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Sphingolipids as biomarkers in the cancer diagnosis and therapy
No full textSfingolipidi predstavljaju važne komponente membrana eukariotskih stanica, stoga imaju ulogu u regulaciji brojnih staničnih signalnih puteva. U ovom završnom radu cilj je utvrditi koji bioaktivni sfingolipidi i njihovi metabolički enzimi mogu služiti kao potencijalni dijagnostički, prognostički i prediktivni biomarkeri u nekoliko tipova raka, uključujući rak jetre, debelog crijeva i dojke. Biomarkeri su po definiciji objektivne, kvantificirajuće karakteristike bioloških procesa stoga su važni za pravovremeno otkrivanje, dijagnozu, prevenciju te uspješno liječenje bolesti. Brojne su studije pokazale značajnu povezanost između metabolizma sfingolipida i kliničkog ishoda bolesti. Takvi rezultati daju veliku važnost sfingolipidima kao biomarkera u dijagnozi i prognozi raka. Kako bi se uspješno otkrila složena patofiziologija uključena u rak te put genetskih promjena stanica raka, potreban je širok raspon različitih sfingolipidnih biomarkera za poboljšanje osjetljivosti i specifičnosti biomarkera potrebne za istraživanje. Pri tome se naglasak stavlja na pro-apoptotički ceramid, pro-proliferitativni sfingozin, ceramid-1-fosfat (C1P), sfingozin-1-fosfat (S1P) te na brojne enzime koji sudjeluju u metabolizmu sfingolipida. Stoga će poremećaj metabolizma i sfingolipidnog reostata u organizmu dovesti do gubitka optimalne razine sfingolipida što upućuje na razvoj kancerogenog stanja.Sphingolipids are important components of eukaryotic cell membranes that have a role in regulating numerous cellular signals. In this final assignment, the aim is to determine which bioactive sphingolipids and their metabolic enzymes can be potential diagnostic, prognostic, and predictive biomarkers in several types of cancer, including liver, colon, and breast cancer. Biomarkers are by definition objective, quantifiable characteristics of biological processes and therefore important for early detection, diagnosis, prevention, and successful treatment of the disease. Numerous studies show a significant association between sphingolipid metabolism and clinical disease outcomes. Such results give big importance to sphingolipids as biomarkers in the diagnosis and prognosis of cancer. Successful determination of complex pathophysiology involved in cancer and genetic change in cancer cells, demands the use of panel of different sphingolipid biomarkers to improve sensitivity and specificity of biomarkers needed for research. Emphasis is on the use of pro-apoptotic ceramide, pro-proliferative sphingosine, ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and numerous enzymes involved in the metabolism of sphingolipids. Therefore, disruption of metabolism and sphingolipid rheostat in the organism will lead to loss of optimal sphingolipid levels leading to the development of a cancerous condition
Proteini ADAR u bolestima ljudi
No full textAdenosine deaminase acting on RNA (ADAR) is a mammalian protein family,
consisting of five enzymes out of which ADAR1 and ADAR2 possess catalytic activity. Their mechanism of action, called A-to-I (adenosine-to-inosine) editing, is a post-transcriptional modification on double-stranded RNA (dsRNA) molecules and occurs in both nucleus and cytoplasm of various human cell. As one of the most abundant post-transcriptional modifications on nucleic acids, A-to-I editing is associated with various pathologic conditions throughout the human body. Because of their effect on dsRNA and subsequent pathogen recognition, ADAR proteins have a pivotal role in biochemical mechanisms of interferon (IFN) induction and regulation of the innate immune response. However, the central role of ADAR proteins in immunological mechanisms means that dysregulation of their activity, be it increase or decrease, can cause some significant disruptions in the human body leading to autoimmune reaction or immunosuppression. The link between ADAR induced A-to-I editing and several cancer types is clearly present. However, in different tumors ADAR has a different role. It can act as a tumour suppressor gene through neoantigen formation or stimulate tumor development as an oncogene. Site-specific editing events in the central nervous system (CNS) are essential for the creation of glutamate and serotonin receptor subunits that make receptors impermeable to calcium. Editing in the CNS facilitates synaptic scaling and plasticity indicating that neural death by excitotoxicity is an evolutionary adaptation in case of ADAR2 failure. Moreover, dysregulation of ADAR activity is present in the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and mental illnesses such as depression, bipolar disorder, schizophrenia and addiction. In a viral infection, depending on the cellular context and virus type, A-to-I editing can act both in a proviral and an antiviral manner. ADAR proteins can influence the course of viral infection in an editing-dependent or editing-independent form. In this review, we use the Epstein-Barr virus (EBV) and Hepatitis C virus (HCV) as exemplary cases to underline the essential role of ADAR in the viral life cycle.Adenozin deaminaza koja djeluje na RNA (ADAR) je porodica proteina sisavaca koja se sastoji od pet enzima od kojih ADAR1 i ADAR2 imaju sposobnost katalitičke aktivnosti. Mehanizam njihovog djelovanja, nazvan A-to-I editing, post-transkripcijska je modifikacija na dvolančanim molekulama RNA (dsRNA) koja se javlja se u jezgri i citoplazmi različitih ljudskih staničnih linija. Kao jedna od najzastupljenijih genetskih modifikacija, A-to-I editing povezan je s različitim patologijama u čitavom ljudskom tijelu. Zbog svog učinka na dsRNA i naknadnog prepoznavanja patogena, ADAR proteini imaju ključnu ulogu u biokemijskim mehanizmima indukcije interferona (IFN) i regulacije urođenog imunološkog odgovora. Međutim, središnja uloga ADAR proteina u imunološkim mehanizmima znači da poremećaj regulacije njihove aktivnosti, bilo to povećanje ili smanjenje, može uzrokovati značajne poremećaje u ljudskom tijelu, odnosno autoimune reakcije ili imunosupresije. Očita je veza između A-to-I editinga induciranog ADAR-om i nekoliko vrsta tumora. Međutim, u različitim tumorima ADAR ima različitu ulogu. Može djelovati tumor supresor gen i stvarati neoantigene ili poticati razvoj tumora kao onkogen. Editing događaji koji su specifični za mjesto središnjem živčanom sustavu (CNS) presudni su za stvaranje podjedinica glutamatnih i serotoninskih receptora koji čine cijele receptore nepropusne za kalcij. Uređivanje u CNS-u olakšava sinaptičko skaliranje, a plastičnost ukazuje da je neuronska smrt ekscitotoksičnošću evolucijska prilagodba u slučaju neuspjeha editinga proteinom ADAR2. Štoviše, poremećaj regulacije ADAR aktivnosti prisutan je kod neurodegenerativne bolesti amiotrofične lateralne skleroze (ALS) te mentalnih bolesti poput depresije, bipolarnog poremećaja, shizofrenije i ovisnosti. U virusnoj infekciji, ovisno o staničnom kontekstu i vrsti virusa, A-to-I editing može djelovati provirusno i antivirusno. ADAR proteini mogu utjecati na tijek virusne infekcije ovisno o uređivanju ili neovisno o uređivanju. U ovom pregledu koristimo Epstein-Baar virus (EBV) i hepatitisa C virus (HCV) kao ogledne slučajeve kako bismo razjasnili ulogu ADAR-a u životnom ciklusu virusa
Analysis of Cornus mas antioxidants
No full textDobro je poznato da biljni fenoli imaju širok raspon pozitivnog djelovanja, ali je manje poznato da su plodovi i lišće hrvatskih drijenova bogati fenolnim spojevima. Određena istraživanja su već pokazala da ekstrakti plodova i ostalih biljnih dijelova drijena imaju snažno antioksidativno, kardioprotektivno, neuroprotektivno i antikancerogeno djelovanje. Zbog toga je cilj ovog rada analizirati plodove, lišće i pulpu drijena te prehrambene prerađevine od drijena poput džemova i sirupa kao pogodnih izvora polifenola za svakodnevnu primjenu.
Uzorci drijena i njegovih prerađevina su prikupljeni sa šireg područja grada Rijeke i u Primorsko-goranskoj županiji. Ekstrakcija biljnih fenola iz biljnih uzoraka drijena i njegovih prerađevina se odvila u otopini metanola. Za određivanje ukupnog udjela fenola korišten je Folin-Ciocalteu test, dok je za određivanje antioksidacijske aktivnosti primijenjena DPPH (2,2-difenil-1-pikrilhidrazil) metoda. Količina fenolnih spojeva i antioksidacijska aktivnost je najveća u uzorcima lišća, dok je ukupni fenolni sastav i antioksidacijska aktivnost najmanja u prehrambenim prerađevinama od drijena.
Razlike u rezultatima između pojedinih uzoraka se mogu pripisati razini zrelosti plodova i lišća, geografskom položaju nasada drijena, vremenu prikupljanja uzoraka i sl.
Analiza polifenolnog sadržaja metanolnog ekstrakta plodova drijena provedena je na spregnutom masenom spektrometru s hibridnom analizatorom (LC-Q-TOF). Usporedbom spektara sa bazama podataka identificirani su tri antocijana (cijanidin 3-glukozid, delfinidin 3-galaktozid i cijanidin 3-O-rutinozid), dva antocijanidina (pelargonidin i cijanidin) i pet fenolnih kiselina (vinska kiselina, jabučna kiselina, kvininska kiselina, galna kiselina i klorogena kiselina).
Ovim radom su dobiveni prvi podaci o ukupnom fenolnom sastavu hrvatskih drijenova te kvantitativni sastav antocijanidina i fenolnih kiselina.It is well known that the plant phenols have a wide range of positive effects, but it is less known that fruits and leaves of Croatian cornelian cherry are rich in phenolic compounds. Certain studies have already proven strong antioxidant, cardioprotective, neuroprotective and anticancer effects of cornelian cherry fruits and other plant parts extracts. Therefore, the aim of this paper was to analyse fruits, leaves and pulp of the Croatian cornelian cherry and food products from cornelian cherry such as jams and syrups as suitable sources of polyphenols for everyday use.
Samples of dogwood and its products were collected from the wider area of the city of Rijeka and on the area of the Primorsko-goranska County. Extraction of the plant phenols dogwood samples and corresponding products were done by use of methanol solution. The Folin-Ciocalteu test was used to determine the total phenol content, while antioxidant activity was evaluated by the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The amount of phenolic compounds and antioxidant activity was highest in leaf samples, while the total phenolic composition and antioxidant activity was lowest in the cornelian cherry food products. Differences in the results between individual samples can be attributed to the level of fruit and leaf maturity, geographical location of cornelian cherry plantations and time of sample collection.
Analysis of the cornelian cherry fruit methanol extracts polyphenolic content was performed by use of a coupled mass spectrometer with a hybrid analyzer (LC-Q-TOF). By comparison of the obtained spectra with the databases, three anthocyanins (cyanidin 3-glucoside, delfinidine 3-galactoside and cyanidin 3-O-rutinoside), two anthocyanidins (pelargonidine and cyanidin) and five phenolic acids (tartaric acid, malic acid, quinic acid, gallic acid and chlorogenic acid) were identified.
This paper provides data on the Croatian cornelian cherry total phenolic composition and the corresponding quantitative composition of anthocyanidins and phenolic acids
Nanonosači za fotosenzibilizatore
No full textPosljednjih nekoliko desetljeća fotodinamička terapija (eng. photodynamic therapy, skraćeno PDT) razvila se kao obećavajući novi tretman u liječenju karcinoma i rješenje u prevladavanju antimikrobne rezistencije. PDT uključuje interakciju pobuđenog fotosenzibilizatora (eng. photosensitizer, skraćeno PS) PS* s molekulskim kisikom ili biološkim supstratom, pri čemu se stvaraju citotoksične reaktivne kisikove vrste (eng. reactive oxygen species, skraćeno ROS). Iako uspješnost PDT-a ovisi o brojnim faktorima vezanima za PS, svjetlo i kisik, ovaj rad usredotočen je na još uvijek aktualne nedostatke PS-a 3. generacije: topljivost u vodi i selektivnost. Budući da se modifikacija PS-a i inkapsulacija u tradicionalne nosače nisu pokazali konačnim rješenjem, znanstvenici su se usredotočili na istraživanje novih tipova nosača. Smatra se kako bi nanonosači mogli riješiti navedene nedostatke PS-a koji, između ostalih, zasad koče potencijalnu široku kliničku primjenu PDT-a. U ovom radu istražene su najvažnije karakteristike različitih tipova biorazgradivih i bionerazgradivih nanonosača i način na koji mogu utjecati na poboljšanje topljivosti i selektivnosti PS-a. Istaknuti su najnoviji napreci u formulaciji svakog tipa i mogućnost njihove primjene u tretmanu karcinoma i rješavanju problema antimikrobne rezistencije.Over the last few decades, photodynamic therapy (PDT) has emerged as a promising new cancer treatment and a solution to overcoming antimicrobial resistance. PDT involves the interaction of excited photosensitizer (PS*) with molecular oxygen or biological substrate, producing cytotoxic reactive oxygen species (ROS). Although PDT's efficacy depends on numerous factors related to PS, light and oxygen, this paper focuses on the ongoing drawbacks of 3rd generation PSs: water solubility and selectivity. Since the modification of PS and the incapsulation into traditional carriers did not prove to be a definitive solution, scientists focused on exploring new types of carriers. It is believed that nanoparticles can solve the aforementioned deficiencies of PS, which, among other things, currently block the potential wide clinical application of PDT. In this paper, the most important characteristics of different types of biodegradable and non-biodegradable nanocarriers are explored and how they can influence the PS solubility and selectivity enhancement. The most recent improvements of the formulation of each type and the possibility of their use in the treatment of cancer and the resolution of antimicrobial resistance are highlighted
Synthesis of 4H-thieno[3,2-c]chromen-4-one derivatives and evaluation of their anti-inflammatory activity on pSTAT1 and pSTAT5 isolated from peripheral blood mononuclear cells
No full textovom radu opisana je sinteza tricikličkih angularnih spojeva, 4H-tieno[3,2-c]kromen-4-ona (7-8, 9a-e) i derivata (5-(2-hidroksi-4-metoksifenil) tiofen-2,4-di-il)dimetanola (5, 6a-d).
Ispitana je protuupalna učinkovitost spojeva 4, 7, 8 i 9a-e in vitro, mjerenjem učinkovitosti inhibicije različitih fosfo-STAT proteina (engl. Signal Transducer and Activatior of Transcription) koji su dio JAK (engl. Janus kinase)/STAT signalnog puta. Spoj 7 (etil 7-hidroksi-4-okso-4H-tieno[3,2-c]kromen-2-karboksilat) pokazao je najbolja inhibicijska svojstva sa vrijednostima IC50 5,0 μM (pSTAT5/GM-CSF/PBMC); IC50 49,5 μM (pSTAT5/IL-2/PBMC) i IC50 10,50 μM (pSTAT1/IFN-/PBMC).Synthesis of tricyclic angular compounds, namely 4H-thieno[3,2-c]chromen-4-one (7-8, 9a-e) and (5-(2-hydroxy-4-methoxyphenyl) thiophene-2,4-diyl)dimethanol (5, 6a-d) derivatives, is described.
Compounds 4, 7, 8 and 9a-e were tested in vitro for their anti-inflammatory activity, by measuring various pSTAT (signal transducer and activator of transcription) inhibition within the JAK (Janus-activated family kinase)/STAT signaling pathway. Compound 7 (ethyl 7-hydroxy-4-oxo-4H-thieno[3,2-c]chromene-2-carboxylate) showed best inhibition properties on inhibition of pSTAT5 in GM-CSF-triggered assay, with IC50 value of 5.0 μM. Inhibition of pSTAT5 in IL-2-triggered assay showed IC50 value of 49.5 μM, while inhibition of pSTAT1 in IFN--triggered assay showed IC50 value of 10.5 μM
Tripyridylporphyrin in photodynamic inactivation of biofilm
No full textLegionella pneumophila Gram-negativna je bakterija koja spada u porodicu Legionellaceae. Uzročnik je pontijačke groznice i legionarske bolesti. Obitava u vodenom okruženju kao što su jezera, rijeke, ali i umjetno stvoreni vodeni sustavi kao što su sustavi s visokim temperaturama vode, sustavi rashladne vode, spa i wellness centri, hoteli, bolnice, vodeni sustavi u stomatološkim ordinacijama. U takvom okruženju stvaraju se biofilmovi koje nastanjuje L. pneumophila i u njima se razmnožava. Biofilmovi su otporni na dezinfekcijska sredstva i termičku obradu vode što otežava eradikaciju legionele. Zbog toga potrebno je razviti nove metode dezinfekcije voda, a jedna od tih metoda je fotodinamička inaktivacija. Fotodinamička inaktivacija temelji se na upotrebi fotosenzibilizatora, u ovom radu 5-(4-oktadec-9-enamidofenil)-10,15,20-tris(3-piridil)porfirina (TMPyP3-C17H33), u aerobnim uvjetima. Izlaganje amfifilnog porfirina izvoru svjetlosti određene valne duljine rezultira stvaranjem singletnog kisika (1O2) i/ili ostalih kisikovih reaktivnih vrsta (ROS) koji dovode do oksidacije i stanične smrti patogena. Cilj ovoga istraživanja bio je ispitati učinkovitost fotodinamičke inaktivacije amfifilnog porfirina na razaranje 6-dnevnog biofilma Legionella pneumophila soj 130b. Korištena je sterilna vodovodna voda, a kao izvor svjetlosti korištena je ljubičasta svjetlost intenziteta 40 mW/cm2. Koncentracije amfifilnog porfirina od 0,176 μM, 0,351 μM, 0,702 μM i 1,404 μM su dovele do uništavanja biofilma L. pneumophila u vodovodnoj vodi. Istraživanjem je dokazano da spoj učinkovito razara bakterijski biofilm pa bi se metoda mogla primijeniti u praksi.Legionella pneumophila is a Gram-negative bacteria of the family Legionellaceae. It is a causative agent of Pontiac fever and Legionnaires' disease. It resides in water environment like rivers, lakes as well as in artificially created water systems like high temperature systems, cooling water systems, spa and wellness centres, hotels, hospitals, water systems in dental offices. In such environment biofilms are formed and inhabited by L. pneumophila that multiplies within them. Biofilms are resistant to disinfectants and thermal processing what makes eradication of Legionella more difficult. Consequently, new methods for water disinfection need to be developed and photodynamic inactivation is one of those methods. Photodynamic inactivation is based on usage of photosensitizer, in this case 5-(4-octadec-9-enamidophenyl)-10,15,20-tris(3-pyridyl)porphyrin (TMPyP3-C17H33) in aerobic conditions. Exposure of amphiphilic porphyrin to light of specific wavelength results in generation of singlet oxygen (1O2) and/or other reactive oxygen species (ROS) that lead to oxidation and cell death of pathogen. Focus of this research was determining effectiveness of photodynamic inactivation of amphiphilic porphyrin on destruction of 6 day-old biofilm of Legionella pneumophila strain 130b. Sterile tap water and violet light with intensity of 40 mW/cm2 were used. Concentration of amphiphilic porphyrin of 0.176 μM, 0.351 μM, 0.702 μM and 1.404 μM led to destruction of Legionella pneumophila biofilm in tap water. This research demonstrated that compound effectively destroys bacterial biofilm therefore this method could be put into practice
The TRIOBP Isoforms and Their Distinct Roles in Actin Stabilization, Deafness, Mental Illness, and Cancer
No full textThe TRIOBP (TRIO and F-actin Binding Protein) gene encodes multiple proteins, which together play crucial roles in modulating the assembly of the actin cytoskeleton. Splicing of the TRIOBP gene is complex, with the two most studied TRIOBP protein isoforms sharing no overlapping amino acid sequence with each other. TRIOBP-1 (also known as TARAor TAP68) is a mainly structured protein that is ubiquitously expressed and binds to F-actin, preventing its depolymerization. It has been shown to be important for many processes including in the cell cycle, adhesion junctions, and neuronal dierentiation. TRIOBP-1 has been implicated in schizophrenia through the formation of protein aggregates in the brain. In contrast, TRIOBP-4 is an entirely disordered protein with a highly specialized expression pattern. It is known to be crucial for the bundling of actin in the stereocilia of the inner ear, with mutations in it causing severe or profound hearing loss. Both of these isoforms are implicated in cancer. Additional longer isoforms of TRIOBP exist, which overlap with both TRIOBP-1 and 4. These appear to participate in the functions of both shorter isoforms, while also possessing unique functions in the inner ear. In this review, the structures and functions of all of these isoforms are discussed, with a view to understanding how they operate, both alone and in combination, to modulate actin and their consequences for human illness
Adverse drug reactions of medicinal products belonging to ATC R03 group – Drugs for obstructive airway diseases
No full textAstma je kronična upalna bolest dišnih putova koju karakteriziraju napadaji kašlja te otežano i čujno disanje. U upalnom procesu sudjeluju razne stanice, uključujući mastocite, eozinofile i T limfocite, a astma je idealan primjer interakcije gena i okoliša. Dijagnoza bolesti se provodi prateći već postojeće smjernice u kojima su opisani prepoznatljivi obrasci respiratornih simptoma. Prema funkciji, lijekovi za liječenje astme se dijele u kontrolne lijekove, lijekove za hitno ublažavanje simptoma i dodatnu terapiju za pacijente s teškom astmom, a prema načinu djelovanja na inhalacijske kortikosteroide, β2-agoniste, antagoniste leukotrienskih receptora, antikolinergike i makrolidne antibiotike. U ovom radu napravljena je analiza prijava sumnji na nuspojave antiastmatika za razdoblje 1.1.1993. – 15.3.2020., dobivenih od HALMED-a. Prema ATK klasifikaciji antiastmatici spadaju u skupinu R – Pripravci koji djeluju na respiratorni sustav, odnosno u podskupinu R03 – Pripravci za liječenje opstruktivnih bolesti dišnih putova. Ukupno je analizirano 1035 prijava, a analizirane su prema životnoj dobi i spolu pacijenata, prema djelatnoj tvari lijeka, klasifikaciji organskih sustava (SOC), najčešće prijavljenim nuspojavama (PT), ozbiljnosti i godinama. Najviše prijava sumnji na nuspojave zaprimljeno je za pacijente životne dobi između 45 do 64 godina (23.9%) i pacijente ženskog spola (50.8%). Najviše prijava sumnji na nuspojave zabilježeno je za lijekove s djelatnim tvarima salbutamol (11.8%), montelukast (11.7%) i deksametazon (10.6%), a najveći broj nuspojava zabilježen je za organski sustav Opći poremećaji i reakcije na mjestu primjene (25.4%). Najčešće prijavljena nuspojava je kašalj. Većina prijava nije ocijenjena ozbiljnima, a u 17 slučajeva došlo je do smrti pacijenta. Najviše slučajeva klasificirano je ozbiljnima prema kriteriju ostala medicinski značajna stanja. Najveći broj prijava sumnji na nuspojave zaprimljen je 2019. godine. Kontinuirana edukacija zdravstvenih radnika i pacijenata/korisnika lijekova o važnosti prijavljivanja sumnji na nuspojave te posljedično, kontinuirano prijavljivanje sumnji na nuspojave pridonosi ocjeni sigurnosnog profila lijekova čime se osigurava da se u prometu nalaze samo oni lijekovi čiji je omjer koristi i rizika primjene pozitivan.Asthma is a chronic inflammatory respiratory disease characterized by coughing episodes, difficult breathing, and wheezing. Various cells participate in the inflammatory process, including mastocytes, eosinophils and T lymphocytes, and asthma is an ideal example of gene-environment interaction. Diagnosis of the disease is carried out following pre-existing guidelines which describe recognizable patterns of respiratory symptoms. According to the function, drugs for the treatment of asthma are divided into control drugs, relievers and additional therapy for patients with severe asthma, and according to the mechanism of action on ICS, β2-agonists, leukotriene receptor antagonists, anticholinergics and macrolide antibiotics. This paper has analysed reports of suspected adverse drug reactions of antiasthmatics from 1.1.1993. to 15.3.2020., obtained from HALMED. According to the ATC classification, antiasthmatics belong to a group R – Respiratory system, i.e. in subgroup R03. A total of 1035 reports were analysed by age and gender of patients, by active substance, system organ class (SOC), most commonly reported adverse drug reactions (PT), seriousness and years. The highest number of adverse drug reaction reports was received for patients aged between 45 and 64 years (23.9%) and for female patients (50.8%). The highest number of adverse drug reaction reports was received for salbutamol (11.8%), montelukast (11.7%) and dexamethasone (10.6%), and the highest number of adverse drug reactions was reported for system organ class General disorders and application site reactions (25.4%). The most commonly reported adverse drug reaction is cough. Most of the adverse drug reaction reports were not assessed as serious, and in the 17 cases the outcome was the patient’s death. Most of the cases are classified as serious according to the criteria “other medically important conditions”. The highest number of adverse drug reaction reports was received in 2019. Continuing education of the healthcare professionals and the patients about the importance of adverse drug reaction reporting and consequently, continuing adverse drug reaction reporting contributes to the evaluation of safety profile of the medicinal products, which ensures that only medicinal products with positive benefit-risk ratio are marketed
Optimizacija protokola za mjerenje ROS-a kod Drosophila melanogaster
No full textOxidative stress is an important concept in biology and medicine and refers to a condition where the levels of reactive oxygen species (ROS) significantly overwhelm the capacity of antioxidant defenses, leading to potential damage to the body. ROS levels fluctuate during a 24-hour cycle due to internal circadian rhythms. ROS molecules play a key role in the organism, as they take part in signaling pathways in the cell, but could also trigger the onset of different pathophysiological conditions. For this reason, it is important to have a method that is able to accurately measure ROS concentrations. In this thesis we tested two commonly used dyes: dihydroethidium (DHE) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), in different buffers and pH levels. We found that DHE is a more sensitive dye for measuring H2O2 concentration in Drosophila melanogaster (D. melanogaster) samples. H2DCF’s inadequacy was explained by the lack of Fe2+/Fe3+ needed for the reaction to occur, since H2DCF-DA does not directly react with H2O2. DHE is both very stable and able to directly react with H2O2. These experiments also showed how there was a significantly higher H2O2 concentration in the head than in the whole body extracts, but we did not notice a difference between samples collected in the morning and evening. The reason for higher ROS level in the head extracts is due to high levels of oxidizable molecules and fairly low levels of antioxidant defense compared to the rest of the body. Our experiments will contribute to a better understanding of the regulation of ROS concentration, both as a function of the time of day and as a function of different environmental stressors or pathologies.Oksidativni stres je važan pojam u biologiji i medicini, a odnosi se na stanje u kojem razine ROS-a značajno nadmašuju sposobnost antioksidativne obrane što dovodi do potencijalnog oštećenja tijela. Razine ROS-a osciliraju tijekom 24-satnog ciklusa zbog unutarnjih cirkadijalnih ritmova. ROS molekule igraju ključnu ulogu u organizmu jer sudjeluju u signalnim putovima u stanici, ali također mogu pokrenuti nastanak različitih patofizioloških stanja. Stoga je važno imati metodu koja može precizno mjeriti koncentracije ROS-a. U ovom radu testirali smo dvije najčešće korištene boje: dihidroetidiij (DHE) i 2',7'-diklorodihidrofluorescin diacetat (H2DCF-DA), u različitim puferima i pH razinama. Otkrili smo da je DHE osjetljivija boja za mjerenje koncentracije H2O2 u uzorcima Drosophila melanogaster (D. melanogaster). Neadekvatnost H2DCF-DA objašnjena je nedostatkom Fe2+/Fe3+ potrebnih za reakciju jer H2DCF-DA ne reagira izravno s H2O2. DHE je vrlo stabilna i sposobna reagirati izravno s H2O2. Ovi su eksperimenti također pokazali kako je u ekstraktima glave bila znatno veća koncentracija H2O2 nego u ekstraktima cijelog tijela, ali nismo primijetili razliku između uzoraka prikupljenih ujutro i navečer. Razlog za višu razinu ROS-a u glavi su visoke razine molekula koje se mogu oksidirati te prilično niske razine antioksidansa u odnosu na ostatak tijela. Naši će eksperimenti će pridonijeti boljem razumijevanju regulacije koncentracije ROS-a kao funkcije doba dana i različitih faktora koji uzrokuju stres iz okoline ili patologija
Characterization of newly produced antibody against CD63 protein in mammals
No full textCD63 je membranski glikoprotein koji pripada superobitelji tetraspanina. Molekularna masa CD63 iznosi 25 kDa, ali može doseći i do 60 kDa zbog različitih stupnjeva glikozilacije ovog proteina. Prisutan je u svim ljudskim stanicama, te ima mnogo potvrđenih i još više ne potvrđenih funkcija. CD63 se u stanici nalazi u endosomalnom sustavu, egzosomima i na površini stanice. Uključen je u signalnu transdukciju koja regulira razvitak, aktivaciju, rast i pokretljivost stanice. Promovira preživljenje stanice, reorganizaciju citoskeleta, adheziju, migraciju, te je uključen u transport proteina. Jako je eksprimiran u ranim fazama raznih tumora, a u kasnijim fazama ga gotovo uopće nema. Ima ulogu i u nekim drugim patološkim stanjima, te bi mogao imati izvanrednu dijagnostičku, prognostičku i terapijsku vrijednost i služiti kao marker za stadije tumora.
Cilj ovog rada je optimizacija imunofluorescencije i karakterizacija novoproizvedenog anti-CD63 protutijela (hCD63.01).
Provedeno je više pokusa imunofluorescencije na HeLa, SW620 i MCF-7 staničnim linijama. Mijenjane su koncentracije sredstva za permeabilizaciju i sredstva za blokiranje, te i samo sredstvo za permeabilizaciju. Za usporedbu je korišteno MEM-259 anti-CD63 protutijelo. Kako bi odredili je li novoproizvedeno protutijelo prošlo kroz promjene tijekom pročišćavanja, provedena je imunofluorescencija sa nepročišćenim supernatantom hCD63.01, te su rezultati uspoređeni sa onima pročišćenog protutijela hCD63.01.
Utvrđeno je da hCD63.01 protutijelo nije značajno promijenjeno tijekom pročišćavanja. Predložen je optimizirani protokol imunofluorescencije sa Tween-om 20 kao sredstvom permeabilizacije. hCD63.01 je u svim pokusima dalo jači signal od MEM-259 komercijalno dostupnog protutijela za CD63., te je moguće da hCD63.01 prepoznaje više formi ovog proteina. Dodatna istraživanja su potrebna za potpunu karakterizaciju i razumijevanje potencijala hCD63.01 novoproizvedenog protutijela.CD63 is a membrane glycoprotein which belongs to a superfamily of tetraspanins. Its molecular weight is 25 kDa but can reach up to 60 kDa depending on the level of glycosylation the protein went through. It is present in all human cell types and has lots of different functions some of which are known and some of which have yet to be proven. CD63 can be found in the cell endosomal system, egzosomes and on the cell surface. It is involved in signal transduction which regulates cell development, activation, growth and motility. It promotes cell survival, reorganization of actin cytoskeleton, adhesion, cell migration and is included in protein transport. It is highly expressed in early stages but almost nonexistent in the later stages of most tumors and has a role in some other pathological states. CD63 could have an excellent diagnostic, prognostic and therapeutical value for different pathological conditions and be used as a stage marker for various tumors.
Aim of this paper is the optimization of immunofluorescence and characterization of a novel anti-CD63 antibody (hCD63.01).
Several immunofluorescence experiments were conducted on HeLa, SW620 and MCF-7 cell lines. Changes in the protocol were made regarding concentration of the permeabilization reagent and the blocking buffer. Different permeabilization reagents were used. Apart from the hCD63.01 antibody, a commercial anti-CD63 antibody (MEM-259)was used for comparison. Supernatant of nonpurified hCD63.01 was also used in order to establish possible changes which may have occurred in the antibody purification process.
It was established that hCD63.01 did not went through significant changes during purification. Optimized immunofluorescence protocol was proposed with Tween 20 as the permeabilization reagent. In every experiment hCD63.01 had a stronger signal than MEM-259 commercially available CD63 antibody. It is possible that hCD63.01 recognizes more forms of CD63. Further studies are needed for the complete characterization and understanding of hCD63.01 antibody’s potential