conteúdos de mRNA e da proteína desse receptor. Não foi possível analisar a expressão do mRNA do gene SSTR5, pois o tratamento das amostras com DNAse causou degradação do RNA. A imunorreatividade para SSTR5 esteve presente em todos os pacientes e não foi alterada pelo tratamento prévio. Não houve diferença estatística na expressão do gene SSTR2 entre os grupos controle (1,253 ± 0,511) e tratado (1,267 ± 0,386) bem como diferença significante da imunoexpressão do SSTR2 entre os grupos. Houve correlação entre os conteúdos de mRNA e da proteína desse receptor (P = 0,021). Não houve correlação entre a expressão dos receptores analisados e a resposta ao octreotide e à cabergolina isoladamente ou em associação. Conclusões: a cabergolina isolada representa opção terapêutica na doença de Cushing persistente ou recorrente. A associação de octreotide na dose estudada por 30 dias não foi mais eficiente em reduzir o cortisol urinário. A resposta a essas drogas não está relacionada à expressão dos receptores SSTR2, SSTR5 e DRD2 / Introduction: The Cushings disease presents high morbimortality. Its treatment of choice is transsphenoidal surgery which has satisfactory results in about 70% of cases. In persistent or recurrent disease, a second transsphenoidal surgery, radiotherapy and bilateral adrenalectomy can be carried through, however, these options present disadvantages as development of hypopituitarism and lifelong dependence on hormone replacement therapy. Presently, no drug has shown efficacy in corticotrophinomas treatment. The most efficient agents are the inhibitors of steroidogenesis which have no effect at pituitary tumor. Objectives: To evaluate isolated octreotide and cabergoline effects and their association on plasma ACTH and urinary cortisol in Cushings disease patients, to correlate this effect with tumoral expression of SSTR2, SSTR5 and DRD2 receptors; to correlate tumoral expression of these receptors by quantitative RT-PCR and immunohistochemistry; to evaluate whether these drugs modifies these receptors expression. Patients and methods: control group with 11 patients (10 women and 1 man) between 21 and 43 years who underwent pituitary surgery with no prior treatment and a treated group with 11 patients (2 men and 9 women) between 22 and 53 years that received the following treatment before surgery: : after three baseline urinary cortisol samples and one plasma ACTH sample, patients received octreotide 100 g, subcutaneous 8/8h for 30 days collecting three urinary cortisol samples and one plasma ACTH. After that, cabergoline was introduced 0,5 mg 3x/week for 30 days collecting three urinary cortisol samples and one plasma ACTH sample. Then, octreotide was associated to cabergoline for another 30 days followed by three urinary cortisol and one plasma ACTH sample. Results: Urinary cortisol concentrations significantly decreased after isolated and combined cabergoline use (P = 0,016 and P = 0,012, respectively). Combined treatment efficacy was not greater than isolated cabergoline administration. Plasma ACTH did not change statistically during treatment and did not correlate with urinary cortisol. The average of DRD2 gene expression was higher in control group (0,776 ± 0,252) in relation to treated group (1,170 ± 0,417) (P = 0,036). It had dissociation between mRNA and protein contents of this receptor. SSTR5 gene mRNA expression was not analyzed due to RNA degradation after DNAse tissue treatment. SSTR5 immunoreactivity was present in all patients and it was not modified by previous treatment. No statistic difference was observed between SSTR2 gene expression in control group (1,253 ± 0.511) and in treated group (1,267 ± 0,386). There was no significant difference in SSTR2 immunoexpression between groups. It had correlation between the mRNA and protein contents of this receptor (P = 0.021). No significant relationship was found between hormonal response to isolated and combined therapy and receptors mRNA expression levels. Conclusions: cabergoline represents therapeutical option in persistent or recurrent Cushings disease. Octreotide-cabergoline association in the studied dosage and for the period of 30 days was not more efficient in eliciting urinary cortisol reduction. The responsiveness to these drugs did not correlate to SSTR2 and DRD2 mRNA expression

Not available

No description available.

Introdução: A doença de Cushing apresenta elevada morbimortalidade. Seu tratamento de escolha é a cirurgia transesfenoidal que possui resultados satisfatórios em cerca de 70% dos casos. Na doença persistente ou recorrente, reabordagem cirúrgica, radioterapia e adrenalectomia bilateral podem ser realizadas, porém, essas opções apresentam como desvantagens o desenvolvimento de hipopituitarismo e a dependência de terapia de reposição. Até o momento, nenhuma droga tem se mostrado eficaz no tratamento do corticotrofinoma. Os esquemas terapêuticos mais eficazes são os inibidores da esteroidogênese que não atuam no tumor hipofisário. Objetivos: avaliar o efeito do octreotide e da cabergolina administrados isoladamente e em associação nas concentrações urinárias de cortisol e plasmáticas de ACTH em pacientes com corticotrofinomas; correlacionar esse efeito com a expressão tumoral dos receptores SSTR2, SSTR5 e DRD2; correlacionar a expressão tumoral desses receptores através de RT-PCR quantitativa e imunohistoquímica; avaliar se o uso prévio dessas drogas altera a expressão desses receptores. Casuística e Métodos: grupo controle composto por 11 pacientes (10 mulheres e 1 homem) entre 21 e 43 anos sem tratamento prévio à neurocirurgia e um grupo tratado formado por 11 pacientes (2 homens e 9 mulheres) entre 22 e 53 anos que receberam o seguinte tratamento antes da cirurgia: coleta de três amostras de cortisol urinário e ACTH plasmático, seguida da introdução de octreotide 100 g, subcutâneo, 8/8h durante 30 dias e nova coleta de três amostras de cortisol urinário e ACTH plasmático. Em seguida, iniciou-se a cabergolina 0,5 mg via oral 3 vezes na semana durante 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. A seguir, o octreotide era associado por mais 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. Resultados: Os valores de cortisol urinário apresentaram queda significante após o uso de cabergolina isolada (P = 0,016) e em associação ao octreotide (P = 0,012). A eficácia do tratamento combinado não foi maior que a da cabergolina isolada. Os valores de ACTH plasmático não revelaram diferença significante durante o tratamento e não se correlacionaram com os valores de cortisol. A média de expressão do mRNA do gene DRD2 foi maior no grupo tratado (1,170 ± 0,417) quando comparada ao grupo controle (0,776 ± 0,252) (P = 0,036). Houve dissociação entre os conteúdos de mRNA e da proteína desse receptor. Não foi possível analisar a expressão do mRNA do gene SSTR5, pois o tratamento das amostras com DNAse causou degradação do RNA. A imunorreatividade para SSTR5 esteve presente em todos os pacientes e não foi alterada pelo tratamento prévio. Não houve diferença estatística na expressão do gene SSTR2 entre os grupos controle (1,253 ± 0,511) e tratado (1,267 ± 0,386) bem como diferença significante da imunoexpressão do SSTR2 entre os grupos. Houve correlação entre os conteúdos de mRNA e da proteína desse receptor (P = 0,021). Não houve correlação entre a expressão dos receptores analisados e a resposta ao octreotide e à cabergolina isoladamente ou em associação. Conclusões: a cabergolina isolada representa opção terapêutica na doença de Cushing persistente ou recorrente. A associação de octreotide na dose estudada por 30 dias não foi mais eficiente em reduzir o cortisol urinário. A resposta a essas drogas não está relacionada à expressão dos receptores SSTR2, SSTR5 e DRD2 / Introduction: The Cushings disease presents high morbimortality. Its treatment of choice is transsphenoidal surgery which has satisfactory results in about 70% of cases. In persistent or recurrent disease, a second transsphenoidal surgery, radiotherapy and bilateral adrenalectomy can be carried through, however, these options present disadvantages as development of hypopituitarism and lifelong dependence on hormone replacement therapy. Presently, no drug has shown efficacy in corticotrophinomas treatment. The most efficient agents are the inhibitors of steroidogenesis which have no effect at pituitary tumor. Objectives: To evaluate isolated octreotide and cabergoline effects and their association on plasma ACTH and urinary cortisol in Cushings disease patients, to correlate this effect with tumoral expression of SSTR2, SSTR5 and DRD2 receptors; to correlate tumoral expression of these receptors by quantitative RT-PCR and immunohistochemistry; to evaluate whether these drugs modifies these receptors expression. Patients and methods: control group with 11 patients (10 women and 1 man) between 21 and 43 years who underwent pituitary surgery with no prior treatment and a treated group with 11 patients (2 men and 9 women) between 22 and 53 years that received the following treatment before surgery: : after three baseline urinary cortisol samples and one plasma ACTH sample, patients received octreotide 100 g, subcutaneous 8/8h for 30 days collecting three urinary cortisol samples and one plasma ACTH. After that, cabergoline was introduced 0,5 mg 3x/week for 30 days collecting three urinary cortisol samples and one plasma ACTH sample. Then, octreotide was associated to cabergoline for another 30 days followed by three urinary cortisol and one plasma ACTH sample. Results: Urinary cortisol concentrations significantly decreased after isolated and combined cabergoline use (P = 0,016 and P = 0,012, respectively). Combined treatment efficacy was not greater than isolated cabergoline administration. Plasma ACTH did not change statistically during treatment and did not correlate with urinary cortisol. The average of DRD2 gene expression was higher in control group (0,776 ± 0,252) in relation to treated group (1,170 ± 0,417) (P = 0,036). It had dissociation between mRNA and protein contents of this receptor. SSTR5 gene mRNA expression was not analyzed due to RNA degradation after DNAse tissue treatment. SSTR5 immunoreactivity was present in all patients and it was not modified by previous treatment. No statistic difference was observed between SSTR2 gene expression in control group (1,253 ± 0.511) and in treated group (1,267 ± 0,386). There was no significant difference in SSTR2 immunoexpression between groups. It had correlation between the mRNA and protein contents of this receptor (P = 0.021). No significant relationship was found between hormonal response to isolated and combined therapy and receptors mRNA expression levels. Conclusions: cabergoline represents therapeutical option in persistent or recurrent Cushings disease. Octreotide-cabergoline association in the studied dosage and for the period of 30 days was not more efficient in eliciting urinary cortisol reduction. The responsiveness to these drugs did not correlate to SSTR2 and DRD2 mRNA expression

No description available.

Methamphetamine (METH) is a potent psychostimulant known to cause cognitive abnormalities and neurodegenerative changes in the brains of METH abusers. One approach for developing therapies for METH abuse is to understand the molecular mechanisms of toxicity of the drug. Investigations in our laboratory and elsewhere have shown that single intraperitoneal injections of METH (30-40 mg/kg of body weight) can cause damage to striatal and cortical monoaminergic systems and induce neuronal apoptosis in the striatum of rodents via activation of endoplasmic reticulum (ER) and mitochondrial death pathways. Hence, the purpose of this thesis was to investigate if toxic binge METH injections can cause ER- and mitochondria-induced stress in the rat striatum. Recent studies have suggested that dopamine (DA) D1 and D2 receptors might mediate neuronal apoptosis in the striatum after single toxic METH doses. We therefore hypothesized that signaling through these two types of DA receptors might activate toxic effects of the binge METH regimen. The role of DA D1 or D2 receptors in METH-induced cell death pathways was thus examined by using pharmacological inhibitors of these receptors. In this dissertation, I report that binge METH regimen caused differential changes in immediate early genes (IEGs) that are known to influence synaptic changes in the brain. METH-induced changed in the expression of the IEGs were dependent on DA D1 receptor stimulation. The second study examined the effects of binge METH on the expression of ER stress- and mitochondrial dysfunction-responsive genes. Pretreatment with the DA D1 receptor antagonist, SCH23390, caused complete inhibition of METH-induced ER and mitochondrial stresses whereas the DA D2 receptor antagonist, raclopride, provided only partial blockade. SCH23390 also blocked METH-induced hyperthermia whereas raclopride failed to do so. Interestingly, both antagonists attenuated METH-induced dopaminergic and serotonergic deficits in the striatum. Moreover, SCH23390 but not raclopride blocked METH-induced serotonergic deficits in cortical tissues. I also found that METH treatment induced upregulation of activin βA mRNA, increased TGF-β and phosphorylated Smad2 proteins in the rat striatum. SCH23390 pretreatment completely blocked all these effects whereas raclopride did not block METH-induced increases in TGF-β expression.

La méthamphétamine (METH) est une drogue stimulante qui peut causer des déficiences des fonctions cognitives et des dommages irréversibles dans le cerveau des utilisateurs. Il est important de comprendre les mécanismes moléculaires de la toxicité de la drogue pour pouvoir développer des traitements pour contrer les effets toxiques de la METH. Plusieurs études dans notre laboratoire et autres ont montré qu’une seule dose élevée de METH (30-40 mg/kg de poids corporel) suffit à endommager l’arborisation terminale des neurones dopaminergiques dans le striatum et le cortex chez les rongeurs, de même qu’elle peut causer l’activation des signaux apoptotiques produits a partir du réticulum endoplasmique (RE) et de la mitochondrie dans le striatum. De ce fait, le but de cette thèse était d’analyser si la dose toxique de 40 mg/kg de METH injectée par faibles doses répétées (4 fois, avec des intervalles de 2 heures), appelée « binge METH », peut aussi causer des stress cellulaires du RE et de la mitochondrie dans le striatum. Des travaux récents ont suggéré que les récepteurs D1 et D2 de la dopamine pourraient être les intermédiaires de l’apoptose des neurones dans le striatum causée par l’administration d’une unique toxique dose de METH. Nous avons alors émis l’hypothèse que les messages cellulaires diriges par la stimulation des récepteurs D1 et D2 de la dopamine pourraient être à l’ origine des effets toxiques du « binge modele ». Le rôle des récepteurs de la dopamine sur l’activation des signaux de l’apoptose a été examiné en utilisant des antagonistes de ces récepteurs. Dans cette dissertation, je donne la preuve que « binge METH » affecte l’expression des immediate early genes de façon différente. Il semble que ces effets soient dépendants de la stimulation du récepteur D1. Un autre volet de cette dissertation a analysé les effets de « binge METH » sur l’expression de gènes impliqués dans la réponse au stress du RE et à l’altération de la fonction de la mitochondrie. Le prétraitement avec l’antagoniste du récepteur D1 de la dopamine, SCH23390, a complètement bloqué l’apparition de ces stress cellulaires après les injections de METH, alors que l’antagoniste du récepteur D2, raclopride, a eu des effets minimes. SCH23390 a aussi bloqué l’effet de METH à causer l’augmentation de la température corporelle des animaux, mais pas raclopride. Cependant, les deux antagonistes ont protégés contre les pertes dans plusieurs marqueurs des neurones de dopamine et sérotonine dans le striatum. De plus, SCH23390, mais non raclopride, a aussi protégé les neurones de sérotonine dans le cortex. Durant mes travaux, j’ai aussi identifié qu’il y a une augmentation de l’ARN messager de activin βA, la protéine TGF-β et Smad2 phosphorylée après les injections de METH. Ces effets sont réduits suite à un prétraitement par SCH23390 ; cependant, raclopride n’a eu aucun effet sur l’expression de TGF-β.En résumé, ces nouvelles données suggèrent que le récepteur D1 joue un rôle prédominant dans la toxicité de la METH. / Methamphetamine (METH) is a potent psychostimulant known to cause cognitive abnormalities and neurodegenerative changes in the brains of METH abusers. One approach for developing therapies for METH abuse is to understand the molecular mechanisms of toxicity of the drug. Investigations in our laboratory and elsewhere have shown that single intraperitoneal injections of METH (30-40 mg/kg of body weight) can cause damage to striatal and cortical monoaminergic systems and induce neuronal apoptosis in the striatum of rodents via activation of endoplasmic reticulum (ER) and mitochondrial death pathways. Hence, the purpose of this thesis was to investigate if toxic binge METH injections can cause ER- and mitochondria-induced stress in the rat striatum. Recent studies have suggested that dopamine (DA) D1 and D2 receptors might mediate neuronal apoptosis in the striatum after single toxic METH doses. We therefore hypothesized that signaling through these two types of DA receptors might activate toxic effects of the binge METH regimen. The role of DA D1 or D2 receptors in METH-induced cell death pathways was thus examined by using pharmacological inhibitors of these receptors. In this dissertation, I report that binge METH regimen caused differential changes in immediate early genes (IEGs) that are known to influence synaptic changes in the brain. METH-induced changed in the expression of the IEGs were dependent on DA D1 receptor stimulation. The second study examined the effects of binge METH on the expression of ER stress- and mitochondrial dysfunction-responsive genes. Pretreatment with the DA D1 receptor antagonist, SCH23390, caused complete inhibition of METH-induced ER and mitochondrial stresses whereas the DA D2 receptor antagonist, raclopride, provided only partial blockade. SCH23390 also blocked METH-induced hyperthermia whereas raclopride failed to do so. Interestingly, both antagonists attenuated METH-induced dopaminergic and serotonergic deficits in the striatum. Moreover, SCH23390 but not raclopride blocked METH-induced serotonergic deficits in cortical tissues. I also found that METH treatment induced upregulation of activin βA mRNA, increased TGF-β and phosphorylated Smad2 proteins in the rat striatum. SCH23390 pretreatment completely blocked all these effects whereas raclopride did not block METH-induced increases in TGF-β expression.

Abstract Adenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A3 receptor (A3R) is the least explored among the four human adenosine receptor subtype members (A1, A2A, A2B and A3) and it is implicated in both neuroprotective and neurodegenerative effects. During the course of this work, the production of the recombinant human A3R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D2 receptor (D2R) was studied. A3R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A3R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A3R expression: 10µg of the monomeric A3R could be purified from one liter of insect cell culture. For successful crystallization thermostability of the A3R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A3R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D2R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D2R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D2R showed that the protein-protein interactions are calcium dependent. / Tiivistelmä Adenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A3 reseptori (A3R) on adenosiinireseptorien neljästä alatyypistä (A1, A2A, A2B ja A3) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin. Tässä työssä arvioitiin sekä ihmisen rekombinantti-A3R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D2 reseptorin (D2R) heteromerisoitumista. Rekombinantti A3 reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA3R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A3R:n tuottoon: noin 10 µg monomeerista A3R:a voitiin puhdistaa litran hyönteissoluviljelmästä. Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A3R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A3R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A3R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille. Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D2R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D2R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D2R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen.