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Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine- induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA- associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

As complexities of addictive behaviors cannot be fully captured in laboratory studies, scientists use simple addiction- associated phenotypes and measure them inlaboratory animals. Locomotor sensitization, characterized by an increased behavioralresponse to the same dose of the drug, has been extensively used to elucidate thegenetic basis and molecular mechanisms of neuronal plasticity. However, to whatextent it contributes to the development of addiction is not completely clear. Wetested if the development of locomotor sensitization to methamphetamine affectsvoluntary self- administration, and vice versa, in order to investigate how two drug-associated phenotypes influence one another. In our study, we used the geneticallytractable model organism, Drosophila melanogaster, and quantified locomotorsensitization and voluntary self- administration to methamphetamine using behavioraltests that were developed and adapted in our laboratory. We show that flies expressrobust locomotor sensitization to the second dose of volatilized methamphetamine, which significantly lowers preferential self- administration of methamphetamine.Naive flies preferentially self-administer food with methamphetamine over plainfood. Exposing flies to volatilized methamphetamine after voluntary self- administration abolishes locomotor sensitization. We tested period null (per01) mutantflies and showed that they do not develop locomotor sensitization, nor do they showpreferential self-administration of methamphetamine. Our results suggest that theremay be partially overlapping neural circuitry that regulates the expression oflocomotor sensitization and preferential self-administration to methamphetamineand that this circuitry requires a functional per gene.

Drug addiction is a persistent brain disease with severe and sometimes fatal consequences. Addictive drugs induce long-lasting neuroadaptations in the functioning of the nervous system and currently there is no efficient pharmacological treatment that can successfully prevent or reverse these changes. As a consequence, addicted individuals often suffer from recurrent relapses, sometimes triggered by environments, situations or stressors that have previously been associated with drug taking.Behavioral neuroscience uses animal models to understand the neurobiological mechanisms which cause and correlate with the development of addiction, and recently the emphasis is on animals that are genetically tractable, such as mice (Mus musculus) and fruit flies, (Drosophila melanogaster). In spite of many obvious differences between humans and Drosophila, similarities at the genetic level and in the basic neuronal physiology, make Drosophila an excellent model organism for the study of many complex human behaviors, including addiction. Discovery that circadian genes influence the development of behavioral sensitization to cocaine in Drosophila led to numerous studies about the role of circadian genes in the regulation of drug-induced behaviors in laboratory mammals and humans. Results show that circadian genes are involved in regulating the behavioral and molecular response to different classes of drugs of abuse. Furthermore, studies in humans show the interconnectivity between the regulation of circadian behavior, mental diseases and addiction, and suggest that behavioral interventions aimed at improving the quality of the circadian behavior will be important in prevention and treatment of addictive behaviors., Ovisnost je o drogama kronična bolest s teškim, nerijetko fatalnim posljedicama. Opojne droge izazivaju dugotrajne neuroadaptivne promjene u funkcioniranju živčanog sustava, a ne postoji učinkovit lijek koji bi ih suzbio ili ispravio. Ovisnici stoga pate od opetovanih recidiva, ponekad izazvanih okolinom, situacijom ili stresom koji je i bio povezan s uzimanjem droge.Bihevioralna neuroznanost koristi modelne organizme za razumijevanje neurobioloških mehanizama koji uvjetuju ili koreliraju s razvojem ovisnosti, a odnedavno naglasak je na životinjama koje su genetski pogodne, kao miš (Mus musculus) ili vinska mušica (Drosophila melanogaster). Unatoč mnogim očitim razlikama između ljudi i Drosophile sličnosti na genetskoj razini i u području bazične neuronske fiziologije čine Drosophilu izvrsnim modelnim organizmom za izučavanje mnogih složenih ljudskih ponašanja, uključujući ovisnost. Otkriće da cirkadijalni geni kontroliraju razvoj bihevioralne senzitizacije na kokain kod Drosophile potaklo je niz istraživanja o ulozi koju cirkadijalni geni imaju u ponašanjima induciranim kokainom kod laboratorijskih životinja i ljudi. Rezultati pokazuju da cirkadijalni geni reguliraju ponašanja i molekularne odgovore na razne vrste opojnih droga. Štoviše, istraživanja na ljudima upućuju na međupovezanost između cirkadijalnog ponašanja, mentalnih oboljenja i ovisnosti, te sugeriraju da bihevioralni zahvati usmjereni na poboljšanje cirkadijalnog ponašanja mogu biti važni u prevenciji i liječenju ovisničkog ponašanja., La drogadicción es una de las enfermedades crónicas con consecuencias graves, a veces fatales. Las drogas adictivas causan cambios neuroadaptivos a largo plazo en el funcionamiento del sistema nervioso y de momento no existe un medicamento eficiente que lo reprimiría o corregiría. Por eso los drogadictos sufren repetidas recaídas, a veces causadas por el entorno, situación o estrés previamente relacionado con el consumo de drogas.Neurociencia conductual usa organismos modelos para comprender mecanismos neurobiológicos que condicionan o se correlacionan con el desarrollo de la adicción, y recientemente el enfoque está en los animales genéticamente convenientes, como ratón (Musmusculus) o mosca del vinagre (Drosophila melanogaster). A pesar de las numerosas diferencias obvias entre los humanos y las moscas del vinagre, la semejanza al nivel genético y aquel de fisiología neuronal básica convierten dicha mosca del vinagre en un excelente organismo modelo para estudiar muchas conductas humanas complejas, incluida la adicción. Al descubrir que los genes circadianos controlan el desarrollo de la sensibilización de comportamiento de la mosca del vinagre en cuanto a la cocaína, se provocó toda una serie de investigaciones sobre el papel que tienen los genes circadianos para los comportamientos inducidos por la cocaína tanto en los animales de laboratorio, como en los humanos. Los resultados indican que los genes circadianos regulan comportamientos y respuestas moleculares a diferentes tipos de drogas adictivas. Es más, las investigaciones en los humanos muestran la interconexión entre el comportamiento circadiano, enfermedades mentales y adicción, y proponen que las intervenciones en el comportamiento enfocadas en la mejora del comportamiento circadiano pueden ser importantes en la prevención y tratamiento de conductas adictivas.

Addiction to psychostimulants (PS), cocaine (COC) and methamphetamine (METH), can be studied in model organisms by analyzing endophenotypes relevant for addiction, such as behavioral sensitization (BS). A group of circadian genes were previously identified as required for development of BS, but their mechanism of action on PS-induced neuronal plasticity is not known. We hypothesize that a potential mechanism involves interaction between PAS domain contained in circadian proteins and elevated levels of reactive oxygen species (ROS). PS increase ROS through oxidation of PS-induced monoamine relase while PAS domain is sensitive to redox potential. Here we present evidence for circadian and redox modulation of BS in Drosophila. We are using BS phenotype to investigate interaction between drug-induced ROS levels and modulation by circadian genes. BS is measured using new high throughput test that allows administration of volatilized PS to a population of individually housed Drosophila and quantification of locomotor activity before and after drug exposure. Repeated administration of the same dose of PS leads to increased locomotor response (BS), but with different time interval between doses for COC or METH. We have confirmed the importance of circadian genes period, Clock and cycle, and we show the requirement for functional dopamine transporter and D1-type dopamine receptor in BS to COC and METH. Size of senzitized response depends on time of the day, indicating circadian modulation of the drug induced response. Single administration of COC or METH increase catalase (CAT), while decrease superoxide-dismutase (SOD), two enzymes responsible for regulating redox state of the cell. Exogenous suppression or induction of oxidative stress abolished BS to COC and METH, indicating that redox status interferes with neuronal plasticity. Our data shows that Drosophila can develop BS to either COC or METH, and that BS is controlled and modulated by functional circadian genes, involves dopaminergic system and is influenced by the redox status. By combining different manipulations in the same animal, such as PS and exogenous pro- and anti- oxidant administration in transgenic animals that allows for spatial and temporal control of relevant genes, we plan to define the interaction between circadian genes and redox state on neuronal plasticity.

Addictive drugs engage mechanisms of neural plasticity to change the function and structure of the brain resulting in addiction. Molecular mechanisms of drug-induced neuroplasticity are only partially understood, and recent evidence suggests involvement of redox regulation. Our hypothesis is that if changes in redox status modulate neuronal plasticity, then this will be evident as a change in a form of neuroplastic change: locomotor sensitization to psychostimulants. To study the mechanisms of neuroplasticity in the lab, we use a model organism, Drosophila melanogaster, and we focus on a simple endophenotype: locomotor sensitization (LS) to cocaine (COC) and methamphetamine (METH) using our newly develop high throughput system, FlyBong. We induce LS using two administrations of the same dose of volatilized drug. To change redox status we feed flies for 16 hrs before drug exposure to one of the following substances: prooxidants hydrogen peroxide or paraquat, and antioxidants quercetin or tempol. To measure effects of treatment on redox status we measure activity of catalase and superoxide dismutase in the whole fly homogenates. We find that exposure to COC and METH alone increases activity of the antioxidant enzyme catalase and decreases activity of superoxide dismutase, while treatment with quercetine does the opposite. Interestingly, when COC or METH are combined with either pro- or antioxidant substances LS is abolished. However, LS is restored when pro- and antioxidants are given together, indicating that a proper balance of redox state is required for LS to develop. Our results indicate that redox state interacts with mechanisms of neural plasticity and that when balance is disturbed, LS does not develop. We are interested in defining those interactions, and defining consequences that redox-induced neuroplastic change have on rewarding aspects of psychostimulants that can be measured in self-administration paradigm.

Addictive drugs engage mechanisms of neural plasticity by changing the function and structure of the brain resulting in addiction. In both vertebrates and invertebrates subset of circadian genes regulates different forms of drug-induced neuronal plasticity. However, description of genetic mechanisms through which circadian genes modulate different drug-induced behaviors is limited, in part due to inadequate behavioral test that can be used in genetic screens in genetically tractable model organisms. To address these issues, we developed new behavioral assays for measuring motor activating and rewarding effects of psychostimulants cocaine (COC) and methamphetamine (METH) in Drosophila melanogaster. Our new assay for measuring locomotor activity after volatilized exposure to psychostimulants (FlyBong) is: high throughput, objective, reproducible and allows analyzing data either on population or individual fly level. FlyBong was validated showing different locomotor sensitization to volatilized COC in circadian mutants per01, ClkJrk and cyc01 ; ry compared to , tim01 or wild type. We show that flies can develop locomotor sensitization to volatilized METH, and response of circadian mutants to METH is distinct from those to COC. We modified two- choice Capillary Feeder (CAFE) assay to test rewarding effect of COC and METH. In assay flies can self-administer food from capillaries that contain sugar solution with or without drug (COC or METH). We show that flies self- administer COC and METH in spite of inherently aversive bitter taste. Preference for COC is higher and longer-lasting compared to METH, which is mostly aversive. COC self- administration is under modulation of gene cyc, while self-administration of METH in addition is modulated by Clk and per. Our results suggest that different circadian genes modulate motor-inducing and rewarding aspects of COC and METH. Different behavioral phenotypes in different circadian mutants to COC and METH can be used for investigation of neural mechanisms and genetic contribution to endophenotypes related to addiction in humans.

Addictive drug self-administration in used on laboratory animals because it models some aspects of addiction and drug consumption can be easily quantified. Drosophila preferentially consumes ethanol–containing liquid, but it could be the consequence of the evolution of Drosophila behavior that was shaped by fermenting food that contains ethanol. It is not known if psychostimulants, such as cocaine and methamphetamine, bitter compounds with no nutritional relevance for Drosophila, can induce preferential consumption. To test if flies will voluntarily self- administer psychostimulants we used the two- choice Capillary Feeder (CAFE) assay, where flies can choose between capillaries containing sweet liquid with or without addition of a psychostimulant. Preference Index (PI) is calculated based on the amount of food consumed each day for several days. In spite of the inherently aversive taste of bitter compounds, PI for both psychostimulants is positive from the first day of exposure, and on average flies show significantly higher PI for methamphetamine. Over the course of 4 days PI for cocaine increases and stays positive, while PI for methamphetamine decreases and becomes negative (aversive). Depending on the concentration, flies consume cocaine-containing food until the lethal outcome, suggesting the loss of behavioral flexibility. This is further supported by the cue-dependent consumption, because changing the location of drug containing capillaries every day or limiting cues leads to smaller PI. In order to obtain cocaine flies overcome addition of further bitter taste, quinine, indicating high motivation for consumption. To further validate our method we are characterizing responses of flies mutant for genes involved in preferential drug consumption in mammals. Above data suggests that Drosophila can develop preferential consumption for nutritionally irrelevant substance. Consumption of cocaine leads to behavioral changes that are commonly observed in mammals, such as loss of behavioral flexibility. Thus, Drosophila can be used as a tool in dissecting neural mechanisms underlying drug-seeking behavior and identification of new genes involved in development of addiction.

Drug addiction is a complex behavior and in the lab it can be studied on model animals as a simple endophenotype, one of which is self- administration. Several addiction-related endophenotypes have been shown in Drosophila melanogaster indicating that flies are good model for studying neuronal mechanisms of addiction. Flies will self-administer ethanol– containing food suggesting that ethanol has a rewarding effect on behavior. Psychostimulants cocaine (COC) and methamphetamine (METH) induce mechanisms of neuronal plasticity evident as locomotor sensitization, however it is not known if flies will voluntary administer COC or METH. Our hypothesis is that if psychostimulants have a rewarding effect on flies, then they will self-administer these drugs. To measure voluntary administration of psychostimulants in Drosophila we use the two- choice Capillary Feeder (CAFE) assay, where flies can choose between capillaries with drug- food or a non-drug food. Preference Index is calculated based on the amount of food consumed each day for several days. Since self- administration is type of operant conditioning, we pare a drug with a cue to measure the influence on the PI value. To study the effects of relapse we measure self-administration after a period of deprivation and we study the motivation for self-administration by combining the drug with an aversive, bitter tasting substance, quinine. We show that PI for COC is positive and increases over consecutive days, while PI for METH is initially significantly higher than for COC, but then decreased over consecutive days. After period of abstinence from COC-containing food flies rapidly return to the same or greater PI, suggesting relapse-like effect. Psychostimulants affect motivation for drug consumption as flies overcame bitter tasting quinine to obtain COC. These results suggest that in Drosophila, as in vertebrates, COC and METH act on neuronal mechanisms regulating rewarding behavior and motivation for drug taking. Further research will be focused on screening for effects of genetic mutations in likely candidate genes, as well as yet unknown functional genes, which regulate rewarding behavior and psychostimulants-induced neuronal plasticity.

Chronic mental illness are some of the leading cause of disability worldwide. They include major depressive disorder, bipolar disorder and schizophrenia. Schizophrenia has the most severe symptoms, with an estimated prevalence of 24 million people in the general population. Chronic mental illness are characterized by both genetic and non-genetic risk factors, however, their underlying biological cause remains relatively unknown. Several candidates have been implicated in protein aggregation pathology of chronic mental illness, including TRIOBP-1, DISC1, CRMP1 and NPAS3. In this thesis, we investigate the effect of environmental stress factors on aggregation of these proteins, when into cultured cells. Initially we were focusing on treatment protocol optimization and settled on stress treatment for 3 hours. The results suggest that TRIOBP-1, DISC1 and NPAS3 show signs of aggregation when following application of stress. However, NPAS3 showed the most interesting results, where it clearly mislocalized from nuclei to the cytoplasm forming insoluble aggregates. The tests were repeated, showing aggregation of NPAS3 after it was treated with sodium arsenide, iron (II) chloride or zinc acetate, as well as potential aggregation when treated with calcium chloride and an MG132 proteasome inhibitor. Environmental stress factors potentially do affect protein aggregation, which can be used as a new approach to investigate the effect on chronic mental illness. Further cell culture research could provide us with initial results and could be used as a model for observing stress in chronic mental illness based on these findings. Further research should be focused on stress treatment protocol modification, as well as the implication of other stress factors, such as heat shock, as the high temperature resulted in a quantitative test failure, where most of the transfected cells showed signs of NPAS3 aggregation., Kronične mentalne bolesti jedan su od vodećih uzroka smetnji širom svijeta. One uključuju depresivni poremećaj, bipolarni poremećaj i shizofreniju. Shizofreniju karakteriziraju nateži simptomi, te je procijenjena prevalencija kod 24 miljuna ljudi u populaciji. Kronične mentalne bolesti karakteriziraju genski faktori i faktori koji nemaju genski uzrok, međutim, njihov temeljni biološki uzrok ostaje relativno nepoznat. Nekoliko kandidata povezanih s patologijom agregacije proteina kod mentalnih bolesti uključuju TRIOBP-1, DISC1, CRMP1 i NPAS3. U ovom diplomskom radu istražujemo učinak stresnih faktora okoliša na agregaciju ovih proteina u staničnim kulturama. U početku smo se usredotočili na optimizaciju protokola za tretiranje stanica stres faktorima i odlučili smo se na tretiranje u trajanju od 3 sata. Rezultati ukazuju da TRIOBP-1, DISC1 i NPAS3 agregiraju nakon tretiranja stres faktorima. Međutim, NPAS3 je pokazao najzanimljivije rezultate, gdje se jasno vidi premještaj iz jezgre stanice u citoplazmu, stvarajući netopljive agregate. Ponovljena testiranja potvrđuju NPAS3 aggregaciju nakon tretiranja s natrijevim arsenidom, željezovim (II) kloridom i cink acetatom, kao i potencijalnu agregaciju nakon tretiranja kalcijevim kloridom i inhibitorom proteasoma MG132. Okolišni stres faktori potencijalno utječu na agregaciju proteina, te se mogu koristiti kao novi pristup istraživanja učinka stres faktora na kronične mentalne bolesti. Daljnja istraživanja na staničnim kulturama mogla bi nam dati početne rezultate i koristiti se kao model za promatranje učinka stresa kod kroničnih mentalnih bolesti na temelju ovih otkrića. Daljenja istraživanja bi trebala biti usmjerena na modifikaciju protokola za tretiranje stanica, kao i korištenje drugih faktora stresa, primjerice toplinski šok, obzirom da je visoka temperatura rezultirala neuspješnim kvantitativnim eksperimentom, gdje je većina transficiranih stanica pokazivala znakove NPAS3 agregacije.

Many molecular mechanisms are involved in the reward system of the brain. Natural rewards include food, mating and socialization. Artificial rewards include psychostimulative substances, such as methamphetamine (METH). Dopaminergic reward system of fruit fly in Drosophila melanogaster can be stimulated by both natural and artificial rewards. Reward system in D. melanogaster is mainly regulated by signal molecules dopamine (DA) and neuropeptide F (NPF) signalling. Positive social experiences are rewarding and increase the levels of DA and NPF in D. melanogaster. Sexual deprivation in D. melanogaster has led to an increased preference for ethanol and is explained through the mechanism of NPF signalling. Negative social experiences lead to increased aggression, lack of sleep, disturbed food intake and mating. It causes many epigenetic changes with the emphasis on DNA methylation. Social isolation increases the preference for psychostimulative substances in many species and in flies an increase in preference to ethanol post social isolation was discovered so far. The aim of this thesis is to analyse the effect of different social experiences on the preferential consumption of METH in D. melanogaster. I analysed the effect of isolation/grouping and its duration, as well as, sexual deprivation, which has not yet been done in D. melanogaster. I included four different groups of flies – isolated mated and virgin and grouped mated and virgin. The FlyCafe experiment allowed the self – administration of METH over the three days of the experiment. Isolated flies show higher METH preference and the preference decreases as the duration of isolation increases. Group housed flies show aversion to METH and this aversion increases with the duration of grouping. Social isolation shows the dominant effect on preference compared to sexual deprivation. The highest change in preference, with regards to duration of isolation/grouping, is present in group housed and mated flies. It indicates the importance of the mating duration in regards to the peak mating capacity in flies. Future experiments should analyse the long-lasting effects of social isolation and sexual deprivation on preferential consumption of METH, to more precisely define environmental effect on the motivation for METH consumption., Mnogi molekularni mehanizmi su uključeni u nagrađujući sustav u mozgu. Prirodne nagrade uključuju hranu, parenje i socijalizaciju. Umjetne nagrade uključuju psihostimulativna sredstva, poput metamfetamina (METH). Dopaminergični nagrađujući sustav vinske mušice u Drosophila melanogaster može se poticati prirodnim i umjetnim nagradama. Nagrađujući sustav u D. melanogaster većinski je reguliran signalizirajućim molekulama dopamina (DA) i neuropeptid F (NPF) signalizacijom. Pozitivna socijalna iskustva su nagrađujuća te povećavaju razinu DA i NPF u mušica. Deprivacija parenja je u D. melanogaster dovela do povećane preference za etanol te je objašnjena mehanizmom NPF signalizacije. Negativna socijalna iskustva dovode do povećane agresije, manjka sna, poremećaja hranjenja te parenja. Dolazi do mnoštva epigenetičkih promjena s naglaskom na promjene u metilaciji DNA. Socijalna izolacija povećava preferencu ka psihostimulativnim sredstivma u raznih vrsta, a u mušica je do sada otkriveno povećanje u preferenci ka etanolu kao posljedica socijalne izolacije. Cilj ovog rada je ispitati utjecaj različitih socijalnih iskustava na preferencijalnu konzumaciju METH-a u D. melanogaster. Ispitan je efekt trajanja izolacije/grupiranja, kao i utjecaj parenja, što do sada nije bilo ispitano u D. melanogaster. U eksperimenut su uključene četiri skupine D. melanogaster – izolirane parene i neparene te grupirane parene i neparene. FlyCafe eksperimentom omogućena im je samoadministracija METH-a kroz tri dana eksperimenta. Izolirane mušice pokazuju veću preferencu ka METH-u te je preferenca niža sa dužim periodom izolacije. Grupirane mušice pokazuju averziju ka METH-u te se averzija povećava s duljinom grupiranja. Socijalna izolacija pokazuje dominantni utjecaj na preferencu u usporedbi s deprivacijom parenja. Najveća promjena u preferenci s obzirom na duljinu izolacije/grupiranja je prisutna kod grupiranih i parenih mušica. To ukazuje na značaj duljine parenja s obzirom na vrhunac sposobnosti parenja u mušica. Budući eksperimenti trebali bi ispitati dugotrajniji utjecaj socijalne izolacije i deprivacije parenja na preferencijalnu konzumaciju METH-a, kako bi se preciznije definirao okolinski utjecaj na motivaciju za konzumiranjem METH-a.

Metamfetanim (METH) je psihostimulans koji dovodi do razvoja ovisnosti. Naši preliminarni rezultati su pokazali da METH dovodi do promjena u broju potomaka mužjaka vinske mušice. Razvojni ciklus je povezan s metabolizmom, a time i sa razvojem reaktivnih oksidacijskih vrsta. METH povećava stvaranje reaktivnih kisikovih vrsta (ROS) i ometa njihovo uklanjanje, uzrokujući oksidativni stres. Kvercetin (QUE) je molekula s poznatim antioksidativnim svojstvima koja bi potencijalno mogla djelovati na promjene u razvojnom ciklusu uzrokovane METH-om. Cilj eksperimentalnog rada je bio ispitati kako promjene u oksidacijskom statusu (QUE) utječu na broj potomaka mužjaka izloženih vMETH-a. METH je administriran grupi mužjaka pomoću FlyBong metode, nakon čega je izdvojeno 20% mužjaka s najvećim lokomotornim odgovorom na METH. Mužjaci su pareni sa netretiranim ženkama na hrani s različitim koncentracijama QUE (0.6 mM i 3.2 mM). Praćen je utjecaj QUE na broj potomaka tijekom tri generacije. Rezultati su pokazali statistički značajno povećanje broja embrija prve generacije u skupini koja je bila izložena METH-u kao i trend povećanog broja čahura i mušica. Utjecaji METH-a se nije prenio na naredne generacije, dok je utjecaj QUE-a bio vidljiv u drugoj, ali ne i trećoj generaciji. Rezultati upućuju na potencijalan transgeneracijski utjecaj QUE na redoks ravnotežu. Kako bi se ovi rezultati statstički potvrdili, eksperiment je potrebno ponoviti s povećanim brojem replika i poboljšanom formulacijom hrane., Methamphetamine (METH) is a psychostimulant that leads to the development of addiction. Our preliminary results showed that METH leads to changes in the number of male fruit fly offspring. The development cycle is connected with metabolism, and thus with the development of reactive oxidation species. METH increases the generation of reactive oxygen species (ROS) and interferes with their removal, causing oxidative stress. Quercetin (QUE) is a molecule with known antioxidant properties that could potentially act on METH-induced changes in the developmental cycle. The aim of the experimental work was to examine how changes in oxidation status (QUE) affect the number of offspring of males exposed to vMETH. METH was administered to a group of males using the FlyBong method, after which 20% of males with the highest locomotor response to METH were selected. Males were mated with untreated females on food with different concentrations of QUE (0.6 mM and 3.2 mM). The influence of QUE on the number of offspring was monitored over three generations. The results showed a statistically significant increase in the number of embryos of the first generation in the group that was exposed to METH, as well as a trend of an increased number of cocoons and flies. The effects of METH were not transmitted to subsequent generations, while the influence of QUE was visible in the second but not the third generation. The results suggest a potential transgenerational influence of QUE on redox balance. In order to statistically confirm these results, the experiment needs to be repeated with an increased number of replicates and an improved food formulation.

Seasonal affective disorder (SAD) is a subtype of major depressive disorder. It is a multifactorial disorder characterised by the seasonal appearance of its symptoms such as depressed mood, hypersomnia or insomnia, increased or reduced appetite... There are two types of seasonal affective disorder, based on the season when the symptoms appear – summer and winter type. The cause of the disorder has not yet been found, but there are a couple of hypotheses that suggest the possible origin of the disorder. They are called the photoperiodic and phase shift hypotheses, also known as the chronobiological hypotheses. Aside from them, the serotonin and melatonin disbalance may also contribute to the disorder. The goal of this thesis was to discover various treatment options for seasonal affective disorder. Well-known and used treatments are light therapy, cognitive behavioural therapy and pharmacotherapy. No treatment is significantly superior to the others, at least not in all aspects. Light therapy gives the fastest results, but cognitive behavioural therapy shows greater potential to lower the chances of relapse in the next season. Pharmacotherapy is mostly used as a combination treatment with either of the other two. Right now only two drugs have been found to work almost as effectively as light therapy – fluoxetine and bupropion, but they also have side effects. Two other treatments are dawn simulation and negative air ion therapy. Their effect is a little weaker but are still used in patients with milder symptoms. Because all mental illnesses are very hard to treat universally, medical doctors prescribe a combinatorial treatment consisting of more therapies to treat the symptoms in the best possible way for each patient individually., Sezonska depresija je podvrsta kliničke depresije. To je multifaktorijalni poremećaj kojeg karakterizira sezonska pojava simptoma kao što su depresivno raspoloženje, hipersomnija ili insomnija, povećan ili smanjen apetit... S obzirom na godišnje doba u kojem se simptomi pojavljuju, razlikujemo zimski i ljetni tip sezonske depresije. Iako je uzrok poremećaja i dalje nepoznat, postoji nekoliko teorija koje predlažu mogući uzrok. Radi se o teorijama fotoperioda i faznih pomaka, zajedno nazvanim i kronobiološke teorije. Neuravnoteženost serotonina i melatonina bi također bi također mogle pridonijeti poremećaju. Cilj ovog rada je proučiti različite metode liječenja ovog poremećaja. Dobro poznati i najčešće korištene metode su terapija svijetlom, kognitivno-bihevioralna terapija i farmakoterapija. Ni jedna metoda se ne smatra najboljom, barem ne u svim aspektima. Terapija svijetlom pokazuje najbrže rezultate, ali kognitivno-bihevioralna terapija ipak pokazuje najveći potencijal u sprečavanju ponovne pojave simptoma u sljedećoj sezoni. Farmakoterapija se uglavnom koristi kao kombinacija sa jednom od dvije prethodno navedene metode. Trenutno su samo dva lijeka uspješna kao i terapija svijetlom, fluoxetin i bupropion, ali oni imaju i svoje nuspojave. Druge dvije metode koje se malo rijeđe koriste za liječenje su simulacija zore i terapija negativnim ionima iz zraka. One imaju malo blaži efekt, ali se zato koriste kod pacijenata sa blažim simptomima. Sve mentalne poremećaje je teško liječiti na jedan univerzalan način, tako i za sezonsku depresiju liječnici pripisuju kombinaciju dostupnih metoda kako bi najbolje tretirali svakog pojedinog pacijenta.

Konzumacija opojnih droga utječe na dopaminsku i serotoninsku neurotransmisiju te je značajan faktor razvoja ovisnosti. Psihostimulansi poput metamfetamina (METH) djeluju na centre u mozgu zadužene za regulaciju procesuiranja nagrade i ugode. Nagrađujući utjecaj droga dovodi do opetovane konzumacije i u konačnici do ovisničkog ponašanja koje se ispituje na laboratorijskim životinjama kao preferencijalna samoadministracija. Drosophila melanogaster poznati je modelni organizam za izučavanje ovisničkog ponašanja. U provedenom istraživanju koristili smo fumin (fmn) mutante Drosopile melanogaster kao modelni organizam za ispitivanje preferencijalne konzumacije METH-a. Zanimalo nas je kakva će biti preferencijalna administracija u tih mušica koje zbog mutacije u genima za dopaminski transporter (DAT) imaju narušenu homeostazu u dopaminskom sustavu. Metoda pomoću koje se ispitivala preferencijalna konzumacija je Fly CAFE (eng. Capillary Feeder) esej u kojemu su mušice imale izbor između šećerne otopine i šećerne otopine s dodatkom METH-a. Dobiveni rezultati upućuju da dopamin (DA) ima ključnu ulogu u preferencijalnoj konzumaciji METH-a. fmn mušice razvijaju odbojan do neutralan odgovor prema šećernoj otopini koja sadrži METH. Također prikazana je značajna statistička razlika u preferenci za METH-om između divljeg tipa (wt) mušica s normalnom funkcijom dopaminskog sustava i fmn s neaktivnim DAT-om. Potrebna su daljnja istraživanja i razumijevanje samoadministracije opojnih droga i njihovog nagrađujućeg djelovanja kako bi se dizajnirali prikladni farmakološki tretmani u liječenju i sprječavanju ovisnosti., The consumption of narcotic drugs affects dopamine and serotonin neurotransmission and is a significant factor in the development of addiction. Psychostimulants such as methamphetamine (METH) act on centers in the brain responsible for regulating reward and pleasure processing. The rewarding influence of drugs leads to repeated consumption and ultimately to addictive behavior that is tested on laboratory animals as preferential self-administration. Drosophila melanogaster is a well-known model organism for studying addictive behavior. In the conducted experiment we used fumin (fmn) mutants of Drosopila melanogaster as a model organism for testing the preferential consumption of METH. We were interested to test the preferential administration in flies that, due to a mutation in the genes for the dopamine transporter (DAT), have disturbed homeostasis in the dopamine system. The method used to test preferential consumption is the Fly CAFE (Capillary Feeder) assay in which the flies had a choice between a sugar solution and a sugar solution with the addition of METH. We found that dopamine plays a key role in the preferential consumption of METH. fmn flies develop an aversive to neutral response to a sugar solution containing METH. A significant statistical difference in preference for METH was also shown between wild type flies (wt) with normal dopamine system function and fmn with with an inactive DAT. Further research and understanding of self-administration of narcotic drugs and their rewarding effects will be useful in design of appropriate pharmacological treatments for the treatment and prevention of addiction.

Zloćudni tumori nastaju zbog poremećaja regulacijskih mehanizama, neophodnih za rast i dijeljenje stanice. Nerijetko su im u podlozi nastanka mutacije u različitim genima, najčešće protoonkogenima i tumorsupresorskim genima. Početak zloćudne preobrazbe povezan je s nastankom klona brzoproliferirajućih stanica, na koji se nastavljaju evolucija i selekcija klonova s najsnažnijim replikacijskim potencijalom. Zloćudni tumori posjeduju deset temeljnih obilježja, među kojima je i reprogramirani metabolizam. Metabolička aktivnost stanica zloćudnog tumora promijenjena je u odnosu na zdrave stanice od kojih je tumor nastao, a naročito se intenzivno istražuje u odnosu na metabolizam glukoze i glutamina. Za razliku od zdravih stanica koje svoje metaboličke puteve preusmjeravaju u glikolizu u anaerobnim uvjetima, stanice zloćudnih tumora sklone su metaboliziranju velikih količina glukoze glikolizom u uvjetima u kojima je opskrba kisikom optimalna. Ovaj se fenomen naziva aerobna glikoliza ili Warburgov učinak. Osim glukoze, stanice zloćudnih tumora imaju i veliku potrebu za glutaminom, koji služi kao izvor dušika u biosintetskim reakcijama. Biosinteza i iskorištavanje serina u metabolički reprogramiranim stanicama smatra se iznimno važnim za njihovu proliferaciju. Prvi enzim biosinteze serina je fosfoglicerat dehidrogenaza (PHGDH), čija je povećana ispoljenost, kao posljedica amplifikacije dijela kromosoma, dokazana u nekim vrstama zloćudnih tumora. Cilj ovog istraživanja bio je provjeriti postojanje stanično specifične ispoljenosti PHGDH na razini transkripta (RT-qPCR), proteina i u odnosu na unutarstanični smještaj proteina (Western blot), u odgovoru na dvije vrste gladovanja, u tri linije stanica podrijetlom od tumora glave i vrata, te debelog crijeva. Rezultati pokazuju da se ispoljavanje ciljnog proteina u sve tri linije stanica pojačava u gladovanju. Ova promjena može (FaDu), i ne mora (Detrot562, HT29) biti praćena povećanjem transkripcijske aktivnost gena PHGDH. Neočekivano je otkriveno da u linijama stanica podrijetlom od tumora glave i vrata (FaDu, Detroit562), PHGDH u gladovanju ulazi u jezgru, dok ovaj fenomen izostaje u stanicama podrijetlom od karcinoma debelog crijeva (HT29)., The occurrence of malignant tumors is consequential to disturbed regulatory mechanisms that are needed for cellular growth and division. Mutations of protooncogenes and tumor suppressor genes are frequently associated with their genetic background. Initiation of malignant transformation relates to the onset of a highly proliferative clone, while its evolution depends on the selection of clones with the most prominent replicative potential. Reprogrammed metabolism is one of ten basic cancer hallmarks. Metabolic activity of cancer cells is changed with respect to the cells from which specific cancer originates. Currently, the most intense research efforts are focused on the metabolism of glucose and glutamine. Contrary to untransformed cells which utilize glycolytic pathways when deprived of oxygen, malignant cells utilize a high amount of glucose through the same pathway, even when sufficient oxygen is available. This phenomenon is known as aerobic glycolyisis or the Warburg effect. In addition to glucose, malignant cells need glutamine, which they use as a source of nitrogen in biosynthetic reactions. Serine metabolism is considered to be of utmost importance for the proliferation of malignant cells. The first enzyme of serine biosynthesis is phosphoglycerate dehydrogenase (PHGDH). This enzyme is highly expressed in some malignant tumors, as a consequence of chromosome amplification. The objective of this research was to explore the cellular specific expression of PHGDH at the level of the gene transcriptional activity (RT-qPCR), and the level and localization of the protein product, respectively, in three cell lines originating from head and neck malignant tumors and colon carcinoma, exposed to two types of starvation. In all three cell lines, exposure to starvation is associated with an increased level of the PHGDH protein. This change may (FaDu) and may not (Detroit562, HT29) be related to transcriptional activity of the PHGDH gene. During starvation, PHGDH unexpectedly translocates into the nucleus of the head and neck originating cell lines (FaDu, Detroit562), while the effect is absent in colon cancer originating HT29 cells.

Methamphetamine (METH) is a potent psychostimulant that causes relapsing substance addiction. Negative consequences of substance abuse are numerous and challenging to study in humans. Fortunately, effects of addictive drugs can be studied in model organisms, such as Drosophila melanogaster or fruit fly. Fruit flies are ideal for this research because their genome is sequenced and extensively studied. Methamphetamine causes elevated reactive oxygen species (ROS) production and disrupts their elimination, leading to oxidative stress. Circadian genes are partially responsible for ROS cycling and in Drosophila four core circadian genes are: period (per), timeless (tim), cycle (cyc) and clock (Clk). In this paper hydrogen peroxide (H2O2) concentration is followed after two exposures to volatilized METH in heads of five fly genotypes: wild type Canton S., per01, tim01, ClkJrk and cyc01. Hydrogen peroxide concentration was measured with dihydroethidium (DHE) stain. Concentration of H2O2 is expected to increase with higher METH exposure, and differences in H2O2 amount should also be noticed between the genotypes. Results show different H2O2 levels among genotypes and this provides insight into the role of circadian genes in the regulation of the oxidative status of Drosophila. The obtained data shows that wild type, per01 and tim01 flies show decrease with first vMETH dose, followed by increase in H2O2 concentration after the second dose. ClkJrk and cyc01 mutants show steady increase in H2O2 concentration with each dose. This shows that irrespective of the mutation METH leads to the increase in the oxidative species and poses questions about the molecular mechanism involved in the regulation of redox state after psychostimulant exposure., Metamfetamin (METH) je snažan psihostimulans koji uzrokuje relapsirajuću ovisnost. Negativne posljedice ovisnosti o drogama brojne su i izazovno ih je izučavati na ljudima. No, učinci droga mogu se vrlo jednostavno inducirati u modelnim organizmima kao što je Drosophila melanogaster ili vinska mušica. Vinske mušice pogodni su organizam za izučavanje u ovakvim istraživanjima jer su njihovi geni sekvencirani i dobro proučeni. Metamfetamin uzrokuje povišenu proizvodnju reaktivnih kisikovih vrsta (ROS) i remeti njihovo uklanjanje, što dovodi do oksidativnog stresa. Cirkadijalni geni su djelomično odgovorni za kruženje ROS-a, a četiri ključna cirkadijalna gena u Drosophile su: period (per), timeless (tim), cycle (cyc) i clock (Clk). Ovaj rad prati koncentraciju vodikovog peroksida (H2O2) u glavama mušica nakon drugog izlaganja volatiliziranom METH-u. Oscilaciju koncentracija pratilo se u pet genotipa mušica (wild type Canton S., per01, tim01, ClkJrk i cyc01) pomoću dihidroetidija (DHE). U rezultatima se očekuju razlike u koncentraciji među genotipovima i porast koncentracije vodikova peroksida sa porastom broja izlaganja METH-u. Rezultati ukazuju na različite razine vodikova peroksida u različitim genotipovima i to pruža uvid u ulogu cirkadijalnih gena u oksidativnom statusu Drosophile. Nadalje, rezultati prikazuju da u wild type, per01 i tim01 mušica, koncentracija H2O2 pada nakon prve, a raste nakon druge vMETH doze, dok u ClkJrk and cyc01 mutanata koncentracija H2O2 raste nakon svake doze vMETH-a. To ukazuje da bez obzira na mutaciju, METH uzrokuje porast oksidativnih vrsta i postavlja pitanja o molekularnim mehanizmima uključenim u regulaciju redoks stanja nakon izlaganja psihostimulansima.

Circadian rhythm is present in almost all living organisms due to the rotation of the Earth and regular light and dark changes. Its role is evident in 24 hour modulation of biological processes such as the sleep/wake cycle. Disrupted circadian rhythm correlates with oxidative stress, which is caused by increased production of Reactive Oxygen Species (ROS). In this thesis, we explored the connection between the circadian rhythm and oxidative state in D. melanogaster. Based on similarity in the responsiveness to volatilized cocaine between flies with mutation in timeless gene and wild type flies, we were interested to see whether these similarities will be paralleled in their redox regulation. An indicator of redox state was the concentration of the oxidative metabolism byproduct H2O2 in head homogenates. For detection of H2O2 we used the dihydroethidium (DHE) staining. Furthermore, we wanted to determine if H2O2 production is regulated by circadian rhythm. Measurements were conducted in flies raised in conditions of 12 hours of light-12 hours of dark (LD) and in conditions of constant dark (DD). We determined that H2O2 production was regulated by circadian rhythm for wt flies. However, H2O2 production in DD conditions was increased overall. In tim mutants there was an indication of circadian regulation of H2O2 production in LD conditions but the rhythm was abolished in DD conditions. wt and tim flies differed significantly in the 24 hour modulation of H2O2 in DD, but less so in LD. Further studies are needed to elucidate the role of tim in the regulation of H2O2 amount and the connection with cocaine responsiveness. Taken together, our data suggest that redox status is regulated in a circadian fashion and that a functional tim gene is essential for maintaining circadian rhythmicity., Cirkadijalni ritam je prisutan u skoro svim organizmima. Nastao je kao posljedica rotacije Zemlje i izmjene dana i noći. Važan je za regulaciju brojnih bioloških procesa kao što je i ciklus spavanja. Narušeni cirkadijalni ritam je često povezivan s pojavom oksidativnog stresa koji nastaje kao posljedica povišene produkcije reaktivnih kisikovih vrsta (ROS). U ovome radu, istražili smo povezanost cirkadijalnog ritma i oksidativnog statusa u D.melanogaster. S obzirom da su wild type i timeless mušice pokazale sličnost u odgovoru na izlaganje volatiliziranom kokainu, zanimalo nas je hoće li sličnost biti prisutna i kod regulacije redoksa. Indikator redoks statusa je bila koncentracija H2O2 u homogenatima glava mušica. H2O2 je nusprodukt oksidativnog metabolizma, a za njegovu detekciju korištena je DHE metoda bojanja. Nadalje, ispitali smo je li proizvodnja H2O2 regulirana cirkadijalnim ritmom. Mjerenja su provedena na mušicama koje su držane u LD ili DD uvjetima. Pokazali smo da je kod wt mušica H2O2 proizvodnja regulirana cirkadijalnim ritmom. Međutim, proizvodnja H2O2 je bila sveukupno povišena u DD uvjetima. Kod tim mutanata, indikacije cirkadijalne modulacije u proizvodnji H2O2 prisutne u LD uvjetima nisu uočene i u DD uvjetima. wt i tim mušice se značajno razlikuju u modulaciji H2O2 koncentracija u DD uvjetima dok je u LD uvjetima razlika značajno manja. Potrebno je još studija koje će odrediti točnu ulogu tim gena u regulaciji H2O2 i koje će definirati njegovu ulogu u procesu odgovora na izlaganje kokainu. Sveukupno, dobiveni podatci sugeriraju da je metabolizam ROS-a povezan s cirkadijalnim ritmom te da je funkcionalni gen tim esencijalan za održavanje cirkadijalne ritmičnosti.

Bipolar disorder is a psychiatric disorder that greatly affects a patient’s day-to-day functioning and is characterised by recurrent manic and depressive episodes. Current mania treatments do not show efficacy for all patients, increasing the need for targeted therapeutic development. The drug development process includes its assessment using applicable animal models, which are sparse for mania in bipolar disorder. An ideal mouse model of mania should fulfil the three validity criteria, as well as display specific endophenotypes comparable to those in human patients. In the thesis, I reviewed some suggested mice models of mania in bipolar disorder, which are either a result of genetic manipulations or induced by drug administration. Single genetic manipulations focused on genes related to the circadian rhythm and dopaminergic, serotonergic, and glutamatergic systems resulted in mice models such as the Clock Δ19 and DAT knockdown mutant mice, which show a limited number of symptoms, predominantly locomotor hyperactivity. In comparison, an amphetamine-induced mouse model of mania showed limited results, suggesting that genetic models are more appropriate. The complex symptomatology of mania has yet to be fully reflected successfully in mouse models, which are mostly based on simple behavioural abnormalities. Regardless, the mutual comparison and assessment of known models allows the establishment of future directions in mouse model of mania development. Highlighting the use of multivariate approaches such as genetic information from human studies, extensive behavioural testing, and objective quantification of behaviours with cross-species paradigms will enable further advancement towards an ideal model of mania., Bipolarni poremećaj je psihijatrijski poremećaj koji uvelike utječe na svakodnevne funkcije pacijenta i karakteriziran je ponavljajućim epizodama manije i depresije. Trenutno dostupni tretmani manije nisu učinkoviti za sve pacijente, uvelike povećavajući potrebu za razvojem ciljanih terapija. Proces razvoja lijekova uključuje njegovo testiranje na odgovarajućim životinjskim modelima koji su rijetki za maniju u bipolarnom poremećaju. Idealni mišji model manije trebao bi zadovoljavati tri kriterija valjanosti, kao i prikazati specifične endofenotipove usporedive s onima zabilježenim kod ljudskih pacijenata. Ovaj rad uključuje pregled nekih od predloženih mišjih modela manije, koji su produkt genetskih manipulacija ili inducirani administracijom lijekova. Pojedinačne genetske manipulacije usredotočene na gene povezane s cirkadijalnim ritmom te dopaminergičkim, serotonergičkim i glutamatergičnim sustavima rezultirale su mišjim modelima kao što su Clock Δ19 i DAT knockdown mutantski miševi, koji pokazuju ograničen broj simptoma, pretežno lokomotornu hiperaktivnost. Za razliku od njih, amfetaminom induciran mišji model manije pokazao je ograničene rezultate, ukazujući na to da su genetski modeli prikladniji za primjenu. Složena simptomatologija manije još nije u potpunosti odražena u mišjim modelima, koji se uglavnom temelje na jednostavnim poremećajima u ponašanju. Bez obzira na to, međusobno uspoređivanje i procjena poznatih modela omogućuje uspostavljanje budućih pravaca u razvoju mišjeg modela manije. Uporaba multivarijantnih pristupa, kao što su korištenje genetskih informacija iz ljudskih istraživanja, razvoj opsežnih testova ponašanja te objektivna kvantifikacija ponašanja u kombinaciji sa razvojem međuvrsnih paradigmi omogućit će daljnji napredak u dizajnu idealnog modela manije.

Vitamin D grupni je naziv za skupinu vitamina topivih u mastima. Smatra se jednim od najstarijih prohormona na Zemlji. Najpoznatije vrste vitamina D su vitamin D2 (ergokalciferol) i vitamin D3 (kolekalciferol). Izvori vitamina D2 su biljnog porijekla dok su izvori vitamina D3 životinjskog porijekla. Aktivni oblik vitamina D je kalcitriol (1,25-dihidroksikolekalciferol). Glavni izvor vitamina D je sunce, točnije UVB zračenje. Pritom treba naglasiti da sunčanje u svrhe sinteze vitamina D treba prakticirati bez korištenja zaštitnog faktora, a opet paziti i na to da ne bismo pocrvenili. U optimalnim uvjetima naša koža može sintetizirati od 10 000- 20 000 IU (od engl. international units, internacionalnih jedinica) vitamina D u 30 minuta, nakon čega sinteza vitamina D prestaje. Osim što igra vrlo važnu ulogu u očuvanju zdravlja kostiju i imunosnog sustava, sve više se ističe njegova korist u brojnim bolestima, a vrlo važnu ulogu ima i tijekom trudnoće i za majku i za fetus te kasnije za novorođenče. Jako puno ljudi diljem svijeta ima hipovitaminozu vitamina D, koja se dijagnosticira mjerenjem koncentracije 25- hidroksivitamina D, 25(OH)D u krvi, točnije u serumu. Radi nedostatka vitamina D u organizmu može doći do raznih komplikacija i oboljenja. S druge strane, prevelike doze vitamina D u organizmu uzrokuju hipervitaminozu pa radi toga treba paziti da se preporučena doza ne prekorači jer u suprotnom može uzrokovati neželjene nuspojave i toksičnost. Kako bi se izbjegao neželjeni utjecaj, vitamin D treba uvijek uzimati pod liječničkim nadzorom., Vitamin D is a group name for a group of vitamins which are soluble in fats. It is considered one of the oldest prohormones on earth. The most common types of vitamin D are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Sources of vitamin D2 are of plant origin, whereas vitamin D3 sources are of animal origin. The active form of vitamin D is calcitriol (1,25-dihydroxycholecalciferol, 1,25(OH)2D). The main source of vitamin D is the sun, more precisely its UVB radiation. It needs to be clear that sunbathing in the means to synthesize vitamin D needs to be done without sunscreen, but avoiding sunburn. In optimal conditions our skin can synthesize from 10 000 – 20 000 IU (international units) of vitamin D in 30 minutes, after that the synthesis of vitamin D naturally stops. In addition to playing a very important role in maintaining bone and immune system health, its benefits in many diseases are increasingly emphasized and it plays a very important role during pregnancy for both mother and fetus and also later for the newborn. A lot of people around the world have vitamin D hypovitaminosis which is diagnosed by measuring the concentration of 25 hydroxyvitamin D, 25(OH)D in the blood, more precisely in the serum. Due to the lack of vitamin D in the body, various complications and diseases can occur. On the other hand, overdoses of vitamin D in the body cause hypervitaminosis, so care should be taken not to exceed the recommended dose because otherwise it can cause unwanted side effects and toxicity. To avoid side effects, vitamin D should always be taken under medical supervision.

Pojam editiranje gena odnosi se na izmjenu željenog nukleotidnog slijeda DNA molekule. Otkriće novih, revolucionarnih metoda genetičkog editiranja, od kojih su trenutno najpopularnije i najkorištenije nukleaze cinkovih prstiju (engl. zinc finger nucleases, ZFN), efektorske nukleaze nalik aktivatoru transkripcije (engl. transcription activator-like effector nucleases, TALEN) te niz međusobno razdvojenih kratkih, klasteriranih, palindromskih ponavljanja povezanih s proteinom 9 (engl. clustered regularly interspaced short palindromic repeat – CRISPR– associated protein 9, CRISPR-Cas9), uspostavilo je naprednu generaciju alata koji imaju ključnu ulogu u molekularnoj biologiji, ali i nizu drugih biotehnoloških disciplina. Međutim, vjerojatno najveći potencijal za primjenu metoda editiranja genoma čovjeka je u medicini zbog pretpostavke da se promjenama u specifičnim genima mogu suzbiti brojne bolesti i poremećaji koji se još uvijek smatraju neizlječivima na razini uzroka. U tijeku je razvoj novih terapijskih mogućnosti, a jedna od trenutno klinički primjenjivih terapija je stanična imunoterapija T limfocitima koji su genetički modificirani kimernim antigenskim receptorom (engl. chimeric antigen receptor, CAR), za liječenje novotvorina. U ovom završnom radu, osim opisane metodologije alata za editiranje genoma te perspektivnih pretkliničkih i kliničkih ispitivanja za otkriće novih strategija liječenja bolesti, razmatraju se i etički aspekti editiranja genoma, kao i opisane smjernice za njegovu primjenu, koja je za sada moguća isključivo na somatskim stanicama. Kako bi se dostigla pouzdana razina učinkovitosti i sigurnosti metoda editiranja gena, potrebno je daljnje usavršavanje u odabiru adekvatnih metoda dostave programabilnih nukleaza u stanice te ustrajanje u razvoju pouzdanijeg in vivo editiranja, a ponajviše u donošenju etičkih i pravnih okvira za razumno, svrsishodno i nemanipulativno korištenje editiranja genoma., The term gene editing refers to the modification of the desired nucleotide sequence of a DNA molecule. Discovery of new, revolutionary methods of genetic editing, of which the most popular and most used are zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeat – CRISPR–associated protein 9, (CRISPR-Cas9), has established an advanced generation of tools that play a key role in molecular biology, as well as a number of other biotechnological disciplines. However, probably the greatest potential for the application of human genome editing methods is medicine due to the assumption that changes in specific genes can suppress a number of diseases and disorders that are still considered incurable at the cause level. New therapeutic options are being developed, and one of the currently clinically applicable therapies is cellular immunotherapy that uses T lymphocytes that are genetically modified by the chimeric antigen receptor (CAR), for the treatment of neoplasms. In this undergraduate thesis, in addition to the described methodology of genome editing tools and perspective preclinical and clinical trials for the discovery of new treatment strategies, the ethical aspects of genome editing are discussed, as well as the described guidelines for its application, which is currently possible only on somatic cells. In order to achieve a reliable level of efficiency and safety of the gene editing methods, further improvement in the selection of adequate methods for delivering programmable endonucleases to cells is needed, but also persistence in the development of more reliable in vivo editing and in adopting ethical and legal frameworks for the reasonable, purposeful and non-manipulative use of genome editing.

Circadian rhythms are oscillations that occur in a 24 hour period as a result of an endogenous biochemical oscillator known as the circadian clock. It regulates physiological and behavioral processes in all mammals. Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) molecule that also acts as a signaling molecule. We wanted to look at the connection between oxidative stress and the involvement of circadian genes to then be able to further investigate the effects of methamphetamine (METH) on the redox states in Drosophila. Disruptions in circadian rhythms are often seen in substance abuse disorders. In order to measure oxidative stress we used fluorescent dihydroethidium (DHE) staining and studied the variations in the levels of H2O2 in the wild type (wt) and circadian mutants ClkJrk, per01, tim01 and cyc01 of Drosophila melanogaster. Furthermore, we wanted to know if there was a difference in levels of H2O2 circulating in the head and body of the fly and whether it was under the influence of the time of day. Our data suggests that H2O2 levels vary between wt and circadian mutants and that in the circadian mutants the concentration of H2O2 varies depending on the time of day, while in wt it does not. More H2O2 is found in the head., Cirkadijalni ritmovi su oscilacije koje se zbivaju u periodu od 24 sata kao rezultat endogenog biokemijskog oscilatora poznatog kao cirkadijalni sat. On regulira fiziološke i bihevioralne procese u svim sisavcima. Vodikov peroksid (H2O2) je jedna od molekula reaktivnih kisikovih vrsta koja ujedno djeluje i kao signalna molekula u mnogim procesima. Htjeli smo vidjeti poveznicu između oksidativnog stresa i cirkadijalnih gena kako bi mogli dalje istražiti utjecaj metamfetamina (METH) na redoks status kod Drosophile. Poremećaji u cirkadijalnim ritmovima se često viđaju kod zloupotrebe substanci. Kako bismo izmjerili oksidativni stres koristili smo se metodom bojanja fluorescentnom dihidroetidij (DHE) bojom te smo mjerili koncentraciju H2O2 koji se nalazi u wild type (wt) mušicama i cirkadijalnim mutantima ClkJrk, per01, tim01 and cyc01 Drosophile melanogaster. Nadalje, htjeli smo vidjeti postoji li razlika u količini H2O2 koji se nalazi u glavi i u tijelu mušica i je li pod utjecajem doba dana. Naši podatci ukazuju na to da količine H2O2 variraju između wt i cirkadijalnih mutanata i da kod cirkadijalnih mutanata koncentracija H2O2 varira ovisno o dobu dana, dok kod wt ne. Veća je koncentracija H2O2 u glavi.

Methamphetamine (METH) is a psychostimulant that affects central nervous system by increasing the concentration of the free dopamine, whose oxidation leads to the permanent damage in the structure and function of neurons, as the consequence of the increased production and accumulation of the radical oxygen species (ROS). By using Drosophila melanogaster as a model organism we have tested impact of an acute dose of volatilized METH (vMETH) on the concentration of hydrogen peroxide (H2O2), in the brain, as one of the ROS and redox status representative. Individual flies of wild type strain CantonS (wt) used in the experiment were exposed to vMETH using the FlyBong method. The concentration of H2O2 was measured in the head tissue extracts, 10 hours after the administration, using dihydroetidium florescent dye. The results have shown that the acute exposure to vMETH does not lead to the change in the concentration of H2O2 in the wt flies compared to the control group. Considering that research have shown connection between administration of the METH and increased expression of period (per) gene with enzyme activity of tyrosine hydroxylase, included in the synthesis of dopamine, I tested whether there is a difference in the concentration of H2O2 after the acute exposure to vMETH between different circadian mutants and wt flies. Circadian mutants of genes per, timeless (tim), Clock (Clk) and cycle (cyc) with alleles per01, tim01, ClkJrk and cyc01 were used in the experiment. per01 and tim01 flies showed the decreased concentration of H2O2 compared to the controls while ClkJrk and cyc01 flies had higher concentration of H2O2 after the acute exposure to vMETH. The concentration of H2O2 is significantly lower in tim01 flies compared to wt flies after the exposure to vMETH while cyc01 flies had a significantly higher concentration than wt flies. Based on the results of this thesis, we suggest the influence of circadian genes on the endogenous concentration of H2O2, which is in the absence of per or tim increased, and in the absence of Clk or cyc decreased compared to wt. The administration of vMETH potentiates the production of H2O2 in the absence of cyc while the absence of tim gene attenuates that effect. The lack of difference in wt flies is surprising and possible mistakes in the method or its use should be considered. By establishing the molecular pathways involving H2O2 and circadian genes new methods for treatment of addiction could be developed., Metamfetamin (METH) je psihostimulans koji utječe na središnji živčani sustav povećavajući koncentraciju slobodnog dopamina, čija oksidacija uzrokuje trajna oštećenja strukture i funkcije neurona kao posljedicu povećane proizvodnje i akumulacije slobodnih kisikovih radikala (ROS-a). Korištenjem Drosophile melanogaster kao modelnog organizma istražen je utjecaj akutne doze volatiliziranog METH-a (vMETH) na koncentraciju vodikovog peroksida (H2O2), u mozgu, kao jednog od predstavnika ROS-a i redoks statusa. U eksperimentu su korištene individualne mušice divljeg tipa CantonS (wt) izložene vMETH–u korištenjem FlyBong metode. Koncentracija H2O2 je mjerena u ekstraktima tkiva glava mušica 10 sati nakon administracije upotrebom dihidroetidij fluorescentne boje. Rezultati su pokazali da akutno izlaganje vMETH-u ne dovodi do promjene u koncentraciji H2O2 kod wt mušica u odnosu na kontrolu. S obzirom na literaturno poznatu povezanost administracije METH-a s povećanom ekspresijom period (per) gena i aktivnosti enzima uključenog u sintezu dopamina, tirozin hidroksilaze, testirano je ima li razlike u koncentraciji H2O2 nakon akutne izloženosti vMETH-u između različitih cirkadialnih mutanata i wt mušica. Za eksperimente su korišteni cirkadialni mutanti u genima per , timeless (tim), Clock (Clk) i cycle (cyc) sa sljedećim alelima: per01, tim01, ClkJrk i cyc01. per01 i tim01 mušice su pokazale smanjenu koncentraciju H2O2 u odnosu na svoje kontrole, dok su ClkJrk i cyc01 mušice imale veću koncentraciju H2O2 nakon akutnog izlaganja vMETH-u. Koncentracija H2O2 je značajno niža kod tim01 mušica u usporedbi s wt mušicama nakon izlaganja vMETH-u, dok cyc01 mušice imaju značajno veću koncentraciju nego wt mušice. S obzirom na rezultate ovoga rada predlažemo utjecaj cirkadialnih gena na endogenu koncentraciju H2O2 koja je u odsustvu per ili tim povećana, a u odsustvu Clk ili cyc smanjena u odnosu na wt. Administracija vMETH-a potiče proizvodnju H2O2 u odsustvu cyc dok odsustvo tim gena sprječava taj efekt. Nedostatak razlike kod wt mušica je iznenađujući te je potrebno razmotriti moguće pogreške u metodi ili njezinom provođenju. Utvrđivanjem molekularnih putova koji uključuju H2O2 i cirkadialne gene mogle bi se razviti nove metode liječenja ovisnosti o METH-u.

Ovisnost o drogama definira se kao kompulzivno uzimanje droge s gubitkom kontrole u ograničavanju unosa. To je kompleksna bolest koju karakteriziraju različiti kognitivni, bihevioralni i fiziološki simptomi koji nastaju kao posljedica opetovane konzumacije droge. Zbog svoje kompleksnosti, ovisnost se u laboratoriju istražuje ispitivanjem jednostavnih ponašanja koja su karakteristična - npr. samoadministracija i lokomotorna senzitizacija. Za laboratorijska istraživanja neophodno je imati životinjske modele, a zbog genetičkih sličnosti između ljudi i životinja te brzog i jeftinog uzgoja, Drosophila melanogaster je vrlo često korišten životinjski model. Cilj našeg istraživanja bio je ispitati postoji li korelacija između dva oblika ponašanja karakteristična za ovisnost da bismo saznali postoje li dijelovi mozga koji zajednički utječu na njihovu regulaciju. Preferencijalna konzumacija droge je oblik ponašanja u kojem jedinke svjesno i aktivno biraju hranu s dodatkom droge naspram one bez droge, a lokomotornu senzitizaciju definiramo kao povećanu lokomotornu aktivnost nakon administracije droge. Preferencijalna konzumacija metamfetamina ispitivana je pokusom samoadministracije (FlyCafe metodom), a lokomotorna senzitizacija ispitivana je FlyBong metodom. Mušice aktivno preferiraju hranu s dodatkom metamfetamina, unatoč gorkom okusu te povećavaju lokomotornu aktivnost uslijed opetovane administracije metamfetamina. Rezultati ukazuju na to da postoji korelacija između ta dva oblika ponašanja, što otvara mogućnost novih istraživanja kako bi se ta korelacija bolje definirala i kako bi se potencijalno otkrili zajednički neuralni mehanizmi koji upravljaju tim ponašanjima., Drug addiction is defined as compulsive drug use with a loss of control in restricting intake. It is a complex disease characterized by various cognitive, behavioral and physiological symptoms that result from repeated drug use. Due to its complexity, addiction is explored in the laboratory by examining the simple behaviors that are characteristic - for example self – administration and locomotor senzitization. For laboratory research it is necessary to have animal models, and because of genetic similarities between humans and animals and also fast and cheap cultivation, Drosophila melanogaster is a very often used animal model. The goal of our study was to examine whether there is a correlation between two addictive behaviors to find out whether there are parts of brain that affect together on their regulation. Preferential drug consumption is a form of behavior in which species consciously and actively choose foods with drug additions versus those without drugs, and locomotor sensitization is increased locomotor activity after drug administration. The preferential consumption of methamphetamine was investigated by the self-administration experiment (FlyCafe method), and locomotor sensitization was investigated by the FlyBong method. Flies actively prefer methamphetamine-supplemented foods despite their bitter taste and increase locomotor activity due to repeated administration of methamphetamine. The results indicate that there is correlation between the two types of behaviors, which opens up the possibility of new research to better define this correlation and to potentially reveal common neural mechanisms that govern these behaviors.

Popularnost fotodinamičke terapije proizlazi iz citotoksičnog učinka koji se postiže na ciljano tkivo, bez uništenja zdravog tkiva. Sastoji se od administracije fotosenzibilne molekule, primjene svjetla odgovarajuće valne duljine i reakcije s molekularnim kisikom. Ključan proces je prijelaz fotosenzibilizatora u tripletno pobuđeno stanje te nastanak reaktivnog singletnog kisika. Zbog toga, modifikacijom svojstava fotosenzibilizatora, nastoji se pojačati efikasnost ove terapije. Traži se molekula visokih kvantnih prinosa tripletnog pobuđenog stanja i singletnog kisika. Meta istraživanja su većinom porfirini od kojih su posebice zanimljivi meso supstituirani porfirini čiji supstituenti znatno mijenjaju vrijednosti istaknutih kvantnih prinosa. S obzirom da se može utjecati na broj i vrstu supstituenta u meso poziciji porfirinskog prstena, to je fokus velikog broja znanstvenika. Pokazani su rezultati uvođenja elektron-donorskih i akceptorskih skupina, silicija, halogenih elemenata, te ugljikovodičnih lanaca različite duljine. Dao se osvrt i na pulsnu fluorescentnu tehniku te tehniku mjerenja udjela porfirina u tripletnom pobuđenom stanju. S obzirom da je derivat s bromom pokazao najveći prinos tripletnog pobuđenog stanja (ΦT = 0.98), a derivat sa silicijem najveći prinos singletnog kisika (Φ∆ = 0.77), obećavajući porfirini su oni u čijoj se meso poziciji nalazi brom te silicij. Dao se uvid i u indirektan utjecaj supstituenata na kvantne prinose fotofizikalnih procesa, na primjeru ugljikovodičnih lanaca u meso poziciji. Lanac sa 22 ugljikova atoma je hidrofoban supstituent koji povećava agregaciju porfirina u vodenom mediju. Na taj način smanjuje njegovu učinkovitost u fotodinamičkoj terapiji (ΦT < 0.08). Nasuprot navedenih zaključaka, rezultati koji se ne mogu sa sigurnošću interpretirati i trebaju dodatna istraživanja su metoksifenilna skupina i atom fluora u meso poziciji., The popularity of photodynamic therapy results from a cytotoxic effect achieved on target tissue without destroying healthy tissue. It is based on administration of a photosensitizer, light of the appropriate wavelength and reaction with molecular oxygen. The key process is transition of the photosensitizer to the triplet excited state and generation of reactive singlet oxygen. Therefore, the goal is to enhance the efficacy of the therapy by modifying properties of the photosensitizer. For this there is a need for a molecule of high quantum yields of a triplet excited state and a singlet oxygen. Target of the current research are porphyrins, of which the most interesting are meso-substitued porphyirins. Their substituents significantly change the value of mentioned quantum yields. Since number and type of substituents is adjustable, this is the focus of many studies and investigations. Here are shown the results of introduction of electrondonor and electron-acceptor groups to the porphyrin ring, as well as alkyl chains, silylation and halogenation. Also, here is given the overview of two techniques, pulse train fluorescence technique and measurement of the porphyrins in triplet excited state. Since the bromine derivative showed the highest quantum yield of triplet excited state (ΦT = 0.98) and silylated derivative the highest quantum yield of singlet oxygen (Φ∆ = 0.77), the promising porphyrins are the ones with bromine and silicon in the meso position. Also, the example of alkyl chains in meso position, gives the insight in the indirect impact of substituents on quantum yields. Alkyl chain with 22 carbon atoms is hydrophobic and it increases the aggregation of porphyrins in aquatic medium. In that way, the efficacy of photosensitizer is decreased (ΦT < 0.08). As opposed to these conclusions, results that cannot be interpreted with same certainty and need more research are metoxyphenyl group and fluorine in meso position.

Cilj završnog rada je opisati proizvodnju te analizirati osnovne parametre kakvoće bijelog vina Belica s područja Kastva. Za proizvodnju navedenog vina koriste se sorta Verdić te autohtone sorte za Hrvatsko primorje; Mejsko belo, Divjaka, Jarbola, Malvazija istarska te sorta Brajkovac koja je u tijeku ispitivanja autohtonosti. Kemijskom analizom je na Odjelu za biotehnologiju Sveučilišta u Rijeci analizirano 13 uzoraka različitih proizvođača Belice iz berbe 2016. Analizirani su osnovni parametri kakvoće vina: stvarna volumna alkoholna jakost, pepeo, ukupna kiselost, hlapiva kiselost, ukupan sumporni dioksid, slobodan sumporni dioksid, relativna gustoća, reducirajući šećer, pH vrijednost, jabučna kiselina te mliječna kiselina. Rezultati analiziranih uzoraka vina su u skladu s vrijednostima Pravilnika o vinu iz 1996. (NN 96/96)., The aim of final work was to describe production and analyse basic quality parameters of white wine Belica from the area of Kastav. The wine is produced from grapes of Verdić and also from autochthonous grapes for subregion Hrvatsko primorje such as Mejsko belo, Divjaka, Jarbola, Malvazija istarska and Brajkovac that is still in ongoing testing for autochthony. 13 specimen of wine Belica from different winemakers from year of harvest 2016 were chemically analysed at the Department of biotechnology at the University of Rijeka. Basic quality parameters including actual alcoholic strength, ash, total acidity, volatile acidity, total sulfur dioxide, free sulfur dioxide, relative density, reducing sugar, pH value, malic acid and lactic acid, were analysed. The results of analysed wine specimen were in accordance with the Croatian wine regulations from year 1996 (NN96/96).

Addiction is characterized by compulsive drug seeking and taking despite the negative consequences. In the laboratory, addiction is studied as set of simple forms of behaviors (endophenotypes), some of which are locomotor sensitization (LS), a phenotype of the motor-activating effects and preferential consumption, a phenotype of rewarding effects. The aim of the thesis is to develop tests for inducing and quantifying cocaine (COC) and methamphetamine (METH) motor-activating and rewarding effect, and investigate genetic mechanisms underlying such neural plasticity. We developed and optimized two new methods, FlyBong for measuring motor-activating effects, and a self-administration method. We show that repeated exposures to volatilized COC and METH induce LS in flies, and that flies prefer COC and METH containing food to regular food. Pharmacodynamics of COC and METH LS and preferential consumption depends on the dopamine transporter. Reduction of dopamine, serotonin and octopamine, did not completely abolish the locomotor sensitization to COC and METH, suggesting the involvement of other monoamines in this processes. Manipulation of dopaminergic and serotoninergic vesicular monoamine transporters (VMAT) using RNA interference (RNAi), indicated that locomotor sensitization and preference to COC and METH depend on functional VMAT. LS to COC depends on several circadian genes, period (per), cycle (cyc) and Clock (Clk), leads to high levels of reactive oxygen species (ROS) production and is susceptible to the exogenous redox perturbation. LS to METH depends only on one circadian gene, Clk, leads to lower levels of ROS and is less susceptible to exogenous redox perturbation. The motor-activation and rewarding effects of COC depend on the circadian gene per, while METH depends on Clk, indicating differential involvement of circadian genes in these two processes. Pre-treatment with H2O2 led to different effects on sensitivity and LS to COC and METH suggesting that H2O2 might function as a neuromodulator, and that its role is drug-specific. Here we developed new behavioral tests and described the effects of COC and METH on two forms of neuronal plasticity. Our new high throughput test for measuring LS is ideally suited for unbiased genetic screen or selection. We described likely circadian and redox influence on behavior that correlates with monoaminergic V regulation. We hypothesize that circadian genes and redox regulation work together to change the brain functioning after consumption of addictive drugs., Jednostavna definicija ovisnosti podrazumijeva kompulzivno traženje i primjenu sredstva ovisnosti unatoč negativnim posljedicama koje ima za organizam. Ovisnost se kod laboratorijskih životinja proučava kao niz jednostavnih ponašanja (endofenotipova) upotrebom optimiziranih bihevioralnih testova koji moraju biti objektivni, reproducibilni i kvantitativni. Ovisnost o psihostimulansima (PS) kokainu (COC) i metamfetaminu (METH) se može razložiti na jednostavnija ponašanja povezana s motoričkim (senzitizacija) i nagrađujućim (preferencijalna konzumacija) učinkom. Cilj ovog doktorskog rada je ispitati utjecaj COC i METH na motorički i nagrađujući učinak povezan s genetskim promjenama koje dovode do neuralne plastičnosti primjenom novo razvijenih testova za induciranje i kvantifikaciju ponašanja. Tijekom mog doktorskog studija razvila sam i optimizirala novu metodu za mjerenje motoričke aktivnosti potaknute primjenom PS koju smo nazvali FlyBong. Upotrebom novog optimiziranog protokola „FlyBong“, utvrđeno je da akutno i opetovano izlaganje Drosophile volatiliziranom COC i METH inducira povećanje motoričkog odgovora. Istražena je farmakodinamička sličnost između mehanizma djelovanja COC i METH te utjecaj monoamina dopamina, serotonina i oktopamina na bihevioralni odgovor kod akutne i opetovane primjene PS. Testirala sam hipotezu da su cirkadijalni geni period, timeless, clock, cycle i pigment-dispersing factor uključeni u endofenotip senzitizacije na COC i METH. Senzitizacija potaknuta s COC nije prisutna kod per01, cyc01 i ClkJrk mutanata, dok senzitizacija na METH nije prisutna kod ClkJrk mutanata. Izmjerila sam utjecaj COC i METH na sistemske biomarkere redoks statusa: katalazu, superoksid dismutazu, vodikov peroksid i reaktivne kisikove vrste čime sam pokazala da COC sistemski inducira veći, a METH manji pomak oksidativnog statusa Testirala sam utjecaj predtretmana s anti- i prooksidansima na motorički odgovor kod akutne i opetovane primjene COC i METH, čime sam pokazala da antioksidansi utišavaju senzitizaciju na METH, dok kod COC senzitizacija nije prisutna. Istražila sam potencijalnu neuromodulatornu i oksido-redukcijsku ulogu vodikovoga peroksida povezanu s predtretmanom koji nije uzrokovao sistemski pomak oksidativnog statusa, ali je utjecao na endofenotip senzitizacije. Testirana je hipoteza korelacije broja cirkadijanih gena i osjetljivosti na redoks status u ovisnosti o PS. Nađeno je da PS koji ovisi o više cirkadijalnih gena te uzrokuje veću sistemsku redoks preturbaciju pokazuje veću osjetljivost na promjenu redoks statusa u endofenotipu VII senzitizacije ako je pred tretman anti- ili prooksidativan, dok PS koji ovisi o manjem broju cirkadijalnih gena, uzrokuje manju sistemsku redoks preturbaciju te pokazuje manju osjetljivost na promjenu redoks statusa više orijentiranu na predtretman s molekulama manjeg antioksidativnog kapaciteta. Kako bi ispitala da li PS utječu ma motivaciju za konzumacijom optimizirala sam test samo-administracije (Capillary Feeding, CAFE) gdje mušice biraju između otopine šećera i otopine šećera pomiješane s PS. Ovim istraživanjem je nađeno da mušice kojima je ponuđen izbor između otopine šećera i otopine šećera pomiješane s PS pokazuju preferenciju prema otopini s PS, koja je posljedica nagrađujućeg efekta PS. Nađeno je da su procesi učenja i pamćenja povezani s periferijalnom konzumacijom te da će mušice preferencijalno konzumirati otopinu s PS unatoč dodatku supstance gorkog okusa. Ukoliko se mušicama koje su izložene PS nekoliko dana za redom, jedan dan ne ponudi PS te ih se nakon toga ponovno izloži izboru između otopine šećera i otopine šećera i PS, mušice će pokazivati jednaku ili veću preferenciju za PS kao što je bila prije perioda apstinencije. Istražen je utjecaj cirkadijalnih gena te receptora i prijenosnika povezanih s farmakodinamičkim djelovanjem COC i METH na nagrađujući učinak. Pokazala sam da se preferencijalna konzumacija COC i METH razlikuje po tipu cirkadijalnih gena koji na nju utječu, a neki od njih kontroliraju motoričku senzitizaciju. Ovi rezultati upućuju na mogući zajednički genetski mehanizam koji je u podlozi preferencijalne konzumacije, kao oblika nagrađujućeg utjecaja PS i motoričke senzitizacije. Ishodi mojeg doktorskog rada rezultirali su razvojem dvije nove objektivne i visokoprotočne metode za kvantifikaciju ponašanja Drosophile koja će omogućiti naredna istraživanja genetske osnove neuralne plastičnosti izazvane PS korištenjem metode genetskog probira ili genetske selekcije. Daljnja istraživanja cirkadijalnih/redoks povratnih spregi potaknutih primjenom COC i METH trebala bi pomoći u razumijevanju mehanizma kojima PS mijenjaju neuronske mreže, te usmjeravanju u nove potencijalne farmakološke smjerove razvoja lijekova za prevenciju i liječenje ovisnost