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Ispitivanje kvalitete morske vode u Luci Baroš i mogućnosti unaprijeđenja kvalitete ili prenamjene
No full textIspitivanje kvalitete morske vode u Luci Baroš provedeno je radi utvrđivanja hidroloških parametara čistoće mora u vanjskom području riječke Luke Baroš u svrhu određivanja mogućeg potencijala urbanističkog rješenja u kategoriji održivog razvoja. Praćenje analitičkih parametara određivanja kakvoće morske vode omogućiti će donošenje odluka u provedbi urbanističko-arhitektonskih rješenja unutar istraživanog područja u rekreativne svrhe. Istraživanje je provođeno tijekom nasumično odabranog tjednog monitoringa na tri lokacije unutar Luke Baroš: na izlazu na otvoreno more (lokacija 1), na najdubljem području pristupa Luci, ispred Lučke uprave Rijeka te u blizini ušća mora s područja riječke Rive u Luku Baroš (lokacija 2) te na mjestu ušća Mrtvog Kanala u Luku Baroš, ispred kompleksa EX port Delta-e (lokacija 3).
Uzorkovanje je provedeno u periodu od 12.kolovoza do 17.kolovoza. Na mjestu uzorkovanja morske vode određena je temperatura. U izuzetim uzorcima morske vode, s tri lokacije, određena je elektrovodljivost, pH, koncentracija otopljenog kisika, koncentracija klorida, nitrata, fosfata, kadmija, anionskih tenzida i kemijska potrošnja kisika. Korišteni su gotovi kitovi za kvantitativnu analizu kemijskih parametara namijenjeni ispitivanju slane vode, radni protokoli proizvođača i uređaji tvrtke Hach Lange, spektrofotometar HL DR 3900 i HQD PM prijenosni uređaj s priključnim elektrodama. Tvrtka Hach Lange je navedene uređaje ustupila za ovu svrhu.
Utvrđeno je kako morska voda ispitivana u Luci Baroš nije prikladna za primjenu u rekreacijske svrhe, zbog povišenih koncentracija anionskih tenzida, kemijske potrošnje kisika te prisutstva kadmija. Većina vrijednosti pokazuje oscilaciju pa rezultati nisu predvidljivi i morska voda u tom području nema trajnu kvalitetu. Rezultati su najznačajnije odstupali 17.kolovoza 2017.g., u odnosu na 16.kolovoz 2017.g.; a najčešće su se u ukupnom zbiru podataka razlikovali rezultati lokacije 2 u odnosu na lokaciju 3 i obrnuto. Mjerenja na datum 16.kolovoz najviše osciliraju u odnosu na druga mjerenja te je zaključeno kako je toga dana morska voda bila najmanje čistoće, radi nerazjašnjenog uzroka. Statističkom obradom podataka utvrđene su značajne razlike kemijskih pokazatelja unutar pojedine lokacije ovisno o datumu uzorkovanja i datumu uzorkovanja za sve tri lokacije istovremeno. Višestrukom regresijskom analizom utvrđeno je da lokacija najviše utječe na promjenu pokazatelja, a tek onda datum uzorkovanja.
Za donošenje pouzdanijeg zaključka potrebno je provesti dugoročni monitoring i dodatna istraživanja. Dobiveni rezultati će biti korisni u zaštiti okoliša na lokalnoj, državnoj i međunarodnoj razini, postavljanju mogućih urbanističko-arhitektonskih rješenja i javnosti u cjelini.
4Determination of quality of seawater in Port Baroš represents experimental process of hydroanalysis of salt water purity in the external area of Port Baroš of Rijeka, for the purpose of defining the potential for urbanictic solutions in the category of sustainable development. Tracking of the analytical parameters in determination of seawater quality will allow decision- making in creating urban-architectonic solutions inside the area of research for recreational purposes. Research was conducted on randomly-selected week in August, by sampling seawater from three locations of the Port: on the exit to open Adriatic sea (location 1), on the deepest public-approachable point (location 2) and in the point of mixing of water from Mrtvi kanal canal and the sea, in front of complex EX port Delta (location 3).
Sampling was conducted from 12th of August to 17th of August. On-site, temperature was measured; and in the samples taken from the three locations: electrical conductivity, pH, dissolved oxygen, chlorides, nitrates, cadmium, anionic surfactants and chemical oxygen demand. Kits for quantitative analysis of salt water, manufacturer`s working protocols and instruments from the company Hach Lange- spectrophotometer HL DR 3900 and HQD PM portable measuring device with probes are used. Company Hach Lange has provided the equipment for this purpose.
It has been determined that the salt water in the Baroš port is not suitable for application in recreational purposes, due to the hightened concentrations of anionic surfactants, chemical oxygen demand and presence of cadmium. Most of the values show constant oscillations so results are not predictable and seawater in that area does not posses a permanent quality. Results differed the most on August 17th in comparison with August 16th, and in the total measurement data, location 2 results differed the most in comparison to the results from location 3 and vice versa. Measurements on August 16th oscillated the most in regard to other measurements and it is concluded that that day the water was of least purity, due to the undetermined cause. Statistical anaysis showed significant differences in chemical values measured per location dependent on date of sampling and anaysis for all three locations at once. Multiple regression analysis proved that location contributes the most to the change of parameters, with date coming second to it.
To solidify the conclusions, it would be necessary to conduct a long-term monitoring and additional research. The results gathered will be useful in environment protection on local, national and international level, by proposing possible urban-architectonic solutions and to the public as whole
IDENTIFICATION OF PROTEIN MARKERS IN METASTATIC LARYNGEAL SQUAMOUS CELL CARCINOMA AND CONTRIBUTION OF NCAM MOLECULE POLYSIALYLATION TO PROGRESSION OF SQUAMOUS CELL CARCINOMA OF THE LARYNX
Get PDFPlanocelularni karcinom grkljana naj.e..i je oblik malignog oboljenja glave i vrata kojeg tijekom progresije karakterizira u.estala pojava metastaza u limfnim .vorovima vrata u ranim fazama razvoja bolesti. Ovaj tip karcinoma obilje.avaju mnogobrojne genetske abnormalnosti i promjene razine ekspresije niza proteinskih i glikoproteinskih molekula koje su neposredno povezane s procesom kancerogeneze. Unato. brojnim otkri.ima molekularni mehanizmi u pozadini patogeneze ovog oboljenja jo. uvijek nisu u potpunosti razja.njeni. Osim toga, sve je vi.e dokaza koji upu.uju na to da je abnormalna ekspresija i glikozilacija proteina posljedica inicijalne maligne transformacije stanica, a aberantna glikozilacija predstavlja jedan od klju.nih doga.aja u invaziji i metastaziranju mnogih malignih oboljenja. U svrhu identifikacije novih proteinskih biljega, koji bi dodatno razjasnili patogenezu metastatskog oblika bolesti i koji bi se mogli koristiti kao potencijalni dijagnosti.ki i prognosti.ki biljezi, u ovomu doktorskom radu je provedeno globalno diferencijalno proteomsko profiliranje uzoraka tkiva primarnih metastatskih tumora i pripadaju.eg tumorom nezahva.enog tkiva. Primijenjena je metoda masene spektrometrije spregnute s teku.inskom kromatografijom i naknadnom bioinfomati.kom analizom. Identificirano je ukupno 289 statisti.ki zna.ajno diferencijalno eksprimiranih proteina u metastatskim tumorima, a validacija odabranih potencijalnih markera je provedena uz pomo. metoda Western blot i 2D imunoblot. U primarnom metastatskom planocelularnom karcinomu grkljana su po prvi puta specifi.no potvr.eni potencijalni proteinski biljezi ladinin-1, antigen diferencijacije monocita- CD 14, lumikan, alfa 4A lanac tubulina- TUBA4A, afamin, ali i s njima povezani stani.ni signalni putevi poput CD44-Ŕ1 integrinskoga signalnog puta induciranog faktorom inhibicije migracije makrofaga-MIF i signalnog puta nuklearnog faktora kapa B. Nadalje, uz pomo. imunohistokemijske analize je u svim uzorcima tkiva primarnih tumora utvr.ena i ekspresija polisijalizirane forme adhezijske molekule neuralnih stanica (engl. PSA-NCAM -Polysialylated neural cell adhesion molecule), onkofetalnoga glikoproteinskog antigena, koji se povezuje s patogenezom i metastatskom diseminacijom nekih izrazito malignih oboljenja. Utjecaj PSA-NCAM na promjenu statusa stani.nih signalnih kaskada, koje su uklju.ene u odr.anje normalne homeostaze tkiva sluznice, bio je negativan .to upu.uje na .injenicu da ekspresija PSA-NCAM doprinosi ranim fazama maligne transformacije planocelularnog karcinoma grkljana.Laryngeal squamous cell carcinoma (LSCC) is the most common form of malignant disease in the head and neck region characterized by the frequent occurrence of metastases in the neck lymph nodes early in the disease progression. This form of cancer is characterised with numerous genetic abnormalities and changes in expression levels of proteins and glycoproteins associated with the process of carcinogenesis. Despite numerous findings molecular mechanism of metastatic disease has not yet been fully elucidated. In addition, there is increasing evidence that abnormal expression and glycosylation of proteins result of initial malignant transformation of cells, and aberrant glycosylation is one of the key events in the invasion and metastasis of many malignant diseases. In addition, there is increasing evidence indicating that abnormal expression and glycosylation of proteins is a result of cellular initial malignant transformation, and aberrant glycosylation is one of the key events in the invasion and metastasis of many malignant diseases. To identify novel protein markers which would further elucidate the pathogenesis of the metastatic disease and that could be also used as potential diagnostic and prognostic biomarkers, the global differential proteomic profiling of primary metastatic tumour and matched tumour non-affected tissues was performed. For proteomic profiling the method of mass spectrometry coupled with liquid chromatography and subsequent bioinformatic analysis was applied. A total number of 289 differentially expressed proteins were identified in metastatic tumors and selected protein markers were further validated by Western blot and 2D-imunoblot method. Potential protein biomarkers Ladinin-1, monocyte differentiation antigen CD-14, lumican, alpha-tubulin 4A chain TUBA4A, afamin and their associated cellular signaling pathways such as CD44-1 integrin induced macrophage migration inhibitory factor MIF and nuclear factor kappa B signaling pathway, were for the first time to our knowledge specifically determined in metastatic LSCC. Furthermore, in all analysed primary tumor tissue samples the expression of polysialylated neural cell molecule (PSANCAM), an oncofetal glycoprotein antigen previously associated with pathogenesis and metastatic dissemination of highly malignant diseases, was determined by immunohistochemical analysis. The impact of PSA-NCAM expression on the cellular signaling cascades involved in maintainance of normal mucosa tissue homeostasis was negative, which indicates that the expression of PSA-NCAM contribute to the initial stages of malignant transformation in laryngeal squamous cell carcinoma
Analysis of icl expression in Mycobacterium smegmatis by using fluorescent time-lapse microscopy
No full textMycobacterium smegmatis se, za razliku od M. tuberculosis, odlikuje brzim rastom i nepatogeno..u .to ga .ini prikladnim za laboratorijska ispitivanja. Ustanovljena va.nost icl gena za patogenost, virulenciju i pre.ivljavanje M. tuberculosis u .ivom organizmu potaknula je prou.avanje ekspresije icl gena M. smegmatis prilikom kultivacije na acetatu, supstratu koji u tijelu doma.ina nastaje Ŕ-oksidacijom lipida te ulazi u anaplerotski glioksilatni ciklus. Djelovanje glioksilatnog ciklusa posredovano je djelovanjem enzima izocitrat liaze (ICL) kojeg kodiraju icl1 i icl2 geni. Metoda time-lapse fluorescentne mikroskopije, uz obradu filmova u BactImAS platformi omogu.ila je prou.avanje dinamike ekspresije icl gena u realnom vremenu (eng. real-time). Pokazana je uskla.enost kolonija, ali i individualnost pojedina.nih stanica. Inicijalni val ekspresije odvijao se istovremeno kod svih bakterija u koloniji, samo 2 sata nakon dodatka acetata. Nakon toga, svaka stanica uspostavila je vlastitu dinamiku ekspresije koja se kod mnogih stanica poja.ala neposredno prije diobe. Metaboli.ki ciklusi koje stanice koriste za pre.ivljavanje na acetatu u po.etku podupiru diobu, ali ne i rast jer je stopa rasta opadala neovisno o razini ekspresije icl gena. Povr.ina sestrinskih stanica razlikovala se vi.e od 1.5 puta u pribli.no 50% slu.ajeva, kao i ukupna fluorescencija (eng. total fluorescence). S druge strane, srednja fluorescencija (eng. mean fluorescence) je kod ve.ine sestrinskih stanica bila podjednaka. U radu je za analizu ekspresije icl gena kori.ten BactImAS softver za obradu i analizu time-lapse filmova, unaprije.ena je funkcija pripreme filogenetskih stabala i specificirane su karakteristike filmova prikladnih za obradu u programu. Podaci dobiveni iz filmova pomo.u BactImAS platforme postavljaju temelje za mnogobrojne analize u budućnosti.Due to its fast growth and non-pathogenic nature, Mycobacterium smegmatis is a convenient experimental model for research of its close cousin, M. tuberculosis. The established relevance of icl genes for M. tuberculosis virulence, pathogenicity and survival in host organisms has encouraged icl gene expression analysis in M. smegmatis during cultivation on acetate, a substrate that is derived from β-oxidation of host lipids after which it enters the anaplerotic glyoxylate cycle. The enzyme isocytrate lyase (ICL), which is coded by icl1 and icl2 genes, participates in the glyoxylate cycle. Time-lapse fluorescent microscopy, along with movie processing in BactImAS software, has enabled analysis of icl gene expression dynamics through time. Here, the individuality of each cell is shown along with synchronicity of bacterial colonies. The initial wave of expression occurs only 2 hours after acetate addition in all cells of the colony. After that, each cell establishes an individual dynamic of expression which is enhanced in most cells before cell division occurs. The growth rate was decreasing despite the level of icl expression so it was concluded that the anaplerotic pathways which enable acetate utilization are used primarily for the establishment of cell divison, not cell growth. The area of sister cells had more than 1.5 time difference in almost 50% of cases, as did the total fluorescence. On the other hand, mean fluorescence values were predominantly equal among sister cells. Working on gene expression analysis also helped to improve BactImAS software for time-lapse movie processing and analysis. The BTree function was enhanced and movie characteristics that are compatible for BactImAS were specified. The amount of data extracted with the use of BactImAS has set a foundation for comprehensive future analysis
Glucosylceramide critically contributes to the host defence of cystic fibrosis lungs
No full textCistična fibroza (CF) je najčešća autosomna recesivna bolest u zapadnim zemljama. Kronične upale i povećana podložnost infekcijama s patogenima poput Pseudomonas aeruginosa su glavne karakteristike cistične fibroze. Pokazano je da ceramid ima centralnu ulogu u različitim plućnim infekcijama, uključujući infekcije s Pseudomonas aeruginosa, te da se nakuplja u plućnom tkivu i stanicama oboljelih od cistične fibroze. Nadalje, ekspresija sfingozina je značajno reducirana u dišnim putevima miševa i ljudi oboljelih od cistične fibroze. No, o ulozi glukozilceramida u cističnoj fibrozi jako se malo zna. U ovom radu pokazujem da su razine glukozilceramida reducirane na apikalnoj površini bronhijalnih stanica i stanica dušnika u CF mišu. Inhalacija CF miševa s glukozilceramidom je zaštitila miševe od infekcije s Pseudomonas aeruginosa dok su ne-inhalirani CF miševi razvili tešku upalu pluća. In vitro studije nisu pokazale direktan učinak glukozilceramida na Pseudomonas aeruginosa. Ovi rezultati indiciraju da glukozilceramid ima potencijal postati nova meta u liječenju cistične fibroze.Cystic fibrosis (CF) is the most common autosomal recessive disorder in Western countries and it is characterized by chronic inflammation and increased susceptibility to infection by pathogens such as Pseudomonas aeruginosa. It has been previously shown that ceramide has a central role in various pulmonary infections, including infections with P. aeruginosa, and is accumulated in lung tissues and cells from CF patients. In addition, sphingosine is severely down-regulated in the airways of CF mice and patients. However, little is known about a potential role of glucosylceramide in cystic fibrosis. Here I demonstrate that levels of glucosylceramide are reduced on the apical surface of bronchial and tracheal epithelial cells in CF mice. Inhalation of CF mice with glucosylceramide protected these mice from infection with P. aeruginosa, while non-inhaled CF mice developed severe pneumonia. However, in vitro studies failed to show a direct effect of glucosylceramide on Pseudomonas aeruginosa. This data indicate that glucosylceramide has a potential as a novel target to treat CF
Investigation of the characteristics of behavioral sensitization in Drosophila melanogaster
No full textBihevioralna senzitizacija predstavlja neasocijativno učenje u kojem ponovljena administracija stimulansa rezultira progresivnim stepenastim povećanjem odgovora. Bihevioralna senzitizacija kod Drosophile može biti kratkotrajna, i trajati kraće od 30 minuta, ili dugotrajna, do dva dana. Pretpostavlja se da su u njihovoj podlozi različiti molekularni mehanizmi. Cilj rada je bio razvoj visokoprotočne metode za razvoj dugotrajne bihevioralne senzitizacije na psihostimulanse kokain i metamfetamin. Svi testovi provedeni su na mužjacima divljeg tipa (wt) Drosophile melanogaster, na soju Canton S, i mutantima period (per) i timeless (tim), od kojih je per prethodno povezan s razvojem bihevioralne senzitizacije na kokain, dok tim nije.
Oralno administrirani metamfetamin kod mušica je izazivao toleranciju, bez obzira na doba dana administracije, koncentraciju ili dužinu trajanja administracije. Nasuprot tome, administracija volatiliziranog kokaina ili metamfetamina dovela je do brzog povećanja lokomotorne aktivnosti koja je bila dozno ovisna (75-1.25μl) i ovisna o dužini trajanja (1-7 minuta). Za administraciju volatiliziranog psihostimulansa razvili smo novu metodu gdje se regulacijom protoka zraka volatilizirana supstanca dostavlja individualnim mušicama u Drosophila Activity Monitoring System-u. Naredni eksperimenti će definirati uvjete za induciranje bihevioralne senzitizacije na kokain i metamfetamin u ovom visokoprotočnom sistemu.
Razvijen je i postupak za izazivanje kratkotrajne bihevioralne senzitizacije na mehanički stres. Mušice su izložene početnom jakom mehaničkom stresu (startle, ST), a potom seriji slabijih senzitizirajućih podražaja (SS1-3). Mušice jače reagiraju na SS podražaje ukoliko su prethodno izložene ST, što je u skladu s prethodno publiciranim rezultatima. Per i tim mušice na ST i SS reagiraju poput wt što pokazuje po prvi puta da ti geni nisu uključeni u regulaciju kratkotrajne bihevioralne senzitizacije, i govore u prilog razlike u mehanizmima indukcije kratkotrajne i dugotrajne bihevioralne senzitizacije.
Naši rezultati su u skladu s dvoprocesnom teorijom (dual-process theory) plastičnosti odgovora na ponovljenu stimulaciju, autora Groves i Thompson, 1970., koja govori da o jačini i trajanju podražaja ovisi razvoj senzitizacije ili habituacije. Oralna administracija (dugotrajan podražaj slabog intenziteta) izazvala je
toleranciju, dok je mehanički stres (kratkotrajan podražaj jakog intenziteta) inducirao razvoj bihevioralne senzitizacije. Karakteristika podražaja dana putem volatiliziranog kokaina zadovoljava uvjete za razvoj bihevioralne senzitizacije što će se provoditi u narednim eksperimentima.Behavioral sensitization represents non-associative learning in which repeated administration of stimulus results in a progressive increase in response. Behavioral sensitization in Drosophila can be short-term (takes less than 30 minutes) or long-term (lasts up to two days). It is assumed that they rely on different molecular mechanisms. The aim of the study was the development of high-throughput method to develop long-term behavioral sensitization to psychostimulants cocaine and methamphetamine. All tests were performed on male wild type (wt) Drosophila melanogaster, of strain Canton S, and mutants period (per) and timeless (tim), of which is per previously associated with the development of behavioral sensitization, while tim is not.
Oral administration of methamphetamine in flies induce tolerance, regardless of the day time, concentration or duration of administration. As opposed to that, the administration of volatilized cocaine or methamphetamine led to a rapid increase in locomotor activity which was dose dependent (75-1.25ěl) and time dependent (1 to 7 minute length). For administration of volatilized psychostimulants we have developed a new method where with regulation of airflow volatilized substance is delivered to individual flies in DAMS (Drosophila Activity Monitoring System). The next experiments will define requirements in this high-throughput system for induction of behavioral sensitization to cocaine and methamphetamine.
Also, we developed a method for the induction of short-term bihevioral sensitization to mechanical stress. Flies were exposed to an initial strong mechanical stress (startle, ST), and then a series of weaker sensitizing stimulus (SS1-3). Flies are more responsive to the SS stimulus if they were previously treated with ST, which is in accordance with previously published results. On ST and SS per and tim flies react like wt which shows for the first time that these genes are not involved in the regulation of short-term bihevioral sensitization, and indicate the differences in the mechanisms of induction of short and long-term behavioral sensitization.
Our results are in line with dual-process theory of plasticity response to repeated stimulation by Groves and Thompson 1970. which tells that development of sensitizaion or habituation depends on the strength and duration of the stimulus. Oral administration (long-terme stimulus od low intensity) caused tolerance, while
the mechanical stress (short-term stimulus of high intensity) induced development of behavioraln sensitization. The characteristic of the stimulus given by volatilized cocaine satisfies conditions for the development of behavioral sensitzation which will be implemented in the next experiments
Analysis of potential protein biomarkers of nephrotoxicity in urine from patients receiving antibiotics
No full textVankomicin je antibiotik koji se koristi za liječenje infekcija uzrokovanih bakterijama koje su otporne na druge antibiotike, no kod nekih pacijenata liječenih tim lijekom dolazi do oštećenja bubrega. Budući da dosadašnji biomarkeri nefrotoksičnosti ne pokazuju dovoljnu osjetljivost i specifičnost, potrebno je pronaći nove biomarkere koji bi omogućili ranu detekciju poremećaja bubrežne funkcije kako bi bilo moguće pravovremenim prekidom terapije spriječiti daljnje oštećenje bubrega. Cilj ovoga rada je bio usporediti proteomske profile urina pacijentica na terapiji vankomicinom sa i bez kliničkih znakova oštećenja bubrega uslijed terapije navedenim antibiotikom s ciljem detekcije potencijalnih proteinskih biomarkera toksičnog učinka vankomicina na bubrege.
Analiza jednodimenzionalnom gel elektroforezom je pokazala da se količina, ali i sastav proteina urina mijenjaju ovisno o danu uzimanja terapije. Nadalje, značajne razlike u proteinskim profilima urina pacijentica sa i bez oštećenja bubrega kao posljedica terapije vankomicinom se javljaju tek nakon trećeg, a naročito su izražene nakon sedmog dana terapije, pri čemu su najveće razlike uočene u rasponu molekulskih masa od približno 25-75 kDa. Dvodimenzionalna gel elektroforeza u kombinaciji sa MALDI-TOF/TOF masenom spektrometrijom je otkrila ekspresiju proteina keratina 1 tipa II, NGAL i retinol-vezujućeg proteina 4 u urinu pacijentice kod koje je terapija vankomicinom uzrokovala poremećaj u radu bubrega, što sugerira da bi ova tri proteina mogla predstavljati potencijalne biomarkere nefrotoksičnosti vankomicina. Daljnje studije su potrebne na većem broju pacijenata kako bi se validirali dobiveni rezultati i ispitao njihov potencijalni dijagnostički i klinički značaj.Vancomycin is an antibiotic used for treating infections caused by bacteria which are resistant to other antibiotics, but in some of the treated patients it causes kidney damage. Since the currently known biomarkers of nephrotoxicity do not show sufficient sensitivity and specificity, it is necessary to find new biomarkers which would enable to discover disorders in kidney function in early stages and stop the therapy at the right time with the aim to prevent further development of kidney damage. The main goal of this study was to compare proteomic profiles of urine samples collected from patients on vancomycin therapy with and without clinical signs of kidney damage detected during the therapy in order to find potential protein biomarkers of vancomycin nephrotoxicity.
Analysis of samples by one-dimensional gel electrophoresis showed that both quantity and composition of urinary proteins change depending on the day of therapy. Furthermore, significant differences in proteomic profiles of urine samples collected from patients with and without kidney damage caused by vancomycin therapy occur only after third, and are especially evident after seventh day of therapy, with the biggest differences observed within a range of molecular weights of about 25-75 kDa. Two-dimensional gel electrophoresis combined with MALDI-TOF/TOF revealed expression of keratin 1 type II, NGAL and retinol-binding protein 4 in urine of patient with kidney damage caused by vancomycin therapy, which suggests that those three proteins could present potential biomarkers of vancomycin nephrotoxicity. It is necessary to conduct further studies including larger number of patients in order to validate these results and to investigate their potential diagnostic and clinical significance
De moscas a humanos: Genes circadianos en la neurogenética de la adicción
No full textDrug addiction is a persistent brain disease with severe and sometimes fatal consequences. Addictive drugs induce long-lasting neuroadaptations in the functioning of the nervous system and currently there is no efficient pharmacological treatment that can successfully prevent or reverse these changes. As a consequence, addicted individuals often suffer from recurrent relapses, sometimes triggered by environments, situations or stressors that have previously been associated with drug taking.Behavioral neuroscience uses animal models to understand the neurobiological mechanisms which cause and correlate with the development of addiction, and recently the emphasis is on animals that are genetically tractable, such as mice (Mus musculus) and fruit flies, (Drosophila melanogaster). In spite of many obvious differences between humans and Drosophila, similarities at the genetic level and in the basic neuronal physiology, make Drosophila an excellent model organism for the study of many complex human behaviors, including addiction. Discovery that circadian genes influence the development of behavioral sensitization to cocaine in Drosophila led to numerous studies about the role of circadian genes in the regulation of drug-induced behaviors in laboratory mammals and humans. Results show that circadian genes are involved in regulating the behavioral and molecular response to different classes of drugs of abuse. Furthermore, studies in humans show the interconnectivity between the regulation of circadian behavior, mental diseases and addiction, and suggest that behavioral interventions aimed at improving the quality of the circadian behavior will be important in prevention and treatment of addictive behaviors.Ovisnost je o drogama kronična bolest s teškim, nerijetko fatalnim posljedicama. Opojne droge izazivaju dugotrajne neuroadaptivne promjene u funkcioniranju živčanog sustava, a ne postoji učinkovit lijek koji bi ih suzbio ili ispravio. Ovisnici stoga pate od opetovanih recidiva, ponekad izazvanih okolinom, situacijom ili stresom koji je i bio povezan s uzimanjem droge.Bihevioralna neuroznanost koristi modelne organizme za razumijevanje neurobioloških mehanizama koji uvjetuju ili koreliraju s razvojem ovisnosti, a odnedavno naglasak je na životinjama koje su genetski pogodne, kao miš (Mus musculus) ili vinska mušica (Drosophila melanogaster). Unatoč mnogim očitim razlikama između ljudi i Drosophile sličnosti na genetskoj razini i u području bazične neuronske fiziologije čine Drosophilu izvrsnim modelnim organizmom za izučavanje mnogih složenih ljudskih ponašanja, uključujući ovisnost. Otkriće da cirkadijalni geni kontroliraju razvoj bihevioralne senzitizacije na kokain kod Drosophile potaklo je niz istraživanja o ulozi koju cirkadijalni geni imaju u ponašanjima induciranim kokainom kod laboratorijskih životinja i ljudi. Rezultati pokazuju da cirkadijalni geni reguliraju ponašanja i molekularne odgovore na razne vrste opojnih droga. Štoviše, istraživanja na ljudima upućuju na međupovezanost između cirkadijalnog ponašanja, mentalnih oboljenja i ovisnosti, te sugeriraju da bihevioralni zahvati usmjereni na poboljšanje cirkadijalnog ponašanja mogu biti važni u prevenciji i liječenju ovisničkog ponašanja.La drogadicción es una de las enfermedades crónicas con consecuencias graves, a veces fatales. Las drogas adictivas causan cambios neuroadaptivos a largo plazo en el funcionamiento del sistema nervioso y de momento no existe un medicamento eficiente que lo reprimiría o corregiría. Por eso los drogadictos sufren repetidas recaídas, a veces causadas por el entorno, situación o estrés previamente relacionado con el consumo de drogas.Neurociencia conductual usa organismos modelos para comprender mecanismos neurobiológicos que condicionan o se correlacionan con el desarrollo de la adicción, y recientemente el enfoque está en los animales genéticamente convenientes, como ratón (Musmusculus) o mosca del vinagre (Drosophila melanogaster). A pesar de las numerosas diferencias obvias entre los humanos y las moscas del vinagre, la semejanza al nivel genético y aquel de fisiología neuronal básica convierten dicha mosca del vinagre en un excelente organismo modelo para estudiar muchas conductas humanas complejas, incluida la adicción. Al descubrir que los genes circadianos controlan el desarrollo de la sensibilización de comportamiento de la mosca del vinagre en cuanto a la cocaína, se provocó toda una serie de investigaciones sobre el papel que tienen los genes circadianos para los comportamientos inducidos por la cocaína tanto en los animales de laboratorio, como en los humanos. Los resultados indican que los genes circadianos regulan comportamientos y respuestas moleculares a diferentes tipos de drogas adictivas. Es más, las investigaciones en los humanos muestran la interconexión entre el comportamiento circadiano, enfermedades mentales y adicción, y proponen que las intervenciones en el comportamiento enfocadas en la mejora del comportamiento circadiano pueden ser importantes en la prevención y tratamiento de conductas adictivas
Neonatal risk mortality scores as predictors for health-related quality of life of infants treated in NICU: a prospective cross-sectional study
No full textPurpose To determine the relationship of Apgar scores, gestational age and neonatal risk mortality scores to healthrelated quality of life (HRQoL) for infants at the age of 8 months treated after birth in neonatal intensive care unit (NICU). Methods All surviving infants treated in two-third level NICUs in Rijeka, Croatia (from August 2013 to August 2014) were included in this prospective, cross-sectional study. For all neonates, the Score for Neonatal Acute Physiology (SNAP), SNAP with Perinatal Extension (SNAP-PE) and their simplified modifications (SNAP II and SNAP-PE II) were calculated. At the corrected age of 8 months, the Pediatric Quality of Life Questionnaire (PedsQL)—infant scale—was completed by parents of surviving infants. Multiple regression analysis was performed in order to assess the value of neonatal risk
mortality scores, Apgar scores and gestational age as possible predictors of HRQoL, measured by questionnaire score. Results A strong correlation has been found between SNAP and 5-min Apgar scores to HRQoL. A positive correlation was also found between gestational age and HRQoL. Conclusion SNAP and 5-min Apgar scores are important outcome indicators, can aid clinicians’ and parents’ decision making on the benefits and burdens of acute medical interventions and help determine quantities of medical treatment. Educated medical staff, effective and efficient medical treatment and a high quality of care which prevent adverse events in the first minute of life should be a priority in efforts to improve the future quality of life
Protein ostrelysine A interactions with liposomes composed by phosphatidylcholine, cholesterol and ceramide phosphoethanolamine
No full textOstreolizin A (u daljnjem tekstu OlyA) je protein niske molekulske mase (̴15 kDa) izoliran iz gljive Pleurotus ostreatus i pripada obitelji egerolizina. Poznato je da u kombinaciji s 59-kDa proteinom pleurotolizinom B (PlyB) tvori pore i da se specifično veže na liposome u čijem se sastavu nalaze sfingomijelin (SM) i kolesterol (Kol), ceramid fosfoetanolamin (CPE) i/ili CPE:Kol, zasićeni glicerofosfolipidi/Kol te da je za vezanje potrebno minimalno 30% kolesterola. Karakteristika OlyA je i vezanje na membranske splavi koje u sastavu također imaju sfingomijelin i kolesterol i time ima potencijalnu primjenu u označavanju membranskih domena bogatih navedenim lipidima. No, pošto je sfingomijelin glavni lipid kralješnjaka, a ceramid fosfoetanolamin beskralješnjaka i pošto se razlikuju jedino skupinama na glavi molekule odlučili smo ispitati interakcije OlyA s ceramid fosfoetanolaminom i kolesterolom i saznati količine ovih lipida potrebne za vezanje proteina. Pošto se CPE nalazi u membranama beskralješnjaka uočena je i potencijalna primjena kombinacije OlyA/PlyB u suzbijanju različitih insekata i parazita.
U tu svrhu smo napravili lipidne filmove rotacionim uparivačem otapala te u prvom dijelu istraživanja preliminarnim testom SDS-PAGE elektroforeze ispitali vezanje OlyA. U ovom, ali i svim ostalim testovima, koristili smo liposome s različitim molnim udjelima CPE, Kol i 1-palmitoil-2-oleil-fosfatidilkolina (POPC), dok su liposomi POPC:Kol (1:1) služili kao negativna kontrola vezanja. Isti test vezanja OlyA, ali i kombinacije OlyA/PlyB smo ponovili i osjetljivijom metodom površinske plazmonske rezonancije koja prati intermolekularne interakcije u stvarnom vremenu. Pokazali smo da je za vezanje potrebno minimalno 5% CPE i više od 30% kol.
U drugom djelu istraživanja proučavali smo permeabilizacijski učinak OlyA/PlyB proteinskog kompleksa na liposome punjene kalceinom i pokazali da se kalcein otpušta iz svih liposoma osim POPC:Kol (mol:mol,
1:1) koji su ionako negativna kontrola. Ovi rezultati ukazuju da smanjenje količine CPE i kolesterola u liposomima smanjuje i permeabilizacijsku aktivnost OlyA/PlyB. Također, stvaranje pora u liposomima načinjenim od lipida koji čine membranu parazita Toxoplasma gondii otkriva mogućnost korištenja OlyA/PlyB kompleksa u suzbijanju navedenog parazita.
U posljednjem dijelu istraživanja razvili smo kolorimetrijski test za određivanje ugrađene količine CPE u liposome,te smo odredili količine CPE, POPC i kolesterola u liposomima, a pomoću metode dinamičkog sipanja svijetlosti odredili veličinu velikih unilamelarnih liposomaOstreolysin A (OlyA) is a low-molecular weight protein (̴15 kDa) isolated from fungus Pleurotus ostreatus and belongs to the aegerolysin family. It is known that in combination with a 59-kDa protein, pleurotolysin B (PlyB), it forms a pore and that it specifically binds to lipid vesicles that are comprised of sphingomyelin (SM) and cholesterol (Chol), ceramide phosphoethanolamine (CPE) and/or CPE:Chol and saturated glycerophospholipids/Chol. Furthermore, at least 30% cholesterol is necessary for binding. Another characteristic of OlyA is that it binds to membrane rafts composed of sphingomyelin and cholesterol and thus has a potential use in labelling membrane domains rich with the aforementioned lipids. Since sphingomyelin is the major lipid in mammals and in invertebrates that is ceramide phosphoethanolamine, and since these molecules differ only in their head groups, we decided to examine the interaction of OlyA with ceramide phosphoethanolamine and cholesterol, and to determine the quantity of these lipids necessary for binding. Since CPE is found in cell membranes of invertebrates a potential application of the combination Olya/PlyB in combating various insects and parasites.
For this purpose, we produced lipid films with the rotary evaporator and in the first research stage we used the SDS-PAGE electrophoresis as a preliminary test to examine the binding of OlyA. In this and all subsequent tests, we used liposomes with varying molar fractions of CPE, Chol and palmitoyl-oleoyl-phosphatidylcholine (POPC), where POPC:Chol (mol:mol, 1:1) served as a negative control. The binding of OlyA, but also of the combination OlyA/PlyB, was further assayed with the sensitive surface plasmon resonance method that tracks intermolecular interactions in real time. We have shown that a minimum of 5% part CPE, and more than 30% part Chol is necessary for the OlyA binding
Anticholinesterase activity of the synthetic derivatives of barettin
No full textBudući da su sesilni morski organizmi, osobito morske spužve, bogat izvor mikroorganizama i bioaktivnih prirodnih spojeva, oni predstavljaju temelj za razvoj potencijalnih lijekova. Inhibitori enzima acetilkolinesteraze, koji imaju širu primjenu u medicini, upravo su jedni od tih lijekova. S obzirom na to da nijedan inhibitor acetilkolinesteraze iz morskog svijeta nije komercijalni proizvod, prikupljanje i analiza morskih organizama vrijedan je pokušaj da se istraže novi prirodni spojevi te njihova potencijalna upotreba u liječenju. Iz farmakološki zanimljivih morskih prirodnih spojeva izoliranih iz spužve Geodia barretti koji su prethodno pokazali sposobnost inhibicije acetilkolinesteraze stvoren je set semisintetskih analoga (spojevi MBC) kako bi se istražilo njihovo farmakološko djelovanje. U istu je svrhu stvoren i set spojeva Kine, analoga bromocionina izoliranog iz morske ascidije Ciona edwardsii. Testirani analozi pokazali su antiacetilkolinesteraznu aktivnost. Analizom Dixonovih dijagrama utvrdilo se da je riječ o reverzibilnim kompetetivnim inhibitorima. Nijedan od analoga nije pokazao inhibitornu snagu kakvu imaju prirodni spojevi baretin i 8,9-dihidrobaretin. Najaktivniji analozi MBC-197 te Kine22 dalje su se testirali na živčano-mišićnom preparatu. Pokazalo se da nijedan od njih u testiranim koncentracijama ne utječe na neuromuskularni spoj ni na membranske potencijale.Sessile marine organisms, especially marine sponges, represent an important basis for potential development of certain drugs since they are rich in microorganisms and bioactive natural products. Such an example are inhibitors of acetylcholinesterase, which are already widely used for medical purposes. None of the acetylcholinesterase inhibitors from the marine realm are yet commercial products, therefore collecting and analysing marine organisms represent a valuable effort to further discover new natural products and their potential medical use. In order to investigate their pharmacological effects, a set of semisynthetic analogs (MBC) was designed out of the marine natural products isolated from the sponge Geodia barretti. These natural compounds had previously shown inhibitory activity towards acetylcholinesterase. Another set of analogs (Kine) was designed out of bromocyonine isolated from the Mediterranean ascidian Ciona edwardsii. Tested analogs have shown acetylcholinesterase-inhibitory activity. Analyses of Dixon plots have revealed these compounds as reversible competitive inhibitors. None of the semisynthetic compounds were as active as isolated compounds barettin and 8,9-dihydrobarettin. The most active analogs MBC-197 and Kine22 underwent further testing on neuromuscular preparation. In tested concentrations, none of them exerted an action on neuromuscular transmission nor affected membrane potentials