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Introduction: Alzheimer's disease (AD) and mild cognitive impairment (MCI) are characterized by different phases of cognitive decline. In AD, there is a progressive impairment of acquired cognitive abilities while subjects with MCI show less severe cognitive disturbances. Cognitive dysfunction develops as a result of the complex interactions between multiple genetic, epigenetic, developmental, and environmental factors. Among many genes involved in cognition is the gene coding for catechol- O-methyltransferase (COMT), the enzyme responsible for the degradation of dopamine, resulting in the modulation of dopaminergic function. Therefore, COMT genetic variants were studied in various dementias characterized by cognitive loss. The aim of the study was to evaluate the possible association between COMT rs6269 and COMT rs4680 polymorphisms and cognitive decline in subjects with AD and MCI. Methods: Cognitive deterioration was assessed with the Mini-Mental State Examination (MMSE) and the Clock Drawing test (CDT) scores in 193 patients with AD and 269 subjects with MCI. COMT rs6269 and COMT rs4680 (Val158Met) were genotyped using the real-time PCR. Multiple linear regressions and Kruskal Wallis ANOVA were used to detect the association of the COMT rs6269 or COMT rs4680 genotypes with cognitive decline. Results: Multiple linear regression showed that COMT rs6269 was significantly associated with MMSE and CDT scores and these effects were affected by diagnosis and COMT rs6269 genotypes and age. In contrast, multiple linear regression revealed that COMT rs4680 was not significantly associated with MMSE or CDT scores, and the only significant effects were found for age and diagnosis. To further evaluate the effect of COMT polymorphisms on cognition, all subjects were subdivided into AA, GA, or GG genotype carriers of the COMT rs6269 or COMT rs4680, respectively, and according to MMSE or CDT scores. Carriers of the GG genotype of the COMT rs6269 had significantly (p=0.007) lower CDT scores than AA carriers, while other genotype groups did not differ significantly according to the CDT or MMSE scores. Conclusion: Although COMT rs4680 (Val158Met) was reported to be related to cognitive loss in various neuropsychiatric disorders, present results revealed a significant association between the other COMT polymorphism, rs6269, with cognitive deterioration in dementia.

Posttraumatic stress disorder (PTSD) is a severe trauma and stress-related disorder associated with different somatic comorbidities, especially cardiovascular and metabolic disorders, and with chronic low-grade inflammation. Altered balance of the hypothalamic-pituitary-adrenal (HPA) axis, cytokines and chemokines, C-reactive protein, oxidative stress markers, kynurenine pathways, and gut microbiota might be involved in the alterations of certain brain regions regulating fear conditioning and memory processes, that are all altered in PTSD. In addition to the HPA axis, the gut microbiota maintains the balance and interaction of the immune, CNS, and endocrine pathways forming the gut-brain axis. Disbalance in the HPA axis, gut-brain axis, oxidative stress pathways and kynurenine pathways, altered immune signaling and disrupted homeostasis, as well as the association of the PTSD with the inflammation and disrupted cognition support the search for novel strategies for treatment of PTSD. Besides potential anti-inflammatory treatment, dietary interventions or the use of beneficial bacteria, such as probiotics, can potentially improve the composition and the function of the bacterial community in the gut. Therefore, bacterial supplements and controlled dietary changes, with exercise, might have beneficial effects on the psychological and cognitive functions in patients with PTSD. These new treatments should be aimed to attenuate inflammatory processes and consequently to reduce PTSD symptoms but also to improve cognition and reduce cardio-metabolic disorders associated so frequently with PTSD.

Alzheimer's disease (AD) is the most prevalent form of dementia, accounting for 60-70% of all dementia cases. It is a progressive and incurable neurodegenerative disorder characterized by the amyloid beta (Aβ) peptide deposition in the amyloid plaques and accumulation of hyperphosphorylated tau protein in the neurofibrillary tangles. It affects neuronal functioning and connectivity, resulting in a progressive loss of brain functions, with the cortex and hippocampus being primarily affected. The current AD therapy is only effective in alleviating the symptoms, whereas ongoing research aims at discovering new disease-modifying treatment strategies. The neurosteroids dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) have been studied for their neuroprotective potential in AD. Although these steroid hormones are abundant in the human blood, they are also produced de novo in neurons and glial cells, where their concentration is 6-8 times higher in comparison to periphery. In our research, we have utilized both genetic and pharmacologically induced mouse model of AD. The triple-transgenic AD (3xTg-AD) mouse is one of the most appropriate animal models of AD, displaying all main histopathological and behavioral features of AD, including age-dependent development of amyloid plaques, neurofibrillary tangles and progressive cognitive decline. The 3xTg-AD mice and C57BL/6 control mice were chronically treated with DHEAS using subcutaneously intrascapulary implanted osmotic pumps. The pharmacologically induced AD model was established by intracerebroventriculary injecting the C57BL/6 mice with Aβ oligomers and chronically administered with DHEA via intraperitoneal injection. Various cognitive and behavioral tests were performed on both models and analyzed using Noldus EthoVision XT software. Upon completion of the treatments and behavioral testing, the mice were euthanized and their brains were harvested for further analysis. Our results suggest that DHEA(S) could potentially serve as a protective agent against cognitive decline and other symptomatic presentations in mouse models of AD. Nevertheless, further validation of these results is necessary and it is imperative to extend our findings to include human blood samples to investigate potential therapeutic strategies of these neurosteroids in the future.

Melanom je jedan od najagresivnijih i najzloćudnijih tumora kože, uz veliku sklonost metastaziranju. Uključenost i važnost signalnog puta Hedgehog-GLI u melanomu te njegova interakcija sa signalnim putem MAPK već je zabilježena u dosadašnjim istraživanjima. U ovom doktorskom radu se nastoje dodatno razjasniti još nedovoljno istražene uloge ovog signalnog puta u melanomu te pronaći nove gene koje on regulira. Sekvenciranjem RNA tri stanične linije s prekomjernom ekspresijom proteina GLI1, GLI2 ili GLI3 dobiveni su podaci da postoji 1 642 ciljna gena koji su preklapajući za GLI1 i GLI2, 23 preklapajuća ciljna gena za GLI2 i GLI3 i samo 9 preklapajućih gena za GLI1 i GLI3. Pronađeno je 607 jedinstvenih ciljnih gena GLI1, 1 080 jedinstvenih gena GLI2 i 37 jedinstvenih gena GLI3. Daljnje bioinformatičke analize su pokazale da je u slučaju uzoraka s prekomjernom ekspresijom GLI1 i GLI2, oko 20-30% gena sa značajno promijenjenom ekspresijom uključeno u signalne puteve Wnt, MAPK i RAS. Nakon detaljne selekcije i analize javnih baza podataka, odabrano je 12 gena za validaciju qPCRom na proširenom panelu od dodatnih 11 staničnih linija melanoma. Validacija qPCR-om je omogućila da se lista dobrih gena kandidata smanji na 6 gena koji su pokazali ujednačeno povećanu ekspresiju u velikom broju staničnih linija. Iako je prvotno planirana validacija odabranih gena u staničnim linijama s potpuno uklonjenim genima GLI1, GLI2, i GLI3, nije bilo moguće uspostaviti spomenute modele koristeći metodu CRISPR/Cas9. Također nije bilo moguće provesti utišavanje gena GLI pomoću siRNA. Iz tih razloga su kao model za smanjenu aktivnost signalnog puta HH-GLI, korištene stanične linije melanoma rezistentne na inhibitor GANT61 te je na njima validirano 6 ciljnih gena. Dodatno su ovi geni validirani na staničnoj liniji s prekomjernom ekspresijom SHH te na sferoidnim kulturama tri stanične linije melanoma (CHL-1, A375 i MEL224), uspostavljenih metodom viseće kapljice u svrhu ovog doktorskog rada. Novoidentificirani ciljni geni proteina GLI koji bi se trebali dodatno istražiti i funkcionalno validirati u melanomu su geni KRT16, KRT17, S100A7, IL1R2 te MRAS., Melanoma is one of the most aggressive and malignant skin tumors, with a high tendency to metastasize. The involvement and importance of the Hedgehog-GLI signaling pathway in melanoma and its interaction with the MAPK signaling pathway have already been reported in previous studies. In this PhD thesis, we are trying to further clarify the role of this signaling pathway in melanoma and to find its new target genes. RNA-sequencing of three melanoma cell lines with overexpressed GLI1, GLI2, or GLI3 revealed that there are 1 642 overlapping target genes for GLI1 and GLI2, 23 overlapping target genes for GLI2 and GLI3, and only 9 overlapping genes for GLI1 and GLI3. There were 607 unique target genes of GLI1, 1 080 unique targets of GLI2, and 37 unique targets of GLI3. Further bioinformatic analysis revealed that in case of GLI1 and GLI2 overexpression, about 20-30% of differentially expressed genes are involved in Wnt, MAPK and RAS signaling pathways. After detailed selection and analysis of several publicly available databases, 12 genes were selected for further validation by qPCR on an expanded panel of additional 11 melanoma cell lines. qPCR validation alowed us to narrow down the gene list to 6 genes that show consistent expression in majority of the cell lines. Although it was planned to conduct target gene validation on melanoma cell lines with completely removed GLI1, GLI2 and GLI3, this could not be achieved using CRISPR/Cas9 method. It was also not possible to achieve silencing of the GLI genes using siRNA. For that reason, melanoma cell lines resistant to GANT61 were used as a model for downregulated activity of the HH-GLI signaling pathway, and 6 target genes were validated on this model. Additionally, these 6 genes were validated on a cell line with stable overexpression of SHH and on spheroid cultures of three melanoma cell lines (CHL-1, A375 and MEL224), established by hanging drop method for the purpose of this thesis. Novel identified target genes of GLI proteins that should be investigated further and functioanlly validated in melanoma are KRT16, KRT17, S100A7, IL1R2 and MRAS.

One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is poorly understood why regenerative potential is lost with evolution and development and why it becomes very limited in adult mammals. Even though our understanding of molecular and cellular mechanisms that promote or inhibit neuronal regeneration is expanding, it is still unclear what are the key differences between the neuronal systems that can and cannot regenerate, and how they can be manipulated to change the outcome of the injury. Our incomplete understanding of molecular events underlying neuronal development and regeneration is the main reason why neurodegenerative diseases and brain and spinal cord injuries are still mostly incurable today. A preferred model to study and reveal the cellular and molecular basis of regeneration is the neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate spinal cord after injury in the first two weeks of their life. After that, the regenerative capacity is abruptly lost: at 14 days in cervical spinal segments and at 17 days in less mature lumbar spinal segments. In this thesis, we used neonatal opossums to study molecular and cellular properties of spinal tissue that has and does not have the capacity to regenerate after injury, to pinpoint the key differences. Using a comparative proteomic approach, for the first time, we identified the proteins unique and differentially distributed in the intact opossum spinal tissue with different regenerative capacities. We have identified a total number of 4735 proteins involved in various cellular processes such as cell growth, proliferation, differentiation, transcription, cell signaling, cytoskeleton and extracellular matrix organization, axon guidance molecules, neurotrophic factors and entire intracellular molecular pathways like mTOR and MAPK signaling pathway. We have also identified many myelin associated proteins, known to act as inhibitors of CNS axon regeneration, which was the positive control for our overall experimental procedure. Most interestingly, we have identified a number of proteins related to different neurodegenerative diseases that change in the opossum spinal cord during the period critical for neuroregeneration. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot, and the changes in the expression of related genes were analyzed by quantitative reverse transcription PCR. Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy. We showed that during the period of development when viii regeneration stops being possible, the substantial number of proteins known to be involved in regeneration and development change their level in the opossum spinal cord. These results upgraded the previous transcriptomic data about the genes differentially expressed in regenerating and non-regenerating opossum spinal cord tissue. Comparison of the data obtained by genomic and proteomic approaches allowed the identification of molecules of interest and narrowed the number of candidates for functional assays. For the first time, we successfully established primary neuronal spinal cell cultures from neonatal opossums of different ages and different regenerative capacities, which represent a novel mammalian in vitro platform, particularly useful to study CNS development and regeneration, and to test the functional role of the candidate molecules, together with the intact neonatal opossum spinal cord preparation. We have also developed the neuroregeneration scratch test to be performed on primary neuronal spinal cell cultures deriving from P5 opossums. Taken together, the results of this thesis contribute to a better understanding of neuronal regeneration in mammals and identify candidate molecules as future targets to promote neuroregeneration in mammalian CNS., Jedan od glavnih izazova suvremene neurobiologije je nemogućnost regeneracije središnjeg živčanog sustava (SŽS) odraslih sisavaca nakon ozljede. Još uvijek nije jasno kako se i zašto sposobnost regeneracije gubi tijekom evolucije i razvoja, te zašto regeneracija kod odraslih sisavaca postaje iznimno ograničena. Iako se naše razumijevanje staničnih i molekularnih mehanizama koji potiču ili inhibiraju regeneraciju zadnjih godina sve više produbljuje, još uvijek je nejasno koje su to ključne razlike između živčanih sustava koji imaju i onih koji nemaju sposobnost regeneracije te kako ih manipulirati sa ciljem da se promjeni ishod ozljede. Naše nepotpuno razumijevanje molekularnih događaja koji su temelj razvoja i regeneracije živčanog tkiva je glavni razlog zašto su neurodegenerativne bolesti, kao i ozljede mozga i leđne moždine danas još uvijek neizlječive. Prikladan model za proučavanje i otkrivanje stanične i molekularne osnove regeneracije su mladi oposumi (Monodelphis domestica). Oposumi su tobolčari koji se rađaju nerazvijeni pa tijekom prva dva tjedna svog života posjeduju jedinstvenu sposobnost potpune regeneracije leđne moždine nakon ozljede. Nakon toga sposobnost regeneracije se naglo gubi: oko 14. dana u vratnom dijelu leđne moždine, te 17. dana u njenom manje razvijenom lumbalnom dijelu. Mlade oposume koristili smo za istraživanje molekularnih i staničnih svojstava tkiva leđne moždine koje ima sposobnost regeneracije nakon ozljede, te onog koje nema, kako bi se odredile ključne razlike. Uporabom komparativne proteomike, po prvi puta, identificirali smo proteine koji su jedinstveni i različito izraženi u tkivu leđnih moždina oposuma koji mogu i onih koji ne mogu regenerirati. Identificirali smo ukupno 4735 proteina koji sudjeluju u staničnom rastu, proliferaciji, diferencijaciji, transkripciji, staničnoj signalizaciji, organizaciji citoskeleta i izvanstaničnog matriksa, molekule koje usmjeravaju aksone, neurotrofne čimbenike i cijele unutarstanične molekularne puteve kao što su mTOR i MAPK signalni put. Također, identificirali smo niz proteina povezanih s mijelinom za koje se zna da djeluju kao inhibitori regeneracije aksona, što je bila pozitivna kontrola za naš cjelokupni eksperimentalni postupak. Kao najvažniji rezultat, otkrili smo da se mnoštvo proteina povezanih s neurodegenerativnim bolestima mijenja u leđnoj moždini oposuma u vremenu kada regeneracija prestaje biti moguća. Različita distribucija odabranih proteina dodatno je potvrđena Western blotom, a promjene u ekspresiji gena analizirane su metodom kvantitativne reverzne transkripcije lančane reakcije polimeraze. Nadalje, imunofluorescentnom mikroskopijom istražili smo staničnu lokalizaciju odabranih proteina. x Pokazali smo da se tijekom razvoja, u trenutku kada regeneracija prestane biti moguća, znatno mijenja razina proteina za koje se zna da kontroliraju regeneraciju i razvoj leđne moždine oposuma. Dobiveni rezultati su nadogradili prethodna istraživanja vezana za gene različito izražene u tkivu leđne moždine oposuma koje ima i onoga koje nema sposobnost regeneracije. Također, usporedbom rezultata komparativne transkriptomike i proteomike moguće je bilo identificirati molekule od interesa i suziti broj kandidata za funkcionalna istraživanja. Po prvi puta, uspješno smo uspostavili primarne stanične kulture leđne moždine oposuma različite dobi i različite regenerativne sposobnosti, koje predstavljaju novu in vitro platformu, posebno korisnu za proučavanje razvoja i regeneracije SŽS-a, kao i za funkcionalna istraživanja molekula kandidata u neuroregeneraciji, zajedno sa preparatom cjelovite leđne moždine oposuma održavanog u kulturi. Također, uspostavili smo test neuroregeneracije koji uključuje mehaničku ozljedu primarnih staničnih kultura dobivenih iz pet dana starih oposuma. Zaključno, rezultati ovog istraživanja doprinose boljem razumijevanju regeneracije živčanog tkiva sisavaca, te otkrivaju potencijalne terapijske molekularne mete koje bi mogle biti interesantne za neuroregeneraciju SŽS-a sisavaca.

Asthma is a common but complex chronic inflammatory disease of the respiratory airways. Serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) are associated with regulation of lung function and immune responses; however, their exact involvement in the pathophysiology of asthma remains unclear. We investigated platelet 5-HT and plasma BDNF concentrations, platelet monoamine oxidase B (MAO-B) activity, and the distribution of genetic variants of BDNF rs6265 (Val66Met) and neurotrophic receptor tyrosine kinase 2 (NTRK2) rs1439050 polymorphisms in 120 asthma patients and 120 healthy subjects, and evaluated their relationship with asthma severity and phenotype. Platelet 5-HT concentrations were lower, whereas platelet MAO-B activity and plasma BDNF concentrations were higher in asthma patients. No differences were observed in these parameters between subjects with asthma subdivided according to asthma severity or phenotype. However, asthma patients with aspirin sensitivity and aspirin-exacerbated respiratory disease had higher plasma BDNF levels. The distribution of BDNF Val66Met and NTRK2 rs1439050 genetic variants showed no differences between subjects with asthma and healthy individuals, or between patients with different asthma severity and phenotype. The BDNF Val66Met polymorphism, but not NTRK2 rs1439050 polymorphism, influenced plasma BDNF levels in healthy subjects only. These results suggest that 5-HT and MAO-B in platelets as well as BDNF in plasma may serve as potential peripheral biomarkers for asthma but not asthma severity., Astma je česta ali kompleksna kronična upalna bolest dišnih putova. Serotonin (5-HT) i moždani neurotrofni čimbenik (BDNF) su povezani s regulacijom funkcije pluća i imunoloških odgovora; međutim, njihova točna uloga u patofiziologiji astme ostaje nerazjašnjena. Istražili smo koncentracije trombocitnog 5-HT-a i BDNF-a u plazmi, aktivnost trombocitne monoaminooksidaze B (MAO-B), i raspodjelu genskih varijanti BDNF rs6265 (Val66Met) polimorfizma i NTRK2 (gen za neurotrofni receptor tirozin kinaza 2) rs1439050 polimorfizma u 120 pacijenata s astmom i 120 zdravih osoba, te istražili njihovu povezanost sa stupnjem težine i fenotipom astme. Koncentracije trombocitnog 5-HT-a bile su snižene, dok su aktivnost trombocitne MAO-B i koncentracije BDNF-a u plazmi bile povišene u pacijenata s astmom. Nisu opažene razlike u ovim parametrima između osoba oboljelih od astme podijeljenih prema težini bolesti ili fenotipu. Međutim, pacijenti s aspirin-osjetljivom astmom i aspirin-pogoršanom respiratornom bolešću imali su povišene razine BDNF-a u plazmi. Raspodjela genskih varijanti BDNF Val66Met i NTRK2 rs1439050 polimorfizama nije pokazala razliku niti između pacijenata s astmom i zdravih osoba niti između oboljelih različitih stupnjeva težine i fenotipova astme. BDNF Val66Met polimorfizam, ali ne i NTRK2 rs1439050 polimorfizam, utjecao je na razine BDNF-a u plazmi samo zdravih ispitanika. Ovi rezultati ukazuju da 5-HT i MAO-B u trombocitima, te BDNF u plazmi mogu poslužiti kao potencijalni periferni biomarkeri za astmu, ali ne i za težinu astme.

Nakupljanje agregata proteina tau u neurofibrilarne snopiće najistaknutije je histopatološko obilježje Alzheimerove bolesti. U zdravim neuronima tau ima karakteristike topljivog proteina te je uglavnom vezan za mikrotubule aksona, dok u neuronima zahvaćenim patologijom tvori agregate i akumulira se u somi i dendritima. Količina agregata i širenje neurofibrilarnih promjena u mozgu u izravnoj su korelaciji s napredovanjem bolesti i stupnjem kognitivnog oštećenja. Glavni rizični čimbenik za nastanak Alzheimerove bolesti je starenje, no točni uzroci agregacije tau još nisu potpuno razjašnjeni. Budući da su putevi agregacije proteina evolucijski konzervirani, radi genetičkog pristupa otkrivanju čimbenika agregacije i toksičnosti proteina tau, u ovom smo radu kao stanični model odabrali kvasac Saccharomyces cerevisiae. Kako bismo bolje razumjeli rane korake u tau patologiji, uspostavili smo i upotrijebili molekularni alat za proučavanje oligomerizacije tau u živim stanicama, temeljen na luminiscenciji, u kojem interakcija dvaju tau proteina rezultira komplementacijom luciferaze NanoLuc i luminiscencijom. Istražili smo dovode li različiti čimbenici, napose proteotoksični stres, do agregacije tau te smo ispitali toksičnost proteina tau u stanicama kvasca u uvjetima stresa. Pokazali smo da proteotoksični stres ne doprinosi oligomerizaciji i toksičnosti tau u stanicama kvasca. Uspostavljeni reporter tau-NanoBiT omogućit će genetički probir čimbenika agregacije i toksičnosti tau proteina u daljnjim istraživanjima., The main histopathological hallmark of Alzheimer’s disease are neurofibrillary tangles - large aggregates of protein tau. In healthy neurons, tau is a soluble protein mostly bound to the microtubules in axons, however in the affected neurons, tau accumulates in the soma and dendrites and forms aggregates. The number of tangles and the spread of neurofibrillary changes in the brain are directly correlated with the progression of the disease. The causes of tau aggregation are not completely clear. Since many molecular pathways of protein aggregation are evolutionarily conserved, we have chosen yeast Saccharomyces cerevisiae as a cell model to study the factors of tau aggregation. To better understand the early steps in tau pathology, we used a molecular tool for studying tau oligomerization in living cells, based on the luminescent reporter NanoBiT, in which tau protein interaction results in complementation of NanoLuc luciferase. We investigated how different factors, especially proteotoxic stress, affect tau aggregation and examined the toxicity of tau in yeast cells under stress conditions. We have shown that proteotoxic stress does not contribute to tau oligomerization and toxicity in yeast cells. The established tau-NanoBiT reporter will enable genetic screens for the aggregation factors and tau protein toxicity in future research.

Dosadašnja istraživanja pokazala su da receptor sličan Toll-u 2 (TLR2) u mozgu sisavaca sudjeluje u neuroinflamaciji dok tijekom razvoja utječe na proliferacijski i diferencijacijski status progenitorskih stanica. Cilj ovog rada je bio utvrditi utjecaj nedostatka TLR2 na izražaj proteina uključenih u sinaptičku plastičnost (neuroplastin) i membranski prijenos iona (ATPaze tipa P) u mišjem mozgu. Provedena je analiza sinaptičkog proteoma, genske i proteinske ekspresije navedenih proteina, njihovog tkivnog i membranskog smještaja, utvrđen je gangliozidom i određena katalitička aktivnost ATPaza u uzorcima moždane kore, hipokampusa i malog mozga mišjeg modela s nedostatkom TLR2 u usporedbi s kontrolnim tkivom. Rezultati su pokazali da je nedostatak TLR2 u mišjem mozgu povezan s: a) značajnim promjenama sinaptičkog proteoma, posebice zastupljenosti proteina nužnih za regulaciju aksonskih projekcija i mijelinizacije, glutamatergičke transmisije i energetskog metabolizma; b) manjim izražajem neuroplastina u hipokampusu i malom mozgu što vjerojatno ukazuje na smanjenje broja sinapsi, dok utvrđena količina ATPaza tipa P upućuje na manji broj sinapsi hipokampusa i povećani broj astrocita u malom mozgu; c) većim izražajem i katalitičkom aktivnošću membranske kalcijeve ATPaze u hipokampusu, što govori u prilog poremećenog transporta kalcijevih iona; d) većim udjelom jednostavnog gangliozida GD3 u hipokampusu te većim udjelom složenih gangliozida GT1b i GQ1b u malom mozgu, što može odražavati promjenu membranske homeostaze i poremećene specifične membranske procese poput proliferacije i ionskog transporta. Zaključno, u ovom radu je dokazano da nedostatak TLR2 u mišjem mozgu utječe na izražaj i funkciju membranskih sustava neophodnih za sinaptičku plastičnost i ionski transport, što predstavlja temelj za daljnje istraživanje novih uloga TLR2 u moždanom tkivu sisavaca., Previous studies have shown that roles of Toll-like receptor 2 (TLR2) in mammalian brain encompass its involvement in neuroinflammation and neurodevelopmental processes such as proliferation and differentiation of neuronal progenitor cells. The aim of this study was to determine whether lack of TLR2 influences on the expression of proteins which participate in synaptic plasticity (neuroplastin) and membrane ion transport (P-type ATPases) in mouse brain. The variations of the synaptic proteome, gene and protein expression of neuroplastin and P-type ATPases as well as their tissue and membrane localization, ATPases catalytic activity, and gangliosidome were analyzed in the tissue samples of cortex, hippocampus, and cerebellum derived from mouse model lacking TLR2 in comparison to control tissue. Results demonstrate that the lack of TLR2 is associated with: a) adjustments in the synaptic proteome related markedly to protein systems involved in regulation of axon projections and myelinization, glutamatergic transmission, and energy metabolism; b) lower expression of neuroplastin in the hippocampus and cerebellum indicating potential loss of synapses, while detected variable content of P-type ATPases may suggest reduced number of synapses in the hippocampus and increased number of astrocytes in the cerebellum; c) higher expression and increased catalytic activity of plasma membrane calcium ATPase in hippocampus arguing for deranged calcium transport; d) increased ratio of simple ganglioside specie GD3 in the hippocampus and complex gangliosides GT1b and GQ1b in the cerebellum which may reflect alteration of membrane homeostasis and specific membrane functions such as proliferation and ion transport. In conclusion, presented findings reveal that the lack of TLR2 in mouse brain effects on the expression and function of membrane systems essential for synaptic plasticity and ion transport, opening new avenues for investigation of yet undescribed roles of TLR2 in the mammalian brain.

Bolest Niemann-Pick tipa C (NPC) je rijetka nasljedna neurodegenerativna bolest nakupljanja slobodnog kolesterola i drugih lipida uzrokovana mutacijama u genu NPC1 ili NPC2, koja pokazuje niz patoloških sličnosti s kompleksnom Alzheimerovom bolesti (AB). Naša prethodna istraživanja su utvrdila povišeno djelovanje ključnog enzima u AB, proteaze BACE1, u NPC modelima. Stoga je cilj ovog doktorskog rada bio istražiti molekularnu pozadinu uočenog efekta te ispitati učinak inhibicije BACE1 na patološke karakteristike bolesti. Također, cilj je bio analizirati lipidom kako bi se utvrdile najranije promjene lipida odgovorne za različitu vulnerabilnost moždanih regija i disfunkciju endosoma u bolesti NPC. Korištene su primarne kulture neurona, kulture organotipskih rezova mozgova te moždane regije hipokampusa i malog mozga miševa koji imaju spontanu mutaciju u genu NPC1 te ne sintetiziraju proteina NPC1 (miševi NPC1) i miševa divljeg tipa (wt, od engl. wild type). U neuronima miševa NPC1 u odnosu na wt detektirana je povećana proteoliza supstrata BACE1- Sez6L i Sez6. Imunocitokemijska analiza je pokazala nakupljanje ovih supstrata u ranim endosomima NPC1 neurona. Promijenjen smještaj Sez6 i Sez6L u ranim endosomima je potvrđen i frakcioniranjem endolizosoma u moždanim regijama hipokampusa i malog mozga miševa NPC1. Interesantno, tretman inhibitorom BACE1 je djelomično popravio patološke značajke bolesti NPC, uključujući smanjenje nakupljanja Sez6 i Sez6L, smanjenje veličine vezikula ranih endosoma te smanjenje ukupne razine proteina tau u neuronima NPC1, kao i smanjenje neuroinflamacije (aktivacije astrocita i mikroglija) u kortiko-hipokamplanim rezovima miševa NPC1. Nadalje, analiza lipidoma je utvrdila da već u asimptomatskoj fazi bolesti postoji značajna razlika u distribuciji i razini određenih lipida u malom mozgu i hipokampusu, te u ranim endosomima miševa NPC1 u odnosu na wt. Ovo istraživanje je utvrdilo disfunkciju proteaze BACE1 i njenih supstrata te njihovu potencijalnu ulogu u patogenezi bolesti NPC. Uočene promjene sastava lipida moždanih regija i ranih endosoma mogu doprinijeti razvoju patoloških promjena u bolesti NPC., Niemann-Pick disease type C (NPC) is a rare hereditary neurodegenerative disorder, caused by mutations in the NPC1 or NPC2 gene. It is characterised by accumulation of free cholesterol and other lipids and has a number of pathological similarities with complex Alzheimer's disease (AD). In our previous studies, increased activity of BACE1, the key enzyme of AD, was found in NPC mouse models. Therefore, the aim of this dissertation was to explore the molecular background of the observed effect and examine the effect of BACE1 inhibition on the pathological characteristics of the disease. In addition, the goal was to determine the earliest lipid changes responsible for the differences in brain region vulnerability and endosomal dysfunction in NPC disease by lipidome analysis. Primary neuronal cell cultures, organotipic brain slice cultures, and hippocampus and cerebellum of mice that have a spontaneous mutation in NPC1 gene and do not synthesize the NPC1 protein (NPC1 mice) and wild type mice (wt) were used. NPC1 neurons showed increased proteolysis of BACE1 substrates- Sez6L and Sez6 compared with wt neurons. Immunocytochemical analysis demonstrated accumulation of the aforementioned substrates in early endosomes of NPC1 neurons. The altered localization of Sez6 and Sez6L in early endosomes was confirmed by endolysosome fractionation in the hippocampus and cerebellum of NPC1 mice. Interestingly, treatment with a BACE1 inhibitor partially ameliorated the pathological features of NPC disease, including reduction in the accumulation of Sez6 and Sez6L, reduction in the size of early endosome vesicles, reduction in total tau protein level in NPC1 neurons, and reduction in neuroinflammation (activation of astrocytes and microglia) in cortico-hippocampal brain slices from NPC1 mice. Lipidome analysis revealed that even in the asymptomatic phase of the disease, there are significant differences in the distribution and content of certain lipids between NPC1 and wt mice in the cerebellum and hippocampus, and in early endosomes. These studies provide evidence for a possible role of the BACE1 protease and its substrates in the pathogenesis of NPC disease. The observed changes in the lipid composition of brain regions and early endosomes may contribute to the development of the pathological features of NPC disease.

Antidepresivi nemaju učinak u svih bolesnika na simptome depresije, uključujući i kognitivne simptome. Za sada ne postoje pouzdani predskazatelji učinka antidepresiva, a jedan od kandidata je moždani neurotrofni čimbenik (BDNF). Njegova je koncentracija snižena u depresivnih bolesnika, dok neki antidepresivi povisuju koncentraciju BDNF-a. Ovo prospektivno, randomizirano, longitudinalno istraživanje uključilo je 121 depresivnog bolesnika, među kojima je 61 bolesnik liječen vortioksetinom, a 60 escitalopramom. Nakon 4 tjedna terapije, unatoč podjednakom poboljšanju simptoma depresije, vortioksetin je izazvao porast koncentracije BDNF-a u plazmi, dok uz terapiju escitalopramom nije zapažena promjena. Oba antidepresiva podjednako su ublažila kognitivnu disfunkciju, osim što escitalopram nije pokazao učinak na testu pažnje. Nadalje, escitalopram je izazvao veći pad koncentracije trombocitnog serotonina u usporedbi s vortioksetinom, pri čemu je veći pad koncentracije serotonina predvidio dobar odgovor na escitalopram. Oba lijeka nisu značajno utjecala na aktivnost trombocitne MAO-B. Rezultati istraživanja upućuju na razlike u biološkom učinku escitaloprama i vortioksetina, koje mogu biti povezane i s kliničkim učinkom. Istraživanje predstavlja doprinos razumijevanju mehanizma djelovanja antidepresiva i naglašava potrebu individualnog pristupa u liječenju., Antidepressants are not effective in all patients in treating depressive symptoms, as well as cognitive dysfunction. So far, there are no established predictors of treatment response, and brain derived neurotrophic factor (BDNF) is potential biomarker candidate. While its concentration has been decreased in depressed patients, some antidepressants increase BDNF levels. This prospective, randomized, longitudinal study included 121 patients with depression: 61 patients were treated with vortioxetine and 60 with escitalopram. After 4 weeks of treatment, despite similar improvement in depressive symptoms, vortioxetine increased plasma BDNF concentration, while escitalopram had no effects. Escitalopram induced larger reduction in platelet serotonin concentration than vortioxetine, whereas greater decrease in serotonin concentration predicted better response to escitalopram. Both antidepressants produced similar improvement of cognitive symptoms, although escitalopram has not affected attention. Both antidepressants have not changed platelet MAO-B activity. Our findings suggest different biological effects of escitalopram and vortioxetine, which may be associated with clinical outcome. This research contributes to better understanding of antidepressant mechanism of action and emphasizes the need for personalized treatment approach.

Ciljevi istraživanja: U modelu jedne umjerene traumatske ozljede mozga (engl. traumatic brain injury, TBI) odrediti pojavnost i opseg izražaja TDP-43 u ipsilateralnom i kontralateralnom korteksu i hipokampusu, usporediti TDP-43 subcelularni izražaj nakon jedne umjerene TBI i blage repetitivne TBI (engl. repetitive mild traumatic brain injury, rmTBI), te odrediti povezanost TDP-43 izražaja s biljezima sinaptogeneze i upale nakon TBI. Materijal i metode: Metodom lateralne ozljede tlakom tekućine (engl. lateral fluid percussion injury, LFPI) izazvana je jedna TBI umjerene jakosti u miša, dok je metodom pada utega izazvana rmTBI. Izražaj proteina od interesa analiziran je u citoplazmatskim i nuklearnim frakcijama ipsilateralnog i kontralateralnog korteksa i hipokampusa u životinja LFPI skupina, te frontalnom korteksu i hipokampusima rmTBI miševa. Histološkim tehnikama praćene su moždane promjene, a imunofluorescentnim tehnikama subcelularna lokalizacija TDP-43 te povezanost s biljezima upale i sinaptogeneze. Rezultati: Ipsilateralni korteks LFPI životinja najoštećenija je moždana struktura koju prati dezintegracija bijele tvari. U ipsilateralnom korteksu pronađena je translokacija TDP-43 u citoplazmu prvog i trećeg dana nakon LFPI u neuronima i mikrogliji. U ipsilateralnom hipokampusu uočen je pad izražaja nuklearnog TDP-43 trećeg dana od jedne umjerene TBI. Četrnaest dana nakon jedne umjerene LFPI, ali i rmTBI uočen je porast izražaja nuklearnog TDP-43 u ipsilateralnom, odnosno frontalnom korteksu. Detektiran je porast izražaja fragmentiranog TDP-35 te značajan porast izražaja fosforiliranih oblika P-TDP-43 i P-TDP-35 u ipsilateralnom hipokampusu četrnaest dana nakon jedne LFPI, ali ne i u hipokampusu rmTBI životinja. U ipsilateralnom korteksu LFPI životinja uočena je povezanost biljega upale NF-κB i TDP-43 trećeg dana u području uz najveće oštećenje te povećan izražaj biljega iNOS prvog dana uz povezanost s TDP-43 u području najvećeg oštećenja. Pad izražaja PSD-95 uz gubitak neurona ipsilateralnog korteksa praćen je pozitivnom korelacijom s TDP-43 prvog, trećeg i četrnaestog dana od LFPI. Porast izražaja proteina APP uočen je subakutno samo u rmTBI skupini životinja, a proteina P-tau samo u LFPI miševa. Zaključak: Jedna umjerena LFPI uzrokuje deregulaciju TDP-43 u ipsilateralnom korteksu i hipokampusu koja utječe na pojedine biljega upale i sinaptogeneze. Navedene promjene ovise o zadobivenoj traumi mozga i od potencijalne su važnosti u farmakoterapiji TBI., Objectives: To determine the incidence and extent of TDP-43 expression of the ipsilateral and contralateral cortex and hippocampus after a single moderate traumatic brain injury (TBI), compare TDP-43 subcellular localization after single moderate TBI and repetitive mild traumatic brain injury (rmTBI), and determine the association of TDP-43 expression with markers of synaptogenesis and inflammation after TBI. Material and methods: Lateral fluid percussion injury (LFPI) was used to induce the single moderate TBI, while the weight drop method was used to cause the rmTBI. The levels of expression of proteins of interest were analyzed in the cytoplasmic and nuclear fractions of the ipsilateral and contralateral cortex and hippocampus in the animals of the LFPI and in the frontal cortex and the hippocampi of rmTBI mice. Histological techniques were used to determine brain changes, and immunofluorescent techniques were used to monitor the subcellular localization of TDP-43 and its association with some markers of inflammation and synaptogenesis. Results: The ipsilateral cortex of the LFPI group is the most damaged brain structure accompanied by white matter disintegration. Also, a translocation of TDP-43 into the cytoplasm was detected on the first and third day after LFPI in the neurons and microglia of the ipsilateral cortex. In the ipsilateral hippocampus, a decrease in the expression of nuclear TDP-43 was found on the third day after one moderate TBI. Fourteen days after LFPI and rmTBI, an increase in nuclear TDP-43 expression was observed in the ipsilateral and frontal cortex, respectively. Significant increases in the expression of TDP-35, P-TDP-43 and P-TDP-35 were detected subacutely only in the ipsilateral hippocampus of the LFPI animals. The association with inflammation marker NF-κB and TDP-43 was found around the area of the greatest cortical damage. Also, significant iNOS expression with the interconnection with TDP-43 was evident in the ipsilateral cortex on the first day after LFPI. The decline in PSD-95 expression with loss of ipsilateral cortical neurons was followed by a positive correlation with TDP-43 on the first, third and fourteenth day after LFPI. An increase in APP protein expression was observed subacutely only in the rmTBI group of animals, and P-tau protein only in the LFPI mice. Conclusion: One moderate LFPI causes deregulation of TDP-43 in the ipsilateral cortex and hippocampus and it is related to some markers of inflammation and synaptogenesis. These changes depend on the type of the acquired brain trauma and are of potential importance in TBI pharmacotherapy.