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Relationship between two methamphetamine-induced behaviors in Drosophila melanogaster: self-administration and locomotor sensitization
Get PDFOvisnost 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
The role of optineurin in autophagy induced by intracellular bacteria and interferon beta
No full textAutofagija je stanični proces kojim se sadržaj za razgradnju putem autofagosoma dostavlja do lizosoma. Autofagija je neselektivna u stanju gladovanja, a selektivna kada se stanica susretne sa štetnim sadržajem poput proteinskih agregata, unutarstaničnih patogena i oštećenih organela. Selektivna može dodatno biti potaknuta citokinima, poput interferona (IFN) β. Sadržaj za razgradnju mora biti ubikvitiniran i prepoznat od strane adaptora autofagije, od kojih su najpoznatiji p62 i optineurin. Adaptori autofagije sadržaj za razgradnju povezuju s lipidiranim LC3 (LC3-II) na autofagosomalnoj membrani. Važnost optineurina u autofagiji pokazana je prilikom infekcije bakterijom Salmonella typhimurium jer gubitak funkcije optineurina vodi smanjenom otklanjanju bakterija putem autofagije te njihovoj povećanoj proliferaciji u HeLa staničnoj liniji. Budući da se radi o imortaliziranoj staničnoj liniji, fiziološki značaj optineurina potrebno je analizirati i u primarnim stanicama. Kako bismo analizirali autofagiju u primarnim stanicama mišjeg modela kojemu nedostaje ubikvitin-vežuća regija optineurina (Optn470T/470T), koristili smo unutarstaničnu bakteriju Francisella novicida. Pokazali smo kako optineurin nema ulogu u kinetici rasta ove bakterije unutar 72 sata nakon infekcije makrofaga dobivenih iz koštane srži te embrionalnih fibroblasta. Konfokalnom mikroskopijom smo zabilježili porast optineurin- i p62- pozitivnih vezikula 24 sata nakon infekcije, što sugerira na indukciju autofagije, ali za razliku od S. typhimurium, F. novicida nije bila unutar autofagosoma. Stoga, nismo mogli jasno odrediti značaj autofagije prilikom te infekcije. Nadalje, kako bismo ispitali ima li nedostatak optineurina utjecaja na selektivnu autofagiju potaknutu s interferonom β, koristili smo Neuro2a KO staničnu liniju u kojoj je CRISPR/Cas9 metodom uklonjen gen za optineurin. Autofagiju smo detektirali pomoću reporterskog RFP-GFP-LC3 konstrukta koji prikazuje protok kroz autofagiju. Kvantifikacijom autofagosoma pokazali smo da KO stanice imaju povišenu autofagiju u bazalnim uvjetima i nakon poticanja autofagije s IFN-β. Nadalje, pokazali smo kako nedostatak optineurina ne dovodi do potpunog bloka autofagije, ali da potencijalno postoji djelomični blok, vidljiv kao nakupljanje p62 i LC3-II Western blot metodom, koji je najviše izražen u bazalnim uvjetima. Zaključno, pokazali smo kako optineurin ne utječe na kinetiku rasta F. novicida čime smo pridonijeli boljem razumijevanju životnog ciklusa ove bakterije. Nadalje, uočili smo mogući djelomični blok autofagije u stanicama bez optineurina što posljedično može utjecati na nakupljane agregata proteina, glavnog obilježja neurodegenerativnih bolesti.Autophagy is a cellular process in which intracellular cargo is sequestered by autophagosomes and delivered to lysosomes. Autophagy is non-selective in times of starvation and selective when cells encounter harmful cargo like protein aggregates, pathogens and damaged organelles. Selective autophagy can be further induced by cytokines, like interferon (IFN) β. Harmful cargo gets ubiquitinated and recognized by autophagy adaptors, e.g. optineurin and p62. Autophagy adaptors deliver the cargo to lipidated LC3 (LC3-II) on autophagosomal membranes. The role of optineurin in selective autophagy was demonstrated in Salmonella typhimurium, where optineurin mutations led to decreased autophagic clearance and increased bacterial proliferation in HeLa cell line. Since this is an immortalized cell line, physiological significance of optineurin needs to be analyzed in primary cells. To analyze autophagy in primary cells from the mouse model lacking the ubiquitin-binding domain of optineurin (Optn470T/470T), we used intracellular bacteria Francisella novicida. We showed that loss of optineurin function does not affect growth kinetics of F. novicida 72 hours post infection in bone marrow derived macrophages and embryonic fibroblasts. We showed increase in numbers of optineurin- and p62-positive vesicles 24 hours post infection by confocal microscopy, suggesting that autophagy was induced to some extent after infection, but unlike S. typhimurium, F. novicida was not detected within autophagosomes. For this reason, the importance of autophagy for this infection it is still unclear. To further test the role of optineurin in selective autophagy induced with interferon β, we used Neuro2a KO cell line in which the optineurin deletion was achieved by CRISPR/Cas9 method. Autophagy was detected with the reporter RFP-GFP-LC3 construct, which allows us to monitor the autophagy flux. Autophagosome numbers were increased in KO cells during basal autophagy and upon induction with IFN-β. Western blot analysis showed that lack of optineurin does not cause a complete block in autophagy. A partial block was detected as a tendency of accumulation of p62 and LC3-II, especially in basal conditions. In conclusion, our results demonstrate that optineurin is not involved in proliferation of F. novicida, leading to better understanding of its life cycle. Furthermore, our results indicate that the lack of optineurin can lead to partial block of autophagy, which could perhaps cause protein aggregation, a main hallmark of neurodegenerative diseases
Rad ne sadrži naslov na drugom jeziku.
No full textOptineurin is a multifunctional ubiquitin (Ub)-binding protein that was proposed to regulate numerous cellular processes including inflammatory signalling. Around 30 mutations in optineurin, which are thought to act by loss-of-function, have been found in patients with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of motor neurons. Because neuroinflammation is one of the earliest and the most consistent neuropathological hallmarks of ALS, we aimed to investigate if the loss of optineurin function leads to heightened neuroinflammation. To assess this, we analysed primary brain cells and tissues from optineurin insufficiency mouse model with C-terminal truncation of Ub-binding region (Optn470T) upon stimulation with inflammatory stimuli and during aging. We showed that optineurin played a key role in activating the Tank binding kinase 1 (TBK1) signalling pathway upon Toll-like receptor (TLR) stimulation and thus upregulated interferon-beta (IFN-β) production in primary neonatal microglia. The repercussions of decreased IFN-β production were seen in diminished activation of the Signal transducer and activator of transcription 1 (STAT1) and disbalance in the expression of several pro- and anti-inflammatory factors including interferon regulatory factor 7 (IRF7), nitric oxide synthetase 2 (NOS2), interleukin 10 (IL-10) and C-XC motif chemokine 1 (CXCL1). Cytokine and chemokine expression was successfully restored upon addition of recombinant IFN-β, suggesting that optineurin-supported IFN-β secretion upon TLR activation is necessary for optimal inflammatory response. Similar phenotype of diminished TBK1 signalling activation has also been found in Optn470T primary neurons. Despite differences in microglial inflammatory response in vitro, similar level of microgliosis was found in wild-type (WT) and Optn470T mice upon short-term systemic LPS application. In contrast, cytokine array analysis of the whole brain lysates from Optn470T mice upon short-term systemic LPS application showed increased production of granulocyte and monocyte chemoattractants. However, Optn470T mice showed diminished infiltration of the peripheral myeloid cells into the brain, suggesting that the role of optineurin in regulation of neuroinflammation in vivo is more complex than expected from the microglial activation patterns in vitro. The analysis of aged Optn470T mice did not show signs of microgliosis and neurodegeneration in comparison to WT control. For these reasons we hypothesize that additional and/or chronic stimuli are needed for manifestation of neurodegenerative disease and/or neuroinflammation in the mouse optineurin insufficiency model.Optineurin je multifunkcionalni ubikvitin (Ub)-vezujući protein za koji je predloženo da regulira niz staničnih procesa, uključujući prijenos signala tijekom upale. U pacijenata s amiotrofičnom lateralnom sklerozom (ALS), neurodegenerativnom bolešću motornih neurona, pronađeno je više od 30 mutacija u optineurinu. Smatra se da te mutacije uzrokuju bolest gubitkom funkcije optineurina. Jedno od najranijih i najkonzistentnijih neuropatoloških obilježja ALS-a je neuroinflamacija. Kako bismo ispitali uzrokuje li gubitak optineurina pojačanu neuroinflamaciju, koristili smo primarne stanice i tkiva izolirane iz mozgova miševa s insuficijencijom optineurina uslijed C-terminalne trunkacije Ub-vezujuće regije (Optn470T) nakon poticanja upale s različitim stimulansima te tijekom starenja. Pokazali smo da optineurin ima ključnu ulogu u aktivaciji signalnog puta TANK-vezujuće kinaze 1 (TBK1) nakon stimulacije receptora sličnih Toll-u (TLR) te da potiče proizvodnju interferona-beta (IFN- β) u primarnim mikroglija stanicama. Smanjena proizvodnja IFN-β rezultirala je smanjenom aktivacijom STAT1 (engl. signal transducer and activator of transcription 1) i poremećajem u ekspresiji nekoliko pro- i anti-inflamatornih gena uključujući interferon regulirajući factor 7 (IRF7), sintetazu dušikovog oksida 2 (NOS2), kemokinski ligand s C-X-C motivom 1 (CXCL1) i interleukin 10 (IL-10). Dodatkom IFN-β, ekspresija citokina i kemokina je uspješno vraćena na razinu WT (engl. wild-type) stanica, ukazujući da je sekrecija IFN-β regulirana od strane optineurina nakon aktivacije TLR-a nužna za optimalni upalni odgovor. Sukladno tome, primarni Optn470T neuroni su pokazali smanjenu aktivaciju TBK1 signalnog puta. Unatoč pronađenim razlikama u primarnim stanicama, akutna sistemska primjena LPS-a rezultirala je jednakim morfološkim znakovima aktivacije mikroglije in vivo. Međutim, analiza citokina u ukupnom lizatu mozga tako tretiranih Optn470T miševa u usporedbi s WT miševima pokazala je povećanu proizvodnju kemokina koji privlače granulocite i monocite na mjesto upale. Usprkos tome, Optn470T miševi su pokazali smanjenu infiltraciju perifernih mijeloidnih stanica u mozak nakon tretmana s LPS-om, što sugerira da je uloga optineurina u regulaciji neuroinflamacije in vivo znatno složenija od njegove uloge u aktivaciji mikroglije in vitro. Nadalje, ostarjeli Optn470T miševi nisu pokazali znakove mikroglioze i neurodegeneracije u odnosu na WT kontrole. Iz tih razloga pretpostavljamo da su u mišjem modelu insuficijencije optineurina za manifestaciju neurodegeneracije i/ili neuroinflamacije potrebni dodatni ili kronični stimulansi
Definiranje karakteristika metamfetamin ovisnog oscilatora u D. melanogaster
No full textKrajem prošlog stoljeća uočeno je da administracija metamfetamina (METH) putem vode za piće izaziva pojavu ritmičnosti kod aritmičnih ţivotinja. Ova pojava ukazuje na prisustvo jedinstvenog cirkadijalnog oscilatora ovisnog o metamfetaminu (MASCO), ali ne i o glavnom cirkadijalnom molekularnom mehanizmu. Iako je od prvog opisa MASCO-a , pa sve do danas provedeno niz eksperimenta na različitim skupinama ţivotinja, anatomska lokacija i točan mehanizam organizacije MASCO-a još uvijek nisu poznati. U dosadašnjim eksperimentima za istraţivanje MASCO-a kao modelni organizmi najviše su korišteni miševi i štakori, no korištenje Vinske mušice (Drosophila melanogaster) uvelike bi olakšalo provedbu eksperimenta zbog jednostavnijeg ţivčanog sustava, lakše genetske manipulacije, kraćeg ţivotnog vijeka i jeftinijeg uzgoja.
U našim eksperimentima proučavan je utjecaj METH-a na ritmičnost mušica, i ispitivanje osnovnih karakteristika MASCO-a. U eksperimentu su korištene mušice divljeg tipa (wt), one s nul mutacijom u dopaminskom transporteru, fumin (fmn) i s nul mutacijom u jednom od glavnih cirkadijalnih gena, period (per01). Lokomotorna aktivnost je najprije mjerena u uvjetima 12 sati svijetla i 12 sati mraka (LD) na standardnoj hrani, a zatim 16 dana u konstantnom mraku (DD) dok su tretirane s 10 μg/mL METH-a. Dobiveni rezultati nisu uspjeli potvrditi postavljene hipoteze da će prisutnost MASCO-a biti vidljiva u promjeni duţine perioda lokomotorne aktivnosti ili u promjeni jačine ritma lokomotorne aktivnosti. Utjecaj tretmana METH-om bio je sličan u wt, fmn i per01 mušica. Buduća istraţivanja u bolje kontroliranim uvjetima mogla bi doprinjeti boljem razumijevanju utjecaja METH-a i cirkadijalnih gena na ritmičnu lokomotornu aktivnost.At the end of the last century it has been noticed that administration of metamphetamine (METH) in drinking water caused rhythmic behavior in arhytmic animals. This phenomenon indicates the presence of a specific metamphetamine sensitive circadian oscillator (MASCO), that is main circadian molecular mechanism independent. Even though many experiments had been conducted since the discovery of MASCO, the right anatomic location and molecular organisation of MASCO remains unknown. In those experiments mice and rats were generally used. Usage of fruit fly (D.melanogaster) would largely improve counduction of experiments because of their simpler nervous system, easier genetic manipulations, short life duration and lower costs.
The main focus of our experiments was the effect of METH on the rythmicity of the flies and on defining the characteristics of MASCO. We used the wild type of flies (wt), flies with a null mutation in dopamine transporter, fumin (fmn) and flies with a null mutation in one of the main circadian genes, period (per01). The fly's locomotor activity was first measured in 12 hours light and 12 hours darkness (LD) on standard food and then, subsequently, 16 days in complete darkness (DD) while being treated with 10 μg/mL METH. Results of the experiments have failed to confirm the main hypotheses, which were that the presence of MASCO will manifest either in changing the length of the period or in changing the power of the rhythm of locomotor acitivity. Effect of the treatment with METH was alike in wt, fmn and per01 flies. Further experiments in more controlled conditions could lead to better understanding of effects of METH and ciracadian genes on rhythmic locomotor activity
Kratkotrajna lokomotorna habituacija i senzitizacija kao oblik jednostavnog učenja kod modelnog organizma vinske mušice Drosophile melanogaster
No full textKratkotrajna lokomotorna habituacija i senzitizacija smatraju se najjednostavnijim oblicima učenja. Iako se smatraju jednostavnim, mehanizam putem kojeg dolazi do tih procesa nije u potpunosti razjašnjen. Habituacija je opisana kao pad u bihevioralnom odgovoru jedinke na opetovano izlaganje bezopasnom blagom podražaju dok je senzitizacija opisana kao rast bihevioralnog odgovora na neugodan jaki podražaj. Cilj ovog rada bio je opisati karakteristike podražaja koji dovode do kratkotrajne lokomotorne habituacije i senzitizacije kod modela vinske mušice Drosophile melanogaster.
Istraživanja su provedena na divljem tipu mužjaka soja Canton S. Mušice su upuhane u staklene cjevčice te su tretirane zračnim udarima različitog intenziteta istog trajanja, te potom zračnim udarima istog intenziteta ali različitog trajanja. Njihova lokomotorna aktivnost zabilježena je pomoću softverskog sustava Loli Track koji omogućuje analizu većeg broja parametara. Parametri koje smo pratili u ovom istaživanju su trajanje aktivnosti vinskih mušica te njihova prijeđena udaljenost nakon izlaganja zračnim udarima.
Dobiveni rezultati sugeriraju kako je kod mušica koje su tretirane zračnim udarima manjeg intenziteta došlo do procesa kratkotrajne lokomotorne senzitizaciju, a one koje su tretirane zračnim udarima jačeg intenziteta habituacije. Nismo uspjeli potvrditi dvije teze. Prvu koja govori kako kod grupe koja je primila stresni mehanički podražaj (nadalje kroz rad ''startle'') brže dolazi do procesa habituacije nego kod one koja prethodno nije primila ''startle'' podražaj. I drugu koja govori kako ponovljeni podražaji istog intenziteta kraćeg trajanja dovode do procesa senzitizacije dok oni istog intenziteta dužeg trajanja do procesa habituacije.Short-term locomotor habituation and sensitization are considered as the simplest forms of learning. Although considered simple, the mechanisms through which these processes occur are not fully understood. Habituation is described as a decline in the behavioral response of an individual due to repeated exposure to a mild stimulus, while sensitizatio is described as a facilitation of a behavioral response to an unpleasant strong stimulus. The aim of this study was to describe the characteristics of stimuli that lead to short-term locomotor habituation and sensitization in the animal model Drosophila melanogaster.
Studies were preformed on wild-type Canton S. males. Flies were blown into glass tubes and treated with air puffs of different intensities but same duration time, and air puffs of same intensity but different duration time. Their locomotor activity was recorded using the Loli Track software system which allows analysis of a number of parameters. The parameters monitored in this study are active time and distance traveled after exsposure to air puffs.
The obtained results suggest that flies which were treated with low intensity air puffs developed sensitization, and those which were treated with high intensity air puffs developed habituation. We have failed to confirm two theses. The first claims that the habituation process occurs faster in the group which recived a startle stimulus than in the one that did not. The other one says that short duration repeated stimuli of the same intensity may lead to sensitization while exsposure to long duration repeated stimuli may lead to habituation
Teratogenic effects of drugs
Get PDFKlinički pokusi na trudnicama se ne provode iz etičkih razloga. Rezultat toga je nedostatak podataka o djelovanju lijekova u trudnoći. Lijekovi mogu proći kroz posteljicu i uzrokovati teratogene nuspojave na embrij/fetus. Tipične manifestacije teratogeneze su smrt fetusa, zastoj rasta, nepravilan razvoj organa te razvoj karcinoma kasnije u životu. Budući da je oko 50% trudnoća neplanirano, postoji velika mogućnost izloženosti fetusa teratogenom lijeku, posebno tijekom prvog tromjesečja. Cilj ovog diplomskog rada je analiza prijava sumnji na teratogeni učinak lijekova na embrij/fetus iz svjetske baze nuspojava VigiBase i europske baze nuspojava EudraVigilance te usporedba podataka s obzirom na prijave iz hrvatske nacionalne baze nuspojava VigiFlow.
Provedena je retrospektivna opservacijska studija prijava sumnji na nuspojave lijekova na embrij/fetus korištenjem podataka iz hrvatske nacionalne baze VigiFlow u organskom sustavu Prirođeni, obiteljski i genetski poremećaji, zaprimljene do ožujka 2018. godine. Podaci su analizirani s obzirom na prijavljene djelatne tvari i prirođene anomalije. Poseban je osvrt dan na sadržaje sažetaka opisa svojstava lijekova (eng. Summary of Product Characteristics, SmPC) te na postojeće dodatne mjere minimizacije rizika (eng. Risk Minimization Measures, MMR) i program prevencije trudnoće (eng. Pregnancy Prevention Programme, PPP).
Prema broju prijava sumnji na nuspojave teratogenih lijekova ističu se lijekovi s djelovanjem na živčani sustav, među kojima antiepileptici: fenitoin, karbamazepin, valproatna kiselina, gabapentin, topiramat i lamotrigin. Od anksiolitika ističe se diazepam. Najčešće prijavljivane kongenitalne anomalije su kardiovaskularne anomalije. Za mnoge prijavljene djelatne tvari sigurnost primjene u trudnoći nije dovoljno istražena te nedostaju dodatna upozorenja u SmPC-u i u uputi o lijeku. Za poboljšanje sigurnosti primjene lijekova u trudnoći i za smanjivanje rizika nastanka kongenitalnih poremećaja uzrokovanih lijekovima, važnu ulogu ima spontano prijavljivanje nuspojava u postmarketinškoj fazi lijeka te konstantan rad na MMR-ovima uključujući i provođenje PPP-a, kao i edukacija zdravstvenih radnika i žena reproduktivne dobi.For ethical reasons, clinical trials on pregnant women are not performed. As a result, there is a lack of data on the effect of drugs on pregnancy. Drugs may pass through the placenta and cause teratogenic side effects on the embryo/fetus. Typical manifestations of teratogenesis are fetal death, growth failure, improper organ development and cancer development later in life. Since about 50% of pregnancies are unplanned, there is a great potential for fetal teratogenic exposure, especially during the first trimester. The objective of the master thesis is to analyze the reports of teratogenic effects of medicinal products on embryo/fetus from global database VigiBase and European database EudraVigilance, as well as to compare with the suspected adverse drug reactions (ADRs) reports from Croatian national database VigiFlow. A retrospective observational study of suspected ADRs reports in system organ class (SOC) Congenital, familial and genetic disorders from Croatian national database VigiFlow, received by March 2018 was performed. Data on ADRs reports were given by HALMED's pharmacovigilance department. Suspected ADRs reports were analyzed with regard to reported active substances and congenital anomalies. A special review is given on Summary of product characteristics (SmPC), additional Risk minimisation measures (RMM) and Pregnancy prevention programme (PPP). According to the number of reported teratogenic side effects, medicinal products with effect on nervous system stand out, especially antiepileptics such as phenytoin, carbamazepine, valproic acid, gabapentin, topiramate and lamotrigine. Of the anxiolytics, diazepam is noted. The most commonly reported congenital anomalies are cardiovascular anomalies. For many reported active substances, safety in pregnancy has not been adequately investigated and there are no additional warnings in SmPC and package leaflets (PL). To improve the safety of drug use in pregnancy and reduce the risk of congenital drug-induced disorders, spontaneous reporting of side effects in postmarketing phase of medicinal product and the constant work on MMRs, including the implementation of PPP, as well as education of health workers and women of reproductive age play an important role
Effect of thymol on the activity of honey bee acetylcholinestearse
No full textPad broja pčelinjih zajednica postao je globalni problem, a jedan od glavnih krivaca je grinja Varroa destructor. To je relativno novi parazit, koji može uzrokovati smrt cijele pčelinje zajednice ako se ne primjene neka od sredstava ili metoda koje su dostupne u borbi protiv varooze. Pčelari kao sredstvo borbe protiv varooze najčešće upotrebljavaju sintetičke tvari, no zbog štetnih učinaka i sve češće rezistentnosti potiče se upotreba prirodnih spojeva s akaricidnim djelovanjem.
Monoterpenski derivat timol, koji se nalazi u sastavu različitih eteričnih ulja dobivenih iz biljaka koje pripadaju obitelji Lamiaceae, je pokazao akaricidno djelovanje. Stoga je cilj ovog istraživanja bio ispitati utjecaj timola na prehranu, smrtnost te enzimatsku aktivnost u mozgu odraslih pčela. Mjereni su biomarker neurotoksičnosti acetilkolinesteraza (AChE) i detoksifikacijski enzim glutation S-transferaza (GST). Prvo je provedeno preliminarno istraživanje s kojim su se odredile koncentracije timola koje će se ispitivati, a zatim se provelo glavno istraživanje s koncentracijama timola od 0.05%, 0.5% i 1% w/w. Preliminarni i glavni pokus trajali su 7 dana, pčele su se čuvale u drvenim kavezima, u inkubatoru te su bile izložene timolu oralno. Tijekom preliminarnog pokusa imali smo dva kaveza, a za vrijeme glavnog pokusa tri kaveza za svaku od koncentracija i kontrolu. Tijekom trajanja jednog i drugog pokusa, svakih 24h mjerila se količina pojedene hrane i broj uginulih pčela. Nakon 7 dana, pčele su bile secirane te su za analizu uzete glave u kojima je izmjerena enzimatska aktivnost GST i AChE. Rezultati našeg istraživanja pokazali su da primjena timola ne utječe na stupanj prehranjivanja pčela te da je u masenom omjeru do 0.5% sigurna za pčele što se tiče preživljavanja. Rezultati vezani za enzimatsku aktivnost AChE i GST su dvojaki s obzirom na to da ako usporedimo preliminarni i glavni pokus postoji razlika u aktivnosti tih enzima pri istim koncentracijama timola. No, pokazali smo da timol ima utjecaj na aktivnost AChE i GST kod pčela i to već pri 0.05% w/w iz čega se može zaključiti da timol utječe na kolinergični sustav pčela te da je došlo do detoksifikacije.The losses of bee colonies has become a global problem, and one of the main reasons is the mite, Varroa destructor. It is a relatively new parasite which can cause the death of the whole bee colony if honeybees are not treated against Varroa. Beekeepers mostly use synthetic substances, but because of their harmful effects and increasingly frequent resistance, the use of natural compounds with acaracidal activity is promoted.
Thymol is a monoterpene found in the essential oils obtained from plants belonging to the Lamiaceae family. It was found to possess acaracidal activity. Therefore, the aim of this study was to examine the influence of thymol on the feeding rate, mortality and enzymatic activity in the brain of adult bees. The activity of the two enzymes, the neurotoxic biomarker acetylcholinesterase (AChE) and the antioxidant glutathione S-transferaze (GST) was measured. Firstly, we conducted preliminary research to determine thymol concentrations and then we performed the main experiment with thymol concentrations of 0.05%, 0.5% and 1% w/w. Preliminary and main trial lasted for 7 days, bees were kept in wooden test cages, in the incubator and were exposed to the thymol orally. During the preliminary experiment we had two replicates for each treatment, and during the main trial three repeats for each concentration and control were conducted. During both experiments, dead honeybees were counted and removed every 24h and the mass of the consumed food was measured. After 7 days, the bees were sectioned and the enzymatic activity of GST and AChE was measured in their heads.
The results of our research have shown that the exposure to thymol has no effect on honeybees feeding rate and that in concentrations up to 0.5% is safe for bees in terms of survival. Results related to the enzymatic activity of AChE and GST are inconsistent; by comparing the preliminary and main experiment there is a difference in the activity of these enzymes at the same concentrations of thymol. However, we have shown that thymol has an effect on AChE and GST activity in bees at 0.05% w/w, from which it can be concluded that thymol affects the cholinergic bee system and that detoxification has occurred
Uloga integraze u sazrijevanju HIV-1
Get PDFIntegrase (IN) is one of the most important enzymes in the replication of the HIV-1 virus, as it integrates viral DNA (vDNA) into the genome of the host cells. However, recent studies have suggested a new surprising role of IN in HIV-1 maturation. If IN is inhibited either by mutations or by allosteric integrase inhibitors (ALLINIs), viral ribonucleoprotein complexes (vRNPs) are often eccentrically localized outside of the capsid (CA). Such viral particles are non-infectious and are blocked at an early stage of the reverse transcription in the target cells, as a result of premature degradation of IN and viral RNA (vRNA) genome, together with the spatial separation of reverse transcriptase (RT) from vRNA.
IN directly binds the vRNA genome in virions by its surface exposed lysine residues, exhibiting a distinct preference for selected vRNA structural elements. In this way, IN provides nucleation points by bridging separate RNA molecules, promoting the effective compaction of vRNPs. These interactions then ensure the proper packaging of vRNPs inside the CA.
Even though the mechanism of action of ALLINIs, an emerging class of anti-HIV drugs, is still not fully understood, it seems that they mostly act by blocking the IN-vRNA interaction, which results in aberrant non-infectious particles.
This non-catalytic role of IN enables the understanding of the HIV-1 maturation process in greater detail, which can be successfully used in the development of new drugs, such as ALLINIs. However, a lot of research still needs to be done, as there is a lot of detail in this IN-driven maturation process that still needs to be explained.Integraza (IN) je jedan od najvažnijih enzima u replikaciji HIV-1 virusa, budući da integrira virusnu DNA (vDNA) u genom stanice domaćina. Međutim, novije studije su predložile novu, iznenađujuću ulogu IN u sazrijevanju HIV-1. Ukoliko je IN inhibirana bilo mutacijama bilo alosteričkim inhibitorima integraze (ALLINI-ji), virusni ribonukleoproteinski kompleksi (vRNP-ovi) su često acentrično lokalizirani izvan kapside (CA). Ove čestice nisu infektivne i blokirane su u ranoj fazi reverzne transkripcije u ciljnim stanicama, kao rezultat preuranjene degradacije IN i virusnog RNA (vRNA) genoma, zajedno s prostornom odvojenošću reverzne transkriptaze (RT) od vRNA.
IN izravno veže vRNA u virionima putem svojih površinski izloženih lizinskih ostataka, pokazujući izrazite preference za određene vRNA strukturne elemente. Na ovaj način IN stvara nukleacijske točke, premošćujući odvojene RNA molekule i unaprjeđujući učinkovito zbijanje RNP-ova. Ove interakcije osiguravaju ispravno pakiranje vRNP-ova unutar
CA.
Iako mehanizam djelovanja ALLINI-ja, skupine anti-HIV lijekova u nastajanju, još uvijek nije do kraja razjašnjen, čini se da oni većinski djeluju blokirajući IN-vRNA interakcije, što rezultira abnormalnim neinfektivnim česticama.
Ova nekatalitička uloga IN omogućava detaljnije shvaćanje procesa sazrijevanja HIV-1, što se može uspješno upotrijebiti u razvoju novih lijekova poput ALLINI-ja. Međutim, potrebno je provesti još mnogo istraživanja jer puno detalja u ovom IN potpomognutom procesu sazrijevanja još uvijek mora biti razjašnjeno
Technology transfer in pharmaceutical industry
Get PDFSvjetska populacija suočava se sa sve većim starenjem stanovništva i povećanim brojem kroničnih oboljenja. Spas za to predstavlja razvitak novih lijekova i terapija. Farmaceutska industrija i njena dostignuća danas stoga predstavljaju ključ za poboljšanje zdravlja i kvalitete života na globalnoj razini. S obzirom na kompleksnost procesa kojim novi lijek dolazi na tržište potrebna je suradnja više različitih industrijskih sektora, a kako bi se cjelokupni proces olakšao i ubrzao provodi se farmaceutski transfer tehnologije.
U farmaceutskoj industriji, jednom od najrazvijenijih industrijskih sektora uopće, transfer tehnologije predstavlja proces prijenosa znanja i tehnologija potrebnih za razvitak novog lijeka s ciljem uspješnog dolaska na tržište. Unatoč čestim izazovnim poslovnim i regulatornim uvjetima pri provedbi istog i s investicijama znatno rizičnijim nego u ostalim industrijskim sektorima, farmaceutska industrija ulaže milijarde dolara i tisuće sati rada educiranog radnog kadra, potičući pritom medicinski napredak s ciljem poboljšanja kvalitete života.
Cilj ovog rada je stoga objasniti pojam i proces transfera tehnologije u farmaceutskoj industriji i objasniti temeljne čimbenike koji uvjetuju konačan uspjeh. U svrhu jasnijeg tumačenja procesa dani su primjeri uspješnih transfera tehnologije u farmaceutskoj industriji. Konačno, provedena je i analiza trenutnog stanja u svijetu s fokusom na Europu kao potvrda teorijskom dijelu i postavljenim hipotezama.The world population is facing the growth of rapidly aging population and an increased number of chronic illnesses. Development of new drugs and therapies present one of the answers for this situation. Pharmaceutical industry and its achievements are crucial in health and quality of life improvement globally. Given the complexity of the process by which a new drug comes into the market, a collaboration between various different industrial sectors is needed. In order to facilitate and accelerate the overall process of drug development and marketing a pharmaceutical technology transfer is implemented.
In the pharmaceutical industry, as one of the most advanced industrial sectors in general, technology transfer is the process of transferring the knowledge and technology needed to develop and place a new drug to the market. Despite the often very challenging business and regulatory conditions needed for successful technology transfer, and with considerably high risks of investments the pharmaceutical industry is investing billions of dollars and thousands of working hours of educated personnel while encouraging medical progress with the purpose of improving the quality of life.
Therefore, the aim of this thesis is to explain the concept and process of technology transfer in the pharmaceutical industry while explaining the underlying factors that make the final success. For the purpose of clear interpretation of the whole process, examples of successful technology transfer case studies in the pharmaceutical industry are given. An analysis of the current situation in the world with the focus on Europe was conducted in order to confirm both theoretical and analytical part, and defined research hypotheses
Chemical composition and antioxidant effect of dog rose essential oil (Rosa canina L.)
No full textRosa canina L. (Rosaceae) jedna je od najraširenijih divljih vrsta roda Rosa. Poznato je bogatstvo ploda R. canina raznim bioaktivnim spojevima kao što su vitamini (posebno vitamin C), fenolni spojevi (osobito flavonoidi), karotenoidi i masne kiseline, s potencijalnim pozitivnim učinkom na ljudsko zdravlje. Cilj ovog istraživanja bio je analizirati hlapljive spojeve ploda R. canina podrijetlom iz Hrvatske. Hlapljivi spojevi zajedno s nehlapljivim spojevima odgovorni su za okus i miris plodova.
Hidrodestilacija je najčešće korištena metoda za izolaciju eteričnih ulja, tj. hlapljivih spojeva. Izolacija hlapljivih spojeva iz ploda R. canina provedena je destilacijom u modificiranoj aparaturi po Clevengeru. U ovom radu, za izolaciju hlapljivih spojeva korištena su tri uzorka: cijeli plod, zasebno sjemenke i zasebno mesnati dio.
Kemijski sastav eteričnih ulja analiziran je plinskom kromatografijom-masenom spektrometrijom (GC-MS). U eteričnom ulju R. canina dobivenom iz cijelog ploda šipurike prevladavao je norizoprenoid vitispiran, a zatim keton 6-metilhept-5-en-2-on. Vitispiran je također identificiran u mesnatom dijelu R. canina, ali u znatno manjoj količini, dok je sadržaj 6-metilhept-5-en-2-ona bio gotovo isti kao u cijelom plodu. Ostale kvantitativno važne komponente u ovom uzorku su C9 - C22 ugljikovodici, te aldehidi heks-2-enal, furfural i hept-2-enal. Eterično ulje izolirano iz sjemenki R. canina obilježeno je visokim sadržajem fenilacetaldehida, srednjim sadržajem benzil-izotiocijanata, te odsustvom vitispirana i 6-metilhept-5-en-2-ona. Što se tiče terpena, tipičnih sastojaka eteričnih ulja, monoterpen limonen je dominirao u svim eteričnim uljima.
Antioksidacijska aktivnost dobivenih uzoraka ispitana je korištenjem DPPH (2,2-difenil-1-pikrilhidrazil) metode. Utvrđena je vrlo slaba antioksidacijska aktivnost (≤12,42 %) svih testiranih uzoraka. Slaba antioksidacijska svojstva eteričnih ulja mogu se pripisati njihovom kemijskom sastavu.Rosa canina L. (Rosaceae) is one of the most widespread wild species of the genus Rosa. It is well known that rosehips of R. canina are rich source of various bioactive compounds such as vitamins (especially vitamin C), phenolic compounds (especially flavonoids), carotenoids and fatty acids, with potential positive effect on human health. The aim of this study was to characterize the volatile compounds of R. canina hips originating from Croatia. The volatile compounds together with non-volatile compounds are responsible for taste and for the flavour of fruits.
Hydrodistillation is the most used method for the essential oils isolation, i.e. volatile compounds. The isolation of volatile compounds from R. canina fruits was performed by distillation in modified Clevenger type apparatus. In this study, three samples were used for the isolation of volatiles: the entire rosehips, seeds only and fleshy pulp only.
The chemical composition of the essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS). The dominating compound in R. canina essential oil, obtained from entire rosehips, was norisoprenoid vitispirane followed by ketone 6-methylhept-5-en-2-one. Vitispirane was also identified in R. canina fruit pulp, but in much smaller amount, while the content of 6-methylhept-5-en-2-one was almost the same like in entire rosehips. Other quantitatively important components in this sample were C9 – C22 hydrocarbons, and aldehydes hex-2-enal, furfural and hept-2-enal. The essential oil isolated from R. canina seeds was characterized by a high content of phenylacetaldehyde, moderate content of benzyl isothiocyanate and absence of vitispirane and 6-methylhept-5-en-2-one. Regarding terpenes, the typical constituents of essential oils, monoterpene limonene was dominated component in all essential oils.
The samples were tested on the antioxidant activity using DPPH (2,2-Diphenyl-1-picrylhydrazyl) method. The antioxidant activity of all tested samples was very low (≤12,42 %). Low antioxidant properties of essential oils can be attributed to its chemical composition