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Utjecaj mediteranske prehrane na depresiju
No full textThe World Health Organization estimates that 3.8% of the world population is affected by some sort of depressive disorder. Major depressive disorder is diagnosed if a person has depressed mood and/or anhedonia for a long period of time, along with at least four other depressive symptoms such as feelings of guilt or worthlessness, lack of energy, poor concentration, appetite changes, psychomotor retardation or agitation, sleep disturbances, or suicidal thoughts. The Mediterranean diet is a mostly plant-based diet, focusing on vegetables, fruits, nuts, beans, whole grains, fish, olive oil and other healthy fats, dairy in smaller amounts, herbs and spices, and moderate intake of red wine. It has been linked to overall improvement of mental and physical health. Some studies suggest that it helps prevent or lower the symptoms of a major depressive disorder. This thesis investigated those studies to gather information from around the world, by the means of different methods, and determine whether most studies do find a connection between the Mediterranean diet and a decrease in depressive symptoms. The results showed that it does generally have a positive effect on lowering depressive symptoms, but they also pointed out the problems with the methods of the studies mentioned, and the need for future investigation of the problem.World Health Organization procjenjuje da 3.8% svjetske populacije boluje od nekog oblika depresivnog poremećaja. Veliki depresivni poremećaj dijagnosticiran je kada osoba ima anhedoniju i/ili je depresivnog raspoloženja dug period, uz još barem četiri simptoma poput osjećaja krivnje ili bezvrijednosti, manjka energije, slabe koncentracije, promjene apetita, psihomotorne retardacije ili agitacije, poteškoća sa snom ili suicidalnih misli. Mediteranska prehrana je prehrana većinski na bazi biljaka s fokusom na povrće, voće, orašaste plodove, cjelovita zrna, ribu, maslinovo ulje i ostale zdrave izvore masti, manje količine mliječnih proizvoda, začinskog bilja i umjerene količine crnog vina. Povezuje se s cjelokupnim poboljšanjem fizičkog i mentalnog zdravlja. Neka istraživanja predlažu da pomaže spriječiti ili smanjiti simptome velikog depresivnog poremećaja. Ovaj rad prikuplja ta istraživanja kako bi se pregledale informacije sa svih strana svijeta, uz raznolike metode istraživanja, i definiralo postoji li zaista u većini slučajeva poveznica između mediteranske prehrane i smanjenja depresivnih simptoma. Rezultati su pokazali kako generalno postoji pozitivan utjecaj mediteranske prehrane na smanjenje depresivnih simptoma, ali također su istaknuli probleme s korištenim metodama i potrebu za budućim, dubljim istraživanjima problema
Analiza potencijalne neuroinflamatorne patologije u leđnim moždinama miševa s insuficijencijom optineurina
No full textAmyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease
characterised by motor neuron loss in the brain, brainstem, and spinal cord.
ALS is caused by mutations in more than 50 genes, including OPTN,
encoding for optineurin, a ubiquitin-binding protein that regulates
inflammatory signalling. It has been hypothesized that ALS-linked
optineurin mutations cause protein loss-of-function, leading to dysregulated
immune response that could trigger ALS. However, the exact mechanism of
disease pathogenesis is still unclear given that optineurin participates in
various other cellular functions including autophagy, cell death and others.
Since most ALS cases display spinal onset, we characterised the potential
neuroinflammatory pathology in the lumbar spinal cord of young adult mice
carrying ALS-like optineurin truncation (Optn470T). We assessed if this
optineurin mutation is by itself sufficient to induce ALS-like neuropathology,
or whether the latter could be provoked by lipopolysaccharide (LPS). We
demonstrated an absence of exaggerated neuroinflammation, TDP-43
aggregation and neurodegeneration in the lumbar spinal cords of Optn470T
mice for both basal and LPS-stimulated conditions. Our results indicate that
optineurin truncation in mice does not induce ALS-like neuropathology in
these settings, suggesting that a short-term inflammatory stimulus is
insufficient to precipitate the disease. It is still an open question if chronic
inflammation or mutation in other ALS-affected genes together with
optineurin truncation could uncover the role of optineurin in
neuropathologyAmiotrofična lateralna skleroza (ALS) je kompleksna neurodegenerativna
bolest karakterizirana gubitkom motoričkih neurona u mozgu, moždanom
deblu i leđnoj moždini. ALS je uzrokovan mutacijama u više od 50 gena,
uključujući i OPTN, koji kodira optineurin, ubikvitin vezujući protein uključen
u regulaciju upalne signalizacije. Pretpostavlja se da mutacije optineurina u
ALS-u uzrokuju gubitak funkcije proteina koje mogu izazvati narušeni
imunosni odgovor te dovesti do razvoja ALS-a. Međutim, točan mehanizam
patogeneze bolesti i dalje nije poznat budući da optineurin sudjeluje u
raznim drugim staničnim funkcijama kao što je autofagija, stanična smrt i
drugi. S obzirom na to da ALS slučajevi pokazuju spinalni oblik bolesti,
okarakterizirali smo potencijalnu neuroinflamatornu patologiju u lumbalnim
dijelovima leđne moždine mladih miševa s trunkacijom optineurina nalik
ALS-u (Optn470T). Ispitali smo ukoliko je mutacija optineurina sama po sebi
dovoljna da izazove neuropatologiju nalik ALS-u ili ukoliko ju se može
izazvati lipopolisaharidom (LPS). Demonstrirali smo odsustvo pretjerane
upale, TDP-43 agregata te neurodegeneracije u lumbalnom dijelu leđne
moždine Optn470T miševa pri bazalnim i LPS stimuliranim uvjetima. Naši
rezultati ukazuju na to da trunkacija optineurina ne dovodi do ALS-slične
neuropatologije u ovim uvjetima te sugeriraju da je kratkotrajni upalni
podražaj nedovoljan kako bi uzrokovao bolest. I dalje je otvoreno pitanje
mogu li kronična upala ili mutacije u drugim ALS-zahvaćenim genima
zajedno s trunkacijom optineurina objasniti ulogu optineurina u
neuropatologiji.
Crosstalk of TDP-43 and Optineurin in Amyotrophic Lateral Sclerosis Models
No full textOptineurin is a multifunctional polyubiquitin-binding protein implicated in
autophagy and inflammatory signalling, processes that have been described as
pathogenic mechanisms in neurodegenerative diseases. Notably, more than 40
mutations in the OPTN gene, which encodes for optineurin, were linked to amyotrophic
lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerations marked
with excessive motor neurons loss, loss of neurons from frontal and temporal lobes,
chronic inflammation, and protein aggregation in the central nervous system (CNS).
However, the pathogenic role of optineurin mutations is still largely unclear. The
autopsies of ALS and FTD patients carrying the optineurin mutations show TAR DNAbinding protein 43 (TDP-43) aggregates, which could elicit its nuclear depletion and lossof-function. Since optineurin acts as an adaptor protein in autophagy and inflammatory
signalling, and chronic inflammation could exacerbate TDP-43 aggregation, here we
tested if optineurin ALS-mimicking mutations could lead to impaired TDP-43
proteostasis, excessive inflammation, and/or inefficient immune responses. Moreover,
as ageing is a major risk factor for ALS and FTD, and untreated young mice carrying
optineurin ALS-linked mutations do not develop the disease, here we investigated
whether ageing could trigger neurological, neuropathological, and/or immunological
alterations. To this end, we used (1) Optn470T mouse model, that mimics loss-of-function
Q398X truncation found in ALS patients both of which express a lower level of truncated
optineurin (henceforth termed optineurin insufficiency) and (2) optineurin deficient
microglial BV2 cells made using CRISPR/Cas9 technology (BV2 Optn KO). We found
elevated basal TDP-43 protein levels in primary mouse Optn470T myeloid cells and
cortical neurons, which were post-translationally regulated. Moreover, we demonstrated
that optineurin deficiency did not sensitize cells to apoptosis upon autophagy inhibition
and that TDP-43 accumulation in Optn470T primary microglia was not caused by an
autophagy block. In contrast, we showed that optineurin insufficiency caused an altered
TDP-43 turnover, which was unaffected by experimental block in autophagy in both
bone marrow-derived macrophages (BMDMs) and primary neurons. To further evaluate
the role of optineurin in inflammation and TDP-43 accumulation, we stimulated BV2 cell
lines and primary microglia with lipopolysaccharide (LPS) to mimic bacterial infection. We observed a significant increase in TDP-43 protein levels in WT cells without changes
in optineurin levels. However, LPS failed to increase already elevated TDP-43 levels in
untreated optineurin-deficient and-insufficient myeloid cells, suggesting that they already
reached a plateau at basal conditions. Moreover, we demonstrated no nuclear depletion
or aggregation of TDP-43 in Optn470T primary microglia in basal state or with LPS.
Characterization of aged Optn470T mice did not show motor or cognitive defects, or
differential TDP-43 insolubility in the whole-brain lysates compared to WT mice. In
addition, we demonstrated enhanced expression of certain cytokines and chemokines in
the CNS during ageing, but with neglectable differences between old WT and Optn470T
mice. Moreover, spleen immunophenotyping uncovered signs of ageing of the immune
system (inflammageing and immunosenescence) in old Optn470T mice that were
comparable to WT mice. These included an increase in memory and regulatory T
lymphocytes, a drop in naïve T lymphocytes, and an increase in the number of
macrophages and neutrophils. However, we showed that macrophages and
conventional dendritic cells (cDC) exhibited increased expression of activation markers
in old Optn470T mice, although we could not link it to any phenotype. Altogether, a
combination of ageing and optineurin insufficiency did not induce ALS and/or FTD-like
immune imbalance and neuropathology in mice. To further evaluate crosstalk between
optineurin insufficiency and TDP-43 we established a new two-hit ALS and/or FTD
model by crossing Optn470T mice with the transgenic mice carrying a human TDP-43
patient mutation (G348C) but did not observe an ALS-like phenotype either. In
conclusion, we showed TDP-43 accumulation in optineurin-insufficient neurons and
microglia. In microglia, the accumulation was not caused by an autophagy block, and it
was unresponsive to inflammation, while in neurons it was likely caused by a block in
autophagy. Furthermore, the Optn470T mouse model during ageing, even when crossed
to mutant transgenic TDP-43 did not show motor or cognitive defects, TDP-43
aggregation, or immunological alterations typical for ALS and/or FTD. Thus, further
research is necessary to elucidate the mechanistic links between optineurin mutations
and TDP-43-mediated pathology.Optineurin je multifunkcionalni ubikvitin-vezujući protein koji ima ulogu u upalnoj
signalizaciji i autofagiji, procesima koji su opisani kao patološki mehanizmi u
neurodegenerativnim bolestima. Više od 40 mutacija u genu OPTN, koji kodira za
optineurin, je povezano s amiotrofičnom lateralnom sklerozom (ALS) i
frontotemporalnom demencijom (FTD), neurodegenerativnim bolestima koje
karakterizira prekomjeran gubitak neurona u motoričkom, temporalnom i
frontotemporalnom korteksu, kronična upala i agregacija proteina. Međutim, uloga
mutacija optineurina u patogenezi ALS-a još je uvelike nejasna. Autopsije pacijenata s
ALS-om i FTD-om koji nose mutacije optineurina pokazale su agregaciju TAR DNAvezujućeg proteina 43 (TDP-43) u središnjem živčanom sustavu koja je usko povezana
s njegovom deplecijom u jezgri i gubitkom funkcije. Budući da optineurin djeluje kao
adaptorski protein u autofagiji i upalnoj signalizaciji, a kronična upala može pogoršati
agregaciju TDP-43-a, u ovom radu smo testirali dovode li mutacije optineurina u ALS-u
do poremećene proteostaze TDP-43-a, pretjerane upale i/ili neučinkovitog imunosnog
odgovora. Štoviše, budući da je starenje glavni čimbenik rizika za ALS/FTD spektar
neurodegenerativnih bolesti, te uz činjenicu da netretirani mladi miševi s mutacijama
optineurina povezanih s ALS-om ne razvijaju bolest, ovdje smo istraživali ukoliko
starenje može potaknuti neurološke, neuropatološke i imunsne promjene. U tu svrhu
smo koristili (1) mišji Optn470T model, koji oponaša Q398X mutaciju optineurina
pronađenu u ALS pacijenatima koju nazivamo proteinskom insuficjencijom zbog manjka
mutiranog proteina i (2) mikroglijalnu BV2 staničnu liniju s nedostatkom optineurina
dobivenu pomoću CRISPR/Cas9 tehnologije (BV2 Optn KO). Utvrdili smo povišene
bazalne razine TDP-43 proteina u primarnim mišjim Optn470T mijeloidnim stanicama i
kortikalnim neuronima čije su razine bile posttranslacijski regulirane. Nadalje, pokazali
smo da nedostatak funkcionalnog optineurina nije povećao osjetljivost stanica na
apoptozu nakon inhibicije autofagije te da blokada autofagije ne izaziva nakupljanje
TDP-43 u primarnoj mikrogliji i BV2 staničnoj liniji. Međutim, pokazali smo da nedostatak
optineurina uzrokuje nakupljanje TDP-43 putem autofagije u primarnim neuronima i
makrofagima. Kako bismo dodatno ispitali ulogu optineurina u upali i nakupljanju TDP43, stimulirali smo primarnu mikrogliju i BV2 staničnu liniju s lipopolisaharidom (LPS) koji oponaša bakterijsku infekciju i uočili smo značajno povećanje razine TDP-43 u WT
stanicama. Međutim, LPS nije uspio povećati već nakupljeni TDP-43 u netretiranim
mijeloidnim stanicama s insuficijencijom optineurina. Štoviše, pokazali smo da TDP-43
ne pokazuje depleciju u jezgri niti agregaciju u Optn470T mikrogliji. Karakterizacija starih
Optn470T miševa nije pokazala motoričke ili kognitivne promjene, niti razliku u topljivosti
TDP-43 u lizatima cijelog mozga u usporedbi s WT miševima. Osim toga, pokazali smo
pojačanu ekspresiju citokina i kemokina u mozgu i leđnoj moždini bez značajnih razlika
između dvogodišnjih WT i Optn470T miševa. Štoviše, imunofenotipizacija slezene otkrila
je znakove upale povezane sa starenjem u Optn470T koji su bili usporedivi s WT
miševima, kao što je povećanje broja memorijskih i regulacijskih T limfocita i pad broja
naivnih, te povećan broj makrofaga i neutrofila. Međutim, pokazali smo da makrofagi i
konvencionalne dendritičke stanice (cDC) pokazuju povećanu ekspresiju aktivacijskih
markera u dvogodišnjim Optn470T miševima. Zaključno, kombinacija nefunkcionalnog
optineurina i starenja nije izazvala imunosnu neravnotežu i neuropatologiju sličnu
ALS/FTD-u kod miševa. Kako bismo dodatno istražili vezu između nefunkcionalnog
optineurina i TDP-43 proteina, uspostavili smo novi model ALS/FTD-a križanjem
Optn470T miševa s transgeničnim miševima koji nose ljudsku TDP-43 mutaciju (G348C)
pronađenu u pacijentima, ali bez uočenog ALS fenotipa do dvije godine starosti.
Zaključno, pokazali smo akumulaciju TDP-43 u neuronima i mikrogliji s nedostatkom
funkcinonalnog optineurina. Akumulacija u mikrogliji nije bila uzrokovana blokadom
autofagije niti je TDP-43 reagirao na upalu, dok je u neuronima i makrofagima uzrok
akumulacije vjerojatno blok u autofagiji. Nadalje, mišji model Optn470T
, čak ni nakon
križanja s mutiranim transgeničnim TDP-43 mišjim modelom, nije pokazao motoričke ili
kognitivne promjene, niti imunosne promjene vezane uz ALS/FTD spektar bolesti. Stoga
su daljnja istraživanja potrebna kako bi se razjasnile mehanističke veze između mutacija
optineurina i patologije posredovane TDP-43-om
Interakcije onkoproteina E6 HPV-a s vezujućim partnerima: Utjecaj na stabilnost i stanične funkcije E6
No full textHuman papillomaviruses (HPVs) are a group of small DNA viruses that cause various
human malignancies, with cervical cancer being the most significant disease associated with a
persistent HPV infection. Only a small number of HPV types has been shown to be responsible
for these malignancies. These HPVs are referred as high-risk (HR) types, with HPV-16 and -18
being the most prominent ones. Two major viral oncoproteins, E6 and E7, directly contribute to
the development of cancers by interfering with various cellular signaling pathways. A number of
HPV-16 variants has been identified in different geographical locations, with some variants
exhibiting higher oncogenic potential than others. The first part of thesis focuses on the analysis
of the HPV-16 E6 D25N L83V variant, which was shown to be strongly associated with the
development of cervical cancer. It was shown that this variant exhibits an increased capacity for
interacting with E6AP ubiquitin ligase and consequently degraded it more efficiently, in
comparison to the other analyzed mutants HPV-16 E6 D25N and E6 L83V. The HPV-16 E6
mutants' abilities to degrade key cellular target proteins, including the p53 tumor suppressor and
PDZ-domain containing substrates, were investigated through in vitro and overexpression
degradation assays. The analyses revealed no significant differences in the degradatory activities
among the evaluated E6 mutant oncoproteins. Furthermore, the second part of this thesis
demonstrates that multiple α-E6 oncoproteins can bind to MAML1 via LXXLL motif, resulting in
an increased α-E6 protein stability. β-E6 oncoprotein stability was also shown to be dependent on
the interaction with MAML1, whilst the absence of MAML1 led to both HPV-8 E6 and HPV-18 E6 oncoprotein rapid turnover at the proteasome. The study proposed a model by which most of
β-E6s interact exclusively with MAML1, whereas it appears that two cellular pools of HR α-E6
are present, one forms a complex with MAML1, while the other one interacts with E6AP. Although
HR α-E6/MAML1 complex does not affect the targeting of cellular substrates such as p53 and
DLG1, co-expression of MAML1 and E6AP with HR α-E6 modulates MAML1's normal cellular
activities leading to a significant increase in cellular proliferation. Silencing MAML1 decreases
wound closure in HeLa cells, suggesting its role in the regulation of cellular migration in HPVpositive cells and maintenance of the transformed phenotype. Overall, this doctoral thesis provides
novel insights into the functions of both α- and β-E6 oncoproteins and their roles in HPV-induced
pathogenesis.Humani papilomavirusi (HPVs) pripadaju skupini malih DNA virusa koji uzrokuju
različita maligna oboljenja, pri čemu je rak vrata maternice najznačajnija bolest povezana sa
dugotrajnom HPV infekcijom. Samo mali broj HPV tipova uzrokuje zloćudne bolesti. Spomenuti
tipovi nazivaju se visokorizičnim (HR), među kojima su HPV-16 i HPV-18 najistaknutiji. Dva
glavna virusna onkoproteina E6 i E7 svojim djelovanjem izravno pridonose razvoju raka djelujući
na različite stanične signalne puteve. Postoje mnoge varijante HPV-16 zastupljene na različitim
geografskim lokacijama te se pokazalo da neke od varijanti imaju veći onkogeni potencijal. Prvi
dio doktorske disertacije usmjeren je na analizu HPV-16 E6 D25N L83V varijante koja je usko
povezana s nastankom raka vrata maternice. Utvrđeno je da varijanta D25N L83V ostvaruje
povećanu interakciju sa ubikvitinskom ligazom E6AP te je posljedično najučinkovitija u poticanju
njezine razgradnje u usporedbi sa ostalim ispitanim mutantima HPV-16 E6 D25N i E6 L83V.
Svojstva mutanata da izazovu razgradnju ključnih staničnih ciljnih proteina, uključujući tumorsupresor p53 i stanične proteine koji sadrže PDZ-domene ispitana su in vitro esejima razgradnje i
esejima razgradnje provedenim korištenjem kulture stanica. Međutim, nisu otkrivene značajne
razlike u aktivnostima razgradnje među ispitivanim HPV-16 E6 mutantima. Drugi dio doktorske
disertacije otkriva da se različiti α-E6 onkoproteini vežu za MAML1 putem LXXLL strukturalnog
motiva, što rezultira povećanom stabilnošću onkoproteina E6. Nadalje, stabilnost onkoproteina βE6 također ovisi o interakciji s MAML1, dok utišavanjem MAML1 dolazi do brže proteasomske
razgradnje onkoproteina HPV-8 E6 i HPV-18 E6 u HPV-pozitivnim stanicama. Studija predlaže molekularni model prema kojemu većina onkoproteina β-E6 stupa u interakciju isključivo s
MAML1, dok su prisutna dva stanična skupa HR α-E6, od kojih jedan tvori kompleks s MAML1,
a drugi stupa u interakciji s E6AP. Iako kompleks HR α-E6/MAML1 nema ulogu u razgradnji
važnih staničnih ciljnih proteina kao što su p53 i DLG1, zajednička ekspresija MAML1 i E6AP sa
HR α-E6 mijenja uobičajene stanične aktivnosti MAML1 dovodeći do značajnog povećanja
stanične proliferacije. Dodatno, utišavanje MAML1 smanjuje „proces cijeljenja rane“ u HeLa
stanicama ukazujući na njegovu ulogu u regulaciji stanične migracije HPV-pozitivnih stanica i
održavanju transformiranog fenotipa. Sveobuhvatno, doktorska disertacija donosi nove uvide o
funkcijama onkoproteina α- i β-E6 i njihovoj ulozi u patogenezi izazvanoj HPV infekcijom
ADAR1 prevents protein kinase R activation in herpes simplex virus 1 infection
No full textAdenozin deaminaza koja djeluje na RNA (ADAR, prema eng. adenosine deaminase acting on RNA) katalizira pretvorbu adenozina u inozin na dvolančanim RNA molekulama. Pokazalo se da ADAR1, član obitelji ADAR proteina, može ciljati i virusne RNA transkripte te utjecati na proces replikacije virusa. Ovisno o virusu, njegova uloga može biti provirusna ili antivirusna. Pro- i antivirusne funkcije ADAR1 enzima dobro su istražene za brojne RNA viruse, ali malo se zna o njihovoj ulozi u replikaciji DNA virusa. U našem istraživanju, koristili smo HEK293 stanice sa stabilnom delecijom ADAR1 gena (ADAR1 KO stanice) kako bi testirali učinak nedostatka ADAR1 u infekciji herpes simpleks virusom tip 1 (HSV-1), široko rasprostranjenom ljudskom DNA virusu. Otkrili smo da je replikacija HSV-1 otežana u stanicama s nedostatkom ADAR1 u usporedbi s kontrolnim stanicama. Dodatno, istražili smo potencijalne mehanizme kojima ADAR1 ostvaruje svoj provirusni efekt. Provjerili smo razine proteina uključenih u signalne puteve urođene imunosti, kao i njihove posttranslacijske modifikacije. Otkrili smo da smanjena replikacija HSV-1 u ADAR1 KO stanicama korelira s pojačanom aktivacijom signalnog puta PKR kinaze, ključne kinaze za aktivaciju urođenih protuvirusnih odgovora domaćina. Zajedno, naši rezultati sugeriraju da ADAR1 sprječava PKR-posredovan prekid translacije proteina te time ispoljava provirusni učinak na infekciju HSV-1.Adenosine deaminase acting on RNA (ADAR) catalyzes the conversion of adenosine to inosine on double-stranded RNA molecules. It has been shown that ADAR1, a member of the ADAR protein family, can target viral RNA transcripts and affect the virus replication process. Depending on the virus, its role can be either proviral or antiviral. The pro- and antiviral functions of ADAR1 have been well studied for numerous RNA viruses, but little is known about their role in DNA virus replication. In our study, we utilized HEK293 cells with a stable deletion of the ADAR1 (ADAR1 KO) to test the effect of ADAR1 deficiency in herpes simplex virus 1 (HSV-1) infection, a widespread human DNA virus. We found that HSV-1 replication is impaired in ADAR1-deficient cells compared to control cells. Additionally, we investigated the potential mechanisms through which ADAR1 exerts its proviral effect. We analyzed the levels of proteins involved in the innate immunity signaling pathways, as well as their post-translational modifications. We found that reduced HSV-1 replication in ADAR1 KO cells correlates with the enhanced activation of the PKR kinase signaling pathway, a key kinase for the activation of host innate antiviral responses. Taken together, our findings suggest that ADAR1 suppresses the PKR-mediated translational shutdown, thus having a proviral effect on HSV-1 infection
Monoclonal antibodies in cancer immunotherapy
No full textMonoklonska protutijela predstavljaju obećavajuću i brzo rastuću vrstu bioloških lijekova koji su jedni od najprodavanijih na tržištu. Dio su imunoterapije, koja je uz kemoterapiju, operaciju i radijaciju jedan od temeljnih načina liječenja raka. Imunoterapija se počela razvijati zahvaljujući napretku u razumijevanju odnosa raka i imunosnog sustava, a samim time slijedio je i razvoj monoklonskih protutijela. Protutijela su glikoproteini koji u organizmu nastaju iz B-stanica i njihova je fiziološka uloga obrana i zaštita organizma od patogena. Djeluju vezivanjem na antigen ili Fc receptor i tako posreduju u raznim izvršnim funkcijama – od poticanja imunosnih stanica na uništenje patogena do opsonizacije patogena i aktivacije komplementa. Monoklonska protutijela prvi su put proizvedena 70-ih godina tehnikom hibridoma što je omogućilo dobivanje velikog broja jednakih protutijela specifičnih za određeni antigen. U terapijske svrhe, ona se koriste kao svojevrsna zamjena za fiziološka protutijela kako bi poboljšala, modificirala ili obnovila napad imunosnog sustava na maligne stanice. Daljnji napredak u tehnologijama za njihovu proizvodnju i humanizaciju, poput korištenja transgeničnih miševa ili phage display-a, olakšao je nastajanje visoko-specifičnih lijekova sa niskom imunogeničnosti. Monoklonska protutijela vežu ciljane tumorske antigene s visokim afinitetom i specifičnošću te uz to imaju sposobnost inducirati protutumorske imunosne odgovore. Dolaze u nekoliko oblika, a mogu se podijeliti u tri kategorije: nekonjugirana, konjugirana i bispecifična. Njihovi raznoliki mehanizmi djelovanja pružaju efektivan medicinski tretman i borbu protiv raka i mnogih bolesti. Međutim, primjena monoklonskih protutijela nosi rizik od stvaranja neželjenih reakcija poput citokine oluje, serumske bolesti i toksičnosti za neke organe. U trenutnim pretkliničkim i kliničkim istraživanjima radi se na svođenju ovih nuspojava na minimum.Monoclonal antibodies are a promising and rapidly growing class of biological drugs that are among the best-selling on the market. They are a part of immunotherapy, which, along with chemotherapy, surgery and radiation, is one of the basic methods of cancer treatment. Immunotherapy began to develop when the understanding of the relationship between cancer and the immune system progressed, and soon after that, the development of monoclonal antibodies followed. Antibodies are glycoproteins that are produced in the body from B-cells and their physiological role is defending and protecting the body from pathogens. They act by binding to an antigen or Fc receptor and thus mediate various executive functions - stimulating immune cells to destroy pathogens, opsonizing pathogens and activating the complement. Monoclonal antibodies were first produced in the 70s by the hybridoma technique, which made it possible to obtain a large number of identical antibodies specific for a certain antigen. For therapeutic purposes, they are used as a kind of substitute for physiological antibodies in order to improve, modify or renew the immune system's attack on malignant cells. Further progress in technologies for their production and humanization, such as the use of transgenic mice or phage display, facilitated the creation of highly specific drugs with low immunogenicity. Monoclonal antibodies bind the target tumor antigens with high affinity and specificity and induce anti-tumor immune responses. They come in several forms, and can be divided into three categories: unconjugated, conjugated and bispecific. Their diverse mechanisms of action provide effective medical treatment and fight against cancer and many diseases. However, the use of monoclonal antibodies carries the risk of adverse reactions such as cytokine storm, serum sickness and toxicity to some organs. In current preclinical and clinical research, efforts are being made to reduce these side effects to a minimum
Trombociti i sepsa: analiza diferencijalne ekspresije gena za trombospondin 1 i ADAR proteine
No full textSepsis is a severe, life-threatening disease that affects multiple organ systems and causes numerous damages throughout the organism. However, the mechanisms of sepsis and the body's defenses are not fully understood. Many studies describe the involvement of the immune system in the development of sepsis, but the involvement of platelets in the development and defense of sepsis is elusive. Moreover, thrombocytopenia is frequent in septic patients and is a marker of poor prognosis and a high risk of death in sepsis. Therefore, in this work, by analyzing the transcriptome of patients suffering from sepsis based on available data, we investigated differences in the levels of selected transcripts potentially important for platelet function and sepsis. We analyzed thrombospondin 1 (TSP1), a major protein component of platelet α-granules, and ADAR (adenosine deaminase acting on RNA, 1-3), a group of proteins not previously studied in platelets. We found that transcript levels for TSP1 or ADAR2 and 3 did not change, whereas they increased significantly only for ADAR1 in platelets from septic patients. Next, we performed in vitro analysis of platelet precursor cells and mouse megakaryocytes for TSP1 and ADAR1. We found that TSP1 expression increases as megakaryocytes differentiate into platelets. Culturing megakaryocytes with interleukin 1α (IL1α, cytokine involved in sepsis) did not change TSP1 levels. Unfortunately, we could not detect ADAR1 in mouse megakaryocytes with available antibodies. Future studies are needed to explain the role of these proteins in platelets and their involvement in sepsis.Sepsa je teška po život opasna bolest koja zahvaća više organskih sustava i uzrokuje brojna oštećenja u cijelom organizmu. Međutim, mehanizmi sepse i tjelesne obrane nisu u potpunosti razjašnjeni. Mnoge studije opisuju uključenost imunološkog sustava u nastanak sepse, no uključenost trombocita u nastanak i obranu od sepse nije u potpunosti jasna. Trombocitopenija je česta u bolesnika sa sepsom i pokazatelj je loše prognoze i visokog rizika od smrti. Stoga smo u ovom radu, analizom transkriptoma pacijenata oboljelih od sepse na temelju dostupnih podataka, istražili razlike u razinama odabranih transkripata potencijalno važnih za funkciju trombocita i sepsu. Analizirali smo trombospondin 1 (TSP1), glavnu proteinsku komponentu α-granula trombocita, i ADAR (adenozin deaminaza koja djeluje na RNA, 1-3), skupinu proteina koji prethodno nisu proučavani u trombocitima. Otkrili smo da se razine transkripta za TSP1 ili ADAR2 i 3 nisu promijenile, dok su se značajno povećale samo za ADAR1 u trombocitima septičkih pacijenata. Zatim smo in vitro analizirali prekursorske stanice trombocita, megakariocita miša, za TSP1 i ADAR1. Otkrili smo da se ekspresija TSP1 povećava kako se megakariociti diferenciraju u trombocite. Uzgoj megakariocita s interleukinom 1α (IL1α, citokin uključen u sepsu) nije promijenio razine TSP1. Nažalost, nismo mogli detektirati ADAR1 u megakariocitima miša dostupnim protutijelima. Buduća istraživanja su potrebna kako bi se objasnila uloga ovih proteina u trombocitima i njihova uključenost u sepsu
Tandemska masena spektrometrija u kombinaciji s ionske pokretljivosti za karakterizaciju kardiolipinske molekularne raznolikost
No full textCardiolipin (CL), is a unique class of membrane phospholipids that is found
almost exclusively in the mitochondrial inner membrane of eukaryotic cells.
CL undergoes substantial remodelling through a highly conserved
deacylation-transacylation pathway, and Barth syndrome is caused by a
deficiency of the CL transacylase, Tafazzin (TAZ). Because of their unique
structure, conducting an in-depth examination of the CL molecular diversity
is challenging from an analytical perspective. However, when travelling
wave ion mobility is combined with high-resolution tandem mass
spectrometry (IM-MS/MS), a rapid and simple multidimensional
characterization of the CL molecular diversity is made feasible due to CL
separation from obscuring singly charged ion signals. Calculating the peak
intensities that overlap the monoisotopic CL ions has been made easier
thanks to the Python script used to automate the procedure. All of the IMMS/MS-related parameters have been tuned to ensure CL separation and
get optimum intensity, fragmentation, and quantitation from the samples.
We demonstrate using IM-MS/MS that mutants of Saccharomyces
cerevisiae lacking in CL remodelling (Cld1ΔTaz1Δ) exhibit a shift towards
CL species with more saturated and shorter acyl chains. Furthermore,
Saccharomyces cerevisiae mutant Taz1Δ also display a shift towards CL
species with more saturated and shorter acyl chains but, the most relevant
finding was that the MLCL/CL ratio has increased in a manner that is
comparable to that seen in individuals with Barth syndrome. Our findings
provide additional evidence that the CL profiles of different mammalian
tissues are distinct from one another. In conclusion, IM-MS/MS is a method
that is a powerful, robust and accurate tool when it comes to isolating CL
species from complicated lipid samples and for identifying CL structural
diversity.Kardiolipin (CL) je jedinstvena klasa membranskih fosfolipida koji se mogu
naći gotovo isključivo u mitohondrijskoj unutarnjoj membrani eukariotskih
stanica. CL je podvrgnut značajnom preoblikovanju putem visoko očuvanog
puta deacilacije-transacilacije, a Barthov sindrom je uzrokovan
nedostatkom CL transacilaze (TAZ). Zbog njihove jedinstvene strukture,
provođenje dubinskog ispitivanja molekularne raznolikosti CL-a izazovno je
iz analitičke perspektive. Međutim, kada se pokretljivost iona putujućih
valova kombinira s tandemskom masenom spektrometrijom visoke
rezolucije (IM-MS/MS), moguća je brza i jednostavna višedimenzionalna
karakterizacija molekularne raznolikosti CL zahvaljujući odvajanju CL od
zamračujućih signala jednostruko nabijenih iona. Izračunavanje vršnih
intenziteta koji preklapaju monoizotopne CL ione olakšano nam je
zahvaljujući softveru Python koji smo koristili za automatizaciju postupka.
Svi parametri povezani s IM-MS/MS podešeni su kako bi se osiguralo
odvajanje CL-a i dobili optimalan intenzitet, fragmentacija i kvantifikacija iz
uzoraka. Uspjeli smo pokazati pomoću IM-MS/MS da mutanti
Saccharomyces cerevisiae kojima nedostaje CL remodeliranje
(Cld1ΔTaz1Δ) pokazuju pomak prema CL vrstama sa zasićenijim i kraćim
acilnim lancima. Nadalje, mutant Saccharomyces cerevisiae Taz1Δ također
je pokazao pomak prema CL vrstama sa zasićenijim i kraćim acilnim
lancima, ali je najrelevantnije otkriće bilo da se omjer MLCL/CL povećao na
način koji je usporediv s onim viđenim kod pojedinaca s Barthovim
sindromom. Naša otkrića pružaju dodatne dokaze da se CL profili različitih
tkiva sisavaca međusobno razlikuju. Zaključno, IM-MS/MS je metoda koja
je snažan, robustan i precizan alat kada je u pitanju izolacija vrsta CL iz
kompliciranih uzoraka lipida i za identifikaciju strukturne raznolikosti CL
Razvoj modelnog Sistema kvasca za ispitivanje utjecaja betaoksidacije na promjenu mitohondrijskih kardiolipina
No full textBarth syndrome is a multi-system genetic disorder that affects the
mitochondria. It is characterized by a wide range of conditions mostly affecting
the heart and skeletal muscles. Symptoms include: cardiomyopathy, skeletal
myopathy, neutropenia, increased urinary excretion of 3-methylglutaconic
acid and weaker motor skills. What causes the disorder is the mutations in the
tafazzin (TAZ) gene, which codes the tafazzin enzyme that, in healthy cells, is
widely present in cardiac and skeletal muscles and takes part in the cardiolipin
remodeling pathway. Cardiolipin is a phospholipid located in the mitochondrial
inner membrane. Unlike other phospholipids, cardiolipin has a specific
structure which allows it to interact with a vary of membrane proteins and
complexes, giving the molecule important functions in proper mitochondrial
energy metabolism. It undergoes a highly conserved and complex synthesis
pathway, which is altered in patients with Barth syndrome. Since there exists
a lack of understanding the importance and functions of cardiolipin synthesis,
Barth syndrome is treated only symptomatically. With improved
understanding of the molecular mechanism and importance of cardiolipin
synthesis and remodeling, new treatment approaches for Barth syndrome can
be evaluated. One such targets may be beta-oxidation. Trimetazadine is a
drug widely used for treating heart conditions, which inhibits beta-oxidation
and in some previous research influences mitochondrial phospholipid
turnover. However, the effect of trimetazidine on cardiolipin synthesis and
turnover is unknown. We are curious to see whether the inhibition of betaoxidation promotes mitochondrial phospholipid turnover and increases
functional cardiolipin content in cells with TAZ deficiencies. Saccharomyces
cerevisiae is a great model organism for studying Barth syndrome because all
enzymatic steps in the cardiolipin synthesis and remodeling pathway are
conserved form yeast to humans.Barthov sindrom je multi-sistemski genetski poremećaj koji zahvaća
mitohondrij. Karakteriziran je širokim rasponom stanja koja uglavnom
zahvaćaju srce i skeletne mišiće. Simptomi uključuju: kardiomiopatiju,
skeletnu miopatiju, neutropeniju, povećano izlučivanje 3-metilglutakonske
kiseline i slabije motoričke sposobnosti. Poremećaj uzrokuju mutacije u
tafazzin (TAZ) genu koji kodira enzim tafazzin koji je, u zdravim stanicama,
velikim brojem prisutan u srcu i skeletnim mišićima te sudjeluje u putu
remodeliranja kardiolipina. Kardiolipin je fosfolipid lociran u unutarnjoj
membrani mithonodrija. Za razliku od drugih fosfolipida, kardiolipin ima
specifičnu strukturu koja mu omogućava interakciju s brojnim membranskim
proteinima i kompleksima, dajući molekuli važne funkcije u pravilnom
mitohondrijskom metabolizmu. Podvrgava se visoko očuvanom i
kompleksnom sintetskom putu, koji je promijenjen u pacijenata koji boluju od
Barthovog sindroma. S obzirom na smanjeno razumijevanje važnosti i funkcije
sinteze kardiolipina, Barthov sindrom se liječi simptomatski. Uz bolje
razumijevanje molekularnog mehanizma i funkcije kardiolipinske sinteze i
remodeliranja, mogu se procijeniti novi pristupi liječenju Barthovog sindroma.
Jedna od meta u liječenju mogla bi biti beta-oksidacija. Trimetazidin je lijek
široke primjene u liječenju srčanih oboljenja, koji inhibira beta-oksidaciju te u
ranije provedenim istraživanjima utječe na promjenu mitohondrijskih
fosfolipida. Unatoč tome, utjecaj trimetazidina na promjenu i sintezu
kardiolipina je nepoznat. Zanima nas vidjeti promovira li inhibicija betaoksidacije promjenu mitohondrijskih fosfolipida i povećanje funkcionalnog
kardiolipina u stanicama s TAZ deficijencijama. Saccharomyces cerevisiae je
odličan modelni organizam za proučavanje Barthovog sindroma zbog činjenice
da su svi koraci sinteze i remodeliranja kardiolipina sačuvani od kvasca do
čovjeka
Three- dimensional cell models of the central nervous system
No full textPovećanje stope smrtnosti na globalnoj razini uzrokovane
neurodegenerativnim bolestima potaknulo je svijet znanosti na kreiranje
znanstvene metode dovoljno jednostavne za izradu, a pritom dovoljno
kompleksne za obuhvaćanje karakteristika središnjeg živčanog sustava
(SŽS). Neadekvatnost postojećih životinjskih i dvodimenzionalnih (2D)
modela korištenih za ispitivanje složenosti patogeneza bolesti koje
zahvaćaju SŽS dovela je do razvoja trodimenzionalnih (3D) staničnih
kultura.
3D stanični modeli SŽS-a generiraju se iz pluripotentnih stanica koje se
daljnjim usmjeravanjem diferenciraju u neurone, astrocite ili
oligodendrocite. Navedene pluripotentne stanice uključuju embrionalne i
neuralne matične stanice, ali i umjetno inducirane pluripotentne matične
stanice. Nakon što su stanice prikupljene iz embrionalnih tkiva ili pak
zdravih ili bolesnih odraslih jedinki, kultiviraju se in vitro u odgovarajućim
uvjetima kako bi se osigurao razvoj, rast i daljnja proliferacija. Ovisno o
kojim stanicama je riječ, dovode se pripadni faktori u staničnu kulturu koji
potiču diferencijaciju stanica do vrste potrebne za istraživanje. 3D modeli
nadalje se formiraju sa ili bez nosača prirodnog ili sintetskog podrijetla koji
može služiti kao strukturna potpora kultiviranim stanicama. Materijali
korišteni nužno moraju biti biokompatibilni kako ne bi došlo do negativnih
utjecaja na stanice kulture ili pak živi organizam.
Laboratorijska metoda izrade 3D modela SŽS-a koristi se u svrhe
proučavanja mehanizama neurodegenerativnih poremećaja te njihovih
nastanaka i napredovanja u organizmu. Izrada modela mehanizama raka
koji pogađaju stanice SŽS-a te ispitivanje potencijalnih farmakoterapija
također ulaze u repertoar uloga koje obnašaju 3D stanični modeli. Od
samoformirajućih sferoida do sofisticiranih organa na čipu, 3D modeli SŽSa odlikuju se različitim stupnjem uniformnosti bioloških karakteristika.
Glavni cilj pri izradi ovih modela je stvoriti sustav što autentičniji stanicama
i tkivima in vivo. Usprkos jedinstvenim svojstvima, 3D modeli odlikuju se i
manjkavostima poput nedostatka vaskularizacije te visokim troškom
proizvodnje usporedno s 2D modelima. 3D staničnim modelima potrebno
je usavršavanje no unatoč tomu od svoje prve uporabe predstavljaju
ulaznicu u svijet istraživanja složeno dizajniranog ljudskog živčanog
sustava.The increase in the global mortality rate caused by neurodegenerative
diseases has prompted the world of science to create a scientific method
that is simple enough to create, yet complex enough to capture the
characteristics of the central nervous system (CNS). The inadequacy of
existing animal and two-dimensional (2D) models used to investigate the
complexity of the pathogenesis of diseases affecting the CNS led to the
development of three-dimensional (3D) cell cultures.
3D cell models of the CNS are generated from pluripotent cells, which are
differentiated into neurons, astrocytes or oligodendrocytes by further
targeting. Pluripotent cells include embryonic and neural stem cells, but
also artificially induced pluripotent stem cells. After the cells have been
collected from embryonic tissues or from healthy or diseased adults, they
are cultivated in vitro under appropriate conditions to ensure development,
growth and further proliferation. Depending on the cells in question,
relevant factors are brought into the cell culture that stimulate the
differentiation of the cells to the type required for research. 3D models are
further formed with or without scaffolds of natural or synthetic origin that
can serve as structural support for cultured cells. The materials used must
necessarily be biocompatible so that there is no adverse interaction with
cultured cells or a living organism.
The laboratory method of creating 3D models of the CNS is used for the
purpose of studying the mechanisms of neurodegenerative disorders and
their development as well as progression in the body. The development of
models of cancer mechanisms that affect the cells of the CNS and the
examination of potential pharmacotherapies also enter the repertoire of
roles played by 3D cell models. From self-forming spheroids to
sophisticated organs-on-a-chip, 3D models of the CNS are characterized by
varying degrees of uniformity of biological characteristics. The main goal
when creating these models is to create a system as authentic as possible
to cells and tissues in vivo. Despite their unique properties, 3D models are
also characterized by shortcomings such as the lack of vascularization and
the high cost of production compared to 2D models. 3D cell models need
improvement, but despite this, since their first use, they represent a ticket
to the world of research into the complexly designed human nervous
system