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Human cumulus cell expression profile of AMH, AMHR2, FSHR and AR genes in predicting oocyte fertilization potential
Get PDFTemelj uspješnosti medicinski pomognute oplodnje je pravilno sazrijevanje jajnih stanica. Na kvalitetu jajne stanice velik utjecaj ima okruženje u kojem se ona nalazi, tzv. mikrookoliš koji uključuje stanice kumulusa i folikularnu tekućinu. Određivanje pojedinih komponenti tog mikrookoliša koje se jednostavno i brzo mogu detektirati, a koje omogućavaju razlikovanje kvalitete jajnih stanica, doprinijelo bi povećanju uspješnosti metoda pomognute oplodnje. Anti-Müllerov hormon (AMH) je glikoproteinski faktor rasta, koji luče stanice kumulusa. S obzirom na njegovu ulogu u folikulogenezi, ovim je doktorskim radom istražen ekspresijski profil ovog gena, i s njim povezanih receptora (AMHR2, FSHR i AR), te njihova korelacija s kvalitetom jajnih stanica u postupku medicinski pomognute oplodnje, odnosno s morfološkim karakteristikama jajnih stanica, zigota i embrija te njihovim fertilizacijskim potencijalom. U istraživanje je uključeno 129 uzoraka stanica kumulusa i 35 uzoraka folikularne tekućine koji su sakupljeni od ukupno 52 pacijentice u postupku medicinski pomognute oplodnje. Primijenjena je metoda relativne kvantifikacije genskih ekspresija istraživanih gena u stanicama kumulusa, enzimatski pojačani dvostruki imunološki test (ELISA) za određivanje koncentracije AMH u folikularnoj tekućini istog folikula te morfološka analiza jajnih stanica, zigota i embrija invertnom mikroskopijom. Rezultati dobiveni istraživanjem po prvi put ukazuju na negativnu povezanost ekspresije AMH i AR gena u stanicama kumulusa s morfološkom kvalitetom pripadajuće jajne stanice, odnosno s njenom zrelošću. Osim navedenog, istraživanjem je utvrđeno i postojanje korelacija među ekspresijama ispitivanih gena: između FSHR gena i svih ostalih istraživanih gena (AMH, AMHR2 i AR), između AMHR2 i AR gena, te između AMH i AR gena. Dobiveni rezultati pridonose razjašnjenju potencijalne veze AMH (i receptora koji su na direktni ili manje direktni način povezani s ovim hormonom) i morfološke kvalitete jajne stanice. Istraživanjem je potvrđena važnost kompleksa kumulusa i oocite, odnosno komunikacije između jajne stanice i njenog mikrookoliša, za razvoj kvalitetne oocite koja je preduvjet za uspješnu oplodnju i razvoj kvalitetnog embrija.The core notion determining the success rate of the medically assisted reproduction is the right growth of the oocytes. The environment, or the so called microenvironment, greatly influences the quality of the oocyte and it includes the cumulus cells and the follicular fluid. Determining the specific components of this microenvironment, which can be easily and swiftly detected, and which enable us to distinguish between good and bad quality oocytes, would contribute to the success rate of the medically assisted reproduction. Anti-Müllerian hormone is a glycoprotein growth factor emitted by the cumulus cells. Considering its role in the folliculogenesis, this doctoral thesis looked into the expression profile of the stated gene and its respective receptors. Their correlation with the oocyte quality in the process of medically assisted reproduction was examined, as well as morphological characteristics of the oocytes, zygotes and embryos together with their fertilization potential. The research included 129 cumulus cells samples and 35 follicular fluid samples which had been harvested from 52 patients during the process of medically assisted reproduction. The method of relative quantification of gene expressions in the cumulus cells for the researched genes was employed, the enzyme - linked immunosorbent assay (ELISA) was used to determine the concentration of AMH in the follicular fluid of the respective follicle, and the morphological analysis of the oocytes, zygotes and embryos was conducted by inverted microscopy. The results obtained in the research for the first time show the negative correlation between the AMH and AR genes expression in the cumulus cells and the morphological quality, i.e. maturity, of the respective egg cell. Furthermore, the research determined the existence of correlations between the expressions of the examined genes as follows: between FSHR genes and all the other researched genes (AMH, AMHR2 and AR), between AMHR2 and AR genes, as well as between AMH and AR genes. The obtained results also contribute to the clarification of the potential connection between AMH (and the receptors directly or less directly linked to this hormone) and morphological quality of the egg cell. The research confirmed the importance of the cumulus - oocyte complex, i.e. of the communication between the egg cell and its microenvironment, for the growth of a high - quality oocyte, which is a prerequisite for a successful fertilization and growth of a high - quality embryo
Conjugation of porphyrins with DL-buthionine sulfoximine and D-erythro sphingosine
No full textFotodinamička terapija (PDT) se sve više upotrebljava u liječenju raka, ali i mnogih drugih bolesti. Da bi PDT bila uspješna, potrebne su tri komponente; fotosenzibilizator (PS), izvor svjetla i kisik. Najčešći PS-i su porfirini, tetrapirolni aromatski spojevi. Do danas su u upotrebi 3 generacije PS-a. Prvu čine prirodni spojevi, drugu sintetski, dok treću generaciju predstavljaju PS-i sintetskog porijekla konjugirani s biološki aktivnim molekulama. Biološki aktivne molekule predstavljene u ovom radu su DL-butioninsulfoksimin (BSO) i D-eritrosfingozin. BSO pripada skupini sulfoksiimina, a glavna uloga ove molekule je inhibicija glutationa (GSH), jednog od najvažnijih antioksidansa stanice. D-eritrosfingozin je produkt raspada ceramida i vrlo je potentan inhibitor enzima protein kinaze C (PKC) i sfingozin kinaze koji su potencijalne mete u liječenju raka. U ovom radu je Adler-Longo metodom sintetiziran 5-(4-aminofenil)-10,15,20-tris(3-piridil)porfirin koji je korišten za daljnje modifikacije i konjugacije. Uspješno izvedenim reakcijama sa sukcinanhidridnom odnosno 1,1'-tiokarbonildi-2(1H)-piridonom (TDP) dobivena je reaktivna skupina porfirina na kojoj su se odvijale konjugacije s D-eritrosfingozinom i BSO. Iako su željene konjugacije izvršene, u ovom radu, dobiveni konjugati nisu do kraja pročišćeni zbog niskih iskorištenja u početnim reakcijama sinteze porfirina. Međutim, stvaranje novih konjugata je spektroskopski potvrđeno i otvara put prema optimizaciji sintetskih puteva i dobivanju čistih konačnih produkata u budućim sintezama.Photodynamic therapy (PDT) is increasingly used in cancer treatment, as well as in treatment of other diseases. For PDT to be efficient, there is a need for three components: photosensitizer (PS), source of light and oxygen. The most common PSs are porphyrins, tetrapirole aromatic compounds. Nowadays, there are three generations of PS that are being used. They are: natural compounds, synthetic compounds and synthetic compounds that are conjugated with biologically active molecules. Biologically active molecules that are part of this paper are DL-buthionine sulfoximine (BSO) and D-erythro sphingosine. BSO belongs to the group of sulfoximines and its main role is inhibition of glutathione (GSH), which is one of the most important cell antioxidant. D-erythro sphingosine is a product of ceramide breakup and it is very potent inhibitor of enzymes protein kinase C (PKC) and sphingosine kinase which are potential target in cancer treatment. In this paper, 5-(4-aminophenyl)-10,15,20-tris(3-pyridyl)porphyrin is synthesized by using Adler-Longo method and used for further modifications and conjugations. Successfully performed reactions with succinic anhydride, and with 1,1′-thiocarbonyldi-2(1H)-pyridone (TDP), yielded a reactive group of porphyrins that are used for conjugations of porphyrins with D-erythrophingosine and BSO. Although the desired conjugations have been performed, new conjugates have not been completely purified due to low yielding condensation reaction in early synthetic steps. However, the formation of new conjugates was confirmed by spectroscopy and it paves the way toward optimisation of the synthetic pathway that will allow the preparation of the purified final products in future synthesis.
High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization
Get PDFCell–cell and cell–matrix interactions are essential to the survival and proliferation of most cells,
and are responsible for triggering a wide range of biochemical pathways. More recently, the
biomechanical role of those interactions was highlighted, showing, for instance, that adhesion
forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small
size, high aspect ratio and anisotropic mechanical response represent a useful model to
investigate the forces involved in the adhesion processes and their role in cellular development.
In this work we explored and quantified, by single cell force spectroscopy (SCFS), the
interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that
the cell adhesion forces are comparable to those found on collagen and bare glass coverslip,
analogously the membrane tether extraction forces are similar to that on collagen but stronger
than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a
reduced proliferation after 36 h was observed. On the contrary both cell morphology and
cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was
characterized by a large number of filopodia and a significant decrease of the cell mobility. The
cytoskeleton organization was characterized by the absence of actin fibers, which were instead
dominant on collagen and flat glass support. Such findings suggest that the mechanical properties
of disordered Si NWs, and in particular their strong asymmetry, play a major role in the
adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion
measurements by SCFS provide out-of-plane forces values consistent with those measured on
conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and
migration processes
Utjecaj alternativne i konvencionalne terapije na promjenu sastava masnih kiselina u jetri dijabetičnih štakora
No full textŠećernu bolest (diabetes mellitus, DM) karakterizira poremećaj metabolizma ugljikohidrata, masti i proteina, a uzrokovana je smanjenim lučenjem ili smanjenom osjetljivošću tkiva na inzulin. Dijabetes tipa 1 (T1D) se odnosi na 5-10% svih slučajeva dijabetesa i obično se javlja u ranijoj životnoj dobi dok je tip 2 (T2D) prisutan u većini slučajeva i to u kasnijoj životnoj dobi. T1D je upalna autoimuna bolest u kojoj -stanice gušterače selektivno uništavaju stanice imunološkog sustava (hipoinsulinemija i hiperglikemija). T2D je povezana s pretilošću i metaboličkim sindromom. Inzulin je nadomjestna konvencionalna terapija u liječenju T1D, ali zbog nuspojava ove terapije je porastao interes za alternativnu fitoterapiju. U terapiji T2D koristi se više vrsta lijekova koji pokrivaju različite mehanizame djelovanja. Obećavajuće rezultate su pokazali bioaktivni fenolni spojevi koji mogu djelovati antioksidacijski, protuupalno, hipoglikemično i hipokolesterolemično. Prednosti mediteranskog stila života (maslina i njeni proizvodi) su poznate stoljećima i korištene u sprječavanje i liječenje različitih metaboličkih sindroma.
U ovom istraživanju sam pratila sastav masnih kiselina u jetri dijabetičnih (T1D) i dijabetičnih/pretilih (T2D) štakora te učinak polifenola lišća masline na sastav masnih kiselina jetre u dijabetičnih (T1D) štakora. T1D je izazvan injiciranjem (i.p.) streptozotocina (SZT), a pretilost/T2D je izazvana visoko-masnom hranom (HFD). Dijabetički štakori (T1D) su tretirani ekstraktom lišća masline (512, 768 i 1024 mg/kg) te inzulinom. Sastav masnih kiselina (FAME) određen je u homogenatu jetre korištenjem plinske kromatografije. Statistička analiza provedena je Mann-Whitney U-testom i Kruskal-Walis testom primjenom programa Statistica.
Značajne razlike masnih kiselina i omjera su pronađene u dijabetičnih štakora (T1D) u odnosu na kontrolnu skupinu (16:1, 18:2, 20:2, 20:4, 20:5, 24:0, 22:6, n-6, n-3, n-6/n-3, desaturaza delta-5 (D5D), 16:1/16:0, 16:1/18:1, 20:4/22:6, 18:2/20:4). Terapija ekstraktom polifenola maslinova lišća značajno mijenja razine 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:2, 20:3, 20:4, 24:0, n-3 jednostruko-nezasićenih (MUFA) masnih kiselina, omjera 18:2/20:4, 16:1/16:0, 16:0/18:0, 16:1/18:1 i n-6/n-3 te stearoil-CoA desaturaze 1 (SCD1) i elongaze 6 (Elovl6) u jetri dijabetičnih štakora. Terapija inzulinom značajno mijenja razinu 16:0, 16:1, 18:2, 20:1, 20:2, 20:3, 24:0, 22:5, n-6 i n-3 masnih kiselina, omjera 18:2/20:4, 16:1/16:0, 16:0/18:0, 16:1/18:1 i n-6/n-3 te Elovl6 i D5D u jetri dijabetičnih štakora. Zabilježena je značajna razlika u razini 16:1, 18:3, 20:4, 24:0, 22:5, 22:6, n-6, n-3 i višestruko-nezasićenih (PUFA) masnih kiselina, omjera 16:1/16:0, 16:1/18:1, 20:4/22:6, n-6/n-3 i PUFA/SFA te desaturaza delta-6 (D6D) u jetri dijabetičnih štakora sa T1D i T2D.
Iz navedenog se može zaključiti da dijabetes tipa 1 i 2 mijenjaju metabolizam masnih kiselina u jetri štakora. Pronađene su značajne razlike u metabolizma masnih kiselina u tipu 1 i 2 dijabetesa. U odnosu na terapiju inzulinom, terapija polifenolima lišća masline značajno mijenja razinu n-6 i MUFA te ključne enzime u metabolizmu masnih kiselina, SCD1 i D6D.Diabetes mellitus (DM) is characterized by a disorder of carbohydrate, fat, and protein metabolism, caused by decreased secretion or reduced tissue sensitivity to insulin. Type 1 diabetes (T1D) refers to 5-10% of all cases of diabetes and usually occurs at an earlier age, while type 2 (T2D) is present in most cases in later life. T1D is an inflammatory autoimmune disease in which β-cells pancreas destroys the immune system cells (hypoinsulinemia and hyperglycemia). T2D is associated with obesity and metabolic syndrome. Insulin is a replacement therapy for T1D, but due to the side effects associated with insulin therapy, there is an increasing interest in alternative therapy especially phytotherapy. In T2D therapy, several types of drugs are used with different mechanisms of action. Bioactive phenolic compounds are a wide-beneficial therapy that can act as antioxidant, anti-inflammatory, hypoglycemic and hypocholesterolemic. The benefits of the Mediterranean lifestyle (olives and its products) have been known a long time and used in prevention and treatment of various metabolic disorders including diabetes. In this study, the effect of treatment with olive leaf polyphenols on the profile of hepatic fatty acids in diabetic/obese rats was studied. T1D was induced intraperitoneally with streptozotocin (SZT, i.p.), and obesity/T2D was caused by consumption of high-fat (HFD). Diabetic rats were treated with leaf leaves extract (512, 768 and 1024 mg/kg) and insulin. The composition of individual fatty acids (FAME) was determined in liver homogenate using gas chromatographic analysis. Statistical analysis was carried out by Mann-Whitney and Kruskal-Wallis test using Statistical Program.
Significant differences in fatty acids and ratios were found in diabetic rats compared to the control group (16: 1, 18: 2, 20: 2, 20: 4, 20: 5, 24: 0, 22: 6, n-6, n -3, n-6 / n-3, delta-5 (D5D), 16: 1/16: 0, 16: 1/18: 1, 20: 4/22: 6, 18: 2/20: 4 ). The therapy with olive leaves polyphenols changed significantly 16: 0, 16: 1, 18: 0, 18: 1, 18: 2, 18: 3, 20: 2, 20: 3, 20: 4, 24: 0, n- 3, mono-unsaturated (MUFA) fatty acids, ratios 18: 2/20: 4, 16: 1/16: 0, 16: 0/18: 0, 16: 1/18: 1 and n-6 / n-3 as well as stearoyl-CoA desaturase 1 (SCD1) and elongase 6 (Elovl6) in the liver of diabetic rats. Insulin therapy changed significantly the 16: 0, 16: 1, 18: 2, 20: 1, 20: 2, 20: 3, 24: 0, 22: 5, n-6 fatty acids, n- 2/20: 4, 16: 1/16: 0, 16: 0/18: 0, 16: 1/18: 1, n-6 / n-3 ratios, and Elovl6 and D5D in liver diabetic rats. Also, a significant difference was found in 16: 1, 18: 3, 20: 4, 24: 0, 22: 5, 22: 6, n-6, n-3 and polyunsaturated fatty acids (PUFA), ratios of 16:1/16: 0, 16: 1/18: 1, 20: 4/22: 6, n-6 / n-3 and PUFA / SFA as well as delta-6-desaturase (D6D) in T1D and T2D rat liver.
It can be concluded from these results that type 1 and 2 diabetes alter the fatty acid metabolism in the rat liver. Significant differences in fatty acid metabolism in type 1 and 2 diabetes were found. In comparison to insulin therapy, therapy with olive leaf polyphenols significantly changed the level of n-6 and MUFA and key enzymes in fatty acid metabolism, SCD1, and D6D
Protocol optimization for feces microbiota protein isolation
No full textU novije vrijeme proteomika mikrobiota fecesa (metaproteomika) zauzima važno područje u istraživanju patogeneze mnogih bolesti, kao i u ranoj dijagnostici. Često se u ovim istraživanjima koriste modeli organizama poput primjerice štakora, a tehnološka platforma koja postaje sve važnija za brzo, sveobuhvatno i precizno definiranje metaproteoma, odnosno ukupnog proteoma mikrobiota, je masena spektrometrija (MS). Sastav mikrobiota fecesa štakora vrlo je kompleksan i obuhvaća različite vrste bakterija, kvasaca te virusa. Stoga je u svrhu proteomskih istraživanja potrebno razvijati efikasne metode za izolaciju različitih vrsta mikrobiota što podrazumijeva i različite načine za razbijanje stanica u svrhu izolacije molekularnih sastavnica iz tih stanica, primjerice ukupnih proteina. Neke od metoda koje se koriste za izolaciju proteina iz mikrobiota, a koje su kompatibilne sa MS uključuju sonikaciju i homogenizaciju keramičkim kuglicama. Uspješnost protokola za pripremu uzorka u proteomskim istraživanjima metaproteoma ključna je za uspješnu i nedvojbenu identifikaciju. U ovom je radu stoga proveden razvoj i tehnička optimizacija protokola za izolaciju ukupnog proteinskog komplementa mikrobiota fecesa štakora. U tu svrhu koristili smo mješavinu bakterijskih kultura i kulture kvasca koja je simulirala sastav mikrobiota fecesa štakora. Stanice mikroorganizama smo razbijali uz kombinacije mehaničkih ili fizikalnih metoda zajedno s kemijskim metodama. Uspješnost pojedinih protokola za izolaciju proteina iz mikrobiota pratili smo kvantitativnim metodama, 1 dimenzionalnom (1D) gel elektroforezom te identifikacijom proteina pomoću MS koja se temelji na matricom potpomognutoj ionizaciji laserskom desorpcijom uz analizator preleta leta (MALDI-TOF MS). Također je provedena optimizacija protokola za digestiju proteina tripsinom na filtru (engl. Filter Aided Sample Preparation – FASP). Najefikasniji protokol za izolaciju proteina bio je temeljen na homogenizaciji stanica kuglicama u kombinaciji s puferom za razbijanje stanica koji sadrži kaotrope te neutralni deterdžent ili ionski deterdžent.Faecal microbiota proteomics (metaproteomics) occupies an important area in the research of many diseases pathogenesis as well as in early diagnosis. Often, in these studies, model organisms such as rats, are used. Moreover, an increasingly important technology platform for rapid, comprehensive and precise definition of the metaproteome, which is a complement of the total microbial proteome, is mass spectrometry (MS). The composition of the rat faecal microbiota is very complex and includes various types of bacteria, yeasts, and viruses. Therefore, for the purpose of proteomic research, it is necessary to develop efficient methods for isolation of different types of microorganisms, which includes different approaches for cell lysis and isolation of molecular constituents from these cells, for example, total protein. Some of the methods used for isolation of microbial proteins that are compatible with MS include sonication and homogenization by ceramic beads. Adequate protocol optimization for metaproteome analysis is crucial for successful and unambiguous identification. Therefore, this thesis presents development and technical optimization of the protocol for the isolation of the total protein complement of the faecal rat microbiota. For this purpose, we used a mix of bacterial and yeast cultures that simulated the composition of the faecal rat microbiota. Microorganisms’ cells were lysed by combinations of mechanical or physical methods and chemical methods. The success of all microbial protein isolation protocols was assessed by protein quantification methods, 1 dimensional (1D) gel electrophoresis and protein identification by MS-based technique known as matrix assisted laser desorption/ionization time of flight (MALDI-TOF MS). Filter Aided Sample Preparation (FASP) protein digestion protocol development and optimization was also performed. The most efficient protein isolation protocol was based on the homogenization of cells using ceramic beads combined with a lysis buffer containing chaotropic agents and a neutral detergent or ionic detergent
Uloga i važnost procesa transfera tehnologije na sveučilištima
No full textU svijetu u kojem tržištem vladaju velike tvrtke, sposobnost inoviranja ima značajan
utjecaj na ekonomski napredak. Ovdje veliku ulogu igraju i sveučilišta koja su u
posljednja dva desetljeća postali jedni od glavnih aktera u komercijalizaciji proizvoda
kroz proces transfera tehnologije. Stoga je cilj ovog rada na razumljiv način dati
osnovne informacije o važnim pojmovima koji se provlače kroz proces transfera
tehnologije – patenta kao jednog od oblika intelektualnog vlasništva, njegovog
licenciranja i sve to uklopiti u proces transfera tehnologije koji se odvija na
sveučilištima. Na taj način će se obraditi uloga i važnost sveučilišta kao institucija.
Također, dat će se i kratak uvid u stanje u području biotehnologije i u Republici
Hrvatskoj. Relevantni podaci u tablicama vrijede za područje Europe, a dobiveni su
pretragom službenih europskih statističkih baza podataka.In a world where the market is dominated by large corporations the ability to innovate
has a significant impact on economy development. Universities have also played a
major role in the past two decades and have become one of the major actors in
product commercialization through technology transfer. Therefore, the aim of this
paper is to provide basic information on one of the terms that are part of technology
transfer – patent, as one of the forms of intellectual property, its licensing and fitting it
into the process of technology transfer at universities. The role and importance of
universities as institutions in this process will also be discussed. A brief overview of
technology transfer in biotechnology and Republic of Croatia will also be given.
Relevant data in the tables are valid for Europe and were collected by searching the
official European statistical databases
Extraction and Quantification of Metabolites from Olive Leaves Cultivated in Istria Region
No full textLišće maslina, predstavlja lako dostupnu i jeftinu sirovinu za izolaciju bioaktivnih spojeva. Ekstrakt lišća masline izrazito je bogat fenolnim spojevima koji imaju farmakološka svojstva, poput antioksidativnih, protuupalnih, antimikrobnih i antivirusnih djelovanja. Oleuropein, kao najzastupljeniji spoj u ekstraktu, pripada skupini sekoiridoida i osim prije navedenih svojstava pokazuje kardioprotektivne i neuroprotektivne učinke, djeluje kao vazodilatator, smanjuje razinu kolesterola i štiti organizam od kognitivnog propadanja. Svrha ovog rada je ekstrakcija, izolacija i kvantifikacija oleuropeina, tirosola, 3-kumarinske kiseline, para-hidroksifeniloctene kiseline, 3-hidroksitirosola i pinorezinola iz tri sorte lišća maslina (Drobnica, Buţa(Labin) i nepoznata sorta (Umag)) s područja Istre. Kvalitativna i kvantitativna analiza ekstrakta lišća maslina provedena je korištenjem kromatografije ultra visoke djelotvornosti (UHPLC). Specifični cilj bio je ispitati utjecaj 3 različita principa ekstrakcije (ekstrakcija s 80%-tnim etanolom pri temperaturi od 70 °C, ekstrakcija s 80%-tnim etanolom pri temperaturi od 78 °C i ekstrakcija s destiliranom vodom pri temperaturi od 90 °C, u trajanju od 4 sata) na koncentraciju fenolnih spojeva u ekstraktu lišća masline i odrediti kojim se principom ekstrakcije i kod koje sorte maslina dobije njihova najveća koncentracija. Dobiveni rezultati su pokazali da koncentracija fenolnih spojeva u ekstraktu lišća masline varira i ovisi o sorti (genetskoj osnovi), starosti masline i uvjetima ekstrakcije. Najveće koncentracije oleuropeina i pinorezinola dobivene su koristeći 80%-tni etanol kao otapalo pri temperaturi od 70 °C, dok su najveće koncentracije 3-hidroksitirosola i tirosola dobivene pri ekstrakciji s destiliranom vodom pri temperaturi od 90°C, za sve tri sorte. Najveća koncentracija oleuropeina iznosi 35,28 mg/g i dobivena je kod stare nepoznate sorte masline s područja Umaga, a najveća koncentracija 3-hidroksitirosola dobivena je kod Drobnice, stare sorte maslinovog lišća s područja Labina i iznosi 2,91 mg/g. Toksičnost i sigurnost otapala koja se koriste u ekstrakciji predstavlja problem u upotrebi ovakvih ekstrakta kao dodataka prehrani, terapeutika i kozmetičkih proizvoda kod ljudi. Stoga su u ovom radu korišteni etanol i voda kako bi dobiveni ekstrakti bili sigurni za upotrebu i poboljšali zdravlje. U budućim istraţivanjima potrebno je optimizirati metode ekstrakcije i ispitati in vitro i in vivo potencijalni terapeutski utjecaj identificiranih komponenti na zdravlje ljudi.Olive leaves represent an easily accessible and inexpensive raw material for the isolation of bioactive compounds. Olive leaf extract is extraordinarily rich in phenolic compounds that have many pharmacological properties, such as antioxidative, anti-inflammatory, antimicrobial and antiviral properties. Oleuropein, as the most common compound in the extract, belongs to secoiridoids and in addition to the above mentioned properties, exhibits cardioprotective and neuroprotective effects, functions as a vasodilator, reduces cholesterol levels and protects the organism from cognitive decline. The purpose of this study is the extraction, isolation and quantification of oleuropein, tyrosol, 3-coumaric acid, para-hydroxyphenylacetic acid, 3-hydroxytyrosol and pinoresinol from three varieties of olive leaf (Drobnica, Buţa (Labin) and unknown variety (Umag)) from the area of Istria. Qualitative and quantitative analysis of olive leaf extract was performed using Ultra-high performance liquid chromatography (UHPLC).The specific aim was to examine the influence of solvent and temperature on the extraction (80% ethanol extraction at 70 °C, 80% ethanol extraction at 78 °C (reflux) and extraction with distilled water at 90 °C, for a period of 4 hours) on the concentration of phenolic compounds in olive leaf extract, and determine which extraction technique results in their highest concentration, as well as from which olive variety. The obtained results have shown that the concentration of phenolic compounds in olive leaves varies and depends on the variety (genetic basis), the age of the olive tree and the applied extraction method. The highest concentrations of oleuropein and pinoresinol were obtained using the 80% ethanol as a solvent at a temperature of 70 °C, while the highest concentrations of 3-hydroxytyrosol and tyrosol were obtained by extraction with distilled water at the temperature of 90 °C, in all three varieties. The highest oleuropein concentration is 35,28 mg/g obtained from old unknown olive variety from the Umag area, and the highest concentration of 3-hydroxytyrosol was obtained in the Drobnica variety, a old varietal olive leaf from the Labin area, amounting to 2,91 mg/g. The toxicity and safety of solvents used in extraction represent a problem in using such extracts as dietary supplements, as well as therapeutic and cosmetic products for human consumption. Therefore, ethanol and water have been used in this study so that obtained extracts could be used to improve human health. In future research it is necessary to optimize the extraction methods and to fully examine the potential therapeutic effect of identified components on human health, in vitro and in vitro
Effects of oxidative stress on development of behavioral sensitization in Drosophila melanogaster
No full textPovećanje dopaminske neurotransmisije inducirano psihostimulansima predstavlja jedan od inicijalnih faktora za razvoj ovisnosti. Budući da oksidativni metabolizam dopamina stvara reaktivne kinone i slobodne radikale, istraživanja sugeriraju da oksidativni stres ima utjecaja na bihevioralne promjene uključujući lokomotornu senzitizaciju. Istraživanja na glodavcima su pokazala kako administracije kokaina (COC) ili metamfetamina (METH) povećavaju oksidativne markere u regiji mozga koja je važna za ekspresiju lokomotorne senzitizacije. Svrha diplomskog rada je definirati ulogu povećanog i smanjenog oksidativnog statusa na razvoj lokomotorne senzitizacije na modelnom organizmu vinske mušice (Drosophila melanogaster).
Mušice su prehranom tretirane s prooksidansima i/ili antioksidansima prije i tijekom administracije volatiliziranog COC ili METH te su praćene promjene u lokomotornoj aktivnosti. Administrirane su dvije iste doze psihostimulansa kroz određeni vremenski interval kako bi pratili razvoj lokomotorne senzitizacije. Mjerenjem oksidativnih markera (aktivnost katalaze CAT i superoksid dismutaze SOD; koncentracija vodikovog peroksida H2O2 i superoksidnog anion radikala O2●- ) ispitivali smo kako smanjenje ili povećanje oksidativnog statusa inducirano tretmanom s prooksidansima i antioksidansima dovodi do promjena u lokomotornoj aktivnosti na ponovljene administracije psihostimulansa.
Razvoj lokomotorne senzitizacije na COC i METH je spriječen kod mušica tretiranih antioksidansima, kao i kod mušica tretiranih s prooksidansima. Dodatno, pokazali smo da kombinirani tretman s prooksidansima i antioksidansima dovodi do obnavljanja lokomotorne senzitizacije na METH. Mjerenjem parametara oksidativnog statusa uočili smo razlike između tretmana, kao i razlike između COC i METH. Naši rezultati sugeriraju da egzogena primjena prooksidansa ili antioksidansa dovodi do promjena u oksidativnom statusu što se očituje na sprječavanje lokomotorne senzitizacije. Identifikacija promjena u oksidativnom statusu koje dovode do promjena u bihevioralnom odgovoru će pomoći objasniti kompleksnost mehanizama ovisnosti te poticati razvoj učinkovitih terapija za svladavanje ovisnosti.Psychostimulants induced increase in dopamine neurotransmission is considered to be one of the initial factors for the development of addiction. Since oxidative metabolism of dopamine produces reactive quinones and free radicals, studies suggest that oxidative stress has implications for behavioral changes including locomotor sensitization. Research on rodents has shown that administration of cocaine (COC) or methamphetamine (METH) increases oxidative markers in the brain region which is important for expression of locomotor sensitization.
The purpose of the master thesis is to define the role of increased and decreased oxidative status on the development of locomotor sensitization in fruit fly (Drosophila melanogaster).
Flies were fed with pro-oxidants and/or antioxidants before and during administration of volatilized COC or METH and changes in locomotor activity were examined. To monitor the development of locomotor sensitization, two doses of psychostimulant were administered over a certain time interval. By measuring oxidative markers (activity of catalase CAT and superoxide dismutase SOD; concentration of hydrogen peroxide H2O2 and superoxide anion radical O2●-), we investigated how decrease or increase in oxidative status, induced by pro-oxidant or antioxidant treatment, leads to changes in locomotor activity after repeated administration of psychostimulants.
Locomotor sensitization to COC and METH is abolished in flies treated with antioxidants, as well as in flies treated with pro-oxidants. In addition, we report that combined treatment with pro-oxidants and antioxidants restores locomotor sensitization to METH. We observed differences between treatments, as well as differences between COC and METH.
Our results suggest that the exogenous exposure to pro-oxidants or antioxidants results in changes in oxidative status, which is evident in the abolished locomotor sensitization. Identifying changes in oxidative status that lead to changes in behavioral response will help explain the complexity of addiction mechanisms and encourage the development of effective therapy for addiction
Differences in the development of behavioral sensitization to cocaine compared to methamphetamine in Drosophila melanogaster
No full textPsihomotorički stimulansi, poput kokaina i metamfetamina utječu na neuronsku mrežu mijenjajući dopaminergičnu i serotonergičnu transmisiju uzrokujući neuroadaptivne promjene moždanih funkcija koje rezultiraju promijenjenim motoričkim sposobnostima. Istraživanja na vinskoj mušici - Drosophili melanogaster pokazuju da opetovana administracija volatiliziranog kokaina izaziva progresivno povećanje lokomotornog odgovora definirano kao lokomotorna senzitizacija. Ponovljenom administracijom, kokain aktivira dopaminsku transmisiju, no posljedice akutne ili ponovljenih administracija volatiliziranog metamfetamina kod Drosophile još nije poznat. Iako postoji nekoliko metoda za mjerenje lokomotorne senzitizacije u Drosophili, niti jedna nije u potpunosti zadovoljila sve uvjete za kvantitativno, reproducibilno, objektivno i visokoprotočno mjerenje.
U ovom diplomskom radu razvili smo i validirali novu visokoprotočnu platformu koja će objektivno i reproducibilno inducirati i na individualnoj razini kvantificirati lokomotornu senzitizaciju Drosophile melanogaster na kokain i metamfetamin. Definirali smo razlike u mehanizmu djelovanja kokaina i metamfetamina te ulogu cirkadijalnih gena i gena dopaminskog sustava u regulaciji razvoja lokomotorne senzitizacije u odgovoru na te psihostimulanse. Naši rezultati potvrđuju kako važnu ulogu u regulaciji lokomotorne senzitizacije na kokain imaju Clock, period i cycle geni, te potencijalno VMAT u dopaminskim i Clock pozitivnim neuronima. Lokomotorna senzitizacija na metamfetamin ovisi o različitim genima od senzitizacije na kokain - Clkjrk te potencijalno D1 receptoru i VMAT u dopaminskim neuronima. Dakle, regulacija senzitizacije na kokain i metamfetamin se razlikuje u genetskim mehanizmima. Utvrdili smo da su za razvoj senzitizacije na oba psihostimulansa važni dopaminski transporteri - DAT te VMAT u period pozitivnim neuronima. Drosophila melanogaster kao modelni organizam u ispitivanju razvoja lokomotorne senzitizacije doprinijet će genetskom probiru i identifikaciji potencijalno ključnih gena koji sudjeluju u regulaciji razvoja bihevioralne senzitizacije na psihostimulanse što će u budućnosti pomoći razumijevanju molekularnih mehanizama u podlozi razvoja ovisnosti.Psychomotor stimulants, such as cocaine and methamphetamine, affect the neuronal network by altering dopaminergic and serotonergic transmission causing neuroadaptive brain function changes resulting in modulation in motor abilities. Research work in Drosophila melanogaster shows that repeated administration of volatilized cocaine leads to a progressive increase in locomotor response defined as locomotor sensitization. With repeated administration cocaine activates dopamine transmission leading to locomotor sensitization, but consequences of acute and repeated administrations of volatilized methamphetamine in Drosophila are not known. Although there are several methods for measuring locomotor sensitization, none fulfills all the criteria being quantitative, reproducible, objective and high-throughput.
In this thesis we have developed and validated a new high-throughput platform that objectively and reproducibly induces locomotor sensitization to cocaine and methamphetamine at population and individual level in Drosophila melanogaster. We defined the differences in the action of cocaine and methamphetamine and the role for circadian genes and the dopaminergic system in regulating the development of locomotor sensitization in response to these psychostimulants. Our results confirm important role for Clock, period and cycle genes in regulating locomotor sensitization on cocaine, and likely VMAT in dopaminergic and Clock positive neurons. Locomotor sensitization to methamphetamine depends on different set of genes than sensitization to cocaine, mainly Clock and likely D1 receptor and VMAT in dopaminergic neurons. Thus, regulation of sensitization to cocaine and methamphetamine differs in genetic mechanisms. We have found that dopamine transporter DAT and VMAT in per positive neurons are important for the development of sensitization to both psychostimulants. Drosophila melanogaster as a model organism for the study of locomotor sensitization will contribute to the genetic screening and identification of potentially critical genes involved in the regulation of behavioral sensitization to psychostimulants, which in the future will help in understanding molecular mechanisms underlying addiction
Gluten and gliadin effects on proliferation of fibroblasts and colon cancer tumour cell lines in vitro
No full textPšenica je jedna od najvažnijih žitarica u današnjoj prehrani koja se koristi
za izradu kruha i mnogih drugih jela te je ključni izvor hrane diljem svijeta.
Najvažniji skladišni protein zrna pšenice, koji se nalazi isključivo u škrobnom
endospermu, je gluten - smjesa glijadina i glutenina. Ove komponente pšenice
izuzetno su opasne za ljude s intolerancijom na gluten, odnosno s
dijagnosticiranom celijakom. Celijakija je bolest koja utječe na oko 1% zapadne
populacija. Unatoč tomu što su do danas otkrivene mnoge bolesti čiji je uzrok
konzumacija glutena u pojedinaca, povezanost konzumacije glutena s nastankom
raka još nije potpuno jasna. Cilj ovog rada bilo je ispitivanje antiproliferativnog
učinka glutena i glijadina na tri odabrane tumorske stanične linije HCT-116,
Caco-2 i SW620 te jednu staničnu liniju fibroblasta HFF u uvjetima in vitro te
analiza eventualne razlike u ekspresiji proteina važnih u patogenezi celijakije
(TGM2) i imunosnom odgovoru (NFkB i RANKL). Niske koncentracije glutena i
glijadina s niskim koncentracijama etanola u otopini djelovale su proliferativno na
sve testirane stanice a učinak etanola u većim koncentracijama pokazao je
antiproliferativni učinak na stanice . Također, utvrđene su manje promjene u
razinama proteina TGM2, NF-kB i RANKL nakon tretmana glutenom i glijadinom
što je u većoj mjeri pratilo efekte u tretmanu s pripadajućim kontrolama etanola.
Jedino je glijadin inducirao statistički značajan pad razine NF-kB nakon 24h
tretmana stanica HCT-116 što se moglo razlučiti od učinka pripadajuće kontrole
etanola.Wheat is one of the most important cereal in today's diet that is used to
make bread and many other foods and is a key source of food all over the world.
The most important storage wheat protein, found exclusively in the starch of
endosperm, is gluten - a mixture of gliadine and glutenine. These wheat
components are extremely dangerous for people with gluten intolerance, or with
diagnosed celiac disease. Celiac disease is a disease that affects about 1% of
Western population. Despite the fact that are many diseases whch are known
that are caused by gluten consumption in individuals, the association of glutenrelated
consumption with cancer is still not clear. The aim of this paper was to
investigate the antiproliferative effect of gluten and gliadine on the three selected
tumor cell lines HCT-116, Caco-2 and SW620 and one cell line of HFF fibroblasts
in in vitro conditions and analysis of possible differences in protein expression
important for celiac disease (TGM2) and immune response (NFkB and RANKL).
Low concentrations of gluten and gliadine with low concentrations of ethanol in
the solution acted proliferatively on all tested cells and the effect of ethanol at
higher concentrations showed antiproliferative effect on the cells. Also, minor
changes in levels of TGM2, NfκB and RANKL levels were observed after gluten
and gliadine treatment, which more closely followed the effects of treatment with
the corresponding ethanol controls. Only gliadine induced a statistically
significant decrease in NfκB levels after 24h treatment in HCT-116 cells that
could be distinguished from the effect of the associated ethanol control