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Dataset of a project: Preparation of N-oxides of pyridylporphyrins, their characterisation and photodynamic activity
No full textImunosno posredovane patologije u mišjem modelu s insuficijencijom optineurina
No full textAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are adult-onset
neurodegenerative diseases characterized by neuronal loss, neuroinflammation and TAR DNA
binding protein 43 (TDP-43) protein aggregation in distinct regions of the central nervous system
(CNS). Both diseases may be caused by mutations in the same genes, one of which is OPTN,
encoding for optineurin. Optineurin is a ubiquitin-binding adaptor protein that regulates various
cellular functions, including inflammatory signaling, autophagy, vesicular and mitochondrial
trafficking. To study its role in disease, a truncation mouse model of optineurin insufficiency that
lacks the ubiquitin-binding region (Optn470T) was generated by our group. Aging is the major risk
factor for neurodegenerative diseases, which strongly affects the immune system and results in a
chronic low-grade inflammation (inflammaging). Since optineurin plays a role in inflammatory
signaling, which was shown to be impaired in ALS/FTD patients, we have performed detailed
analyses of Optn470T mice during aging, which include evaluations of motor and cognitive abilities,
neuropathology (neuronal loss, TDP-43 aggregation and lipofuscin accumulation) and
neuroinflammation. We observed that aging of Optn470T mice did not exaggerate the decrease in
motor and cognitive abilities found in wild-type (WT) mice. This was corroborated by the absence
of neuropathology and exaggerated neuroinflammation in aged Optn470T mice. Given that
peripheral immune cells are also affected in ALS patients and during aging, we analyzed T cell
polarization. We found a decreased percentage of tumor necrosis factor (TNF)- and interleukin
(IL)-17A-producing CD4+ helper T cells and an increased percentage of TNF-producing CD8+ T
cells in Optn470T mice in comparison to WT mice. One of the new potential ALS hallmarks is
decreased phagocytosis of microglia and macrophages, reported during aging and in ALS patients.
Here, we reported a decreased percentage of phagocytic bone-marrow derived macrophages
(BMDMs) from aging mice, which was unaffected by optineurin insufficiency. To test if additional
genetic hits will precipitate the disease phenotype, we crossed Optn470T mice with TDP-43G348C
mice, where the latter have been reported to develop ALS/FTD-like symptoms. We successfully
confirmed the generation of Optn470T/TDP-43G348C, which showed lower expression levels of TDP
43G348C transgene compared to TDP-43G348C mice, suggesting a potential protective mechanism of
Optn470T truncation. TDP-43 was previously reported to directly regulate inflammatory nuclear
factor (NF)-B-mediated signaling, which we did not observe in our experiments in BMDMs. In contrast, lipopolysaccharide (LPS)-stimulated BMDMs from Optn470T/TDP-43G348C mice have
shown unaffected NF-B and decreased TANK-binding kinase (TBK1) signaling when compared
to BMDMs from TDP-43G348C mice. Aged Optn470T/TDP-43G348C mice have shown decreased
body weight and improved motor and cognitive abilities compared to TDP-43G348C mice of the
same age. We also analyzed these mice in inflammatory conditions, which were reported to
aggravate ALS/FTD phenotype. A three-day LPS administration resulted in a significant body
weight loss and higher mortality of TDP-43G348C mice, which was rescued in Optn470T/TDP
43G348C mice. LPS-administered Optn470T/TDP-43G348C mice have shown decreased numbers of
splenocytes, B cells, classical dendritic (cDC) and natural killer cells. Lastly, we observed an
increase in chemokine (C-C motif) ligand 2 (CCL2) and IL-10 serum levels in TDP-43G348C and
Optn470T/TDP-43G348C mice, respectively. Altogether, the presence of Optn470T truncation was
insufficient to cause ALS/FTD-like neuropathology during aging. In contrast, Optn470T truncation
has shown a protective effect in the presence of TDP-43G348C transgene in the basal state and during
inflammation in aged mice, which could have been caused by decreased TDP-43G348C transgene
levels in Optn470T/TDP-43G348C mice. Analysis of neuropathology and neuroinflammation in the
hippocampus (region responsible for memory) and denervation of neuromuscular junctions will
be performed to confirm this.Amiotrofična lateralna skleroza (ALS) i frontotemporalna demencija (FTD) su
neurodegenerativne bolesti odraslih ljudi koje su karakterizirane gubitkom neurona,
neuroinflamacijom i agregacijom TAR DNA-vezujućeg proteina 43 (TDP-43) u različitim
dijelovima središnjeg živčanog sustava (SŽS). Obje bolesti mogu biti uzrokovane mutacijama istih
gena, jedan od kojih je OPTN, koji kodira protein optineurin. Optineurin je ubikvitin-vezujući
adaptorski protein koji regulira razne stanične funkcije, uključujući upalnu signalizaciju,
autofagiju, vezikularno i mitohondrijalno prometovanje. Kako bismo izučili ulogu optineurina u
bolesti, naša istraživačka skupina generirala je mišji model insuficijencije optineurina (Optn470T)
kojem nedostaje ubikvitin-vezujuća regija. Starenje je glavni faktor rizika neurodegenerativnih
bolesti koji znatno utječe na imunosni sustav te rezultira kroničnom upalom slabog intenziteta,
odnosno „upalnim starenjem“. S obzirom na ulogu optineurina u upalnoj signalizaciji, koja je
poremećena u ALS/FTD pacijenata, proveli smo detaljnu analizu Optn470T miševa tijekom starenja.
Evaluirali smo simptome ALS/FTD-a analizirajući motoričke i kognitivne sposobnosti,
neuropatologiju (gubitak neurona, TDP-43 agregacija i akumulacija lipofuscina) i
neuroinflamaciju. Zamijetili smo da starenje Optn470T miševa nije pogoršalo gubitak motoričkih i
kognitivnih sposobnosti pronađenih u miševima divljeg tipa (WT). Ove nalaze potvrdili smo
nedostatkom neuropatologije i pojačane neuroinflamacije u starim Optn470T miševima. S obzirom
da ALS i starenje također utječu na periferne imunosne stanice, analizirali smo polarizaciju T
stanica. Pronašli smo smanjeni postotak CD4+ pomoćničkih T stanica koje proizvode faktor
nekroze tumora (TNF) i interleukin (IL)-17A te povećan postotak CD8+ T stanica u Optn470T
miševima u odnosu na WT miševe. Jedan od novih potencijalnih obilježja ALS-a je smanjena
fagocitoza mikroglije i makrofaga koja je pronađena tijekom starenja i u ALS pacijenata. Ovdje
smo pronašli smanjeni postotak fagocitirajućih makrofaga deriviranih iz koštane srži (BMDM
ovi) starih miševa, koja nije bila poremećena u prisustvu insuficijencije optineurina. Kako bismo
testirali ukoliko će dodatne genetske mutacije doprinijeti razvoju bolesti, proveli smo križanje
Optn470T i TDP-43G348C miševa, gdje je za potonjeg pokazano da razvija ALS/FTD simptome.
Uspješno smo potvrdili generaciju Optn470T/TDP-43G348C miševa, koji su pokazali nižu razinu
ekspresije TDP-43G348C transgena u usporedbi s TDP-43G348C miševima, što je potencijalni
protektivni mehanizam Optn470T trunkacije. Pronađeno je kako TDP-43 direktno regulira upalnu signalizaciju posredovanu nuklearnim faktorom (NF)-B, što mi nismo zamijetili u našim
eksperimentima s BMDM-ovima. Suprotno tome, BMDM-ovi iz Optn470T/TDP-43G348C miševa
stimulirani lipopolisaharidom (LPS-om) su pokazali normalnu NF-B te smanjenu aktivaciju
TANK-vezujuće kinaze 1 (TBK1) signalizacije u usporedbi s BMDM-ovima iz TDP-43G348C
miševa. Ostarjeli Optn470T/TDP-43G348C miševi pokazali su nižu tjelesnu masu te unaprijeđene
motoričke i kognitivne sposobnosti u usporedbi s TDP-43G348C miševima iste dobi. Ove miševe
smo također analizirali u upalnim uvjetima za koje je pokazano da pogoršavaju ALS/FTD fenotip.
Trodnevna administracija LPS-a rezultirala je značajnim gubitkom tjelesne mase i visokom
smrtnošću TDP-43G348C miševa, koje su bile značajno ublažene u Optn470T/TDP-43G348C miševima.
U Optn470T/TDP-43G348C miševa administriranih LPS-om pronađen je niži broj splenocita, B
stanica, klasičnih dendritičkih stanica (cDC) i prirodnih stanica ubojica. Na kraju smo pronašli
povećanje kemokinog C-C liganda 2 (CCL2) i interleukina-10 u TDP-43G348C i Optn470T/TDP
43G348C miševima. Sveukupno, prisustvo Optn470T trunkacije je nedovoljno za izazivanje ALS/FTD
neuropatologije tijekom starenja. Međutim, Optn470T trunkacija je pokazala protektivni učinak u
prisustvu TDP-43G348C transgena u bazalnom stanju i tijekom upale u starih miševa, što bi moglo
biti uzorkovano smanjenim razinama TDP-43G348C transgena u Optn470T/TDP-43G348C miševima.
Analiza neuropatologije i neuroinflamacije u hipokampusu (regiji odgovornoj za memoriju) te
denervacije neuromišićnih spojnica će biti provedene kako bismo to potvrdili
Optimisation of coagulation factor IX purification from human plasma
No full textTradicionalne metode proizvodnje terapeutskih proteina često u upstream procesu uključuju
uzgoj staničnih linija, sakupljanje stanica, staničnu lizu, precipitaciju proteina, filtraciju i druge
korake. U downstream procesima, procesima pročišćavanja terapeutskih proteina, koristi se
kromatografija. Ključan korak optimizacije procesa čišćenja terapeutskih proteina iz
kompleksnih smjesa, bila bi implementacija kromatografskih nosača na koje se mogu direktno
primjeniti uzorci kompleksnih karakteristika, bez potrebe za dodatnom obradom i/ili
predrazrijeđivanjem uzorka. U ovom radu to je demonstrirano na primjeru pročišćavanja
koagulacijskog faktora IX IZ nerazrijeđene ljudske plazme sa odstranjenim krioprecipitatom.
U tradicionalnom postupku čiščenja faktora IX, uzorak plazme se razrjeđuje, nakon čega
slijede dva koraka kromatografije ionske izmjene. Kao posljednji korak koristi se afinitetna
kromatografija na nosaču sa heparinskim ligandom životinjskog porijekla. Rezultati ovog
istraživanja pokazuju da je na monolitne polimetakrilatne kromatografske nosače moguće
direktno nanositi kompleksne biološke uzorke, a da pritom ne dolazi do neželjenih
biokemijskih i biofizikalnih promjena ljudske plazme. To svojstvo monolitnih
polimetarkrilatnih nosača iskorišteno je za pročišćavanje koagulacijskog faktora IX direktno iz
plazme sa odstranjenim krioprecipitatom na DEAE kationskim izmjenjivačima. Također,
utvrđeno je da se kao alternativa kromatografskim nosačima sa imobiliziranim heparinskim
ligandom, u daljnjem pročišćavanju faktora IX mogu upotrijebiti kromatografski nosači sa
kovalentno vezanim sulfatnim skupinama. Implementacija prethodna dva koraka predstavlja
osnovu za novi (semi)kontinuirani proces proizvodnje u svrhu čišćenja i izolacije terapijskog
pripravka koagulacijskog faktora IX.Traditional methods used in the upstream production of therapeutic proteins often include
cell lines, cell harvesting and lysis, protein precipitation, filtration, and other steps.
Chromatography is usually used in the downstream processes. A key step towards optimizing
the process of therapeutic proteins purification from complex mixtures, would be the
implementation of chromatographic supports to which samples with complex characteristics
can be directly applied, without the need for additional processing and/or pre-dilution of the
sample. Using the example of undiluted human plasma, it was demonstrated that it is possible
to directly apply complex biological materials to monolithic polymethacrylate
chromatographic supports, without unwanted biochemical and biophysical changes in the
biological material. This property of monolithic polymethacrylate supports was used to purify
coagulation factor IX directly from plasma with cryoprecipitate removed. In the traditional
purification process of factor IX, the last step is affinity chromatography on a support with a
heparin ligand of animal origin. In our experiments, it was established that as an alternative
to chromatographic carriers with immobilized heparin ligand, chromatographic carriers with
covalently bound sulfate groups can be used. The implementation of the previous two steps
represents the basis for a new (semi)continuous production process for the purpose of
cleaning and isolating the therapeutic preparation of coagulation factor IX
Zbirka zadataka iz analitičke kemije
No full textZbirka zadataka iz analitičke kemije pripremljena je u skladu s nastavnim programom kolegija „Analitička kemija“ koji se izvodi na Fakultetu biotehnologije i razvoja lijekova Sveučilišta u Rijeci. Namijenjena je studentima (studenticama) prve godine preddiplomskog studija Biotehnologija i istraživanje lijekova i drugih srodnih studija.
Sadržaj Zbirke zadataka prilagođen je izvođenju auditornih vježbi seminarske nastave u okviru nastavnog programa kolegija. Zbirka sadrži teorijski dio, riješene zadatke i zadatke za vježbu s rješenjima.
Zadaci u Zbirci, podijeljeni u šest poglavlja, prikupljeni su i odabrani iz međunarodno provjerenih izvora i osobne arhive s ciljem veće uspješnosti studenata u rješavanju zadataka auditornih vježbi
Interakcija optineurina i TDP-43 u staničnim modelima amiotrofične lateralne skleroze
No full textAmyotrophic lateral sclerosis (ALS) is neurodegenerative disease caused by mutations in more than 30 different genes, which act by different and often still unclear pathogenic mechanisms. Two major features mark all ALS cases: insoluble protein aggregates in neurons and glia, and neuroinflammation. Aggregates in the majority of ALS cases, regardless of genetic cause, comprise TAR DNA-binding protein 43 (TDP-43), an RNA/DNA-binding protein with functions in splicing, mRNA metabolism and miRNA production. In contrast, OPTN gene is a rare cause of ALS. It encodes a multifunctional adaptor protein optineurin (Optn) proposed to regulate many cellular processes like autophagy, inflammation, Golgi maintenance and vesicular trafficking. Recent data from our laboratory have shown that both Optn insufficiency and deficiency caused an increase in endogenous TDP-43 levels in myeloid cells (microglia and macrophages), which was unexpectedly not caused by an autophagy block. Inflammatory stimulation by a lipopolysaccharide (LPS), which was previously shown to stimulate TDP-43 aggregation, elevated TDP-43 levels in WT but could not increase already elevated TDP-43 levels in cells lacking functional optineurin. Notably, no aggregates were formed in neither WT nor optineurin insufficient or deficient cells. Here generated a new mouse model carrying transgenic TDP-43G348C and truncated Optn470T/470T to test if transgenic TDP-43 causes greater proteotoxic stress than endogenous TDP-43 and leads to signs of ALS pathology in vitro or in vivo. We investigated transgenic TDP-43 levels in bone marrow-derived macrophages (BMDM) and observed higher TDP-43 levels in Optn470T/470T/TDP-43G348C compared to WT/TDP-43G348C BMDM. Transgenic TDP-43 thus did accumulate in Optn insufficient cells, but not because of an autophagy block. Also, we observed that transgenic TDP-43 levels cannot be additionally increased upon LPS stimulation in Optn470T/470T/TDP-43G348C BMDM, in contrast to WT cells. Notably, transgenic TDP-43 was not cytotoxic in unmanipulated BMDM but caused apoptosis after LPS stimulation in both optineurin sufficient and
insufficient cells. Immunofluorescent analysis showed that transgenic TDP-43 was not depleted from the nucleus and did not aggregate upon LPS stimulation in BMDM. In addition, we investigated spinal cords of transgenic mice model and observed no signs of ALS pathology (neuronal loss and TDP-43 aggregation) in neither WT nor Optn470T/470T 8-month-old mice. In conclusion, in this study, we showed that transgenic TDP-43 has elevated protein levels in Optn insufficient BMDM, just like it was previously shown for endogenous TDP-43, but this is not caused by an autophagy block and does not result in cytotoxicity. Moreover, optineurin insufficiency did not precipitate the pathology in TDP-43G348C mouse model, at least not at the early stage of disease. Further studies are necessary to investigate pathology in Optn470T/470T/TDP-43G348C model at later stages.Amiotrofična lateralna skleroza (ALS) je neurodegenerativna bolest koju uzrokuju mutacije u više od 30 gena, koji djeluju prema različitim, i još uvijek nejasnim, patogenim mehanizmima. Dva glavna obilježja označavaju sve slučajeve ALS-a: netopljivi proteinski agregati, u neuronima i glija stanicama, i neuroinflamacija. Agregati u većini ALS slučajeva, neovisno o genetskom uzroku, sadrže TAR DNA-vezujući protein 43 (TDP-43), RNA/DNA-vezujući protein čije su funkcije prekrajanje, metabolizam RNA i miRNA. Suprotno tome, OPTN gen je rijedak uzrok ALS-a. Gen kodira multifunkcionalni adaptor protein optineurin (Optn), za koji se smatra da regulira mnoge stanične procese poput autofagije, upale, održavanja Golgija i vezikularnog transporta. Nedavni rezultati iz našeg laboratorija pokazali su da insuficijencija i deficijencija Optn uzrokuju povećanje razina endogenog TDP-43 u mijeloidnim stanicama (mikrogliji i makrofagima), koje, neočekivano, nije uzrokovano blokadom autofagije. Stimulacija upale sa lipopolisaharidom (LPS), koja je ranije pokazala da stimulira agregaciju TDP-43, povećala je razine TDP-43 u WT, ali nije uspjela povećati već povećane razine TDP-43 u stanicama bez funkcionalnog Optn. Značajno, agregati nisu formirani niti u WT niti u Optn insuficijentnim ili deficijentnim stanicama. U ovom radu proizveli smo novi mišji model sa transgeničnim TDP-43G348C i skraćenim Optn470T/470T kako bismo istražili uzrokuje li transgenični TDP-43 veći proteotoksični stres od endogenog TDP-43 i dovodi li do znakova patologije ALS-a in vitro ili in vivo. Testirali smo razine transgeničnog TDP-43 u makrofagima dobivenim iz koštane srži (BMDM) i uočili smo veće razine TDP-43 u Optn470T/470T/TDP-43G348C nego u WT/TDP-43G348C BMDM. Transgenični TDP-43 se nakupljao zbog insuficijencije Optn, a ne blokade autofagije. Također, uočili smo da se razine transgeničnog TDP-43 ne mogu dodatno povisiti stimulacijom LPS-om u Optn470T/470T/TDP-43G348C BMDM, za razliku od WT stanica. Transgenični TDP-43 nije bio citotoksičan u netretiranim stanicama, ali je uzrokovao apoptozu nakon stimulacije LPS-om u stanicama oba genotipa. Imunofluorescentna analiza nije
pokazala nuklearnu depleciju niti agregaciju transgeničnog TDP-43 nakon stimulacije LPS-om u BMDM. Pored toga, testirali smo leđne moždine transgeničnog mišjeg modela i nismo uočili patološke znakove ALS-a (gubitak neurona i TDP-43 agregati) niti u WT niti Optn470T/470T miševima starim 8 mjeseci. Zaključno, u ovom radu pokazali smo da transgenični TDP-43 ima povećane razine proteina u Optn insuficijentnim BMDM, kao što je to prethodno prikazano za endogeni TDP-43, ali to nije uzrokovano blokadom autofagije i nije rezultiralo citotoksičnošću. Štoviše, Optn insuficijencija nije ubrzala patologiju TDP-43G348C mišjeg modela, barem ne u ranom stadiju bolesti. Dodatna istraživanja su potrebna kako bi se istražila patologija u Optn470T/470T/TDP-43G348C modelu u kasnijim stadijima
Metabolic Profiling of Colorectal Adenocarcinoma Using Mass Spectrometry Imaging
No full textKolorektalni karcinom čini više od 90 % malignih tumora debelog crijeva, a učestalost ove bolesti u postupnom je porastu. Najrašireniji oblik je adenokarcinom, koji je, između ostalog, rezultat složenih metaboličkih interakcija u stanicama karcinoma na koje utječe i mikrookoliš. Biokemijska svojstva zloćudnih stanica među pacijentima značajno se razlikuju, a putem konvencionalnih histopatoloških pristupa teško ih je detaljnije opisati i klasificirati. Posljedica toga jednaki su terapijski postupci u bolesnika s različitom molekularnom podlogom bolesti. Stoga, cilj ovog istraživanja bio je vizualizirati i identificirati specifične endogene metabolite kolorektalnog adenokarcinoma i susjednog peritumorskog tkiva upotrebom slikovne spektrometrije masa (MSI od eng. Mass Spectrometry Imaging) kod deset ispitanika. MSI je izvedena primjenom matricom potpomognute ionizacije laserskom desorpcijom s analizatorom vremena preleta (MALDI-TOF od eng. Matrix-Assisted Laser Desorption/Ionization-Time Of Flight) u pozitivnom i negativnom načinu snimanja. Dobiveni rezultati MSI tehnikom pokazali su jasnije razlikovanje morfoloških obrazaca tumorskog i peritumorskog tkiva u odnosu na nativni i hematoksilin-eozin (H&E) metodom obojani preparat. Slikovna analiza na temelju m/z signala dobivenih snimanjem u negativnom načinu bolje je razlikovala vrstu tkiva i stanica te pružila informativnije raspodjele iona metabolita u odnosu na pozitivan način snimanja. Analiza glavnih komponenti (PCA od eng. Principal Component Analysis) jasno je klasificirala tumorske i peritumorske regije za pojedine ispitanike na temelju razlika u njihovim m/z signalima. Uočeno je da molekularne komponente većih masa bolje razlikuju navedene regije. Primjenom t-testa nije identificiran statistički značajan m/z signal svojstven tkivima svih ispitanika, što ukazuje na heterogenost tumorskih i peritumorskih regija među svim uzorcima. Kao dominantni spojevi u obje vrste tkiva detektirani su ceramidi, što bi moglo ukazati da su promjene u metabolizmu ceramida usko povezane s nastankom i napredovanjem kolorektalnog adenokarcinoma. Ovo istraživanje potvrđuje da nema univerzalnih markera adenokarcinoma kolona iz grupe malih molekula te upućuje na potrebu za personalizacijom dijagnostike i, shodno tome, terapijskog pristupa kolorektalnom adenokarcinomu.Colorectal carcinoma represents more than 90 % of malignant tumors of the colon, and the incidence of this disease is gradually increasing. The most common form is adenocarcinoma, which is, among other things, the result of complex metabolic interactions in cancer cells that are also affected by the microenvironment. The biochemical properties of malignant cells differ significantly among patients, and it is hard to describe and classify them in more detail through conventional histopathological approaches. The consequence is equal therapeutic procedures in patients with different molecular background of diseases. Therefore, the aim of this study was to visualize and identify specific endogenous metabolites of colorectal adenocarcinoma and surrounding peritumoral tissue using Mass Spectrometry Imaging (MSI) in ten patients. MSI was performed using Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) in positive and negative mode. The results obtained by the MSI technique showed a clearer difference between the morphological patterns of the tumor and peritumoral tissue in relation to the native preparation and preparation stained by hematoxylin-eosin (H&E) method. Image analysis based on m/z signals obtained by negative ion mode better distinguished tissue and cell type and provided a more informative distribution of ionized metabolites compared to positive ion mode. Principal component analysis (PCA) clearly classified tumor and peritumor areas based on differences in their m/z signals in the individual sample analysis. It has been observed that the molecular components of higher masses better differentiate these areas. No common statistically significant m/z signal was found in all subjects using the t-test, indicating heterogeneity of tumor and peritumor regions among all samples. Ceramides have been identified as dominant compounds in both tissue types, which may mean that changes in ceramide metabolism are closely related to the development and progression of colorectal adenocarcinoma. This study confirms that there are no universal tumor markers from the group of small molecules and requisit the need for personalization of diagnostics and, consequently, therapeutic approach to colorectal adenocarcinoma
Characterization of CO releasing metallosurfractant aggregates intended for biomedical applications
No full textOd početka istraživanja terapeutskih sposobnosti CO, postojala je potreba za
ciljanom dostavom ove terapije. Zbog toga, razvili su se razni kemijski
spojevi koji otpuštaju CO. Među njima su izuzetni metalni karbonili , zbog
velikog kapaciteta prijenosa CO. Mijenjajući ligande vezane za metal u
koordinacijskom centru, poboljšala se permeabilnost u biološkim sustavima.
Ovi karakterizirani spojevi, koji mogu imati metalni dio u polarnom ili
hidrofobnom dijelu molekule, nazivaju se metalosurfraktanti. Zbog povećane
amfifilnosti, grade razne supramolekularne agregate u vodenim sredinama.
U prijašnjim istraživanjima, metalosurfraktant sa molibdenom je pokazao
miješivost sa fosfatidilkolinom u agregate poznate kao metalosomi- sa
fizikalno-kemijskim svojstvima, zbog kojih su prikladniji za primjenu u
biološkim sustavima, u odnosu na agregate od čistih metalosurfraktanta.
Glavni cilj ovog projekta, bio je opisati interakciju metalosoma, koji sadrže
metalosurfraktant PCOL10, sa stanicama. Sekundarni cilj, bio je razvijanje
metodologije za istraživanje svojstva metalosurfraktanta i metalosoma sa
alatima računalne kemije.
Nakon razvitka metodologije za proizvodnju PCOL10 metalosoma sa
kontroliranim veličinama metalosoma, opisana su fizička i kemijska svojstva
pod uvijetima relevantnima za inkubaciju sa stanicama. Zatim, stanične
kulture ljudskih fibroblasta su inkubirane s metalosomima, te potom
analizirane s microFTIR spektroskopijom u Alba sinkrotronu. Na temelju
toga, opisana je ovisnost internalizacije metalosoma u stanicama, u odnosu
na trajanje inkubacije, koncentraciju metalosoma i njihovu veličinu.
Eksperimenti inkubacije u stanicama, pokazali su kako je internalizacija
PCOL10 metalosoma u stanicama, uglavnom ovisna o vremenu inkubacije i
koncentraciji. Laboratorijski dio rada obavljen je na Autonomnom Sveučilištu
u Barceloni, u laboratoriju Lipidno-baziranih sustava dostave lijekova.Since the start of research into the therapeutic abilities of CO, systems for
targeted delivery and release have been needed. Because of this, the
category of CO releasing molecules includes numerous types of compounds.
Among them, metal-carbonyl compounds are distinguished because of their
high CO carrying capacity. These compounds were further optimized to
improve specific targeting, by modifying the groups connected to the metal.
The results, were compounds that can either include the metal into the
polar, or hydrophobic group, called metallosurfractants. Their improved
amphiphilic character, allows them to form supramolecular structures in an
aqueous medium. In previous research, a molybdenum based
metallosurfractant has shown miscibility with phosphatidylcholine that forms
vesicles known as metallosomes, with improved physicochemical properties
compared to pure metallosurfractant aggregates. The principal aim of this
project was characterizing the interaction of PCOL10-metallosomes with
cells. The secondary aim was to develop a workflow that models the
properties of metallosurfactants and metallosomes, using computational
chemistry.
First, after developing a reproducible method for preparing PCOL10
metallosomes, the physical and chemical properties under conditions
relevant for cell incubations were described. Finally, the metallosomes were
incubated in cultures of human fibroblasts and analyzed using microFTIR
spectroscopy at Alba synchrotron. The dependence of cell internalization
upon incubation time, metallosome size and concentration, was described.
The cell incubation studies showed that cell internalization of PCOL10
metallosomes depends mainly on incubation time and concentration. All
laboratory research was carried out at the Autonomous University of
Barcelona, at the Lipid-based drug delivery systems laboratory
Stabilnost galij-dopamin nanočestica u različitim vodenim medijima
No full textAntibiotic resistance is slowly becoming a serious problem in global public health due to antibiotic overuse, and due to a significant rise in multi-drug resistant bacteria. A lot of research has been put into finding antibacterial alternatives. Metal ions and metal-based nanoparticles are currently being studied as an alternative to antibiotics. Among various metals with antibacterial properties, gallium (Ga) ions and their nanoparticles have attracted attention due to their antibacterial properties. However, due to the low bioavailability of gallium ions, and the unique physicochemical properties of nanoparticles, gallium nanoparticles (Ga NPs) might be a better alternative compared to ions. Factors that can influence the antibacterial efficiency of Ga NPs are yet to be researched. In this work, the change in concentration of Ga NPs and their dissolution at different temperatures (20 °C and 37 °C) was measured in various aqueous media: distilled water, phosphate saline buffer (PBS), and bacterial growth medium (Mueller Hinton Broth, MHB). Determination of gallium ions concentration and Ga NPs was performed by inductively coupled plasma mass spectrometry (ICP-MS). Dissolution and ICP-MS ion release profiles of Ga NPs in different aqueous solutions and at different temperatures were observed and compared. Results suggest that the rates of dissolution differ in all of the investigated solutions and that temperature affects dissolution. Ga NPs at lower temperatures are more stable in all three solutions and have fewer statistical deviations. At higher temperatures, dissolutions went over 100 % in PBS and MHB. Even though Ga NPs are very stable in distilled H2O, distilled water has low dissolution rates, which might not be ideal for the antibacterial purpose of Ga NPs. Dissolution of Ga NPs is consistent, slowly increasing over 24 hours, at 20 °C. Around 80 % dissolution is detected in PBS and almost 100 % in MHB after 24 h, at lower temperatures. The effects of aqueous media on Ga NPs dissolution demonstrate that both PBS and MHB enable favorable dissolution, especially at 20 °C.Otpornost na antibiotike polako postaje ozbiljan problem u svjetskom javnom zdravstvu zbog prekomjerne uporabe antibiotika i značajnog porasta bakterija koje su otporne na više vrsta lijekova. Puno je istraživanja uloženo u pronalaženje antibakterijskih alternativa. Metalni ioni i nanočestice bazirane na metalima trenutno se proučavaju kao alternativa antibioticima. Među raznim metalima s antibakterijskim svojstvima, ioni galija (Ga) i njegove nanočestice privukli su pozornost zbog antibakterijskih svojstava. Međutim, zbog niske bioraspoloživosti iona galija i jedinstvenih fizikalno-kemijskih svojstava nanočestica, nanočestice galija (engl. gallium nanoparticles, Ga NP) mogle bi biti bolja alternativa nego ioni. Čimbenici koji mogu utjecati na antibakterijsku učinkovitost Ga NP tek treba istražiti. U ovom radu, promjena koncentracije Ga NP i njihovo otapanje na različitim temperaturama (20 °C i 37 °C) mjerit će se u različitim vodenim medijima: destilirana voda, fosfatni pufer i medij za rast bakterija (Mueller Hinton Broth, MHB). Određivanje koncentracije galijevih iona i Ga NPs provedeno je pomoću spektrometrije masa uz induktivno spregnutu plazmu (ICP-MS). Promatrani su i uspoređeni profili otapanja nanočestica i otpuštanja Ga iona u različitim vodenim otopinama i na različitim temperaturama. Rezultati pokazuju da se otapanja Ga NP razlikuju u svim ispitivanim otopinama, te da temperatura utječe na otapanje. Ga NP na nižim temperaturama stabilnije su u sve tri otopine i rezultati imaju manja statistička odstupanja. Pri višim temperaturama dobivene su vrijednosti otapanja preko 100 % u PBS i MHB. Iako su Ga NP vrlo stabilne u destiliranoj H2O, destilirana voda ima niske stope otapanja, što možda nije idealno za antibakterijsku svrhu Ga NP. Otapanje Ga NP je dosljedno, polako se povećava tijekom vremenskog raspona od 24 sata, na 20 °C. Oko 80 % otapanja detektirano je u PBS-u i gotovo 100 % u MHB-u nakon 24 sata, na nižoj temperaturi. Učinci vodenih medija na otapanje Ga NP pokazuju da i PBS i MHB omogućuju povoljno otapanje nanočestica, posebno na 20 °C
Karakterizacija agregacije DISC1 proteina u neuropsihijatrijskim poremećajima
No full textTo date, the pathophysiology of psychiatric disorders remains poorly understood. While
traditionally investigated using genetics, disrupted protein homeostasis has been
suggested as a biological basis for these diseases in at least a subset of patients. Several
proteins previously implicated by genetic studies have been shown to form insoluble
aggregates in such illnesses, including Disrupted in Schizophrenia 1 (DISC1). DISC1 is
a cytosolic, multi-functional scaffolding protein involved in numerous disease-related
neuronal pathways. Due to its large interaction network, it has many reported roles,
including, but not limited to, brain development, maturation, migration and proliferation
of neuronal cells, synapse regulation and mitochondrial trafficking. However, delineation
of the domain structure of DISC1 has only recently been attempted, using highthroughput analysis in Escherichia coli. This approach identified four distinct stable
regions, named D, I, S and C. Here, we used both empirical and theoretical data to confirm
and refine the domain structure of DISC1 when expressed in mammalian cells, as well as
test the functionality of each region through protein-protein interactions. We then utilised
this knowledge to identify the exact part of DISC1 responsible for its aggregation.
Moreover, we created Drosophila lines expressing human DISC1, to assess behavioural
phenotypes corresponding to symptoms often linked with psychiatric disorders.
First, we attempted to refine the borders of DISC1 by combining previously published
bioinformatics data on DISC1 domains with the recent empirical data. Constructs
encoding variants of D, I and C regions were cloned, with borders modified based on
theoretical predictions. These were transfected into HEK293 cells and tested for stability
via proteasome inhibition assay. Results obtained by immunoblotting implicated that
modified versions of D and C regions have improved stability over their empirical
counterparts, with evidence that the I region may not represent a stable folded region by
itself. The D, S and C domains were further shown to be functional in isolation, although
only the C region was capable of forming interactions with known protein binding
partners.
In order to investigate which structural region(s) might be responsible for its aggregation,
fragments of DISC1 were expressed in SH-SY5Y neuroblastoma cells and their
localization patterns viewed by fluorescent microscopy. Single regions exhibited clear
II
cytoplasmic localization with no aggregate formation. In contrast, the combination of D
and I region showed clear signs of aggregation, with full length DISC1 used as a positive
control. Based on this, we hypothesized that the unstructured region between these two
regions, D and I, is responsible for DISC1 aggregation propensity, which was verified
using further truncation constructs.
Finally, we created transgenic Drosophila lines expressing human DISC1 on both the
second and third chromosome by using the Gal4-UAS system, which allows selective
activation of any cloned gene in a variety of neuronal clusters. We assessed flies’ abilities
to climb vertical surfaces, as well as locomotor activity and sleep patterns, which are often
prone to disturbances in neuropsychiatric disorders. Our results signified that locomotor
activity and sleep in Drosophila are affected by both the DISC1 insertion and protein
expression, in correlation to symptoms reported by psychiatric patients.
With this thesis, we confirmed and refined the domain structure of DISC1, a known risk
factor for mental disorders. Additionally, our results provided an insight into its
aggregation as an important emerging aspect of DISC1 pathology, as well as the direct
effect of DISC1 expression on the behavioural phenotypes seen in Drosophila animal
models. Together, our data will aid in elucidation of pathogenic mechanisms underlying
psychiatric disorders, and generation of new, high-quality animal models to study
behavioural consequences and relevance of DISC1 aggregation for major psychiatric
disorders.Unatoč velikim znanstvenim napretcima postignutim tijekom posljednjih desetljeća,
patofiziologija psihijatrijskih poremećaja još uvijek nije u potpunosti razjašnjena. Dok se
tradicionalni pristup istraživanja primarno fokusirao na genetske aspekte bolesti, nova
saznanja ukazuju na poremećaje u homeostazi proteina kao biološku bazu ovih bolesti.
Štoviše, nekolicina proteina prethodno impliciranih u genetskim studijama
neuropsihijatrijskih poremećaja također imaju sposobnost stvaranja toksičnih, netopljivih
proteina u mozgovima pacijenata, uključujući i Disrupted in Schizophrenia 1 (DISC1)
protein.
DISC1 je citosolični, multifunkcionalni protein uključen u velik broj neuralnih procesa
kritičnih za normalno obnašanje kognitivnih i tjelesnih funkcija. Zahvaljujući širokoj
mreži interakcijskih proteina, DISC1 ima ulogu u razvoju mozga, maturaciji, migraciji i
proliferaciji neuronalnih stanica, regulaciji sinapsi te mitohondrijskom transportu. No
unatoč važnosti ovog proteina, tek je nedavno, uz pomoć visokopropusnih tehnologija,
razotkrivena njegova strukturalna organizacija. Ovim su pristupom identificirane četiri
stabilne regije proteina, nazvane D, I, S i C. U ovome radu koristili smo kombinaciju
empirijskih i teorijskih podataka kako bismo potvrdili i usavršili strukturu domene DISC1
proteina u stanicama sisavaca te testirali funkcionalnost svake regije kroz interakciju sa
drugim proteinima. Nadalje, identificirali smo točnu regiju DISC1 proteina odgovornu za
njegovu agregaciju te proizveli linije vinskih mušica koje eksprimiraju ljudski DISC1
kako bismo evaluirali fenotipe ponašanja koji su u skladu sa čestim simptomima viđenim
u psihijatrijskim poremećajima.
Za potrebe optimizacije granica DISC1 regija kombinirali smo prethodno publicirana
bioinformatička saznanja sa nedavnim eksperimentalnim otkrićima. Klonirali smo
modificirane konstrukte koji kodiraju varijante D, I i C regija na temelju teorijskih
predviđanja te ih transficirali u HEK293 stanice. Potom smo testirali stabilnost istih
inhibicijom funkcije proteasoma. Dobiveni rezultati ukazali su na to da modificirane
verzije D i C regije imaju povećanu stabilnost u usporedbi sa eksperimentalno dobivenim
konstruktima, kao i da I regija eksprimirana u izolaciji ne predstavlja stabilnu regiju.
Nadalje, demonstrirali smo da su D, S i C regije funkcionalne u izolaciji, iako je samo
potonja demonstrirala sposobnost interakcije sa poznatim veznim partnerima.
IV
Kako bismo istražili koje strukturalne regije imaju sposobnost agregacije eksprimirali
smo fragmente DISC1 proteina u SH-SY5Y stanicama neuroblastoma te pratili njihovu
lokalizaciju koristeći fluorescentnu mikroskopiju. Izolirane regije demonstrirale su
citoplazmatsku lokalizaciju s izostankom agregacije. Suprotno tome, kombinacija D i I
regije demonstrirala je jasne znakove agregacije, u korespondenciji sa agregacijom
vidljivom pri ekspresiji nefragmentiranog DISC1 proteina korištenog kao pozitivna
kontrola. Temeljem ovih rezultata, pretpostavili smo kako je nestrukturirani dio između
D i I regije zaslužan za sklonost DISC1 proteina ka formaciji agregata, što smo potvrdili
daljnom ekspresijom fragmentiranih konstrukata te regije.
Naposlijetku, proizveli smo transgenične linije vinskih mušica koje eksprimiraju ljudski
DISC1 protein na drugom i trećem kromosomu, koristeći Gal4-UAS sistem koji
omogućava selektivnu aktivaciju bilo kojeg kloniranog gena u različitim skupinama
neurona. Testirali smo sposobnost transgeničnih linija pri uspinjanju vertikalnih površina
te lokomotornu aktivnost i obrasce spavanja, čiji su poremećaji česta pojava u
neuropsihijatrijskim bolestima. Dobiveni rezultati ukazali su na to kako insercija DISC1
gena i ekspresija proteina obje utječu na lokomotornu aktivnost i spavanje u vinskih
mušica, u korelaciji sa simptomima zabilježenim u psihijatrijskih pacijenata.
Ovim doktorskim radom potvrdili smo i optimizirali strukturu domene DISC1 proteina,
važnog rizičnog faktora za razvoj mentalnih poremećaja. Nadalje, naši rezultati pružaju
uvid u agregaciju ovog proteina kao potencijalnog patofiziološkog mehanizma bolesti,
kao i izravan utjecaj ekspresije DISC1 gena na bihevioralne fenotipove viđene u modelu
vinske mušice. Naši će podaci pridonijeti razjašnjenju patogenih mehanizama koji su
temeljni uzrok psihijatrijskih poremećaja, kao i generaciji novih životinjskih modela
visoke kvalitete koji će omogućiti proučavanje bihevioralnih posljedica DISC1
ekspresije, kao i važnost DISC1 agregacije, u psihijatrijskim poremećajima
Agregacija, paralelna agregacija i koagregacija proteina u kroničnim mentalnim bolestima
No full textChronic mental illnesses (CMIs), including schizophrenia (SZ),
bipolar disorder (BiPD), and major depressive disorder (MDD), present
a substantial burden on individuals and healthcare systems worldwide.
CMIs exhibit complex etiologies involving a mixture of genetic and
environmental factors. While protein aggregation is a well-established
hallmark of neurodegenerative diseases, its role in CMIs remains poorly
understood. Key questions remain unanswered, such as whether
aggregation occurs uniformly across brain regions, whether different
proteins aggregate within the same individuals, what drives this
aggregation, and how it may contribute to behavioral symptoms. This
thesis aims to address these knowledge gaps by investigating the
causes and effects of protein aggregation in CMIs, with a focus on the
potential for co-aggregation of key candidate proteins.
This research utilizes post-mortem human brain samples, in vitro
cell models, and a transgenic Drosophila model. Analysis of human
brain samples revealed distinct patterns of insolubility and aggregation
for CRMP1, DISC1, NPAS3, and TRIOBP-1 across different brain regions
linked to SZ, MDD, and suicide. Notably, these proteins were found to
co-aggregate within the same individuals, including some healthy
controls, suggesting either shared physiological mechanisms or
differential pathological thresholds. The extent and location of
aggregation varied between brain regions and individuals, showing that
it does not follow a consistent pattern based on diagnosis.
Moreover, the link between protein aggregation, genetic
mutation, and environmental susceptibility was investigated using
wild-type and mutant forms of NPAS3 in cell culture experiments. While
previous research showed that mutation alone can cause NPAS3
aggregation, my findings demonstrated that loss of nuclear localization
and potential aggregation can also occur under physiological and stress
conditions, even in the absence of mutation. This suggests that these
changes may be less dependent on genetic alterations than previously
assumed.
Region-specific analysis further revealed that the PAS1 domain
strongly influences NPAS3 localization in cells, promoting its retention
in the cytoplasm rather than the nucleus. This mislocalization may
reduce NPAS3's functional activity and increase its susceptibility to aggregation. Additionally, CRMP1 showed potential for co-aggregation with DISC1 and TRIOBP-1 in cells, echoing findings from human brain
analyses. Additionally, the Drosophila model expressing human DISC1
variants shows potential for revealing behavioral and molecular
alterations. However, the model requires thorough validation due to
lack of expression control.
These findings establish that protein aggregation in CMIs is a
heterogeneous, region-dependent process, with multiple proteins
aggregating within the same individuals. It also provides new evidence
that co-aggregation may be a contributing molecular mechanism in
CMIs. Moreover, it highlights how both genetic and environmental
stressors can influence aggregation. This study is limited by variability
in brain tissue quality, protein aggregation detected in controls, and
reliance on overexpression models in cell culture. Also, the Drosophila
model showed leaky gene expression and requires further validation.
Taken together, these factors highlight the need for improved models
and normalization methods in future research. Nevertheless, the
results from this thesis lay the groundwork for future research into
protein aggregation as a potential biomarker or therapeutic target,
offering novel molecular insights into the pathophysiology of CMIs.Kronične mentalne bolesti (KMB), uključujući shizofreniju (SZ),
bipolarni poremećaj (BiPD) i kliničku depresiju (KD), predstavljaju
značajan teret za pojedince i zdravstvene sustave diljem svijeta. KMB
imaju složenu etiologiju koja uključuje kombinaciju genetskih i
okolišnih čimbenika. Iako je agregacija proteina dobro poznata
značajka neurodegenerativnih bolesti, njezina uloga u KMB još uvijek
nije dovoljno istražena. Ključna pitanja ostaju neodgovorena: događa
li se agregacija ravnomjerno u različitim regijama mozga, agregiraju li
različiti proteini unutar istih pojedinaca, što pokreće agregaciju i kako
ona može pridonijeti razvoju simptoma. Ova disertacija ima za cilj
popuniti te praznine u znanju istraživanjem uzroka i posljedica
agregacije proteina u KMB, s posebnim naglaskom na mogućnost
koagregacije ključnih kandidata proteina.
Opisano istraživanje uključuje analize post-mortem uzoraka
ljudskog mozga, in vitro stanične modele i transgenični model vinske
mušice (Drosophila). Analiza ljudskog moždanog tkiva otkrila je
različite obrasce netopljivosti i agregacije proteina CRMP1, DISC1,
NPAS3 i TRIOBP-1 u različitim regijama mozga povezanim sa SZ, MDD
i
suicidalnim ponašanjem. Analizirani proteini koagregirali unutar
određenih pojedinaca sa dijagnozom i u kontrolnim uzorcima,
upućujući na zajedničke fiziološke mehanizme ili različite patološke
pragove. Opseg i lokalizacija agregacije varirali su među regijama
mozga i pojedincima te agregacija nije bila dosljedna ni specifična za
dijagnozu.
Nadalje, povezanost agregacije proteina, genetskih mutacija i
osjetljivosti na okoliš istražena je pomoću divljeg i mutiranog oblika
proteina NPAS3 u staničnim kulturama. Iako su prethodna istraživanja
pokazala kako sama mutacija može uzrokovati agregaciju NPAS3, moji
rezultati pokazuju kako gubitak nuklearne lokalizacije i potencijalna
agregacija mogu nastupiti i pod fiziološkim i stresnim uvjetima, čak i
bez mutacije. Stoga opisane promjene mogu biti manje ovisne o
genetici nego što se prethodno mislilo. Analiza regija proteina NPAS3
dodatno je pokazala kako PAS1 domena snažno utječe na lokalizaciju
NPAS3 u stanicama, potičući njegovu lokalizaciju u citoplazmi umjesto
u jezgri. Opisana promjena u lokalizaciji može smanjiti funkcionalnu
aktivnost NPAS3 i povećati njegovu sklonost agregaciji. Također,
pokazala sam kako CRMP1 može koagregirati s DISC1 i TRIOBP-1 u stanicama, što je u skladu s rezultatima analize uzoraka ljudskog mozga. Transgenični model vinske mušice koji eksprimira ljudski
DISC1 pokazuje potencijal za otkrivanje bihevioralnih i molekularnih
promjena, ali zahtijeva detaljnu validaciju zbog nedostatka kontrole
ekspresije.
Opisani rezultati potvrđuju heterogenost i specifičnost agregacija
proteina u KMB, pri čemu više proteina može agregirati unutar istih
pojedinaca. Disertacija također nudi nove dokaze kako koagregacija
može biti molekularni mehanizam koji doprinosi razvoju KMB te ističe
utjecaj genetskih i okolišnih čimbenika na agregaciju. Ovo istraživanje
ima ograničenja, uključujući varijabilnost kvalitete tkiva, agregaciju
proteina kod kontrola te prekomjernu ekspresiju proteina u staničnim
kulturama. Također, Drosophila model pokazuje manjak kontrole
ekspresije proteina, zbog čega zahtijeva dodatnu validaciju.
Sveukupno, korišteni modeli i metode zahtjevaju značajnu modifikaciju
u budućim istraživanjima. Unatoč tome, rezultati ovog rada postavljaju
temelje za daljnja istraživanja agregacije proteina kao potencijalnog
biomarkera ili terapijskog cilja, nudeći nove molekularne uvide u
patofiziologiju KMB