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Ozone therapy
No full textTerapija ozonom (ozonoterapija) je terapija koja se već dugo proučava i primjenjuje. Ozon je bezbojni plin oštra mirisa, alotropske modifikacije kisika (O3) koji prirodno nastaje u atmosferi (stratosferi i troposferi). U medicini, terapija ozonom koristi se u svrhu dezinfekcije i liječenja stanja, dezinficirajući područje oko njega i poboljšavajući na taj način unos kisika i imunosni sustav. Svojim snažnim oksidacijskim djelovanjem dokazano ima koristan učinak u svrhu dezinfekcije i liječenja raznih patoloških stanja jer poboljšava unos kisika. Primjenjuje se u liječenju dermatoloških, zaraznih i nerodegenerativnih bolesti, bolesti krvožilnog sustava, u liječenju nekih vrsta raka i ortopediji. Osim primjene u medicini, ozon se primjenjuje u prehrambenoj industriji, zaštiti okoliša (sanitacija prostora, obrada otpada i otpadnih voda).
Usprkos mnogobrojnoj primjeni, s ozonom treba biti na oprezu zbog njegove toksičnosti. Svrha ovoga rada bila je prikazati karakteristike ozona, njegovu primjenu, rezultate terapija u kojima je njegova učinkovitost potencijalna te opisati mehanizam njegova terapeutskog djelovanja.Ozone therapy is a therapy that has been studied and used for a long time. Ozone is a colourless gas with a pungent odour, an allotropic modification of oxygen (O3) that occurs naturally in the atmosphere (stratosphere and troposphere). In medicine, ozone therapy is used to disinfect and treat diseases, disinfecting the environment to improve oxygen uptake and the immune system. With its strong oxidizing effect, it has been proven to have a beneficial effect for the purpose of disinfection and treatment of various pathological conditions as it improves oxygen uptake. It is used in the treatment of dermatological, infectious and non-degenerative diseases, diseases of the vascular system, in the treatment of some cancers and in orthopaedics. In addition to medical applications, ozone is also used in the food industry and in environmental protection (hygiene, waste and wastewater treatment).
Despite its many uses, caution should be exercised with ozone because of its toxicity. The purpose of this work was to present the properties of ozone, its application, the results of therapies in which its effectiveness is possible and to describe the mechanism of its therapeutic action
Development of LC-QQQ method for analysis of phenolic components and methods for determination of antioxidant potential in honey samples
No full textMed je kompleksni prirodni proizvod koji se od davnina koristi zbog svog blagotvornog djelovanja na zdravlje ljudi. Sastav meda uvelike ovisi o botaničkom i geografskom porijeklu, klimatskim uvjetima te uvjetima skladištenja. Med je bogat fenolnim komponentama koje djeluju kao prirodni antioksidansi i posljednjih godina dobivaju posebnu pozornost zbog potencijalnog blagotvornog učinka na ljudsko zdravlje. Osim toga, fenolne komponente mogu biti korištene kao biomarkeri biljnog i geografskog porijekla meda. Iz tog razloga, glavni cilj ovog diplomskog rada je razviti spektrofotometrijske metode za analizu fenola i antioksidativne aktivnosti te metodu na bazi spektrometrije masa za apsolutnu kvantifikaciju individualnih fenola. Svaka metoda testirana je na 6 različitih uzoraka medova. Za ispitivanje antioksidativne aktivnosti, korištene su ABTS (2,2'-azino-bis(3-etilbenzotiazoline-6-sulfonska kiselina)) i DPPH (2,2-difenil-1- pikrilhidrazil) metode. Ispitana je korelacija između navedenih metoda i uočena je pozitivna korelacija (R2=0,8643) između ukupnih fenola i ukupnih flavonoida (R2=0,7630) i antioksidativne aktivnosti analizirane ABTS metodom. Uspješno je razvijena metoda ekstrakcije fenolnih komponenti iz meda koristeći ekstrakciju na čvrstoj fazi. Također je uspješno razvijena LC-QQQ metoda za apsolutnu kvantifikaciju 12 fenolnih komponenti u uzorcima medova te su metodom dodatka standarda prije ekstrakcijskog postupka minimalizirani efekt matriksa i gubitak dijela fenola radi odabranog ekstrakcijskog postupka. Razvijene metode pokazale su se djelotvorne bez obzira na vrstu meda te su pokazale značajan napredak u kvantitativnoj analizi fenola budući je smanjen negativan efekt komponenti na ionizaciju uzorka.Honey is a complex natural product that has been used since ancient times because of its beneficial effects on human health. The composition of honey depends on the botanical and geographical origin, climate and storage conditions. Honey is rich in phenolic compounds that can act as natural antioxidants and have been of particular interest in recent years due to their potential positive effects on human health. In addition, phenolic compounds can be used as biomarkers of honey's plant and geographical origin. The aim of this thesis is the development of spectrophotometric methods for the analysis of phenolic compounds in honey and determination of their antioxidant activity. Additionally, methods based on mass spectrometry for absolute quantification of individual phenols were developed. Each method was tested on six different honey samples. The ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) methods were used to test the antioxidant activity of honey. A positive correlation of total phenols (R2=0,8643) and flavonoids (R2=0,7630) with the antioxidant activity determined using the ABTS method was observed. Furthermore, the methods for extraction of phenolic compounds from honey samples using solid phase extraction (SPE) and LC-QQQ for absolute quantification of twelve phenolic compounds were developed. Matrix effect and partial loss of phenolic compounds due to the selected extraction procedure were minimized with the standard addition method in which a mixture of phenolic standards was added before the extraction procedure. The developed methods proved to be effective regardless of the type of honey and showed significant progress in the quantitative analysis of phenolic compounds in honey
Identification of cortex cells during postnatal development of M. domestica
No full textLjudski mozak karakterizira izuzetno razvijeni korteks, u kojem se nalazi 170 milijardi stanica. Broj neurona i glija stanica SŽS-a opada s fiziološkim starenjem zbog progresivnog smanjenja regeneracijskog kapaciteta progenitorskih stanica. Obzirom da je proces neuroregeneracije vrlo biološki sličan razvojnim procesima, istraživanja kortikalnog razvoja na životinjskim modelima predstavljaju obećavajući pristup u regenerativnoj medicini. Laboratorijski model oposuma, Monodelphis domestica posjeduje
mogućnost regeneracije SŽS-a tijekom prva 2 tjedna života te se stoga smatra izrazito pogodnim za neuroregenerativne studije. Stanična karakterizacija i lokalizacija postnatalnog oposuma je nedovoljno istražena, te su jedini dostupni literaturni podaci o ukupnom broju stanica i neurona kvantificirani na dobi starijoj od postnatalnog dana (P) 18. Primjenom metode izotropnog frakcionatora (IFR) i fluorescentne mikroskopije proveli smo istraživanje kvantifikacije apsolutnog broja stanica, broja neurona i
„ne-neuralnih“ stanica na korteksima oposuma različitih postnatalnih dana 6, 18 i 34. Potvrdili smo vremenski period u kojem dolazi do procesa neurogeneze (P18) čiji se završetak nadopunjava s izražajem procesa gliogeneze (P18-P34). Pomoću IHC metode i fluorescentne mikroskopije lokalizirali smo heterogene stanične populacije korteksa oposuma postnatalnih dana 6, 17
i 30. Koristili smo imunohistokemijske markere SOX2, SOX9, GFAP i NeuN koji su nam ukazali na različitu lokalizaciju i proces migracije stanica tijekom kortikogeneze. Marker matičnih stanica SOX2 i marker nezrelih astrocita SOX9 imunoobilježili su stanice ventrikula. Uočili smo migraciju SOX9+ stanica prema vanjskom dijelu korteksa pri P30. Marker GFAP ukazao nam je na lokalizaciju zrelih astrocita pri P30 po cijelom području korteksa.The human brain is characterized by an extremely developed cortex, which contains 170 billion cells. The number of CNS neurons and glial cells decreases with physiological aging due to a progressive decrease in the regenerative capacity of progenitor cells. Since the process of neuroregeneration is biologically very similar to the developmental processes, studies of cortical development in animal models represent a promising approach in regenerative medicine. A laboratory model of the opossum, Monodelphis domestica possesses the ability to regenerate the CNS during the first 2 weeks of life and is therefore considered extremely suitable for neuroregenerative studies. Cellular characterization and localization of the postnatal opossum are insufficiently investigated, and the only available data on the total number of cells and neurons is that quantified at the age older than a postnatal day (P) 18. Using the isotropic fractionator (IFR) method and fluorescence microscopy, we quantified the number of neuronal and “non-neuronal” cortical cells of different postnatal opossums aged 6, 18, and 34 days. We confirmed the time period in which the neurogenesis process (P18) occurs, and the completion of which is succeeded by the gliogenesis process (P18 – P34). With the IHC method and fluorescence microscopy, we localized heterogeneous cell populations of the opossum cortex of postnatal days 6, 17, and 30. We used immunohistochemical markers SOX2, SOX9, GFAP, and NeuN which indicated different localization and cell migration during corticogenesis. The SOX2 stem cell marker and the SOX9 immature astrocyte marker showed a localization at the ventricular zone. We observed migration of SOX9 + cells toward the outer cortex at P30. The GFAP marker indicated the localization of mature astrocytes at P30 throughout the cortex
Genetsko preklapanje opsesivno-kompulzivnog poremećaja i depresije
No full textObsessive-compulsive disorder and major depressive disorder are among the most common mental disorders globally and are frequently co-diagnosed. They are both highly complex, heterogenous and a result of both environmental and genetic factors. This review focuses on the genetics behind these disorders and their overlap. When researching OCD, the genes that have most commonly been associated with the disorder are ones involved in glutamate, serotonin, and dopamine transmission. Such genes include SLC1A1, DLGAP3 and DRD4. In MDD genetics, genes related to neurotransmission, like TPH2, are also relevant but so are genes involved in several other biological pathways, like BDNF, LHPP, SIRT1 and NEGR1 which are involved in mitochondrial biogenesis, cell adhesion or neuronal survival. When researching the overlap between OCD and MDD, common mechanisms, shared genes and treatment effects must all be taken into consideration. Besides irregularities in neurotransmission, reduced amygdala and hippocampal volumes have also been observed in both OCD and MDD patients, as well as abnormalities in the regulation of the HPA axis. That implies shared genes between the two disorders may be involved in those mechanisms. 5- HTTLRP, a functional polymorphism of the SLC6A4 gene has most consistently been associated with both disorders and it affects the serotonin transport. Another gene relevant to both OCD and MDD involved in the same pathway is HTR2A, while FKBP5 is a gene affecting HPA axis regulation and shows great promise in researching the overlap of these disorders. The shared treatment effects, such as SSRIs and neuromodulation therapy, further confirm the idea of shared genes. These and future findings about the genetic overlap between OCD and MDD will help develop better and more effective treatment for people suffering from both these disorders.Opsesivno-kompulzivni poremećaj i depresivni poremećaj među najčešćim su mentalnim poremećajima na globalnioj razini, te su često zajedno dijagnosticirani. Oboje su vrlo kompleksni, heterogeni poremećaji koji nastaju kao rezultat okolišnih i genetičkih faktora. Ovaj se rad fokusira na genetiku oba ova poremećaja i njihovo preklapanje. Pri istraživanju OKP-a, geni najčešće povezivani s ovim poremećajem su oni vezani uz prijenos glutamata, serotonina i dopamina. Neki od tih gena su SLC1A1, DLGAP3 I DRD4. U genetici depresivnog poremećaja, izuzev gena vezanih za neurotransmisiju, poput TPH2, također su relevantni i geni vezani za druge biološke puteve poput mitohondrijalne biogeneze, adhezije stanica i neuronalnog preživljavanja. Neki od tih gena su BDNF, LHPP, SIRT1 i NEGR1. Prilikom istraživanja preklapanja ova dva poremećaja, zajednički mehanizmi, geni i efekti liječenja moraju biti uzeti u obzir kako bi dobili što bolju predodžbu. Izuzev nepravilnosti u neurotransmisiji, smanjen volumen amigdale i hipokampusa uočen je kod pacijenata oboljelih od oba poremećaja, kao i abnormalnosti u regulaciji HPA osi. To ukazuje kako je vjerojatno da će zajednički geni biti oni vezani za te mehanizme. 5-HTTLPR, funkcionalni polimorfizam SLC6A4 gena koji djeluje na transport serotonina najčešće je povezivan s oba poremećaja. HTR2A je također gen koji djeluje na isti mehanizam, dok je FKBP5 gen povezan s regulacijom HPA osi te se smatra vrlo obećavajućim za buduća istraživanja. Slični pristupi liječenju, poput SIPPS-a i neuromodulacije, dodatno potvrđuju ideju o postojanju zajedničkih gena između OKP-a i depresivnog poremećaja. Ova I buduća saznanja o genetskom preklapanju ova dva poremećaja pomoći će razvitku novih i učinkovitijih lijekova za osobe koje boluju od OKP-a i depresivnog poremećaja
Antioxidant activity and stability of 6-amino-6-deoxy-L-ascorbic acid and N-methyl-6-amino-6-deoxy-L-ascorbic acid and their potential use in the cosmetic industry
No full textL-askorbinska kiselina (vitamin C) i njeni derivati imaju važnu fiziološku funkciju u ljudskom organizmu. Uz upotrebu kao dodatak prehrani, na tržištu postoji mnoštvo topikalnih formulacija L-askorbinske kiseline i derivata koji potiču sintezu kolagena, učvršćuju kožu, spriječavaju i djeluju povratno na promjene uzrokovane hiperpigmentacijom te općenito poboljšavaju izgled kože. U kozmetičkim se proizvodima također koristi kao antioksidans, podešivač pH vrijednosti te fotoprotektivni agens. Zbog osjetljivosti na vanjske čimbenike poput temperature, prisutnosti kisika, svjetlosti i pH, L-askorbinska se kiselina u formulacijama stabilizira agensima poput vitamina E i ferulične kiseline. Uz kemijsku stabilizaciju, važna je također stroga kontrola uvjeta tijekom formulacije (razina kisika, pH, i sl.). Osim kontrole uvjeta proizvodnje i dodatka stabilizirajućih agenasa, kemijske su modifikacije molekule dovele do razvoja stabilnih derivata poput natrijevog askorbil fosfata i magnezijevog askorbil fosfata. Iako su navedeni derivati stabilniji od L-askorbinske kiseline, nedostaje im snažniji farmakološki učinak. U ovom radu ispitana je antioksidativna aktivnost i stabilnost novosintetiziranih derivata 6-amin-6-deoksi-L-askorbinske kiseline i N-metil-6-amin-6-deoksi-L-askorbinske kiseline, kemijskim metodama ABTS i DPPH. Uz navedeno, ispitani su optimalni uvjeti skladištenja te je MS/MS analizom određena pH vrijednost pri kojoj derivati zadržavaju stabilnost. Rezultati su pokazali da novosintetizirani derivati posjeduju antioksidativnu aktivnost usporedivu s L-askorbinskom kiselinom te su stabilni u kiselom mediju tijekom 30 dana. Potvrdom antioksidativne aktivnost i stabilnosti derivata, napravljen je temelj za daljnja biološka in vitro ispitivanja.L-Ascorbic acid (vitamin C) and its derivatives perform important physiological functions in the human organism. Except dietary supplements, numerous topical formulations containing L-ascorbic acid and its derivatives are available on the market. These formulations induce collagen synthesis, strengthening of skin tissue, reduction in hyperpigmentation spots and improvement of overall skin health. L-ascorbic acid has also been used as antioxidant, pH adjuster and photoprotecting agent in cosmetic products. Due to it's sensitivity to external factors such as temperature, presence of oxygen, light an pH, L-ascorbic acid is combined with stabilizing agents such as vitamin E and ferulic acid. Along chemical stabilization, strict control of production conditions is required (oxygen level, pH, etc.). Except controlling of production conditions and adding stabilizing agents, chemical modifications of this molecule have led to stable derivatives development. Well known L-ascorbic acid derivatives, such as magnesium ascorbyl phosphate and sodium ascorbyl phosphate are more stable than L-ascorbic acid but they lack the pharmacological activity of L-ascorbic acid. In this thesis, the antioxidant activity and stability of 6-amino-6-deoxy-L-ascorbic acid and N-methyl-6-amino-6-deoxy-L-ascorbic acid have been examined using ABTS and DPPH assays. In addition, optimal storing conditions, as well as the pH at which new synthesised derivatives maintain the stability, were determined using mass sepctrometry. Results have shown the similarity in antioxidative activity between the new synthesised derivatives and L-ascorbic acid. In addition, these derivatives showed good stability in acidic medium during 30 days. By proving the antioxidative activity and antioxidative stability of synthesised L-ascorbic acid derivatives, we made a base for a further biological tests
Uloga DTNBP1/dysbindin-1 u shizofreniji
No full textSchizophrenia is complex neurological disease that is present in 1% of the general population. It is known as a multifactorial disease because of the simultaneous influence of genetic and environmental factors. Some of the symptoms that are recognized are hallucinations, delusions, loss of energy and motivation and cognitive dysfunctions like working memory dysfunction and dysfunctions in learning. A major problem in treating schizophrenia is its heterogeneity and patients do not respond the same to the same treatment. There is also little to no effect in treating cognitive dysfunction with available medications. We still have limited knowledge of the core of pathology of disease and more research are needed to find the best treatment. In the past years lots of research was performed on genetic risk factors for schizophrenia, and one of the most researched genetic factors is the DTNBP1 gene. The DTNBP1 gene encodes for dysbindin-1 protein. In the brain, dysbidnin-1 is mostly expressed in the hippocampus and prefrontal cortex. Studies on post-mortem schizophrenia cases found that there is a decrease in dysbindin-1 expression in brain. These suggest the involvement of dysbindin-1 in the pathology of disease. Dysbindin-1 is known to be a component of the BLOC-1 complex in brain and therefore regulates several functions in the brain including vesicular trafficking following by regulation of neurotransmitter release, neural growth, and transcriptional regulation. Studies on “sandy” mice, with a mutation in the Dtnbp1 gene also show promising results in its involvement in developing schizophrenia-like symptoms. Researching dysbindin-1 and other genetic risk factors functions in the brain and their binding partners is important in finding new potential targets for future treatment of schizophrenia.Shizofrenija je složena neurološka bolest koja je prisutna u 1% opće populacije. Poznata je kao multifaktorijalna bolest zbog istodobnog utjecaja genetskih čimbenika i čimbenika okoliša. Neki od simptoma koji definiraju ovu bolest su halucinacije, zablude, gubitak energije i motivacije te kognitivne disfunkcije poput disfunkcije pamćenja i disfunkcije u učenju. Glavni problem u liječenju shizofrenije jest njena heterogenost i pacijenti ne reagiraju jednako na isti tretman. Liječenje dostupnim lijekovima je također slabo učinkovito na poboljšanje kognitivnih funkcija. Još uvijek postoji ograničeno znanje o srži patologije bolesti i potrebno je više istraživanja kako bi se pronašlo najbolje liječenje. U posljednjih nekoliko godina provedeno je mnogo istraživanja za otkrivanje genetskih čimbenika, a jedan od najistraženijih genetskih čimbenika je gen DTNBP1. Disbindin-1 se najviše eksprimira u hipokampusu i prefrontalnom korteksu. Studije na postmortalnim slučajevima shizofrenije otkrile su da dolazi do smanjene ekspresije disbindin-1 proteina u mozgu. Zbog tih rezultata sugerira se utjecaj disbindina-1 u patologiji bolesti. Poznato je da je disbindin-1 komponenta BLOC-1 kompleksa u mozgu i stoga regulira nekoliko funkcija u mozgu, uključujući transport vezikula, što direktno utječe na regulaciju oslobađanja neurotransmitera. Također regulira razvoj neurona i transkripciju. Studije na "sandy" miševima s mutacijom na genu Dtnbp1 također pokazuju obećavajuće rezultate u njegovoj ulozi u razvoju simptoma sličnih shizofreniji. Istraživanje disbindina-1 i drugih genetskih čimbenika te njihovih funkcija u mozgu i proteina za koje se vežu, važno je u pronalaženju novih potencijalnih meta za buduće liječenje shizofrenije
Synthetic, structural and stability studies in the series of thyreostatic methimazole
No full textU sklopu ove disertacije provedene su sintetske, strukturne i stabilitetne studije u redu
tireostatika metimazola (1). Primjenom kemo- i regioselektivnih kemijskih transformacija
pripremljene su nove, ali i neke poznate srodne supstancije metimazola, tj. njegova poznata ali
i nepoznata potencijalna onečišćenja. Posebno je studirana stabilnost metimazola u kloriranim
otapalima poput diklormetana i dikloretana, sveprisutnih u kemiji i farmaceutici. Dobiveni
premošteni bis metilenski i etilenski derivati su izolirani i strukturno karakterizirani, a
provedena je karakterizacija i transformacija njihovih čvrstih formi. U okviru toga poseban je
naglasak stavljen na utvrđivanje kemizma stvaranja 1,2-bis[(1-metil-1H-imidazol-2-il)tio]etana
(3a) koji uključuje pokušaj detekcije i izolacije možebitnog tiranijevog (episulfonijevog)
međuprodukta (8). Temeljem provedenih sintetskih, spektroskopskih, termalnih, difrakcijskih
i računalnih studija, uključujući i mjerenja kinetike, međuprodukt 8 nije pronađen već njegov
izomer 7-metil-2H,3H,7H-imidazol[2,1-b]tiazol-4-ijum klorid (7a), a prema našim saznanjima,
to je prvi poznati stabilni izomer tiiranijevih iona. Ujedno je utvrđeno da 3a nastaje direktno
nukleofilnom supstitucijom primarno stvorenog međuprodukta klor etil derivata, 2-
[(kloretil)tio]-1-metil-1H-imidazol, 6) s metimazolom.
Također su pripravljeni i karakterizirani oksidacijski i metilacijski derivati 1 te u kontekstu
poznatih onečišćenja metimazola B i C pripravljeni su i standardizirani njihovi uzorci.
U okviru predformulacijskih istraživanja provedena je studija stabilnosti 1 u čvrstom stanju, a
u prisutnosti odabranih pomoćnih tvari. Pritom je utvrđena visoka stabilnost 1 u planiranim
farmaceutskim formulacijama, ali su pronađeni i karakterizirani novi, dosad nepoznati, dimer i
trimer metimazola, kao njegova potencijalna onečišćenja. Ujedno je i predložen kemizam
njihova stvaranja.
Rezultati provedenog istraživanja upotpunjuju trenutne spoznaje o metimazolu (1) te daje
iscrpan uvid u svojstva njegovih strukturnih analoga, kao potencijalnih onečišćenja 1. To
pridonosi povećanju kakvoće, sigurnosti i učinkovitosti metimazola kao djelatne tvari tako i
njegovih finalnih doznih oblika.Within this dissertation, synthetic, structural and stability studies in the series thyrostatic
methimazole (1) were performed. By applying chemo- and regioselective chemical
transformations, new, but also some known related substances of methimazole were prepared,
ie. its known but also unknown potential impurities. In particular, the stability of methimazole
in chlorinated solvents such as dichloromethane and dichloroethane, ubiquitous in chemistry
and pharmaceuticals, has been studied. The obtained bridged bis methylene and ethylene
derivatives were isolated and structurally characterized, and the characterization and
transformation of their solid forms was performed. In this context, special emphasis is placed
on the chemistry of 1,2-bis [(1-methyl-1H-imidazol-2-yl) thio] ethane (3a) formation which
includes an attempt to detect and isolate a possible thiiranium (episulfonium) intermediate (8).
Based on the conducted synthetic, spectroscopic, thermal, diffraction and computational
studies, including kinetic measurements, intermediate 8 was not found but its isomer 7-methyl2H, 3H, 7H-imidazole [2,1-b] thiazol-4-ium chloride (7a) is to our knowledge the first known
stable isomer of thiiranium ions. It was also found that 3a was formed directly by nucleophilic
substitution of the primarily formed intermediate chlorine ethyl derivative, 2-[(chloroethyl)
thio]-1-methyl-1H-imidazole, 6) with methimazole.
Oxidation and methylation derivatives of 1 have also been prepared and characterized, and in
the context of known methimazole impurities B and C, samples have been prepared and
standardized.
As part of the preformulation studies, a solid state study of 1 was performed in the presence of
selected excipients. High stability of 1 in the planned pharmaceutical formulations was
determined, but also a new unknown, dimer and trimer of methimazole were found and
characterized, as its potential impurities. At the same time, the chemistry of their formation was
proposed.
The results of the study complements the current knowledge about methimazole (1) which
provides a comprehensive insight into the properties of its structural analogues, as potential
impurities. This contributes to increasing the quality, safety and efficacy of both methimazole
and its final dosage forms
The effect of trypsin and EDTA solutions on the stability of surface markers of MEF, B12, RAW264.7 and DC 2.4 cells at different time points
No full textJedan od prvih koraka prilikom analize adherentnih stanica na protočnom citometru je njihovo odljepljivanje od podloge i dobivanje stanične suspenzije, međutim ovaj postupak može utjecati na kompoziciju i integritet molekula ispoljenih na samoj površini stanice. Kako bismo to izbjegli, odlučili smo ispitati utjecaj otopina tripsin i EDTA na izražaj određenih proteina, s obzirom na vrijeme inkubacije. Proteini čija razina je praćena su molekule MHC I, m04 te CD80 na čiji izražaj utječe mišji citomegalovirus (MCMV) kako bi izbjegao prepoznavanje od stanica imunosnog sustava, a njihova regulacija i ekspresija predmet su intenzivnog istraživanja u Centru za proteomiku. Razine navedenih molekula proučavane su na stanicama MEF, B12, DC 2.4 i Raw264.7 s i bez infekcije MCMV-om. Dobiveni rezultati analizom na protočnom citometru pokazali su da je izražaj MHC I i m04 značajno smanjen samo na inficiranim stanicama RAW264.7 nakon tretmana otopinom EDTA 10 minuta, a razina CD80 na neinficiranim i inficiranim stanicama DC2.4 tretmanom s otopinom tripsin 10 minuta. Dobiveni rezultati ukazuju na to da je bitno na vrijeme zaustaviti djelovanje otopina tripsin i EDTA kako bi se dobili točni i pouzdani rezultati u budućim eksperimentima. Osim toga, usporedila se i razina navedenih molekula na stanicama te je pokazana veća razina MHC I i m04 na stanicama RAW264.7 i DC 2.4 što ukazuje na to da je izražaj virusom izmijenjenih MHC I na njihovoj površini vjerojatno veći nego na stanicama MEF i B12. Kakav utjecaj na imunosni sustav ima ovaj fenomen treba još ispitati.One of the first steps in the analysis of adherent cells on a flow cytometer is their detachment from the substrate and obtaining a cell suspension, however this procedure can affect the composition and integrity of the molecules expressed on the cell surface. To avoid this, we decided to examine the effect of trypsin and EDTA solutions on the expression of certain proteins, given the incubation time. Proteins whose levels were monitored are the molecules MHC I, m04 and CD80, the expression of which is influenced by murine cytomegalovirus (MCMV) to avoid recognition by immune system cells, and their regulation and expression is the subject of intensive research at the Center for Proteomics. Levels of these molecules were studied on MEF, B12, DC 2.4, and Raw264.7 cells with and without MCMV infection. The results obtained by flow cytometer analysis showed that the expression of MHC I and m04 was significantly reduced only on infected RAW264.7 cells after treatment with EDTA solution for 10 minutes, and CD80 levels on uninfected and infected DC2.4 cells after treatment with trypsin solution for 10 minutes. The obtained results indicate that it is important to stop the action of trypsin and EDTA solutions on time in order to obtain accurate and reliable results in future experiments. In addition, the level of these molecules on the cells was compared and a higher level of MHC I and m04 was shown on RAW264.7 and DC 2.4 cells, which indicates that the expression of virus altered MHC I on their surface is probably higher than on MEF and B12 cells. What effect this phenomenon has on the immune system remains to be investigated
Roles of ADAR1 protein in herpes simplex virus 1 replication
No full textInfekcija virusom herpes simpleksa 1 (HSV-1) uzrokuje nekoliko bolesti, u rasponu od kožnih, oralnih i genitalnih infekcija do fatalnog encefalitisa (1). Unatoč napretku protuvirusnih terapija koje su značajno smanjile morbiditet i mortalitet HSV-a kod imunokompromitiranih osoba, i dalje je od velikog interesa pobliže razumijevanje elemenata koji pridonose virulenciji HSV-1. Iz tog razloga postoji velika potreba za detaljnom analizom, različitih faktora domaćina koje virus koristi za promicanje infekcije kao i faktora obrane koji djeluju protivno HSV-1 infekciji (2). Jedan od potencijalnih faktora koji utječe na HSV-1 virulenciju je adenozin deaminaza koja djeluje na RNA (ADAR1). Njegova funkcija A-u-I uređivanja dvolančanih RNA nedavno je prepoznata kao ključan proces u označavanju dsRNA kao vlastite, prevenirajući tako aktivaciju urođenog imunološkog odgovora te djelujući na razvoj i ishod imunološki posredovanih bolesti i infekcija. Prethodna istraživanja pokazala su da, ovisno o specifičnim interakcijama virusa i domaćina, ADAR1 može djelovati protuvirusno, ali i provirusno. Do danas, njegova uloga u HSV-1 infekciji nije istražena, stoga je cilj ovog rada bio odrediti utjecaj ADAR1 enzima na replikaciju HSV-1. U svrhu ostvarenja tog cilja korištene se stanice s utišanom ekspresijom ADAR1 te stanice negativne kontrole, kod kojih je praćena razina faktora urođenog imunološkog odgovora i virusnih proteina. Dobiveni rezultati ukazuju na protuvirusni učinak ADAR1 tijekom rane faze HSV-1 infekcije s obzirom da se njegovim utišavanjem značajno povećala razina neposredno ranih virusnih proteina te faktora uređenog imunološkog odgovora eIf-2α, čija aktivnost potiče ekspresiju virusnih gena. S druge strane, utišavanje ekspresije ADAR1 nije dovelo do značajne promjene u replikaciji virusa između ispitivanih stanica, stoga je pretpostavljeno da njegova protuvirusna aktivnost nema dovoljno snažan učinak koji bi uzrokovao veći poremećaj u replikaciji HSV-1. Zaključno, rezultati ovog rada potvrđuju ulogu ADAR1 tijekom HSV-1 infekcije te ukazuju na ADAR1 kao potencijalnu metu za pojačavanje protuvirusnog imunološkog odgovora.Herpes simplex virus type-1 (HSV-1) infection can cause several diseases, ranging from cutaneous, oral and genital infections to fatal encephalitis (1). Despite advances in antiviral therapies that have significantly reduced HSV morbidity and mortality, a closer understanding of the elements contributing to HSV-1 virulence remains of great interest. For this reason, there is a great need for a detailed analysis of the host factors that the virus uses to promote infection as well as defense factors that act against HSV-1 infection (2). One of the potential factors affecting HSV-1 virulence is ADAR1. A-to-I editing by ADAR1 has recently been recognized as a key process in marking dsRNA as self, thus preventing activation of the innate immune response and affecting the development and outcome of immune-mediated diseases and infections. Previous research has shown that depending on different virus-host combinations, ADAR1 can have both antiviral and proviral effects. To date, its role in HSV-1 infection has not been evaluated, therefore the aim of this study was to investigate the role of ADAR1 in HSV-1 infection. To achieve this goal, cells with downregulated ADAR1 expression and negative control cells were used to examine the level of innate immune factors and viral proteins. The obtained results indicate the antiviral effect of ADAR1 during the early phase of HSV-1 infection since its downregulation significantly increased the level of immediate-early viral proteins and translation initiation factor eIf-2α, which promotes the expression of viral genes. On the other hand, we did not observe a difference in virus replication when ADAR1 was downregulated, therefore this data suggests that its antiviral activity is not strong enough to cause HSV-1 replication defect. In summary, the results of this study confirm the role of ADAR1 during HSV-1 infection and pointing towards ADAR1 as a potential target to boost antiviral immune response
Otpornost na agregaciju mutanata proteina TRIOBP-1 povezanog sa schizofrenijom
No full textSchizophrenia is a chronic mental illness, characterized by severe symptoms such as delusions, hallucinations, disorganized speech, lack of motivation, and unpredictable behavior. The development of schizophrenia is caused by genetic and environmental factors. The complexity of these factors has made it difficult to develop new methods for the successful treatment of patients. Recent research has shown that disruption of proteostasis may also impact on the progression of schizophrenia. Disruption in proteostasis causes certain proteins to misfold and therefore aggregate if the cell fails to degrade them. Aggregation is a process in which large insoluble structures known as aggregates are formed. So far, five proteins have been identified that may aggregate in schizophrenia. Among these five is TRIO and F-actin-binding protein (TRIOBP-1), the protein of interest in this thesis. Using C-terminally truncated constructs, it was discovered that the critical region for aggregation of TRIOBP-1 is located in the central section of the protein. As a next step, different truncated variants of TRIOBP-1 were expressed in neuroblastoma cells and immunofluorescent microscopy was used as a way to visualize aggregation. In this way, the critical region for aggregation was narrowed down to a sequence of less than 10 amino acids. To expand and verify this, the next step was to use full-length constructs with a small deletion from their center. In this thesis, four such constructs were used. Unexpectedly, all four of them formed aggregates in cells. This finding indicates that either the PH domain, or an optional 59 disordered amino acids at the N-terminus have effect on TRIOBP-1’s aggregation propensity which was previously thought to be very unlikely. The next step is to generate plasmids lacking the optionally translated N-terminal unstructured region, and look into the effect it may have on aggregation. By generating a TRIOBP-1 mutant with the minimal number of mutations required to prevent aggregation, we will be able to generate model systems for studying TRIOBP-1 aggregation. This will allow us to better understand the role of TRIOBP-1 in the progression of schizophrenia.Shizofrenija je kronična mentalna bolest koju karakteriziraju teški simptomi kao što su zabluda, halucinacije, neorganiziran govor, nedostatak motivacije i nepredvidivo ponašanje. Razvoj shizofrenije uzrokovan je genetskim i okolišnim čimbenicima. Složenost ovih čimbenika otežala je razvoj novih metoda za uspješno liječenje pacijenata. Nedavna istraživanja pokazala su da poremećaj proteostaze također može utjecati na napredovanje shizofrenije. Poremećaj proteostaze uzrokuje pogrešno savijanje određenih proteina i s time stvaranje agregata ako ih stanica ne razgradi. Agregacija je proces u kojem se formiraju netopive velike strukture poznate kao agregati. Do sada je identificirano pet proteina koji mogu stvarati agregate u shizofreniji. Među tih pet je i TRIO and F-actin-binding protein (TRIOBP-1), protein od interesa u ovom radu. Koristeći C-terminalno skraćene konstrukte, otkriveno je da se kritična regija za agregaciju TRIOBP-1 nalazi u središnjoj regiji proteina. Kao sljedeći korak, različite skraćene varijante TRIOBP-1 izražene su u stanicama neuroblastoma, a agregacija je vizualizirana pomoću imunofluorescentne mikroskopije. Na taj se način kritična regija za agregaciju suzila na niz od manje od 10 aminokiselina. Da bi se ovo potvrdilo, sljedeći korak je bio upotreba konstrukata pune duljine s malim delecijama iz njihovog središta. U ovom su radu korištena 4 takva konstrukta. Neočekivano, sva 4 konstrukta su tvorila agregate u stanicama. Ovo otkriće ukazuje da ili PH domena ili 59 opcinalnih nestrukturiranih aminokiselina na N-terminalnom kraju utječu na sklonost agregacije TRIOBP-1, za koju se ranije smatralo obrnuto. Sljedeći korak je stvaranje plazmida kojima nedostaje opcionalno translatirana N-terminalna nestrukturirana regija, te razmotriti učinak koji ona može imati na agregaciju. Generiranjem TRIOBP-1 mutanta s minimalnim brojem mutacija potrebnih da se spriječi agregacija, moći ćemo generirati modelne sustave za proučavanje TRIOBP-1 agregacije. To će nam omogućiti da bolje razumijemo ulogu TRIOBP-1 u napredovanju shizofrenije