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Neutralization of mouse cytomegalovirus variants by antibodies
No full textRazvoj cjepiva protiv humanog citomegalovirusa (HCMV) javnozdravstveni je prioritet obzirom da virus globalno uzrokuje ozbiljne posljedice kao što su povećana smrtnost kod imunosuprimiranih pojedinaca i teške neurološke posljedice kod novorođenčadi, poput gubitka sluha i mentalne retardacije. Velik broj cjepiva za citomegalovirus je testiran u pretkliničkim i kliničkim istraživanjima, no trenutno nema odobrenog cjepiva i terapija je usmjerena na suzbijanje virusa i smanjenje simptoma infekcije. Razlog za to se djelomično nalazi u činjenici da je humoralni odgovor na infekciju citomegalovirusom nedovoljno istražen. Infekcija mišjim citomegalovirusom je najčešći korišteni model za infekciju humanim citomegalovirusom. U ovom radu korištene su ženke, 8 – 12 tjedana starosti, imunizirane s divljim tipom virusa (WT MCMV), te njihovo potomstvo, neonatalni miševi inficirani s različitim sojevima i različitim virusnim dozama. Titar sojeva virusa u organima neonatalnih miševa određen je pomoću testa virusnih čistina, a neutralizacijski kapacitet transplacentarno prenesenih protutijela određen je uz pomoć neutralizacijskog testa. Fenotipska analiza mikroglije provedena je pomoću protočne citometrije. Rezultati ovog diplomskog rada pokazuju učinkovitu neutralizaciju različitih varijanti mišjeg citomegalovirusa protutijelima prenesenim od majke inficirane divljim tipom virusa, što za posljedicu ima inhibiciju replikacije virusa u različitim organima. Daljnja istraživanja su potrebna za bolje razumijevanje uloge protutijela u sprječavanju širenja virusa, za njihovu dugoročnu učinkovitost protiv različitih varijanti te usporedbu rezultata između modela i kliničkih istraživanja.The development of a vaccine against human cytomegalovirus (HCMV) is a public health priority, as the virus globally causes serious consequences such as increased mortality in immunosuppressed individuals and severe neurological sequelae in newborns, such as hearing loss and mental retardation. A large number of vaccines for cytomegalovirus have been tested in preclinical and clinical research. However, currently, there is no approved vaccine, and therapy is aimed at suppressing the virus and reducing infec tion symptoms. The reason for this is partly due to the fact that the humoral response to cytomegalovirus infection has not been sufficiently investigated. Infection with murine cytomegalovirus (MCMV) is the most commonly used model for human cytomegalovir us infection. Females, 8 12 weeks old, immunized with the wild type virus (WT MCMV), along with their offspring, were used for this thesis, while neonatal mice were infected with different virus strains and different viral doses. The virus titer was determ ined using the plaque assay, while the neutralization capacity was determined using the neutralization assay in the presence of immune serum or serum from non immunized animals. Phenotypic analysis of the expression of MHC molecules on microglia was conduc ted using a flow cytometer. The results of this thesis show the effective neutralization of different variants of murine cytomegalovirus by antibodies transferred from a mother infected with the wild type virus, which results in the inhibition of viral rep lication in different organs. Further research is required to better understand the role of antibodies in preventing the spread of the virus, their long term effectiveness against different variants, and to correlate results obtained in mouse models and clinical studies
The role of NK and ILC1 cells in the control of murine cytomegalovirus during ontogeny
No full textLjudski citomegalovirus (HCMV) je jedan od najrasprostranjenijih virusa diljem svijeta. Njegova raširenost predstavlja veliki problem u zdravstvenom sektoru zbog toga što može nakon infekcije ostaviti ozbiljne posljedice, pogotovo ukoliko dođe do prijenosa virusa s trudnice na plod, čime uzrokuje prirođenu infekciju HCMV-om koja može imati i letalan ishod. Imunosni sustav u prenatalnom i ranom postnatalnom razdoblju nije razvijen kao u odraslih te je nedostatak zrelosti razlog tome što infekcije u tom periodu mogu uzrokovati ozbiljne posljedice. Mehanizmi kako nerazvijeni imunosni sustav, odnosno prirođena imunost, štiti plod i novorođenčad od infekcija različitim patogenima su slabo istraženi. Za potrebe istraživanja HCMV-a koristi se mišjicitomegalovirus (MCMV) s kojim dijeli mnoge sličnosti u temeljnoj biologiji i patogenezi, te zbog toga infekcija miševa MCMV-om predstavlja najčešće korišteni model infekcije citomegalovirusom. U ovom istraživanju koristili smo transgenične miševe, pristup uklanjanja imunoloških stanica monoklonskim protutijelima, te rekombinantni MCMV kako bi istražili ulogu stanica NK i ILC1 u nadzoru infekcije MCMV-om u novookoćenih miševa. Transgenične miševe smo genotipizirali PCR-om, prisustvo stanica NK i ILC1 odredili smo metodom protočne citometrije, dok smo testom virusnih čistina odredili količinu virusa u ispitivanim organima. Rezultati ovog rada pokazali su kako stanice urođene imunosti, stanice NK i ILC1, imaju važnu ulogu u nadzoru infekcije MCMV-om u jetri novookoćenih miševa. Daljnja istraživanja će pružiti bolje razumijevanje neonatalnog imunosnog sustava.Human cytomegalovirus (HCMV) is one of the most widespread viruses worldwide. It is a major problem in healthcare because the infecion can leaveserious consequences, especially if the virus is transmitted from the pregnant woman to the fetus, i.e., if it causes congenital HCMV infection that can havea fatal outcome. The immune system in the prenatal and early postnatal period is immature as compared to adults, and the immaturity is the reason why infections in that period can result in serious consequences. The mechanisms by which an underdeveloped immune system, i.e. innate immunity, protects the fetus and newborns from infections by various pathogens have been poorly investigated. To study HCMV, murine cytomegalovirus (MCMV) is often used, which shares many biological similarities and pathogenesis; therefore, infection of mice with MCMV is the most commonly used model of cytomegalovirus infection. In this study, we used transgenic mice, an immune cell depletion approach with monoclonal antibodies, and recombinant MCMV to investigate the role of NK cells and ILC1 cells in the control of MCMV infection in newborn mice. The transgenic mice were genotyped by PCR, the presence of NK and ILC1 cells was determinedby flow cytometry method, while the levels of virus in the examined organs was determined by the plaque assay. The results of this work showed that innate immune cells, NK cells and ILC1, play an important role in controlling MCMV infection in the liver of newborn mice. Further research will provide a better understanding of the neonatal immune system
Analiza proteinskih karakteristika i patogenosti varijanti optineurina u amiotrofičnoj lateralnoj sklerozi i glaukomu primjenom bioinformatike
No full textAmyotrophic lateral sclerosis (ALS) is a severe, rapidly progressive neurodegenerative disease that affects upper and lower motor neurons. ALS is characterized by high genetic heterogeneity – so far 17 confirmed or “gold standard” genes and 100 potential risk factors - have been linked to this disease. One of the confirmed genes is OPTN, which encodes for optineurin, a multifunctional, ubiquitin binding adaptor protein proposed to act in various biological processes. OPTN mutations have also been found in glaucoma, another neurodegenerative disease, encompassing a group of eye conditions in which the optic nerve is damaged. The aim of this work was to examine optineurin protein features and to compare OPTN variants present in ALS and glaucoma with naturally occurring variants through different in silico programs. We also examined if the mutations were more frequent in the evolutionary conserved regions of optineurin. The OPTN variants and subsequent protein changes were compiled by expert literature curation and taken from UniProt, OMIM, ClinVar and gnomAD databases. In silico species alignment analysis of optineurin domain conservation was done using ClustalOmega. Our results showed that OPTN variants in both ALS and glaucoma predominantly affect coiled-coil regions of optineurin, but this is comparable to their length. However, ALS variants were enriched in the zinc finger (ZF) domain. They were curiously not enriched in one of the main functional domains of optineurin, the ubiquitin-binding region of ABIN and NEMO (UBAN), but clustered around it. Pathogenicity of OPTN mutations patient mutations was predicted using Polyphen-2, SIFT and PROVEAN programs. Although prediction of pathogenicity differed between the programs for variants present in both ALS and glaucoma, they were consistent in assigning K59N, R83C, Q314L, M447R, E478G, K557T, D564H, L568S, H571Q as damaging protein changes present in ALS, and in assigning E50K as damaging in glaucoma. Eight different species representing major vertebrate genera were taken for in silico species alignment analysis of the optineurin conservation. This analysis showed that 89% of predicted pathogenic variants reported in ALS patients mapped to the conserved regions of OPTN, suggesting that they are more likely to be pathogenic. The highest degree of the conservation, both total and partial, was observed in the UBAN and ZF regions of optineurin. The optineurin E50K glaucoma-linked variant mapped to conserved regions as well. Although variant E322K is located on the conserved position of the optineurin protein, it is predicted to be benign according to Polyphen-2, SIFT and PROVEAN analysis. This in silico research of OPTN variants represents the basis for the further in vivo and in vitro investigations and could possibly help conceive future experimental directions relevant for uncovering optineurin function in neurodegenerative diseases.Amiotrofična lateralna skleroza (ALS) je teška, brzo progresivna neurodegenerativna bolest koja zahvaća gornje i donje motoričke neurone. ALS karakterizira visoka genetska heterogenost s do sada 17 potvrđenih ili ''gena zlatnog standarda'' te >100 potencijalnih faktora rizika povezanih s ovom bolešću. OPTN gen kodira za optineurin, višenamjenski, adaptorski protein koji veže ubikvitin te za koji se pretpostavlja da djeluje u raznim biološkim procesima. Mutacije OPTN gena također su pronađene u još jednoj skupini neurodegenerativnih bolesti – glaukomu – u kojima dolazi do oštećenja i gubitka neurona vidnog živca. Cilj ovog rada bio je usporediti značajke varijanti proteina optineurina i OPTN gena prisutnih u ALS-u i glaukomu s prirodnim varijantama putem različitih in silico programa. Također smo ispitali jesu li mutacije pronađene u pacijentima bile češće u evolucijski očuvanim regijama optineurina. OPTN varijante navedene su stručnim pregledom literature i preuzete iz baza podataka UniProt, OMIM, ClinVar i gnomAD. In silico analiza sličnosti vrsta i očuvanje domene optineurina među vrstama analizirana je pomoću programa ClustalOmega. Naši rezultati pokazuju da OPTN varijante i kod ALS-a i glaukoma pretežno zahvaćaju zavijene zavojnice (Coiled-coil) optineurina, ali to je usporedivo s njihovom duljinom. Međutim, ALS varijante obogaćene su u domeni cinkova prsta (engl. Zinc Finger, ZF). Suprotno očekivanjima, varijante nisu bile obogaćene u ubikvitin-vezujućoj regiji ABIN i NEMO proteina (engl. ubiquitin-binding region of ABIN and NEMO, UBAN), koja predstavlja glavnu funkcionalnu domenu optineurina, ali su bile grupirani oko nje. Patogenost OPTN mutacija je predviđena pomoću programa Polyphen-2, SIFT i PROVEAN. Iako se predviđanje patogenosti djelomično razlikovalo među programima i za varijante u ALS-u i glaukomu, programi su bili dosljedni u označavanju K59N, R83C, Q314L, M447R, E478G, K557T, D564H, L568S, H571Q kao štetne promjene proteina u ALS-u te E50K kao štetne u glaukomu. Osam različitih vrsta koje predstavljaju glavne rodove kralježnjaka uzeto je za in silico analizu sličnosti između vrsta te za ispitivanje očuvanja optineurina i utjecaja konzerviranosti na pojavu mutacija. Analiza sličnosti između vrsta pokazala je da je 89% predviđeno patogenih varijanti optineurina prisutnih u bolesnika s ALS-om mapirano na očuvane regije, što sugerira njihovu patogenost. Najviši stupanj očuvanosti (potpuni i djelomični) uočen je u UBAN i ZF regijama optineurina. Optineurin E50K varijanta, pronađena kod pacijenata s glaukomom, također je smještena u očuvanim regijama. Iako se varijanta E322K nalazi na očuvanom položaju proteina optineurina, prema Polyphen-2, SIFT i PROVEAN analizi je benigna. Zaključno, ovo in silico istraživanje OPTN varijanti predstavlja temelj za daljnja in vivo i in vitro istraživanja i isto bi moglo pomoći u osmišljavanju budućih eksperimenata relevantnih za razumijevanje funkcije optineurina u neurodegenerativnim bolestima
Uloga PI(4,5)P2 i PI4P u sazrijevanju megakariocita, stvaranju protrombocita i funkciji trombocita
No full textDuring megakaryocyte (MK) maturation there is an extensive formation of the demarcation membrane system (DMS) which serves as a membrane reservoir for future platelets (PLTs). During the DMS formation, there is an active transport of vesicles from the Golgi apparatus to the DMS suggesting that lipids and proteins that are necessary for DMS growth originate from the Golgi apparatus. Once mature, MKs release PLTs into the bloodstream where upon encounter with a vessel wall injury PLTs adhere, activate, and aggregate which results in clot formation. The most important lipid that regulates the anterograde Golgi trafficking is the phosphatidylinositol-4-monophosphate (PI4P) and its levels at the Golgi apparatus are controlled to a great extent by the SACM1L phosphatase. In this study, we show that PI4P localizes to the Golgi apparatus and the plasma membrane (PM) of immature MKs while in mature MKs is mostly localized to the PM. We demonstrate that the Golgi pool of PI4P that is controlled by the SACM1L phosphatase is necessary for MK maturation and proplatelet formation because the exogenous expression of wild-type, but not catalytically inactivated SACM1L, retains the dispersion of the Golgi apparatus during MK maturation and results in a decrease of proplatelet formation. In addition, we show that the PM pool of PI4P that is controlled by the PI4KIIIα is also necessary for proplatelet formation since pharmacological inhibition of this kinase decreases proplatelet formation.
PLT activation is accompanied by massive shape change, and an important role in actin reorganization has phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] that is, among other enzymes, regulated by the OCRL phosphatase. Mutations in OCRL cause Lowe syndrome (LS) and it has been shown that, among other symptoms, LS patients can have bleeding problems. Here, we firstly show that for the visualization of PI4P and PI(4,5)P2 in PLTs, a modification of the existing staining protocol is needed and that the best staining of the PM pool of these lipids is obtained when permeabilizing the cells with 0.5% saponin for 5 minutes. Next, we show that the pharmacological inhibition of OCRL in human PLTs leads to an impaired spreading on three different matrices (glass, fibrinogen, and collagen) and that OCRL-inhibited PLTs spread in fibrinogen instead of actin stress fibres, form actin nodules. These actin nodules colocalize with proteins important for actin dynamics (ARP2/3 complex, vinculin, SNX9) and are sites of active signalling. The impaired actin reorganization is mediated by the myosin light chain (MLC) signalling since OCRL-inhibited PLTs have decreased MLC phosphorylation. Furthermore, OCRL-inhibited PLTs have impaired microtubular reorganization, shown by a retained microtubular coil during PLT activation which is accompanied by higher levels of acetylated tubulin. Interestingly, OCRL inhibition does not affect PLT degranulation or integrin activation. Finally, we show that
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the OCRL KO mice also have impaired PLT spreading shown by an increased number of PLTs forming filopodia, and a decreased number of PLTs forming lamellipodia.
Taken together, these results contribute to a better understanding of the role of PI4P during MK maturation and PI(4,5)P2 during PLT activation. They also show the importance of different phosphoinositide pools in MK maturation and proplatelet formation, and they give an insight into the molecular mechanism of impaired PLT activation in LS patients.Megakariociti (MK) prilikom sazrijevanja stvaranju demarkacijski sustav membrana (DMS) koji služi kao rezervoar membrana za buduće trombocite. Za vrijeme stvaranja DMS-a, vezikule iz Golgijevog aparata konstantno putuju prema DMS-u, navodeći na zaključak da lipidi i proteini koji su potrebni za rast DMS-a nastaju u Golgijevom aparatu. Kad MK sazriju, u krvotok otpuštaju trombocite. Trombociti, u kontaktu s oštećenim endotelom krvne žile, adheriraju na mjesto ozljede, aktiviraju se te agregiraju što dovodi do stvaranja krvnog ugruška i zaustavljanja krvarenja. Jedan od najvažnijih lipida koji reguliraju anterogradni transport vezikula od Golgijevog aparata je fosfatidilinozitol-4-monofosfat (PI4P) te su njegove razine u Golgijevom aparatu u velikoj mjeri regulirane djelovanjem SACM1L fosfataze. U ovom istraživanju, pokazali smo da se PI4P u nezrelim MK nalazi u Golgijevom aparatu i na plazma membrani (PM) dok se u zrelim MK uglavnom nalazi na PM. Pokazali smo da PI4P koji se nalazi na Golgijevom aparatu, a čije su razine regulirane djelovanjem SACM1L fosfataze, je potreban za sazrijevanje MK i stvaranje protrombocita jer egzogena ekspresija divljeg tipa, ali ne katalitički ne-aktivnog tipa SACM1L fosfataze ne dozvoljava raspršenje Golgijevog aparata prilikom sazrijevanja MK i dovodi do smanjenog stvaranja protrombocita. Također, pokazali smo da PI4P koji se nalazi na PM, a čije je stvaranje regulirano djelovanjem PI4KIIIα je isto nužan za stvaranje protrombocita jer farmakološka inhibicija ove kinaze smanjuje postotak MK koji stvaraju protrombocite.
Prilikom aktivacije, trombociti mijenjaju svoj oblik, a u procesu reorganizacije aktina važnu ulogu ima fosfatidilinozitol-4,5-bisfosfat [PI(4,5)P2] kojeg, između ostalih enzima, regulira OCRL fosfataza. Mutacije OCRL fosfataze dovode do Lowe sindroma (LS) te je pokazano da, između ostalih simptoma, LS pacijenti mogu imati problema s krvarenjem. U ovom istraživanju smo pokazali da je za vizualizaciju PI4P i PI(4,5)P2 u trombocitima potrebno modificirati postojeći protokol te da je za optimalnu vizualizaciju ovih lipida na PM za permeabilizaciju potrebno koristiti 0.5% saponin 5 minuta. Nadalje, pokazali smo da farmakološka inhibicija OCRL-a u ljudskim trombocitima dovodi do smanjene aktivacije trombocita na različitim matricama (staklo, fibrinogen i kolagen) te da OCRL inhibirani trombociti aktivirani na fibrinogenu umjesto aktinskih stres niti stvaraju aktinske nodule. Ovi aktinski noduli kolokaliziraju s proteinima koji su važni za dinamiku aktina (ARP2/3 kompleks, vinculin, SNX9) te su mjesta aktivne signalizacije. Poremećena reorganizacija aktina je posredovana putem signalizacije lakog lanca miozina (MLC) jer OCRL inhibirani trombociti imaju smanjenu fosforilaciju MLC. Također, OCRL inhibirani trombociti ne reorganiziraju mikrotubule prilikom aktivacije već zadržavaju mikrotubularnu zavojnicu te imaju povišene razine acetiliranog tubulina. Unatoč nepotpunoj aktivaciji, OCRL inhibicija ne utječe na degranulaciju i aktivaciju integrina. Konačno, pokazali smo da se trombociti
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OCRL KO miševa također ne aktiviraju u potpunosti, odnosno da stvaraju više filopodija, a manje lamelipodija.
Zajedno, ovi rezultati doprinose boljem razumijevanju uloge PI4P-a tijekom sazrijevanja MK i PI(4,5)P2 tijekom aktivacije trombocita. Također, ovi rezultati ukazuju na važnost različitih izvora fosfoinozitida tijekom sazrijevanja MK i stvaranja protrombocita i daju uvid u molekularni mehanizam nepotpune aktivacije trombocita u LS pacijenata
Effect of optineurin deficiency on IFN-β-induced autophagy
No full textUčinci različitih neurodegenerativnih bolesti na živčane stanice međusobno se bitno razlikuju, međutim nakupljanje unutarstaničnih, toksičnih agregata krivo smotanih proteina i posljedično gubitak funkcije stanica, zajednička su obilježja svih neurodegenerativnih bolesti. Osnovni razlog prisutnosti agregata nije poznat u velikoj većini slučajeva, ali dio mutacija u genima povezanim s neurodegenerativnim bolestima veže se uz autofagiju. Autofagija je evolucijski očuvani proces kojim se putem lizosoma razgrađuje citoplazmatski sadržaj poput proteinskih agregata, patogena i oštećenih organela. Pritom adaptori autofagije prepoznaju označeni sadržaj za razgradnju i dostavljaju ga do vezikula s dvostrukom membranom koje se zovu autofagosomi. Autofagosomi se spajaju s lizosomima što dovodi do stvaranja autolizosoma gdje se sadržaj razgrađuje. U adaptore autofagije ubrajaju se i dva proteina čije su mutacije pronađene u bolesnika s neurodegenerativnom bolešću amiotrofičnom lateralnom sklerozom (ALS), optineurin (OPTN) i sekvestosom 1 (SQSTM1 ili p62). Za OPTN je poznato da osim kao adaptor autofagije, sudjeluje u još dva koraka autofagije: inicijaciji autofagije i sazrijevanju autofagosoma. Stoga smo u ovom radu htjeli ispitati utjecaj OPTN-a na autofagiju u staničnoj liniji neuroblastoma N2a, koristeći N2a stanice u kojima je OPTN uklonjen CRISPR-Cas9 metodom. Analizirali smo bazalnu autofagiju i autofagiju induciranu s interferonom (IFN)-β, citokinom za koji je nedavno pokazano da regulira autofagiju u neuronima. Koristili smo standardne testove za praćenje autofagije poput mjerenja količine markera autofagosoma LC3-II (engl. microtubule associated protein light chain 3), adaptora p62, i OPTN-a Western blot metodom, te brojanje LC3 pozitivnih vezikula imunofluorescencijom ili fluorescentno označenim LC3 konstruktom. Iz naših rezultata zaključujemo da u OPTN-deficijentnim stanicama postoji djelomični blok autofagije u bazalnim uvjetima kojeg vidimo kao povećani broj i veličinu autofagosoma te povećano nakupljanje LC3-II. IFN-β je u N2a stanicama inducirao autofagiju, ali nismo uočili nakupljanje OPTN i p62 što može značiti da nisu adaptori autofagije potaknute s IFN-β te da djeluju na autofagiju u nekom drugom koraku. Nadalje, moguće je i da su razgrađeni prije naše analize te da pokuse treba modificirati. Zbog djelomičnog bloka u autofagiji pri nedostatku OPTN-a, moguće je da iz tog razloga dolazi do nakupljanja proteinskih agregata u ALS-u. U budućim pokusima važno je stoga dokazati da opaženi poremećaj autofagije dovodi do agregacije proteina.The effects of different neurodegenerative diseases on neurons substantially differ, but the accumulation of intracellular toxic aggregates of misfolded proteins and the consequent loss of cell function are common features of all neurodegenerative diseases. The underlying reason for the presence of aggregates is not known in the vast majority of cases, but some genes mutated in neurodegenerative diseases are linked to autophagy. Autophagy is an evolutionarily conserved process by which noxious cytoplasmic contents such as protein aggregates, pathogens, and damaged organelles are broken down in lysosomes. Autophagy adaptors recognize the cargo labeled for degradation and deliver it to double-membrane vesicles called autophagosomes. Autophagosomes fuse with lysosomes leading to the formation of autolysosomes where the cargo is degraded. Autophagy adapters also include two proteins whose mutations have been found in patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS), optineurin (OPTN) and sequestosome 1 (SQSTM1 or p62). In addition to its adaptor role, OPTN has also been described to participate in two other autophagy steps: autophagy initiation and autophagosome maturation. Therefore, in this paper, we examined the effect of OPTN on autophagy in the N2a neuroblastoma cell line, using N2a cells in which OPTN was removed by the CRISPR-Cas9 approach. We analyzed basal autophagy and autophagy induced by interferon (IFN)-, a cytokine recently shown to regulate autophagy in neurons. We used standard tests to monitor autophagy such as measuring the amount of LC3-II (microtubule associated protein light chain 3), p62 adaptor, and OPTN by Western blotting, and counting LC3 positive vesicles by immunofluorescence or via a fluorescently labeled LC3 construct. We observed a partial block of basal autophagy in the OPTN-deficient cells manifested as an increased number and size of autophagosomes and increased accumulation of LC3-II. IFN-β induced autophagy in N2a cells, but we did not observe the accumulation of OPTN and p62 which may mean that they do not serve as autophagy adaptors upon IFN-β-treatment or that they act on autophagy in some other step(s). Furthermore, it is possible that they were degraded before our analysis and that our experimental approach needs to be modified. Due to the partial block in autophagy in the absence of OPTN, it is possible that this leads to an accumulation of protein aggregates in ALS. In future experiments, it is therefore important to test if this observed autophagy dysregulation in OPTN-deficient cells leads to protein aggregation
Stavovi odgajatelja o korištenju vanjskog prostora kao mjesta učenja
No full textU fokusu ovog istraživanja je vanjski prostor kao mjesto učenja djece rane i predškolske dobi. Kada govorimo o učenju djece te dobi presudna je njihova znatiželja koja je u ranom razdoblju izražena u igri, istraživanju okoline i rezultatima njihovih akcija. Djeca su prirodno motivirana za fizičku aktivnost. Ona se povezuju s okolinom, prirodom i samima sobom te su dio promjena koje se kontinuirano događaju u prirodi. Svjesni smo prilika koje im nude aktivnosti na otvorenome i kroz koje spontano i kontinuirano stječu nova iskustva koristeći sva osjetila. Motiviranost za učenje i rad na otvorenome pridonosi razvoju usmjerenosti pažnje, socijalnih vještina, komunikaciji, suradnji i rješavanju konflikata te potiče razvoj svih vještina i sposobnosti u ranoj i predškolskoj dobi. Cilj nam je ispitati stavove odgajatelja o važnosti i preprekama za slobodnu igru i učenje djece na otvorenome s obzirom na razinu prihvaćanja suvremene paradigme i s obzirom na sociodemografske varijable. Pretpostavka je da odgajatelji koji se priklanjaju tradicionalnoj obrazovnoj paradigmi manje prihvaćaju mogućnosti te u manjoj mjeri uviđaju važnost korištenja vanjskoga prostora od odgajatelja koji gaje suvremeni pogled na dijete, njegov odgoj i obrazovanje. Istraživanje je potvrdilo da su odgajatelji koji se priklanjaju suvremenoj obrazovnoj paradigmi ujedno i oni koji u većoj mjeri prihvaćaju i prakticiraju boravak djece na otvorenome. Međutim, istraživanjem se utvrdilo da na te stavove i praksu ne utječe duljina radnoga staža odgajatelja, već prvenstveno njihova razina formalnog obrazovanja. Naime, utvrđeno je da odgajatelji sa završenim preddiplomskim i diplomskim studijem imaju pozitivnije stavove o važnosti boravka djece na otvorenome, te u većoj mjeri osiguravaju kvalitetne uvjete za odvijanje slobodne igre djece na vanjskom prostoru od odgajatelja sa završenom srednjom školom.The focus of this research is outdoor spaces as a place of learning for children of early and preschool age. When we talk about the learning of children of that age, their curiosity is crucial, which is expressed in the early period in play, research of the environment, and in the outcomes of their actions. Children are naturally motivated for physical activity. They connect with the environment, nature and themselves, and they are part of the changes that are continuously happening in nature. We are aware of the opportunities offered to them by outdoor activities, through which they spontaneously and continuously gain new experiences using all the senses. Motivation to learn and work outdoors contributes to the development of attention, social skills, communication, cooperation, and conflict resolution; it encourages the development of all skills and abilities in early and preschool age. Our goal is to examine preschool teachers' attitudes about the importance and barriers to children’s free play and learning outdoors with respect to the level of acceptance of the modern paradigm and with respect to sociodemographic variables. It was assumed that preschool teachers who adhere to the traditional educational paradigm are less receptive to opportunities and less aware of the importance of using outdoor space than preschool teachers who cultivate a modern view of the child, his upbringing and education. The research confirmed that preschool teachers who adhere to the modern educational paradigm are also those who to a greater extent accept and practice children's stay outdoors. However, research has shown that these attitudes and practices are not influenced by the length of service of preschool teachers, but primarily by their level of formal education. Namely, it was found that preschool teachers with bachelor's and master's degrees have more positive attitudes about the importance of children staying outdoors, and to a greater extent provide quality conditions for free play of children outdoors, than preschool teachers who completed only high school
Sklonosti odgojitelja neuromitovima u odgoju i obrazovanju
No full textRazvoj tehnologije omogućio je i razvoj znanosti, njezine discipline i dostupnost iste. Nova istraživanja iz područja neuroznanosti daju i nova objašnjenja o funkcioniranju mozga i uma, ponašanju i učenju što uvelike doprinosi i novijim spoznajama u odgoju i obrazovanju. Naglašena je aktivna uloga učenika u samom procesu učenja te iako još uvijek postoje razlike između rezultata istraživanja i primjene istih u odgojno obrazovnom radu, postignut je veliki napredak u razumijevanju onoga što pomaže i potiče djecu u procesu učenja. Pogreške u razumijevanju znanstvenih tvrdnji rezultiraju stvaranjem obrazovnih neuromitova što dovodi do neadekvatne primjene u praksi i negativnih posljedica na obrazovanje.
Ovim se istraživanjem na uzorku od 149 hrvatskih odgojitelja ranog i predškolskog odgoja i obrazovanja nastoje ispitati sklonosti vjerovanja u obrazovne neuromitove te utvrditi postoji li povezanost između općeg znanja iz neuroznanosti, sociodemografskih karakteristika sudionika i sklonosti vjerovanja u neuromitove.The development of technology also enabled the development of science, its discipline, and its availability. New neuroscience research also provides new explanations about the functioning of the brain and mind, behavior, and learning, which contribute to new knowledge in education. The active role of students in the learning process itself is emphasized, and although there are still differences between research results and their application in educational work, great progress has been made in understanding what helps and encourages children in the learning process. Misunderstandings of scientific claims result in the creation of educational neuromyths, which lead to inadequate application in practice and negative consequences for education.
This research on a sample of 149 Croatian early childhood and preschool teachers tries to examine the tendency to believe in educational neuromyths and to determine whether there is a connection between general knowledge of neuroscience, the sociodemographic characteristics of the respondents, and the tendency to believe in neuromyths
Stanično-specifična uloga optineurina u TLR4 ili ER stresom posredovanoj aktivaciji NF-κB puta
No full textAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which leads to motor neuron death in the brain and the spinal cord. In 2010 the first optineurin mutations were found in the ALS patients, and the initial biochemical analyses in cell lines suggested that optineurin truncation or mutation in the ubiquitin binding region result in exaggerated nuclear factor κB (NF-κB) activation. Such excessive NF-κB activation and subsequent inflammation were reported to result in neuronal cell death. However, several follow-up studies in primary innate immune cells from optineurin deficiency or insufficiency models could not reproduce these findings. To further test this issue, we decided to induce NF-κB activation with paclitaxel [Toll-like receptor 4 (TLR4) agonist] and tunicamycin [endoplasmic reticulum (ER) stress inducer] to observe if optineurin will exert its role as a negative regulator. These stimuli could mimic some of the stresses that central nervous system (CNS) cells are exposed to during the neurodegenerative process. Microglial (BV2) and neuronal (N2A) optineurin wild-type (WT) and knock-out (KO) cells were used in this research. Optineurin KO BV2 cells exhibited a diminished p65 phosphorylation and TNF-α secretion upon paclitaxel treatment but stronger inhibitor of κB-α (IκB-α) degradation, while N2A KO cells exhibited slightly higher NF-κB activation as detected by p65 phosphorylation. Tunicamycin treated optineurin KO BV2 cells also revealed slightly higher IκB-α degradation and diminished p65 phosphorylation, but TNF-α secretion was not different. Notably, optineurin KO N2A cells also exhibited a slight tendency of stronger NF-κB activation as evidenced by p65 phosphorylation. Results obtained from N2A cells are in correlation with previous studies done on neuronal cell lines, while the ones obtained in BV2 cells differ from results done on primary microglia. Taken together, we propose that optineurin has cell-specific effects, acting as a negative regulator of NF-κB activation in BV2 and N2A cells in response to paclitaxel. It must be noted that these results are obtained in cell lines so they should be used as a preliminary information for subsequent studies on primary cells or in vivo models.Amiotrofična lateralna skleroza (ALS) je fatalna neurodegenerativna bolest koja dovodi do smrti motoričkih neurona u mozgu i leđnoj moždini. Prve mutacije optineurina pronađene su u pacijenata oboljelih od ALS-a 2010. godine. Prvotne biokemijske analize u staničnim linijama pokazale su da trunkacija ili mutacija ubikvitin vežuće regije optineurina dovodi do pojačane aktivacije puta nuklearnog čimbenika κB (NF-κB). Pretjerana NF-κB aktivacija te posljedična upala mogu dovesti do neuralne stanične smrti. Međutim, nekoliko studija napravljenih na primarnim stanicama urođene imunosti izoliranih iz modela deficijencije ili insuficijencije optineurina nisu mogle reproducirati rezultate dobivene na staničnim linijama. U svrhu ispitivanja tih kontradikcija, odlučili smo inducirati NF-κB aktivaciju s paklitakselom [agoinst receptora sličnom Toll-u (TLR4)] i tunikamicinom [izaziva stres endoplazmatskog retikuluma (ER)] kako bismo ispitali hoće li optineurin djelovati kao negativan regulator. Navedeni stimulusi mogu imitirati stresore kojem su stanice središnjeg živčanog sustava izložene prilikom procesa neurodegeneracije. U ovom istraživanju koristili smo migroglijalne (BV2) i neuronalne (N2A) stanične linije divljeg tipa (eng. wild-type, WT) i optineurin deficijentne (eng. knock-out, KO). BV2 KO stanice su pokazale smanjenu fosforilaciju p65 aktivaciju i sekreciju TNF-α, no jaču razgradnju inhibitora κB-α (IκB-α) nakon tretmana s paklitakselom. U N2A KO stanicama tretman paklitakselom rezultirao je blago pojačanom NF-κB aktivacijom što je zabilježeno fosforilacijom p65. BV2 KO stanice tretirane s tunikamicinom su pokazale jaču razgradnju IκB-α i smanjenu fosforilaciju p65, no razlika u izlučivanju TNF-α nije zabilježena. N2A KO stanice pokazale su tendenciju blago pojačane NF-κB aktivacije nakon tretmana tunikamicinom što je praćeno fosforilacijom p65. Rezultati dobiveni na N2A stanicama se slažu s prethodnim studijama provedenim na neuronalnim staničnim linijama. Međutim, rezultati dobiveni na BV2 stanicama se ne slažu s onim dobivenim na primarnoj mikrogliji. Uzimajući sve rezultate u obzir, predlažemo da optineurin ima stanično-specifični, te da ima potencijalnu ulogu kao negativan regulator NF-κB aktivacije u BV2 i N2A stanicama. Moralo bi se uzeti u obzir kako su ovi rezultati dobiveni iz staničnih linija te bi se pritom trebali koristiti kao preliminarna informacija za buduće studije koje bi se provele na primarnim stanicama ili u in vivo modelima
Infection of astrocytes with mouse cytomegalovirus
No full textCitomegalovirus (CMV) je široko rasprostranjen virus u ljudi čija je karakteristika uspostava cjeloživotne latencije. Kod zdravih osoba infekcija je uobičajeno asimptomatska, ali u osoba s oslabljenim i nezrelim imunosnim sustavom može uzrokovati po život opasne infekcije. Kongenitalna infekcija CMV-om je najčešća transplacentarno prenosiva prirođena infekcija koja može utjecati na razvoj mozga i uzrokovati trajne neurološke poremećaje. Dosad je poznato da in vitro CMV može inficirati većinu staničnih vrsta mozga do određene razine, međutim, nije jasno vrijedi li to in vivo i je li ta infekcija produktivna. Glavni cilj ovog diplomskog rada bio je na mišjem modelu kongenitalne infekcije utvrditi inficira li CMV astrocite in vivo i podržavaju li astrociti uspješnu replikaciju virusa. U svrhu istraživanja korišteni su miševi i mišji citomegalovirus (MCMV) divljeg tipa, ali i inducibilni Cre/loxP reporterski sustav koji se sastoji od rekombinantnog MCMV-flox virusa te specifičnog transgeničnog soja miševa, u kojem je Cre rekombinaza aktivna samo u astrocitima. Nakon infekcije novorođenih miševa i prikupljanja organa u određenim vremenskim točkama, provedena je analiza infekcije astrocita imunohistokemijskom metodom, te virusna titracija mozgova pomoću metode određivanja virusnih čistina (plakova). Dobiveni rezultati su pokazali da MCMV uspješno inficira astrocite in vivo. Štoviše, na vrhuncu infekcije u mozgu, trećina inficiranih stanica su bili upravo astrociti. Osim toga, dokazano je da je infekcija astrocita in vivo produktivna te da astrociti doprinose širenju CMV infekcije u mišjem mozgu. Ova saznanja doprinose boljem razumijevanju neuropatogeneze kongenitalne CMV infekcije i otvaraju nova pitanja o utjecaju infekcije na funkciju astrocita i o ulozi ostalih staničnih vrsta u mozgu tijekom infekcije.Cytomegalovirus (CMV) is a widespread virus, characterized by the establishment of lifelong latency. In healthy individuals the infection is typically asymptomatic, however, in immunocompromised persons and in congenitally infected children CMV infection can lead to a life-threatening condition. Congenital CMV infection is the most common intrauterine infection that can affect brain development and cause permanent neurological disabilities. So far, it was clear that CMV can infect most brain cell types in vitro to a certain level, but whether this is also true in vivo and whether the infection is productive remains unknown. Thus, the main objective of this thesis was to determine whether CMV infects astrocytes in vivo and whether astrocytes support successful viral replication, using a mouse model of congenital infection. In this research we have used wild-type mice and mouse cytomegalovirus (MCMV), but also a conditional Cre/loxP reporter system, consisting of recombinant MCMV-flox virus and transgenic mouse strain in which Cre recombinase is active only in astrocytes. After MCMV infection of newborn mice and organ harvesting at certain time points, analysis of astrocyte infection was performed using immunohistochemical staining, as well as determination of brain virus titers by plaque assay. The results obtained from this study confirmed that MCMV successfully infects astrocytes in vivo, moreover, at the peak of infection in the brain, a third of the infected cells were astrocytes. In addition, it has been shown that astrocytes support productive CMV infection in vivo and that they contribute to virus spread throughout the brain. These findings contribute to a better understanding of the neuropathogenesis of congenital CMV infection and are raising new questions about the impact of infection on astrocyte function and the role of other brain cell types during infection
MALDI-MS i LC-MS karakterizacija različitih nusprodukata tijekom sinteze antivirusnih peptid-porfirin konjugata na čvrstom nosaču
No full textThe latest discoveries of promising antiviral drug candidates have brought in
focus so-called peptide-drug conjugates (PDCs). PDCs are class of molecules
formed by linkage, either directly or through a linker, of a drug cargo to a
carrier peptide. Among the various classes of PDCs, the ones with antiviral
properties are drawing lot of attention due to their remarkable pharmacological
potential to fight various viral diseases – one of the biggest humankind health
concerns today. Herein we have performed solid-phase peptide synthesis
(SPPS) of one PDC, with proven in vitro antiviral activity against the ZIKA
virus: PPIX-P1 (VQQLTKRFSL-amide), and characterized the main impurities
in its, and another PDC’s of also proven in vitro anti-ZIKA virus activity: PPIX
P3 (AGILKRW-amide), final purified sample by using Matrix-Assisted Laser
Desorption Ionization – Mass Spectrometry (MALDI-MS) and Liquid
Chromatography – Mass Spectrometry (LC-MS) analytical techniques. First, we
have performed SPPS of peptide P1 and analyzed it by LC-MS and MALDI-MS
to
ensure the credibility of its synthesis. Secondly, conjugation of
protoporphyrin IX (PPIX) with the synthesized peptide at the peptide’s N
terminus was done and the newly synthesized conjugate also analyzed by LC
MS and MALDI-MS analytical techniques. Furthermore, the synthesized
conjugate was cleaved from the solid support and purified by preparative C18
high performance liquid chromatography (HPLC). Obtained pure samples of
PPIX-P1, and PPIX-P3; synthesized and purified in the same methodology,
were then characterized by LC-MS and LC-MS/MS analyses, along with their
impurities. By the MS/MS analysis of some impurities found in the PDCs’
purified samples (m/z 521.7 in case of PPIX-P1 and m/z 473.7 in case of PPIX
P3) we were able to conclude that: (i) peptide part of the conjugate is the
origin of the corresponding impurity and (ii) C-terminal part of the peptide is
more prone to modifications during the synthetic process of the conjugate.
Therefore, in order to achieve higher conjugate quality, the future synthesis
should include some adjustments related to the synthesis of the conjugate’s
peptide part.Najnovija otkrića obećavajućih kandidata za nove antivirusne lijekove dovela
su nedavno u fokus takozvane peptid-lijek konjugate (eng. peptide- drug
conjugates, PDCs). Peptid-lijek konjugati klasa su molekula koje nastaju
povezivanjem, izravno ili preko spojnika (eng. linker), tereta lijeka na peptid
nosač. Među različitim klasama peptid-lijek konjugata, u posljednje vrijeme
veliku pažnju privlače oni s antivirusnim svojstvima zbog svog izvanrednog
farmakološkog potencijala u borbi protiv raznih virusnih bolesti – jedne od
najvećih zdravstvenih briga čovječanstva danas. U okviru ovih nedavnih
znanstvenih otkrića, izvršili smo sintezu peptida na čvrstoj fazi (eng. solid
phase peptide synthesis, SPPS) jednog peptid-lijek konjugata, s dokazanom in
vitro antivirusnom aktivnošću protiv ZIKA virusa: PPIX-P1 (VQQLTKRFSL
amid), te karakterizirali glavne nečistoće u njegovu, i još jednom peptid-lijek
konjugatu s također dokazanom in vitro anti-ZIKA virus aktivnošću: PPIX-P3
(AGILKRW-amid), konačnom pročišćenom uzorku korištenjem analitičkih
tehnika matriksom asistirana laserska desoprcija ionizacijom – masena
spektrometrija (eng. Matrix-Assisted Laser Desorption Ionization – Mass
Spectrometry, MALDI-MS) te tekućinska kromatografija – masena
spektrometrija (eng. Liquid- Chromatography – Mass Spectrometry, LC-MS).
Najprije smo izvršili sintezu peptida P1 na čvrstom nosaču i analizirali ga
MALDI-MS i LC-MS analitičkim tehnikama kako bismo potvrdili vjerodostojnost
njegove sinteze. Potom smo učinili konjugaciju protoporfirina IX (PPIX) sa
sintetiziranim peptidom na njegovom N-terminusu, a novosintetizirani
konjugat također zatim analizirali pomoću LC-MS i MALDI-MS analitičkih
tehnika. Nadalje, sintetizirani konjugat bio je skinut sa čvrstog nosača i
pročišćen
preparativnom C18 tekućinskom kromatografijom visoke
učinkovitosti (eng. High Performance Liquid Chromatography, HPLC). Dobiveni
čisti uzorak peptid-porfirin konjugata PPIX-P1 te PPIX-P3 ( sintetiziranog i
pročišćenog istom metodologijom) te nečistoće u njima karakterizirali smo
potom LC-MS i tekućinska kromatografija – tandemna masena spektrometrija
(eng. Liquid Chromatography - Tandem Mass Spectrometry, LC-MS/MS)
analizama. Temeljem rezultata analiza nečistoća u uzorcima peptid-porfirin
konjugata (m/z 521.7 u slučaju PPIX-P1 i m/z 473.7 u slučaju PPIX-P3)
tandemnom masenom spektrometrijom (eng. Tandem Mass Spectrometry,
MS/MS) mogli smo zaključiti da je: (i) peptidni dio konjugata porijeklo
odgovarajuće nečistoće i (ii) C-terminalni dio peptida skloniji modifikacijama
tijekom sintetskog procesa konjugata. Stoga, kako bi se postigla njihova veća
kvaliteta, potrebno je uvesti određene prilagodbe u peptidnom dijelu sinteze
peptid-porfirin konjugata