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Liver detoxification with PMAO2 zeolite after aluminum chloride intoxication
No full textAluminij je metal 13. skupine periodnog sustava elementa te nema fiziološku ulogu u
organizmu. Povezuje se s nizom bolesti poput: Alzheimerove bolesti, Parkinsonove
bolesti, Downovog sindroma, autizma itd. Sve veće globalne klimatske promjene
dovele su do povišenja razine aluminija u okolišu i ulazak u biota. Trenutno ne postoji
metoda za njegovo uklanjanje iz okoliša ili za detoksikaciju živih organizama pa tako i
čovjeka. Postojeća pak terapija protiv trovanja aluminijevim solima je desferioksamin.
Kako isti ima veliki broj nuspojava, zadnjih godina se istražuje mogućnost korištenja
alternativnih načina detoksikacije aluminija u živim organizmima, primjerice
korištenjem zeolita klinoptilolita koji su poznati ionski izmjenjivači, adsorbensi te
otpuštaju ortosilicijevu kiselinu što je u kontekstu detoksikacije aluminija također
izuzetno važno svojstvo. U ovom doktoratu istražena su fizikalno-kemijska svojstva
prirodnih zeolitnih klinoptilolitnih materijala, tribomehanički aktiviranog klinoptilolita
TMAZ, dvostruko tribomehanički aktiviranog zeolita PMA i dvostruko tribomehanički
aktiviranog oksigeniranog zeolita PMAO2 te je kao kontrolni spoj korišten sintetski
zeolit A. Fizikalno-kemijska svojstva zeolitnih materijala bitna su za objašnjenje
interakcije s ionima i biomolekulama u biološkim sustavima te su u ovom doktoratu
provedeni eksperimenti infracrvene spektroskopije s Fourierovom transformacijom,
određivanje zeta potencijala i pH zeolitnih suspenzija u vodi i modelnim otopinama
crijeva i želuca, in vitro kapacitet vezanja teških metala za zeolitne materijale,
određivanje veličine čestica dinamičkim raspršenjem svjetlosti, analiza sastava i
izgleda površine materijala metodom XPS/EDS. Svi su istraženi zeolitni klinoptilolitni
materijali pokazali značajan kapacitet vezanja kationa olova u modelnoj otopini
želuca i crijeva te arsena, kroma i nikla u modelnoj otopini crijeva. Nadalje, svi su
zeolitni klinoptololitni materijali pokazali detoksikacijska svojstva na aluminij in vivo
na modelu intoksiciranih štakora subkroničnim dozama aluminijevog klorida.
Mehanizam djelovanja ovog svojstva istražen je za PMAO2 analizom jetre životinja
metodom DNA mikročipova koja je pokazala aktivaciju gena Sfrp1,Cry1, Rassf8 i
supresiju gena Nr1d2 u tkivu jetre koji su uključeni u mehanizme detoksikacije. Ovi
rezultati govore u prilog primjeni zeolita klinoptilolita u medicinske svrheAluminum is a metal in the 13th group of the periodic table of elements and has no
physiological role in the body. It has been linked to a number of diseases such as
Alzheimer's, Parkinson's, Down syndrome, autism, etc. Increasing global climate
change has led to an increase in the concentration of aluminum in the environment
and its entry into the biota. Currently, there is no method to remove it from the
environment, or to detoxify living organisms, including humans. The existing therapy
for aluminum salt poisoning is desferrioxamine. As it has large number of side
effects, in recent years possibility of using alternative ways of detoxification of
aluminum in living organisms has been investigated, for example by using
clinoptilolite zeolites which are known ion exchangers, adsorbents and orthosilicic
acid realising compounds which is also extremely important in the context of
aluminum detoxification. Therefore, the physicochemical properties of natural zeolite
clinoptilolite materials tribomechanically activated clinoptilolite TMAZ, double
tribomechanically activated zeolite PMA and double tribomechanically activated
oxygenated zeolite PMAO2 were investigated in this doctorate and synthetic zeolite
A was used as a control compound. Physicochemical properties of zeolite materials
are important for explaining interactions with ions and biomolecules in biological
systems and in this doctorate were performed experiments with Fourier transformed
infrared spectroscopy, determination of zeta potential, pH of zeolite suspensions in
model solutions of intestine and stomach as well as in water, in vitro binding capacity
of heavy metals, determination of particle size by dynamic light scattering and
surface analysis of the materials by XPS / EDS. All investigated clinoptilolite
materials showed a significant binding capacity of lead cations in model solution of
stomach and intestines and arsen, chromium and nickel in the model solution of the
intestines. Furthermore, all zeolite clinoptilolite materials showed detoxifying
properties on aluminum in vivo in a model of intoxicated rats by subchronic doses of
aluminum chloride. The mechanism of action of this property was investigated for
PMAO2 analysis of animal liver by DNA microchip method which showed activation
of Sfrp1, Cry1, Rassf8 genes and supression of Nr1d2 genes in liver tissue involved
in detoxification mechanisms. These results support the use of clinoptilolite zeolite for
medical purposes
Stanično specifične glikozilacije proteina CD63 otkrivene novoproizvedenim mišjim monoklonskim protutijelima
No full textCD63 is an evolutionary conserved transmembrane glycoprotein which is ubiquitously expressed in mammalian cells and abundantly located in late endosomes and lysosomes. It has no enzymatic activity, but exerts its functions by interacting with other proteins to act on a wide range of cellular processes like cytoskeletal organisation, cell adhesion and motility. At least some of these CD63 functions might explain the association of CD63 with cancer metastasis seen in clinical settings. However, for a deeper understanding of CD63 biology, the research community requires novel antibodies suitable for a systematic biochemical characterisation of CD63. This study provides characterisation of two newly produced mouse monoclonal antibodies directed against mouse CD63: mCD63.07 and mCD63.08. Both antibodies bind to a linear epitope located at the extracellular portion of CD63 and are highly specific and sensitive in CD63 detection by immunoblot and immunofluorescence analyses based on signal comparison between wild type and CD63 knockout mouse fibroblasts. Moreover, the knockout validated antibodies were able to distinguish multiple CD63 glycoforms in several murine cell lines of different origins indicating that CD63 might have cell-type specific forms and functions. Finally, with the newly developed immunofluorescence protocol including protein denaturation, reduction and deglycosylation, novel antibodies detected significant CD63 amounts in cellular parts other than late endosomes and lysosomes.Taken together, the newly developed antibodies might become a valuable tool to study CD63 biology thanks so the their two main features: ability to detect multiple CD63 glycoforms and to identify potentially novel cellular localisation of CD63.Tetraspanin CD63 je ubikvitarno izražen protein u sisavaca koji se većinom nalazi u kasnim endosomima i lizosomima. CD63 nije enzimski aktivan, on svoje mnogobrojne funkcije izvršava preko interakcija s drugim proteinima te pritom sudjeluje u širokom rasponu staničnih procesa poput citoskeletne organizacije, stanične adhezije i pokretljivosti. U kliničkim je postavkama uspostavljena veza između CD63 i metastatskog potencijala tumora, za koju mogu biti odgovorne gore spomenute funkcije CD63. Međutim, za potpunije razumijevanje biologije CD63 proteina, istraživačkoj su zajednici potrebna nova protutijela pogodna za sustavnu biokemijsku karakterizaciju CD63 proteina. Ovim su radom karakterizirana dva novoproizvedena mišja monoklonska protutijela usmjerena protiv mišjeg CD63: mCD63.07 i mCD63.08. Na temelju usporedbe signala između mišjih fibroblasta divljeg tipa i mišjih fibroblasta CD63 knockout tipa, utvrđeno je da se oba protutijela vežu na linearni epitop smješten na izvanstaničnom dijelu CD63 i da su vrlo specifična i osjetljiva u detekciji CD63 imunofluorescentnom i imunoblot metodom. Nadalje, knockout validirana protutijela mogu razlikovati višestruke glikoforme CD63 u nekoliko mišjih staničnih linija različitog podrijetla, što ukazuje da CD63 može imati stanično specifične forme i funkcije. Naposlijetku, s novorazvijenim protokolom imunofluorescencije, koji uključuje denaturaciju proteina, redukciju i deglikozilaciju, nova protutijela detektirala su značajne količine CD63 u odjeljcima stanice koji nisu kasni endosomi i lizosomi. Na temelju dobivenih rezultata, novorazvijena antitijela mogla bi postati vrijedan alat za proučavanje biologije CD63 zahvaljujući njihovim dvjema glavnim značajkama: sposobnosti otkrivanja više glikoformi CD63 i identificiranja potencijalno nove stanične lokalizacije CD63
In vitro razvoj i regeneracija primarnih staničnih kultura korteksa oposuma Monodelphis domestica
No full textCentral nervous system (CNS) sustained injuries in mammals often result in severe and irreversible damage, with CNS regeneration being limited due to the restricted regenerative potential of neurons. Yet, CNS regeneration is possible in embryos, and its regenerative potential stops very early after birth. Monodelphis domestica, the grey short-tailed opossum, is a pouch-less marsupial born at a very immature state, equivalent to embryonal day (E)13 in rats or E12.5 in mice. The initial development of their cortical plate occurs at postnatal day (P)3-5 and gliogenesis begins around P18. Moreover, these opossums have an extraordinary ability to fully regenerate their spinal cord up to two weeks after birth, with their regenerative ability declining with the rise of non-neuronal cells. Therefore, they are a great animal model to study CNS development and regeneration potential in vitro. Primary cell cultures derived from their cortices were obtained and examined at different developmental ages, P6 and P17. The cellular composition of primary cortical cell cultures was best characterized in P6 opossums with immunofluorescence microscopy. TUJ1+ neuronal population rose from 60% at days in vitro (DIV)1 to its peak 84% of neurons at DIV5, and further declined to 44% at DIV13. Astrocytes showed maturation from DIV5 to DIV13, accounting for 28% to 32% of GFAP+ cells, respectively. Neuronal and non-neuronal cells’ regenerative potential within the CNS was examined with an induced injury in both P6 and P17 cortical cultures, performed with a scratch assay. P6 cultures did show a better regenerative performance, as neurons regenerated and grew their axons across the cut site, forming growth cones (GCs) which differed in size with age. Astrocytes polarized their processes along the cut site or migrated to the cut, resembling glial scar formation. Opossum primary cortical cultures provide, therefore, an additional and novel source of mammalian cells for in vitro investigations.Ozljede središnjeg živčanog sustava (SŽS) kod sisavaca često rezultiraju teškim i nepovratnim oštećenjima, pri čemu je regeneracija SŽS-a ograničena zbog ograničenog regenerativnog potencijala neurona. Ipak, regeneracija SŽS-a moguća je u embrijima, a regenerativni potencijal prestaje ubrzo nakon rođenja. Monodelphis domestica, sivi kratkorepi oposum, je tobolčar bez pravog tobolca čiji su mladunci rođeni vrlo nerazvijeni, ekvivalentno embrionalnom danu (E)13 kod štakora ili E12.5 kod miševa. Početni razvoj njihove kortikalne ploče događa se postnatalnog dana (P)3-5, dok gliogeneza počinje pri P18. Nadalje, oposumi imaju izvanrednu sposobnost potpune regeneracije svoje leđne moždine i do dva tjedna nakon rođenja, pri čemu njihova regenerativna sposobnost opada s porastom ne-neuronskih stanica. Stoga, oposumi su izvrstan životinjski model za proučavanje razvoja SŽS-a i regenerativnog potencijala in vitro. Primarne stanične kulture pripremljene iz korteksa ispitane su u različitim razvojnim dobima, P6 i P17. Stanični sastav primarnih kortikalnih staničnih kultura najbolje je karakteriziran kod P6 oposuma s imunofluorescentnom mikroskopijom. TUJ1+ neuronska populacija porasla je sa 60% pri in vitro danu (DIV)1 do vrhunca - 84% neurona pri DIV5, s padom na 44% pri DIV13. Astrociti sazrijevaju sa razvojem, od DIV5 do DIV13 njihova je populacija porasla sa 28% na 32% GFAP+ stanica. Regenerativni potencijal neuronskih i ne-neuronskih stanica unutar središnjeg živčanog sustava ispitan je s induciranom ozljedom u kortikalnim kulturama P6 i P17, simuliranom sa testom reza. P6 kulture su pokazale bolji regenerativni potencijal, pri čemu su neuroni regenerirali i izrasli aksone kroz mjesto reza, stvarajući konuse rasta različitih veličina ovisno o dobi. Astrociti su polarizirali, pri čemu su se njihovi izdanci izdužili okomito na mjesto reza ili su migrirali na mjesto, predstavljajući stvaranje glijalnog ožiljka. Primarne kortikalne kulture dobivene iz oposuma je nov izvor stanica sisavaca koji doprinosi in vitro istraživanjima
Project proposal for equipping a laboratory for indoor cultivation and processing of industrial hemp
No full textUnatoč tome što je znanstveno dokazano da kanabinoidi iz konoplje
pozitivno djeluju na brojna stanja i bolesti, pristup proizvodima koji ih
sadrže je još uvijek vrlo ograničen i skup. Razlog tome je nedostatak znanja
i infrastrukture za proizvodnju preparata od konoplje koji zadovoljavaju
standarde dobre prerađivačke prakse na industrijskoj skali. Da bi se riješio
ovaj problem potrebno je proširiti bazu informacija o tehnološkom procesu
proizvodnje konoplje te izgraditi infrastrukturu koja je održiva i
konzistentna u proizvodnji preparata od konoplje.
U ovom radu predstavljena je metodologija projektnog menadžmenta te su
istraženi tehnološki aspekti procesa proizvodnje i prerade iz relevantne
znanstvene i stručne literature. Na temelju prikupljenih informacija je
sastavljen projektni prijedlog za opremanje laboratorija za unutrašnji uzgoj
i preradu konoplje koji zadovoljava sve potrebne standarde kvalitete u
proizvodnji. Laboratorij iz projektnog prijedloga je vertikalno integriran, pa
je stoga održiv i konzistentan u proizvodnji sigurnih proizvoda provjerene
kvalitete na industrijskoj skali.
Cilj ovog rada je sastaviti projektni prijedlog za opremanje laboratorija za
unutrašnji uzgoj i preradu konoplje korištenjem metodologije projektnog
menadžmenta i znanja o tehnološkom procesu proizvodnje i prerade
konoplje. Svrha rada je da sastavljeni projektni prijedlog za opremanje
opisanog laboratorija može poslužiti kao referenca budućim projektima koji
će se baviti proizvodnjom i preradom konoplje za kozmetičke i medicinske
svrhe.Despite the fact that cannabinoids from hemp have been scientifically
proven to have a positive effect on numerous conditions and diseases,
access to products containing them is still very limited and expensive. The
reason for this is the lack of knowledge and infrastructure for the production
of hemp preparations that meet the standards of good processing practice
on an industrial scale. In order to solve this problem, it is necessary to
expand the information base on the technological process of hemp
production and build infrastructure that is sustainable and consistent in the
production of hemp preparations.
In this master thesis, the methodology of project management and the
technological aspects of the production and processing of hemp are
investigated from the relevant scientific and professional literature and the
gathered data is presented. Based on the gathered data, a project proposal
for equipping a laboratory for indoor cultivation and processing of hemp
that meets all the necessary quality standards in production was drawn up.
The laboratory is vertically integrated and is therefore sustainable and
consistent in the production of safe products of proven quality on an
industrial scale.
The goal of this master thesis is to compile a project proposal for equipping
a laboratory for indoor cultivation and processing of hemp using the project
management methodology and knowledge of the technological process of
hemp production and processing. The purpose of the compiled project
proposal is to potentially serve as a reference for future projects that will
engage in the production and processing of hemp for cosmetic and medical
purposes
Evaluation of active sites of enzymes and their comparison to catalytically active peptides involved in ester hydrolysis
No full textRational design of proteins and peptides is a growing field that requires knowledge of protein function down to the amino acid level. Understanding protein geometry and amino acid composition is crucial for understanding molecular and supramolecular chemistry in protein design. In this thesis, the focus is put on active sites of natural enzymes and the drivers of catalytic function in said active sites. The analysis of natural enzyme active sites was done with the purpose of having a set of criteria for building synthetic peptides with identical catalytic function and to broaden enzyme understanding. A data set was built to analyze enzymes from the EC 3.1. (Hydrolases acting on ester bonds) subclass looking at both amino acid content and geometry of active site residues. Patterns in existing data were searched by statistically analyzing 96 esterases. In this process, 22 enzymes with known catalytic triads were selected for further evaluation. Based on the primary structure of the selected enzymes, the composition profiles were created for: (1) the full sequence, (2) the “long active site” including the residues between the first and the last active amino acid, (3) the “short active site” containing only active residues and (4) the “catalytic loop” which looked at 4 residues from each side of the sequence from the catalytic residues. To analyze the dataset two approaches were used. The first approach was based on composition and chemical property analysis. The second approach consisted in using crystal structure PDB files obtained via X-ray, only considering a resolution below 3,0 Å and in measuring the residue geometries in PyMol. The results showed that the distances within which natural enzymes function are less than 1Å in variation and that the angles conform within a 10% variation in the interquartile range compared to the average angle. Moreover, the qualitative amino acid analysis showed an increase of non-polar and a decrease in basic, hydroxylic and polar residues near the catalytically active residues. This suggests a strict functional geometry for the optimal enzymatic activity as well as a specific local amino acid content tolerated in active sites. These findings will allow the development of algorithms for prediction and optimization of enzyme functions and of new theoretical models of modular peptide design.Racionalni dizajn proteina i peptida je polje koje zahtijeva razumijevanje funkcije proteina do razine aminokiselina. Ovo znanje obuhvaća razumijevanje geometrije i sastava aktivnih aminokiselinskih ostataka. Ovaj rad će se fokusirati na aktivna mjesta prirodnih enzima i pokretače katalitičke funkcije u njima. Izvršena analiza ima za svrhu razradu seta kriterija za izgradnju sintetičkih peptida s istom katalitičkom funkcijom, te općenito produbljivanje razumijevanja enzima. Izgrađen je skup podataka EC 3.1. podklase (hidrolaze esterskih veza) kako bi se analizirao sastav i geometrija aminokiselina aktivnog mjesta. Uzorci u podatcima istraženi su analizom 96 esteraza. Tijekom tog procesa, 22 enzima izdvojeno je na kriteriju posjedovanja katalitičke trijade za daljnju analizu. Temeljeno na primarnoj sekvenci odabranih enzima stvoreni su profili sastava za (1) cijelu sekvencu enzima, (2) “dugo aktivno mjesto” koje je uključivalo sve aminokiseline od prvog do zadnjeg aktivnog bočnog lanca, (3) “kratko aktivno mjesto” koje je sadržavalo isključivo bočne lance katalitički aktivnih aminokiselina i (4) “katalitičku petlju” koja je sadržavala 4 aminokiseline sa svake strane aktivnog bočnog lanca. Korištena su dva pristupa od kojih su prvim analizirani sastavi i kemijska svojstva dok su se u drugom koristile kristalne strukture PDB datoteka dobivenih X-zrakama rezolucija ispod 3,0 Å kako bi se izmjerile geometrije bočnih lanaca. Rezultati su pokazali kako su udaljenosti aktivnih bočnih lanaca unutar kojih prirodni enzimi vrše funkciju manji od 1Å variacije te kutevi između njih ne variraju više od 10% od prosječnog kuta aktivne aminokiseline u svom interkvartalnom rasponu. Nadalje, kvantitativna analiza aminokiselina blizu aktivnih bočnih lanaca pokazuje povećanje non-polarnih i smanjenje u bazičnim, hidroksilnim i polarnim aminokiselinama. Ovi rezultati prikazuju strogu funkcionalnu geometriju za optimalnu enzimatsku aktivnost, kao i postojanje specifičnog aminokiselinskog sastava koji aktivna mjesta toleriraju. Ovo istraživanje može potencijalno razviti algoritme za predviđanje i optimizaciju enzimatskih funkcija i nove teoretske modele za modularni dizajn peptida
Povezanost mikrobioma i neurodegenerativnih bolesti: kako prioni izolirani iz mikrobioma utječu na razvoj Alzheimerove bolesti
No full textThe idea that Alzheimer’s disease (AD) has an origin in the gut has been widely accepted based on crosstalk between microbiota, gut and brain. To the hypothesis of the present work, gut microbiota could be a source of a major number of amyloids. This work used two of the best studied bacterial amyloids; (i) curli, produced by Escherichia coli and (ii) Sup35p, produced by Saccharomyces cerevisiae. Curli has beneficial roles for bacteria by helping them to form biofilms and to resist destruction by physical or immune factors, while at the same time share similarities inside tertiary structure with central nervous system’s (CNS) amyloids that are responsible for forming toxic Aβ plaques in AD. Under certain circumstances bacterial derived amyloids can leak from GI tract and accumulate at the systemic and brain level. In order to discern which peptides from human microbiome are able to influence human proteins aggregation, this work implemented proteome-wide bioinformatic tools and in vitro assays to follow which putative amyloid core sequence can lead the formation of amyloid aggregates and the cell-to-cell transmission of the aggregated conformation. Production of amyloid fibrils through CDAG system based on curli was achieved with all 12 tested peptides and their existence was proved by phenotype evaluation in the liquid and on plates containing the amyloid binding dye (Congo red, CR) as well as under Transmission electron microscopy (TEM). The Sup35p prion system, that regulates translation termination in Saccharomyces cerevisiae was used to test the ability of the amyloid cores, to propagate the amyloid aggregated conformation between yeast cells. Finally, bacterial and yeast systems provided simplified models for studying the conserved mechanisms of amyloid formation, degradation, and function as well as connection between bacterial amyloids and amyloid structures in brain formed during neurodegeneration.Unatrag nekoliko godina povezanost intestinalnog sustava s nastankom neurodegenerativnih bolesti kao što je Alzheimer postaje sve istraženije područje temeljeno na komunikaciji između mozga i crijevnog sustava te crijevnog mikrobioma. Prema hipotezi ovog rada, crijevni mikrobiom čini potencijalni rezervoar velike količine amiloida. Shodno tome, u ovom radu su korištena dva najbolje proučena amiloida; (i) curli, koje proizvodi Escherichia coli te (ii) Sup35p, koje proizvodi Saccharomyces cerevisiae. Curli imaju korisnu ulogu u formiranju bakterijskog biofilma i zaštiti od fizičkih ili imunoloških oštećenja stanice, dok istovremeno dijele sličnosti s tercijarnom strukturom amiloida koji se nakupljaju u središnjem živčanom sustavu čineći toksične Aβ plakove- jedne od glavnih patoloških markera Alzheimerove bolesti. Pod određenim okolnostima amiloidi bakterijskog podrijetla mogu „curiti“ iz gastrointestinalnog (GI) trakta i akumulirati se u mozgu. Kako bi se razlučilo koji peptidi izolirani iz ljudskog mikrobioma mogu utjecati na agregaciju ljudskih proteina, ovaj rad implementirao je bioinformatičke alate za analizu cijelog proteoma te in vitro ispitivanja kako bi se utvrdilo koja sekvenca amiloidne jezgre može dovesti do stvaranja amiloidnih nakupina i prijenosa agregiranih konformacija s jedne na drugu stanicu. Proizvodnja amiloidnih fibrila putem CDAG sustava postignuta je kod svih 12 testiranih peptida, a njihov potencijal je dokazan procjenom fenotipa na mikrobiološkim pločama i u tekućini koje sadrže boju za vezanje amiloida (engl. Congo red, CR). Proizvodnja amiloidnih agregegata je također vizualizirana transmisijskom elektronskom mikroskopijom (TEM). Sup35p prionski sustav, koji regulira prekid translacije u Saccharomyces cerevisiae, korišten je za testiranje sposobnosti amiloidnih jezgri da propagiraju amiloidne nakupine između stanica kvasca. Zaključno, sustavi bakterija i kvasaca omogućili su proučavanje očuvanih mehanizama stvaranja, razgradnje i funkcije amiloida te promaknuli saznanja o povezanosti bakterijskih amiloida s amiloidnim formacijama koje nastaju tijekom neurodegeneracije
Experimental models for multiple sclerosis
No full textMultipla skleroza (MS) je autoimuna bolest koja uzrokuje demijelinizaciju aksona središnjeg živčanog sustava (SŽS). Na razvoj bolesti utječu različiti genetički i okolišni čimbenici, stoga MS spada u multifaktorijalne složene bolesti. Autoreaktivne stanice imunosnog sustava uzrokuju autoimuni odgovor u SŽS-u koji rezultira razaranjem mijelinske ovojnice te je prijenos akcijskog potencijala niz neuron usporen. Uz demijelinizaciju, glavne značajke bolesti su i neurodegeneracija aksona koja nastupa u kasnijim fazama bolesti, glioza te upala u SŽS-u. Simptomi bolesti uključuju gubitak motoričkih, senzoričkih i autonomnih funkcija. Bolest se po intenzitetu simptoma i njihovom razvoju u vremenu dijeli na: klinički izolirani sindrom (CIS), relapsno-remitirajuću multiplu sklerozu (RRMS), primarno-progresivnu multiplu sklerozu (PPMS) i sekundarno-progresivnu multiplu sklerozu (SPMS). RRMS je najčešći oblik MS-a. Za istraživanje složene bolesti poput MS-a potrebni su odgovarajući in vivo animalni modeli. Glavni animalni model istraživanja MS-a je eksperimentalni autoimuni encefalomijelitis (EAE). Kao i MS, karakterizira ga autoimuni odgovor te demijelinizacija i oštećenja aksona u SŽS-u. Najčešće životinje u kojima je induciran EAE jesu miševi i štakori. Postoji nekoliko različitih metoda indukcije EAE-a, no najčešće su aktivna i pasivna imunizacija životinja. Za razliku od CD8+ citotoksičnih T limfocita u MS-u, glavni nositelji patogeneze EAE-a su CD4+ pomagački T limfociti. To čini EAE model vrlo korisnim u istraživanju njihovih efektorskih funkcija te regulacijskih T limfocita. Nadalje, EAE modeli važni su u detekciji potencijalnih antigena koji sudjeluju u autoimunome odgovoru te mehanizmima kojima dolazi do upale i demijelinizacije. Najveći nedostaci EAE-a u istraživanju MS-a su lokalizacija lezija u kralježničnoj moždini, za razliku od dominantnih moždanih lezija u pacijentima koji boluju od MS-a. te nedostatak progresivnosti simptoma. EAE je glavni animalni model MS-a koji je zaslužan za dobivanje velike većina saznanja o patofioziologiji bolesti te razvoju terapije za pacijente koji boluju od MS-a.Multiple sclerosis (MS) is an autoimmune disease, which causes demyelination of axons in central nervous system (CNS). Many different genetic and enviromental factors affect disease develompent, hence MS belongs to multifactorial, complex diseases. Autoreactive immune cells cause autoimmune response in CNS which results in myelin sheath destruction and this slowes down action potential conduction along neurons. Beside demyelination, another elements of MS are neuroaxonal degeneration which occurs in later stages of the disease, gliosis and CNS inflammation. Disease symptoms include loss of motor, sensory and autonomic functions. Disease is divided, by its symptoms intensity and development through the time, to: clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and secondary progressive multiple sclerosis (SPMS). RRMS is most common type of MS. For the research of complex diseases, like MS, animal in vivo models are required. The main animal model for MS research is experimental autoimmune encephalomyelitis (EAE). Just like MS, it is characterised by autoimmune response, demyelination and axonal damage in CNS. Most used animals in EAE research are mice and rats. There are several different methods of EAE induction, but the most common are active and passive immunisation of animals. Unlike CD8+ cytotoxic T lymphocytes in MS, main carriers of the EAE pathogenesis are CD4+ T helper lymphocytes. That makes EAE very useful in their effector functions and regulatory T cells research. Furthermore, EAE models are important in detection of potential antigens, which participate in autoimmune response and mechanisms which induce inflammation, and demyelination. Main disadvantages of EAE as a model of MS research are localization of lesions in spinal cord, unlike dominant brain lesions in MS patients, and lack of symptoms progression. EAE is the main animal model of MS which is responisble for the vast majority of pathophysiologic knowledge about the disease and for development of MS therapy
Spectrophotometric and mass spectrometry based methods for determination of the differences between the two wine production processes
No full textVino je alkoholno piće u čijem se kemijskom sastavu ističu fenolni spojevi sa
svojim organoleptičkim, antioksidativnim i protuupalnim svojstvima.
Tehnološki proces proizvodnje vina uvelike utječe na sastav fenolnih
komponenti istih, stoga su inovativne tehnologije od velike važnosti pri
isticanju proizvoda na konkurentnom tržištu. Dozrijevanje vina u morskim
vruljama tehnološki je proces proizvodnje vina kojim se, uz atraktivan izgled
butelje, potencijalno postiže promjena kemijskog sastava istih. Butelje vina
odležane u vruljama nerijetko dosežu visoke cijene na tržištu, stoga je cilj
ovoga rada ispitati postoji li razlika u antioksidativnom potencijalu i
kvalitativnom i kvantitativnom sastavu fenolnih komponenti između uzoraka
vina odležanih u vrulji i podrumu. Spektrofotometrijskim metodama i
metodama spektrometrije masa analizirano je i uspoređeno trideset uzoraka
bijelih vina sorte Malvazija, od kojih je petnaest uzoraka dozrijevano u
podrumu i petnaest uzoraka u morskoj vrulji u periodu od godinu dana.
Provedene spektrofotometrijske metode bile su metode redukcije kationskih
radikala za mjerenje antioksidativnog potencijala, Folin-Ciocalteu metoda za
određivanje ukupnih fenola i metoda određivanja ukupnih flavonoida, dok su
metode spektrometrije masa činile LC-QQQ metoda (s provedenom LCMS/MS kvalitativnom analizom i MS scan analizom) i MALDI-TOF fingerprint
analiza. Rezultati provedenih analiza uspoređeni su statističkim testovima s
ciljem ispitivanja značajnosti razlika podataka. Spektrofotometrijskim
metodama pokazalo se kako se antioksidativni potencijal i razina ukupnih
fenola i flavonoida značajno ne razlikuju između dviju ispitanih skupina.
Nadalje, rezultati dobiveni LC-MS/MS analizom pokazali su da se vina
dozrijevana u vrulji i podrumu značajno razlikuju po razini šest flavonoidnih
spojeva, no ukupan se zbroj fenolnih spojeva značajno ne razlikuje između
dviju ispitanih skupina. Međutim, razlika je postojana te je kao takva
poticajna za buduće analize uzoraka i optimizaciju tehnike dozrijevanja vina
u morskoj vrulji. Ovaj diplomski rad prvi je koji opisuje proces dozrijevanja
vina u morskoj vrulji te analizira i uspoređuje uzorke vina iz vrulje i podruma.Wine is a popular alcoholic beverage and phenolic compounds stand out
among the various chemical compounds in wine with organoleptic,
antioxidant and anti-inflammatory properties. The technological process of
wine production affects the composition of their phenolic components, so
innovative technologies that can influence the phenolic compounds in wine
are of great importance in this competitive market. The maturation of wine
in sea springs is a novel technological process that, in addition to adding an
attractive appearance to the wine bottle, potentially influences the wine’s final
chemical composition. Furthermore, bottles of wine aged in sea springs often
reach higher market prices. Therefore, the aim of this thesis is to determine
the antioxidant potential and phenolic composition of wine samples aged
either in sea springs or traditional cellars. Thirty samples of Malvasia white
wines were analyzed using spectrophotometric and mass spectrometry
methods, of which fifteen samples were matured in traditional cellars and
fifteen samples were matured in a Northern Adriatic sea spring. All wines
were aged for over one year prior to analysis. Spectrophotometric analysis of
antioxidant potential and total phenols and flavonoids in the wine samples
was performed using the cation radical reduction and Folin-Ciocalteu
methods, respectively. Using a LC-QQQ mass spectrometry method, phenols
in the wine were analyzed by quantitative LC-MS/MS analysis, qualitative MS
scan analysis and MALDI-TOF fingerprint. The results were analyzed with
statistical tests to determine significance differences between the two
maturation processes. The antioxidant potential and total phenol and
flavonoid levels did not differ significantly between the two maturation
processes. However, LC-MS/MS analysis showed that wines matured in the
sea spring differ significantly from cellar-matured wines in the concentration
of six flavonoid compounds, but the total sum of phenolic compounds did not
differ significantly between the two groups. This is encouraging for future
optimization of wine maturation techniques in sea springs. This thesis is the
first to describe the process of wine maturation in a sea spring and to analyze
and compare wine samples matured in sea springs and cellars
Olive leaf extract (OLE) alters the excitotoxicity of glutamate by modulating glutamate receptors in the rat brain after induced experimental autoimmune encephalomyelitis (EAE)
No full textGlutamat (Glu) je glavni ekscitatorni neurotransmiter središnjeg živčanog sustava. Učinkovitost prijenosa Glu ovisi o pravilnoj funkcionalnosti i ekspresiji mnogih receptora i transportera, smještenih i na neuronima i na glijalnim stanicama. Povećanje izvanstaničnog Glu uzrokuje poremećenu sinaptičku signalizaciju koja dovodi do neuronske ekscitotoksičnosti i smrti. Aberacije u ekspresiji glutamatnih receptora, transportera ili metabolizirajućih enzima otkrivene su u životinjskim modelima multiple skleroze (MS), tj. eksperimentalnom autoimunom encefalomijelitisu (EAE). U ovoj studiji istraživali smo učinak polifenola lista masline na Glu ekscitotoksičnost.
U radu su korišteni Dark Agouti (DA) štakori na kojima je istražen učinak terapije ekstraktom lista masline (TOLE) kod EAE. Životinje su podijeljene u tri glavne skupine: kontrolna skupina, skupina kojoj je izazvan EAE i koja je žrtvovana 20-ti dan nakon izazvanog EAE (EAE) te skupina kojoj je izazvan EAE i koja je liječena terapijom OLE (1 g/kg, i.p.) 10 dana u kontinuitetu i žrtvovana 20-ti dan nakon izazvanog EAE (EAE+OLE). Uz to, skupina EAE+OLE je od početka do kraja pokusa, umjesto vode za piće dobivala čaj lišća masline (1,5% w/v, ad libitum). Klinički tijek bolesti praćen je u obje skupine do 20. dana nakon indukcije EAE. Razina glutamatnih receptora i transportera (GLUR1, NMDAR1, NMDAR2, GLUR5, GRID2, GLAST1, GLT1), antioksidacijskih enzima (SOD1, SOD2), biljega demijelinacije/remijelinacije (MBP, TREM2, VAMP2), neurodegeneracije (NeuN, SIRT1) i biljega aktivacije mikroglije (IBA1) određena je imunoblotom u lizatu velikog mozga u štakora. Dodatno, na tkivima ugrađenim u parafin je provedeno imunofluoresentno bojanje GLAST, GLT1, SIRT1 i IBA1.
Naši rezultati pokazuju da TOLE oslabljuje intenzitet simptoma izazvan EAE i odgađa klinički tijek bolesti. Dodatno, povećana aktivnost EAAT2/GLT1 glutamatnog transportera, povećana ekspresija SOD2 antioksidacijskog enzima, očuvan integritet mijelina kroz ekspresiju MBP i TREM2, aktivirana mikroglija (IBA1) te očuvani neuroni (SIRT1) pronađeni su u skupini EAE+OLE 20-ti dan nakon izazvanog EAE u mozgu štakora. Ova studija upućuje da bi TOLE mogla povoljno utjecati na tijek liječenja MS-a.Glutamate (Glu) is the principal excitatory neurotransmitter of the central nervous system. Extracellular Glu increase causes aberrant synaptic signalling leading to neuronal excitotoxicity and death. Glu transmission efficiency depends on the correct functionality and expression of many receptors and transporters, located both on neurons and glial cells. Aberrations in the expression of glutamate receptors, transporters, or metabolizing enzymes were detected in multiple sclerosis (MS) animal models, i.e., experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the effect of olive leaf polyphenols on Glu excitotoxicity.
Dark Agouti (DA) rats were used to investigate the effect of olive leaf extract therapy in EAE. Animals were divided into three major groups: control group, EAE-induced group sacrificed on day 20 after EAE induction (EAE), and EAE-induced group treated with OLE therapy (1 g/kg, i.p.) 10 days continuously and sacrificed on the 20th day after induced EAE (EAE+OLE). In addition, the EAE+OLE group consumed olive leaf tea instead of drinking water (1,5% w/v, ad libitum) from the beginning to the end of the experiment. The clinical course was monitored in both groups until the 30th day after EAE induction. The level of glutamate receptors and transporters (GLUR1, NMDAR1, NMDAR2, GLUR5, GRID2, GLAST1, GLT1), antioxidant enzymes (SOD1, SOD2), demyelination/remyelination markers (MBP, TREM2, VAMP2), neurodegeneration (NeuN, SIRT1) and activation markers of microglia (IBA1) were determined by immunoblotting in the lysate of the cerebrum in the rat. Additionally, immunofluorescent staining of GLAST, GLT1, SIRT1 and IBA1 was performed on paraffin-embedded tissues.
Our results demonstrated that TOLE attenuated the intensity of EAE-induced symptoms and delays the clinical course of the disease. Furthermore, increased activity of EAAT2/GLT1 glutamate transporter, increased expression of SOD2 antioxidant enzyme, preserved myelin integrity through MBP and TREM2 expression, activativated microglia (IBA1) and preserved neurons (SIRT1) were found in the EAE20+OLE group 20th day after induced EAE in rat brain. This study suggests that TOLE may have a positive impact on the course of treatment of MS
Immunopathogenetic mechanisms of HIV-1 inefection
No full textHIV-1 član je porodice Retroviridae, a ujedno i jedan od najrasprostranjenijih lentivirusa današnjice s poprilično visokom godišnjom stopom prevalencije u svijetu. Kao rezultat zoonotskih prijenosa s primata postao je i humani patogen koji se prenosi preko tjelesnih tekućina, izazivajući u zaražene osobe imunodeficijenciju i narušavanje cjelokupnog zdravstvenog stanja. Izrazito velika mutabilnost HIV-1 genoma predstavlja i najveću prepreku njegovom iskorjenjivanju. Osnovnu građu sferičnog HIV-1 viriona čine ovojnica s ugrađenim šiljcima, kapsida i nukleoproteinski kompleks kojeg čine RNA i proteini važni za njegovu replikaciju. Klasificirano je ukupno 9 HIV-1 gena koji se, prema funkcijama, dijele u strukturne i pomoćne. Gag i env kodiraju proteine ovojnice i kapside, a pol enzimske proteine. Geni tat i rev zaslužni su za regulaciju genskog izražaja, dok vif, vpr, vpu i nef pospješuju virusnu imunopatogenezu. Nakon fuzije viriona s membranom domaćina, ulaska te reverzne transkripcije i integracije novosintetizirane DNA u genom domaćina, slijedi sinteza virusnih proteina i sklapanje novih virusnih čestica. Novonastali virioni potom pupaju, tj. izlaze iz stanice, čime započinje proces diseminacije. U obrani organizma od HIV-1 djeluje urođeni imunosni sustav (dendritičke i NK stanice, makrofagi te citokinska mreža), stečena imunost (CD4+ i CD8+ limfociti T i limfociti B), te tzv. intrinzični dio imunosnog sustava (APOBEC3G, TRIM5α, teterina i SAMHD1). Najvažniju ulogu u imunoevaziji virusa ima njegov pomoćni protein Nef koji, između ostalog, smanjuje izražaj MHC-I molekula (molekule glavnog sustava tkivne podudarnosti) usmjeravajući ih u razgradnju kako bi se izbjeglo razaranje zaraženih stanica od strane citotoksičnih limfocita T. Nef također potiče i aktivaciju transkripcijskih faktora, a time i replikaciju virusa. Ostali mehanizmi izbjegavanja imunosnog odgovora podrazumijevaju mutiranje ili maskiranje epitopa, latenciju, greške reverzne transkriptaze i dr. Zbog sve veće HIV-1 prilagodbe nužno je razviti što učinkovitiji lijek ili cjepivo kako bi se zaustavilo širenje ili poboljšala kvaliteta zaraženih.HIV-1 belongs to the Retroviridae family and is one of the most widespread lentiviruses with a high annual prevalence rate. As a result of zoonotic transmission from primates, it has evolved into a human pathogen that is transmitted through body fluids and causes immunodeficiency and impaired general health in infected individuals. One of the main obstacles to viral eradication is the high mutation rate of the genome. The spherical HIV-1 virus consists of an envelope with built-in spikes, a capsid, and a nucleoprotein complex containing RNA molecules and proteins important for replication. A total of 9 genes have been classified, which are divided into structural and auxiliary genes according to their function. Gag and env code for envelope and capsid proteins, pol for enzyme proteins. The tat and rev genes are responsible for regulating gene expression, while vif, vpr, vpu, and nef promote viral immunopathogenesis. Infection of the host cell begins with fusion of the virion to the host membrane, entry, reverse transcription, and integration of the newly synthesized DNA into the host genome. After the synthesis of late viral proteins, including structural proteins, begins, new virions bud from the infected cell and are disseminated throughout the body. Immune defense mechanisms against HIV-1 include the innate immune system (dendritic cells, NK cells, macrophages, and cytokine network), the adaptive immune response (CD4+ and CD8+ T lymphocytes and B lymphocytes), and the intrinsic immune system (APOBEC3G, TRIM5α, tetherin, and SAMHD1). The most important role in evading the immune system is played by the accessory HIV-1 protein Nef. One of its most important roles is to downregulate MHC-I molecules so that they are degraded. This prevents lysis of infected cells by cytolytic T cells (CTLs). Nef also promotes activation of transcription factors and thus viral replication. Other immune defence mechanisms mediated by HIV-1 include mutations and/or masking of epitopes, latency, reverse transcriptase errors, and others. Due to the increasing adaptation of HIV-1, it is necessary to develop the most effective drug or vaccine to stop the spread of the virus or improve the quality of life of those infected