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The use of the AI tool AlphaFold 2 for protein structure modeling
No full textProteins are biological macromolecules composed of amino acids linked by peptide bonds. Their three-dimensional (3D) structures are still challenging to determine and the number of proteins with resolved tertiary structures is rather small compared to the number of known protein sequences. The 3D structures of proteins are essential for understanding their function, and thus biological processes orchestrating health and diseases. The 3D protein structure allows us to identify "binding pockets" and functionally relevant regions of the protein. Nowadays innovative approaches have been developed for fast determination of protein conformations. These include computer algorithms that predict the 3D structure of the protein from its polypeptide primary sequence. In this thesis, we use AlphaFold 2, an open-source software that uses available protein datasets and artificial intelligence (AI), to predict the 3D structure of proteins. In this study, AlphaFold 2 structure models were analyzed for randomly generated amino acid sequences and for well-known industrial biocatalysts halohydrin dehalogenases HheC and HheA. The random sequences were generated by the tool RandSeq, while the FASTA inputs for HheA and HheC were formed from the crystal structures 1ZMO and 1ZMT, respectively, downloaded from the Protein Data Bank. The AlphaFold 2 conformations were analyzed using PyMOL and ChimeraX visualization software. While AlphaFold 2 could not reliably predict the structures of random sequences, as expected, the structures of the enzymes HheA and HheC in their monomeric and tetrameric states were predicted with high reliability. However, structural peculiarity like the entry of the C-terminal tail into the diagonal subunit of the HheC tetramer was not predicted. This study shows that AlphaFold 2 structures can be good starting conformations for molecular dynamics simulations while their use for molecular docking calculations should be taken with caution.Proteini su biološke makromolekule sastavljene od aminokiselina povezanih peptidnim vezama. Njihove trodimenzionalne (3D) strukture još uvijek je teško odrediti, a broj proteina s rješenim tercijarnim strukturama prilično je malen u usporedbi s brojem poznatih proteinskih sekvenci. 3D strukture proteina bitne su za razumijevanje njihove funkcije, a time i bioloških procesa koji upravljaju zdravljem i bolestima. 3D proteinska struktura omogućuje nam identificiranje "veznih džepova" i funkcionalno relevantnih regija proteina. Danas se razvijaju inovativni pristupi za brzo određivanje konformacija proteina, a to uključuje i računalne algoritme koji predviđaju 3D strukturu proteina iz primarne sekvence polipeptida. U ovom diplomskom radu koristimo AlphaFold 2, softver otvorenog koda koji koristi dostupne skupove podataka o proteinima i umjetnu inteligenciju (AI) za predviđanje 3D strukture proteina. U ovoj studiji pomoću AlphaFolda 2 predviđene se strukture za nasumično generirane sekvence aminokiselina i za dobro poznate industrijske biokatalizatore halohidrin dehalogenaza HheC i HheA. Nasumične sekvence generirao je alat RandSeq, dok su FASTA ulazi za HheA i HheC formirani iz kristalnih struktura 1ZMO, odnosno 1ZMT, preuzetih iz baze Protein Data Bank (PDB). Predviđene konformacije analizirane su pomoću softvera za vizualizaciju PyMOL i ChimeraX. Iako AlphaFold 2 nije mogao pouzdano predvidjeti strukture nasumičnih sekvenci, kao što se i očekivalo, strukture enzima HheA i HheC u njihovim monomernim i tetramernim stanjima predviđene su s visokom pouzdanošću. Međutim AlphaFold 2 nije predvidio strukturnu osobitost ulaska C-terminalnog repa u dijagonalnu podjedinicu tetramera HheC. Ova studija pokazuje da strukture predviđene AlphaFoldom 2 mogu biti dobre početne konformacije za simulacije molekulske dinamike, dok njihovu upotrebu za izračune molekulskog uklapanja treba uzeti s oprezom
Isolation and characterization of primary fibroblasts from skin and lung of wild-type mice and Sti-heterozygous mice
No full textTijekom životnog vijeka organizma sisavaca postepeno se narušava homeostaza proteina koja uzrokuje slabljenje većine staničnih funkcija, što rezultira bolestima koje su povezane sa starenjem i smrću. Međutim, vrlo malo se zna o izvanstaničnim i staničnim čimbenicima koji dodatno ubrzavaju poremećaj homeostaze proteina tijekom procesa starenja. U ovom radu karakterizirani su poremećaji homeostaze proteina u primarnim stanicama izoliranim iz divljeg tipa miševa C57BL/6 na specifičnim stadijima njihovog postnatalnog životnog ciklusa te su uspoređeni s poremećajima homeostaze proteina u istim staničnim vrstama izoliranim iz Sti-heterozigotnih miševa, koji uslijed mutacije enzima alanil-tRNA sintetaze ugrađuju aminokiselinu serin umjesto alanina u svoje proteine. Izolirani su fibroblasti iz tkiva kože i pluća miševa divljeg tipa i Sti-heterozigotnih miševa starosti 3 i 6 mjeseci. Poremećaj homeostaze proteina očituje se nakupljanjem slobodnih kisikovih radikala, ROS-a koji može oštetiti brojne makromolekule i uzrokovati poremećaj njihove funkcije. U izoliranim fibroblastima određena je količina ROS-a. U fibroblastima Sti-heterozigotnih miševa starosti 6 mjeseci nakuplja se veća količina ROS-a u odnosu na fibroblaste miševa divljeg tipa iste starosti. Dobiveni rezultati upućuju da uslijed pogrešaka u sintezi proteina dolazi do narušavanja homeostaze proteina.During the lifetime of the mammalian organism, protein homeostasis is gradually disrupted, causing the weakening of most cellular functions, resulting in diseases associated with aging and death. However, not much is known about extracellular and cellular factors that further accelerate the disruption of protein homeostasis during the aging process. This work describes protein homeostasis disorders in primary cells isolated from wild-type C57BL/6 mice and Sti-heterozygous mice at specific stages of their postnatal life. Sti-heterozygous mice are carrying the mutation of the enzyme alanyl-tRNA synthetase which caused incorporation of amino acid serine instead of alanine in its protein. Primary fibroblasts were isolated from the skin and lung of wild-type mice and Sti-heterozygous mice that are 3 and 6 months old. Protein homeostasis disorders is reflected by the accumulation of reactive oxygen species, ROS, which can damage a number of macromolecules and cause disruption of their function.The amount of ROS was determined in isolated fibroblasts. Fibroblasts isolated from the Sti-heterozygous mice in age 6 months accumulate a higher amount of ROS compared to fibroblasts from wild-type mice of the same age. The obtained results indicate that errors in protein synthesis lead to disrupted homeostasis of proteins
Novel comprehensive in vitro model of acquired resistance to carboplatin as a tool to identify new targets of ovarian cancer therapy
No full textRak jajnika je ginekološka bolest koja, uslijed nedostatka specifičnih simptoma i farmakogenetskih biljega (biomarkera), često biva otkrivena u kasnim stadijima, kada se bolest već proširila i na druge organe. Malobrojne bolesnice koje odgovore na primarnu terapiju vrlo često razviju otpornost na lijekove. Kako bismo bolje razumjeli taj ključni problem uspješne terapije, razvili smo model stanica postepenog razvoja otpornosti raka jajnika na karboplatinu, sastavljenog od roditeljskih stanica i sedam varijanti sa stabilnom rastućom otpornosti na karboplatinu. Svaka varijanta, uključujući i roditeljske stanice, karakterizirana je s obzirom na otpornost na najčešće korištene kemoterapeutike u terapiji raka jajnika, razine biljega epitelno-mezenhimske tranzicije i metastatskog potencijala. Nadalje, nakon izmjerene ekspresije gena u svim varijantama stanica korištenjem dva pristupa analizirali smo dobivene vrijednosti bioinformatičkim metodama. Dobivene liste značajno promijenjenih gena (DEG) i gena čija ekspresija korelira s odgovorom na karboplatinu (CCORG) uspoređene su u kontekstu signalnih puteva, interakcija proteina i bioloških procesa. Izdvojeni su potencijalno interesantni geni čija je uloga u procesima razvoja otpornosti i epitelno-mezenhimske tranzicije analizirana in vitro metodama, a njihov značaj kao mogućih farmakogenetskih biljega u klinici bioinformatičkim analizama informacija u dostupnim bazama podataka. Dodatnim eksperimentima korištenjem specifičnih inhibitora pojedinih proteina od interesa ispitana je važnost izdvojenih signalnih puteva u razvoju otpornosti i pokretljivosti stanica. Predstavljeni model, zajedno s dva različita pristupa analizi ekspresije gena doprinio je razumijevanju razvoja otpornosti i omogućio detekciju gena čija uloga dosad nije bila opisana u kontekstu otpornosti i pokretljivosti stanica te smo time pokazali međusobnu povezanost ova dva fenomena potaknuta lijekom.Due to the lack of specific early-stage symptoms and biomarkers, ovarian cancer is often diagnosed in late stages, when the disease has already spread. A small number of patients that positively respond to chemotherapy often develop drug resistance upon therapy. To tackle the problem, we developed high-grade serous ovarian cancer (HGSOC) model composed of variants with differing degrees of resistance to carboplatin. All variants, including the parental cells, were characterized in terms of carboplatin and paclitaxel sensitivity, markers of epithelial-mesenchymal transition and metastatic properties (migration and invasion). To understand the differences, we measured the expression of genes in all established variants and analysed it by two different bioinformatics approaches. Resulting differentially expressed gene (DEG) and carboplatin-correlating gene (CCORG) lists were compared in terms of signalling pathways, protein interactions and biological processes. Potentially interesting genes were selected and analysed in terms of their role in resistance development and metastasis by in vitro experiments. Their prognostic and predictive clinical value was examined by in silico approaches. Additional experiments using inhibitors were used to examine the role of selected signaling pathways. Presented cell model, along with two distinct approaches to gene expression analysis gave us a valuable insight in resistance development mechanisms and enabled us to select novel genes with reported functions in drug resistance and drug-induced epithelial-mesenchymal transition
Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids
No full textGlucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11β-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRRs). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concentration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behavior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11β-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is associated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of binding of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined using the molecular mechanics Poisson–Boltzmann surface area approach, indicating that resveratrol could affect CYP3A activity
Ko-agregacija proteina TRIOBP-1 i NDE1 te njihova značajnost u mentalnim bolestima
No full textSchizophrenia is a chronic mental illness, that affects people worldwide, and presents a significant challenge within the field of mental health. Because of its global rise in prevalence, the imperative to comprehend its underlying molecular mechanisms becomes increasingly vital. The complexity of this and other chronic mental illnesses presents significant diagnostic and treatment challenges, emphasizing the need for innovative approaches to address their intricate nature. Recent insights, as well as the focus of this thesis, aim to shed light on the occurrence of protein aggregation in the context of schizophrenia, offering new perspectives alongside genetics and environmental factors. This thesis focuses on the co-aggregation of TRIOBP-1 and NDE1, both of which play crucial roles in cellular processes and neurobiology and delves into their interaction, aiming to unravel their contributions to the disorder. Previous studies acknowledge NDE1's interactions with aggregating proteins like TRIOBP-1 and DISC1, underscoring its significance in this context.
To investigate this co-aggregation further, we employed a range of methodologies. Plasmids were expressed in neuroblastoma cells, and their interactions were visualized using immunofluorescent microscopy. Western blot analysis confirmed the presence and expression levels of proteins. Additionally, we utilized an insoluble protein purification assay to validate the observed aggregates. Notably, our experiments revealed surprising findings, such as NDE1-inducing aggregation in mutant TRIOBP-1 and the co-aggregation of Flag-NDE1 and EGFP. These results shed light on the complex ways in which NDE1 can interact with other proteins.
This research unveiled intricate protein interactions, emphasizing the ongoing need for a deeper understanding of this disorder. By unravelling the links between protein aggregation and schizophrenia, studies like this pave the way for more accurate diagnostics and innovative treatment approaches. These insights hold the promise of enhancing the quality of life for individuals with schizophrenia and driving further progress in this area.Schizofrenija je kronična mentalna bolest koja utječe na ljude diljem svijeta i predstavlja značajan izazov u području mentalnog zdravlja. S obzirom na globalni porast njezine prevalencije, postaje sve važnije razumjeti njezine temeljne molekularne mehanizme. Složenost ove i drugih kroničnih mentalnih bolesti stvara znatne izazove u dijagnostici i liječenju, što ističe potrebu za inovativnim pristupima za razumijevanje njihove složene prirode. Nedavna otkrića, kao i fokus ovog rada, imaju za cilj staviti fokus na pojavu agregacije proteina u kontekstu schizofrenije, nudeći nove perspektive uz genetiku i čimbenike okoliša. Ovaj rad usredotočuje se na koagregaciju TRIOBP-1 i NDE1, od kojih oba igraju ključne uloge u staničnim procesima i neurobiologiji, te istražuje njihovu interakciju kako bi se razotkrili njihovi doprinosi tom poremećaju. Prethodne studije potvrđuju interakcije NDE1 s agregirajućim proteinima poput TRIOBP-1 i DISC1, ističući njegovu važnost u tom kontekstu.
Kako bismo dalje istražili ovu koagregaciju, koristili smo niz metodologija. Plazmide smo izrazili u stanicama neuroblastoma te smo njihove interakcije vizualizirali upotrebom imunofluorescentne mikroskopije. Western blot analiza potvrdila je prisutnost i razine izraženih proteina. Nadalje, koristili smo esej purifikacije netopivih proteina kako bismo potvrdili promatrane agregate. Značajno je da su naši eksperimenti otkrili iznenađujuće rezultate, poput NDE1 koji potiče agregaciju mutiranog TRIOBP-1 i koagregaciju Flag-NDE1 i EGFP-a. Ti rezultati stavljaju fokus na kompleksne načine na koje NDE1 može interagirati s drugim proteinima.
Ovo istraživanje otkrilo je kompleksne interakcije proteina, naglašavajući stalnu potrebu za dubljim razumijevanjem ovog poremećaja. Razotkrivanjem veza između agregacije proteina i schizofrenije, istraživanja poput ovog otvaraju put za precizniju dijagnostiku i inovativne pristupe liječenju. Ovi uvidi obećavaju poboljšanje kvalitete života osoba sa schizofrenijom i potiču daljnji napredak u ovom području
Pathogenesis of autoimmune hemolytic anemia
No full textAutoimuna hemolitička anemija (AIHA) heterogena je skupina bolesti uzrokovana napadom vlastitih protutijela, autoprotutijela, na antigene eritrocita. Dijeli se na primarnu AIHA, nastalu kao posljedica imunološke reakcije, te sekundarnu, nastalu uslijed poremećaja imunološke regulacije. Incidencija AIHA iznosi 1 do 3 slučaja na 100 000 osoba godišnje, a dijagnosticira se direktnim (DAT) i indirektnim (IAT) antiglobulinskim testom. Na temelju dijagnostičkih testova i osobina autoprotutijela, AIHA se dijeli na toplu (wAIHA), hladnu (cAIHA) i miješanu. Nadalje, hladna se AIHA, prema etiopatogenezi, dijeli na bolest hladnih aglutinina (CAD) te sindrom hladnih aglutinina (CAS). Paroksizmalna hladna hemoglobulinurija (PCH) rijedak je oblik autoimune hemolitičke anemije u kojemu se autoprotutijela vežu na eritrocite na niskim temperaturama, a komplement aktiviraju pri visokim temperaturama. Uz aktivaciju sustava komplementa i stvaranje membranskog napadajućeg kompleksa (MAC), hemoliza može biti uzrokovana aktivacijom ADCC mehanizma ili fagocitozom eritrocita od strane makrofaga. Poremećaji gena regulacije imunosnog sustava, infekcije, okoliš, lijekovi i tumorske tvorbe mogući su uzroci nastanka AIHA. Obzirom na heterogenost bolesti i različite imunopatogene mehanizme nastanka AIHA, terapije se mogu uvelike međusobno razlikovati. Prva su linija liječenja glukokortikosteroidi, potom slijede rituksimab, ali i splenektomija. Imunosupresivni lijekovi, ciklosporin, azatioprin ciklofosfamid, čine treću liniju terapije. U tijeku su klinička ispitivanja novih terapija koje uključuju inhibiciju fagocitoze eritrocita (fostamatinib, nipocalimab), inhibiciju Brutonove tirozin kinaze (rilzabrutinib i ibrutinib), inhibiciju proteasoma (bortezomib), anti CD38 monoklonska protutijela (daratumumab, isatuximab), inhibiciju mTOR (sirolimus) i inhibiciju proteina komplementa (ekulizumab, pegcetakoplan, sutimlimab).Autoimmune hemolytic anemia (AIHA) is a heterogeneous group of diseases
caused by the attack of endogenous antibodies (autoantibodies) on
erythrocyte antigens. It is divided into primary AIHA, which results from an
immune response, and secondary AIHA, which is caused by disorders of
immune regulation. The incidence of AIHA is 1 to 3 cases per 100,000
people per year, and it is diagnosed by direct (DAT) and indirect (IAT)
antiglobulin tests. Based on the diagnostic tests and the characteristics of
the autoantibodies, AIHA is divided into warm (wAIHA), cold (cAIHA), and
mixed forms. In addition, cold AIHA is subdivided into cold agglutinin
disease (CAD) and cold agglutinin syndrome (CAS), depending on the
etiopathogenesis. Paroxysmal cold hemoglobinuria (PCH) is a rare form of
autoimmune hemolytic anemia in which autoantibodies bind to erythrocytes
at low temperatures and activate the complement system at high
temperatures. With activation of the complement system and formation of
the membrane attack complex (MAC), hemolysis may be caused by
activation of the ADCC mechanism or phagocytosis of erythrocytes by
macrophages. Disorders of genes regulating the immune system, infection,
environment, drugs, and tumor formation are possible causes of AIHA.
Considering the heterogeneity of the disease and the different
immunopathogenic mechanisms of AIHA, therapies may differ widely. The
first line of treatment is glucocorticosteroids, followed by rituximab, but also
splenectomy. Immunosuppressive drugs, cyclosporine, azathioprine, and
cyclophosphamide, make up the third line of therapy. Clinical trials of new
therapies include erythrocyte phagocytosis inhibition (fostamatinib,
nipocalimab), Bruton tyrosine kinase inhibition (rilzabrutinib and ibrutinib),
proteasome inhibition (bortezomib), monoclonal antibodies to CD38
(daratumumab, isatuximab), mTOR inhibition (sirolimus), and complement
protein inhibition (eculizumab, pegcetacoplan, sutimlimab)
Olfactory changes and reorganization of brain neural circuitry following spinal cord injury
No full textOzljeda kralježnične moždine (OKM) traumatski je događaj s devastirajućim posljedicama za fizičko i psihičko stanje pacijenata. Sve je više dokaza kako OKM ne utječe samo na motorne funkcije, već i na osjetilne i autonomne funkcije te tako smanjuje kvalitetu života ljudi koji žive s ozljedom. OKM se povezuje i sa gubitkom osjeta mirisa i promjenama u moždanoj neuronskoj arhitekturi, stoga je bitno odrediti povezanost ozljede kralježnične moždine i olfaktorne funkcije. Prvi cilj ovog istraživanja bio je koristiti intranazalnu administraciju tracera aglutinin pšeničnih klica konjugiran peroksidazom hrena (WGA-HRP) kako bi se promatrali osjetilni neuroni olfaktornog trakta kroz olfaktorni epitelij do olfaktornog bulbusa u mozgu štakora. WGA-HRP signal uspoređivan je između zdravih štakora i štakora s OKM. Osim WGA-HRP tracera, promatrao se i intenzitet imunofluorescencije dekarboksilaze glutaminske kiseline (GAD67), neuronske jezgre (NeuN) i aktivacija mikroglije. Provedene su imunohistokemijske metode bojenja sagitalnih i koronarnih rezova olfaktornog bulbusa zdravih štakora i onih s OKM. Rezultati su na kraju analizirani pomoću fluorescentnog mikroskopa te se uspoređivao intenzitet fluorescencije WGA-HRP tracera, GAD67 i mikroglije između zdravih i štakora s OKM. Rezultati pokazuju kako postoje razlike u intenzitetu WGA- HRP signala, te je signal slabiji nakon ozljede kralježnične moždine. Također, postoje i razlike u GAD67 signalu, te je on jači nakon ozljede kralježnične moždine. Mikroglije su također emitirale više fluorescencije nakon OKM, te se njihova morfologija promijenila iz neaktivnog stanja u aktivno. Ovi podaci ukazuju na promjene u olfaktornoj arhitekturi nakon OKM. Prijenos živčanih podražaja od olfaktornog epitelija preko olfaktornog bulbusa do mozga je smanjen, a aktivacija mikroglija pokreće upalne procese lučenjem citokina u olfaktornom bulbusuSpinal cord injury (SCI) is a traumatic event with devastating consequences for the physical and mental condition of patients. There is increasing evidence that SCI affects not only motor functions, but also sensory and autonomic functions and thus reduces quality of life of people living with the injury. SCI is also associated with loss of smell and changes in the brain neural circuity, so it is important to determine the relationship between spinal cord injury and the olfactory function. The first goal of the study was to use intranasal administration of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) to trace sensory neurons of the olfactory tract from the nasal olfactory epithelium to the olfactory bulb in the rat brain. The WGA-HRP tracing was compared between healthy rats and rats with spinal cord injury. In addition to WGA-HRP tracing, the goal of the study was to examine the pattern and intesity of glutamic acid decarboxylase (GAD67) immunofluorescence, neural nuclei (NeuN) staining as well as activation of microglia. Immunohistochemical staining methods were performed on sagittal and coronal sections of the olfactory bulbs harvested from healthy rats and those with spinal cord injury. The results were finally analyzed using a fluorescent microscope. Fluorescence intensity of WGA-HRP tracer, GAD67 and microglia was compared between healthy and SCI rats. The data showed that there are differences in WGA- HRP signal intensity between SCI and control animals, with the signal being weaker after the injury. There are also differences in the GAD67 signal, with it being stronger following SCI. Microglia also emitted more fluorescence after SCI with their morphology shifted from inactive to an active state. These data indicate changes in olfactory architecture following SCI. The transmission of nerve stimuli from olfactory epithelium through the olfactory bulb to the brain is reduced, and microglial activation triggers inflammatory processes by secreting cytokines within the olfactory bulb
Immunopathogenesis of anaphylactic reaction
No full textAnafilaktičke reakcije spadaju u najozbiljniji oblik preosjetljivosti tipa I, a
karakterizirane su masovnom degranulacijom mastocita i sistemskim
otpuštanjem primarnih i sekundarnih medijatora poput histamina i
leukotriena, čije djelovanje može uzrokovati fatalan ishod u vidu
kardiovaskularnog ili respiratornog kolapsa. Okidači koji uzrokuju
anafilaktičku reakciju su razni i uključuju lijekove (uglavnom β-laktamske
antibiotike), hranu (npr. kikiriki), te otrov kukaca iz roda Hymenoptera. S
obzirom na etiologiju, anafilaksija može biti posredovana imunim
mehanizmima ili se definira kao anafilaktoidna reakcija. U slučaju
imunoloških uzroka, uglavnom je zastupljen alergijski oblik koji je ovisan o
IgE protutijelima, dok su rjeđe prisutni oblici posredovani IgG protutijelima
i aktivacijom sustava komplementa. S druge strane, anafilaktoidna reakcija
podrazumijeva nealergijsku etiologiju, čija je patogeneza slabije
razjašnjena. Neovisno o uzrocima, bolest se klinički može manifestirati u
više različitih fenotipova. Oni uključuju reakcije nalik tip I reakcijama,
reakcije nalik citokinskoj oluji, miješane reakcije te reakcije uzrokovane
komplementom. Anafilaktičku reakciju posreduju medijatori koji se dijele
na primarne i sekundarne. Primarni medijatori uključuju histamin, triptazu,
kimazu te proteoglikane, dok sekundarni uključuju čimbenik aktivacije
trombocita (PDGF), cistenil leukotriene (LTR), citokine, kemokine te faktore
rasta. Histamin je jedan od najvažnijih medijatora, a uzrokuje kliničke
simptome kao što su crvenilo kože, glavobolja, opstrukcija dišnih puteva,
hipotenzija i tahikardija putem H1 i H2 receptora. Većina drugih medijatora
uzrokuje slične simptome, i mehanizam njihovog djelovanja se također
intenzivno istražuje.
Samo liječenje anafilaktičih reakcija se najčešće izvodi administracijom
adrenalina, dok se za dugoročnu primjenu proučavaju humanizirana
monoklonalna protutijela koja se vezuju za IgE i/ili smanjuju izražaj
njegovog receptora, poput omalizumaba i ligelizumaba.Anaphylactic reaction is the most severe form of type I hypersensitivity
reaction. Anaphylaxis is characterized by massive degranulation of mast
cells and systemic release of primary and secondary mediators such as
histamine and leukotrienes, the effects of which can cause a fatal outcome
in the form of cardiovascular or respiratory collapse. The triggers of
anaphylactic reaction are diverse and include drugs (especially β-lactam
antibiotics), foods (e.g., peanuts), and venom from Hymenoptera insects.
In terms of etiology, anaphylaxis can be mediated by immune mechanisms,
or it can be defined as an anaphylactoid reaction. Among immunologic
causes, the allergic form, which depends on IgE antibodies, is most
prevalent, whereas forms mediated by IgG antibodies and activation of the
complement system are less common. An anaphylactoid reaction, on the
other hand, indicates a nonallergic etiology, the pathogenesis of which is
less clear. Regardless of the cause, the disease can manifest clinically in
several different phenotypes. These include Type I-like reactions, cytokine
storm-like reactions, mixed reactions, and complement-mediated reactions.
The mediators that play an important role in triggering anaphylactic
reactions are divided into primary and secondary mediators. Primary
mediators include histamine, tryptase, chymase, and proteoglycans,
whereas secondary mediators include platelet-activating factor, cysteinyl
leukotrienes, cytokines, chemokines, and growth factors. Histamine causes
clinical symptoms such as flushing, headache, airway obstruction,
hypotension, and tachycardia via H1 and H2 receptors. Most other primary
mediators cause similar symptoms, whereas secondary mediators primarily
cause an increase in endothelial permeability, vasodilation, and, in the case
of platelet-activating factors, cardiovascular collapse. Treatment of
anaphylactic reactions is usually by administration of epinephrine, while
humanized monoclonal antibodies such as omalizumab and ligelizumab,
based on the principle of binding to IgE or attenuating IgE receptor
expression, are being investigated for long-term use
Kritički pregled zdravstvenih prednosti konzumiranja kombuche
No full textKombucha, a fermented beverage originating from the Manchuria region in
Northeast Asia over 2000 years ago, is produced by fermenting sweetened tea
with a symbiotic consortium of bacteria and yeasts known as SCOBY. The
resultant kombucha tea is rich in organic acids, polyphenols, sugars, vitamins,
minerals, etc. Organic acids, such as acetic, gluconic, glucuronic and lactic
acids, contribute to the beverage’s acidity and purported health properties.
The beverage's antioxidant, antimicrobial, anticancer, hepatoprotective and
anti - inflammatory effects have been documented, backed by in vitro and in
vivo studies on rodents. Phenolic compounds, vitamins and microbial enzymes
are thought to contribute to these effects. Kombucha's microorganisms
include yeasts and bacteria (acetic and lactic acid bacteria). However,
potential health risks like excessive acidity, lead contamination and
contraindications for certain individuals may exist. The potential benefits of
kombucha's various organic compounds, though promising, require further
research and clinical trials for confirmation.Kombucha je fermentirano piće koje potječe iz Kine prije više od 2000 godina.
Proizvodi se fermentacijom zaslađenog čaja uz SCOBY. Rezultirajući čaj od
kombuche bogat je organskim kiselinama, polifenolima, šećerima,
vitaminima, mineralima, itd. Organske kiseline doprinose njegovoj kiselosti i
zdravstvenim svojstvima. Dokazani su antioksidativni, antimikrobni,
antikancerogeni, hepatoprotektivni i protuupalni učinci ovog pića, poduprti in
vitro i in vivo istraživanjima na glodavcima. Polifenoli, vitamini i ostali spojevi
doprinose tim učincima. Mikroorganizmi prisutni u kombuchi uključuju kvasce
i bakterije. Međutim, postoje rizici kao što su pretjerana kiselost, trovanje
olovom i kontraindikacije za određene rizične osobe. Daljnja istraživanja u
svrhu testiranja svojstava kombuche na ljudsko zdravlje su potrebna
Reprogramming of skin homeostasis and regeneration using young cells micro-transplatation
No full textOčekivani životni vijek drastično se povećao tijekom posljednjih nekoliko desetljeća diljem svijeta,
pri čemu je povećana pojava brojnih kroničnih bolesti. Starenje je složen proces koji uključuje
progresivno smanjenje funkcije tkiva i gubitak regenerativnog kapaciteta. U osnovi toga je stanična
senescencija, stanje stabilnog zastoja staničnog ciklusa koje se javlja kao odgovor na oštećenje i
akumulaciju staničnog stresa. Starenjem se povečava učestalost nastanka senescentnih stanica, te se
stoga posljednjih godina razvijaju različite strategije njihove inhibicije i eliminacije. Ovim
istraživanjem sam htjela pokazati da će promjenom omjera mladih i starih stanica u koži štakora
Fischer 344 doći do promjene tkivnog mikrookoliša i smanjenja efekta sekretornog fenotipa
senescentnih stanica (engl. Senescence-associated secretory phenotype, SASP) na okolne stanice i
tkiva. Dobiveni rezultati pokazuju kako mikrotransplatacija mladih stanica u kožu štakora pozitivno
utječe na revitalizaciju niše matičnih stanica i povećanje ukupnog broja stanica u tkivu te sam ujedno
pokazala dugotrajnost tog efekta. Pokazana je i promjena u genskoj ekspresiji u tkivu kože sa
starenjem, te promjena profila ekspresije nakon tretmana. Zaključno, pokazala sam da sam uspješno
smanjila sekretorni fenotip senescentnih stanica, promijenila homeostazu starog tkiva i uspješno
regenerirala kožu.Life expectancy has increased dramatically over the past few decades worldwide, with an increase in
the occurrence of numerous chronic diseases. Aging is a complex process that involves a progressive
decrease in tissue function and loss of regenerative capacity, underlined by cellular senescence, a
stable state of cell cycle arrest that occurs in response to damage and accumulation of cellular stress.
Aging increases the frequency of occurrence of senescent cells, and for this reason, various anti-aging
strategies have been developed in recent years. In this work, I wanted to show that by changing the
ratio of young vs. old cells in the skin of Fischer 344 rats, we can change the tissue microenvironment
and a reduce the effect of senescence-associated secretory phenotype (SASP). The obtained results
show that the micro-transplantation of young cells in the skin of rats has a positive effect on the
revitalization of the niche of stem cells and an increase in the total number of cells in the tissue, and
at the same time I have demonstrated that this effect persist for longer period of time. Changes in
gene expression in skin tissue with aging, or changes as a result of treatment, were also shown. In
conclusion, I have shown that I successfully reduced the secretory phenotype of senescent cells,
changed the homeostasis of old tissue and successfully regenerated the skin