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Highly regulated intracellular calcium entry affects numerous cellular physiological events. External regulation of intracellular calcium signaling presents a great opportunity for the artificial regulation of cellular activity. Calcium entry can be mediated by STIM proteins interacting with Orai calcium channels; therefore, the STIM1-Orai1 pair has become a tool for artificially modulating calcium entry. We report on an innovative genetically engineered protease-activated Orai activator called PACE. CAD self-dimerization and activation were inhibited with a coiled-coil forming peptide pair linked to CAD via a protease cleavage site. PACE generated sustained calcium entry after its activation with a reconstituted split protease. We also generated PACE, whose transcriptional activation of NFAT was triggered by PPV or TEV protease. Using PACE, we successfully activated the native NFAT signaling pathway and the production of cytokines in a T-cell line. PACE represents a useful tool for generating sustained calcium entry to initiate calcium-dependent protein translation. PACE provides a promising template for the construction of links between various protease activation pathways and calcium signaling.

Cilj ovog istraživanja je bio utvrditi suradljivost postmenopauzalnih bolesnica s ranim invazivnim hormonski osjetljivim rakom dojke na adjuvantnu terapiju s nesteroidnim aromataznim inhibitorima, vitaminom D i kalcijem (ako je to propisano od onkologa) u svakodnevnoj kliničkoj praksi u Hrvatskoj. Cilj je također bio utvrditi da li se onkolozi u Hrvatskoj pridržavaju smjernica o koštanom zdravlju te smjernica o kliničkom praćenju bolesnica s ranim rakom dojke. U prospektivno, neintervencijsko istraživanje je uključeno 438 bolesnica koje su uzimale nesteroidni aromatazni inhibitor anastrozol ili letrozol te su praćene u prosjeku 23.5 (±4.9) mjeseci. Tijekom istraživanja, bolesnic su u prosjeku imale 5.3 pregleda. Suradljivost bolesnica procijenjena je primjenom metode samoizvještaja. Bolesnica se smatrala suradljivom ako je prema samoizvještaju uzela ≥80% propisane doze inhibitora aromataze, vitamina D i kalcija. U našem istraživanju utvrdili smo visoki stupanj suradljivosti na aromatazne inhibitore, odnosno od ukupnog broja bolesnica suradljivo na aromatazne inhibitore je bilo 425/438 (97%) bolesnica, a nesuradljivo je bilo njih 13 (3%). Kalcij i vitamin D su propisani od strane onkologa u samo 329/438 (75.1%) bolesnica. Bolesnica suradljivih na kalcij i vitamin D je bilo 291/329 (88.4%), a nesuradljivih 38/329 (11.6%). Denzitometrija skeleta je napravljena kod samo 180/438 (41.1%) bolesnica. Osteoporoza je utvrđena kod 24 bolesnice (5.5%), imajući na umu da 258/438 (59%) bolesnica nije imalo napravljenu denzitometriju. Bifosfonati su propisani kod 54/438 (12.3%) bolesnica, dok je samo 19 (35.2%) imalo osteoporozu. S druge strane, neke bolesnice s osteoporozom nisu primale bifosfonate. Ovi rezultati pokazuju da je kod značajnog dijela bolesnica u našem istraživanju (36.5%) ugroženo koštano zdravlje zbog nepridržavanja smjernica sa strane onkologa (24.9% bolesnica nije imalo propisanu terapiju s kalcijem i vitaminom D) te, s druge strane, zbog loše suradljivosti samih bolesnica na kalcij i vitamin D (11.6% onih kojima je propisan kalcij i vitamin D nisu bile suradljive). Podatak da je samo 41.1% bolesnica imalo napravljenu denzitometriju skeleta, koja je obvezna kod svih bolesnica koje uzimaju aromatazne inhibitore, te da neke bolesnice s osteoporozom ne uzimaju bifosfonate, također pokazuje da se onkolozi slabo pridržavaju smjernica o koštanom zdravlju. Kompromitirano koštano zdravlje povezano je sa značajnim smanjenjem kvalitete života te značajno povećava troškove liječenja. Analiza je također pokazala da se onkolozi slabo pridržavaju smjernica o praćenju bolesnica s ranim rakom dojke jer su se tijekom kliničkog praćenja redovito radile suvišne i nepotrebne pretrage: RTG pluća, ultrazvuk abdomena i tumorski biljezi CEA i CA 15-3, iako to nije propisano u hrvatskim niti u međunarodnim smjernicama. Bolje pridržavanje smjernica sa strane onkologa o praćenju može značajno smanjiti troškove, te poboljšati kvalitetu života bolesnika, a bez negativnog utjecaja na preživljenje., The aim of this analysis was to evaluate adherence of postmenopausal patients with early invasive hormone-dependent breast cancer to treatment with adjuvant non-steroidal aromatase inhibitors, vitamin D and calcium (if they were prescribed by their oncologists) in every day clinical practice in Croatia. The aim of this analysis was also to evaluate adherence of Croatian oncologists to the bone health guidelines and to the follow-up criteria as suggested by the current national and international guidelines for women with breast cancer receiving adjuvant endocrine therapy. In this prospective, noninterventional study a total of 438 postmenopausal patients receiving adjuvant endocrine treatment with non-steroidal aromatase inhibitors anastrozol or letrozol were included. Patients were followed up for an average 23.5± 4.9 months. Average number of oncological visits was 5.3. Patients who took ≥80% of the prescribed dose were considered adherent. In our study self-reported adherence to aromatase inhibitors was 97% (425/438 patients) and there were only 13 (3%) non-adherent patients. Vitamin D and calcium were prescribed to only 329/438 (75.1%) patients at some point during the study. Self-reported adherence to vitamin D and calcium was 88.4%, and there were 38/329 (11.6%) non-adherent patients. Osteoporosis was diagnosed in 24 patients (5.5%) of the total study population, bearing in mind that 258/438 (59%) patients did not have densitometry. Bisphosphonates were prescribed to 54/438 (12.3%) patients, whilst only 19 (35.2%) of those had osteoporosis. On the other hand, there were still patients with osteoporosis who were not treated with bisphosphonates. These results shows that in significant number of our patients (36.5%) bone health was compromised because of lack of oncologists' adherence to the bone health guidelines (24.9% of patients did not have vitamin D and calcium prescription) and because of patientsʼ non-adherence to vitamin D and calcium (11.6%). Densitometry was performed in only 180/438 patients (41.1%) and this data also shows as above, bad compliance to the bone health guidelines by oncologists. Compromised bone health is often associated with deteriorated patients' quality of life, and increased medical costs what is important issue in already overstretch health care budgets in transitional countries. Our analysis among postmenopausal women with breast cancer receiving adjuvant endocrine therapy also shows that more unnecessary and unproven follow-up procedures (chest radiograph, abdominal ultrasound, tumor markers CEA and CA 15-3) were done compared to the guidelines’ recommendations. The use of diagnostic tests not proven to increase survival during standard follow-up of early breast cancer patients, is not cost-effective and has become a large, unnecessary burden for overstretched and limited transitional countriesʼ budgets. Improved adherence to the current guidelines can substantially cut medical costs, reduce risks and improve patient quality of life without adversely affecting outcome.

Napitak od kombuche predstavlja popularni napitak od fermentiranog čaja, funkcionalnu namirnicu s raznim dobrobitima poput antibakterijskog i antioksidativnog svojstva te podupiranja imunosnog sustava i metabolizma. Dobrobiti kombucha napitka kriju se u bioaktivnim komponenama koje sadrži, a one uključuju aminokiseline, vitamine, polifenole, vlakna i organske kiseline. Među njima, prisutni su i važni biogeni elementi kao što su željezo, mangan i cink. Ti elementi neophodan su dio enzima ključnih u održavanju različitih biokemijskih procesa u organizmu, stoga je njihova dnevna konzumacija važna za optimalno zdravlje čovjeka. Zbog načina proizvodnje i vrste početnih sastojaka korištenih u proizvodnji, kombucha napitci su po svome sastavu raznoliki. U ovome radu prvi su se puta tehnikom masene spektrometrije s induktivno spregnutom plazmom (ICP-MS) analizirali minerali kalcija, željeza, magnezija, cinka i mangana u pet različitih uzoraka kombucha napitka, od kojih je jedan bio kontrolni uzorak. Analiza uzoraka omogućila je praćenje promjena koncentracija minerala u svakome uzorku te uvid u utjecaj različitih dodataka u uzorcima na koncentraciju samih minerala. Kombucha drink is a popular drink made of fermented tea. This functional food has various health benefits including antibacterial and antioxidant properties and a role in supporting the immune system and metabolism. The benefits of the kombucha drink are founded on the bioactive components it contains, which include amino acids, vitamins, polyphenols, fibers, and organic acids. Among them, important biogenic elements such as iron, manganese, and zinc are also present. As a functional part of various enzymes, these elements are crucial in maintaining various biochemical processes in the body, therefore their daily consumption is important for optimal human health. Kombucha drinks are diverse in their composition due to differences in the production method and the type of used ingredients. In this paper, the minerals calcium, iron, magnesium, zinc, and manganese were, for the first time, analyzed with the inductively coupled plasma mass spectrometry method (ICP-MS) in five different samples of a kombucha drink, one of which was used as a control sample. The sample analysis enabled the monitoring of the change in mineral concentrations in each sample and observing the influence of different additives in the samples on the concentration of the minerals themselves.

Aktivnost mnogih citosolnih proteinov je odvisna od koncentracije kalcija. Eden izmed pomembnih znotrajceličnih proteinov, ki se odziva na kalcijeve ione, je α-aktinin-4. α aktinini so citoskeletni proteini iz družine spektrinov, ki igrajo povezovalno vlogo med različnimi celičnimi strukturami. Sestavljajo jih štiri regije in sicer aktin vezavna domena, paličasta regija s štirimi spektrinskimi ponovitvami, povezovalna regija “vrat” in kalmodulinu podobna domena. α aktinine delimo na dve skupini glede na njihovo tkivno specifičnost. Mišični izoobliki, α-aktinina 2 in 3, ne vežeta Ca2+-ionov. Po drugi strani ne-mišični izoobliki, α aktinina-1 in -4, vežeta Ca2+-ione, kar vpliva na njuno strukturo in posledično na prostorsko ureditev z njima povezanih aktinskih filamentov. Aktivno obliko α-aktinina-4 predstavlja antiparalelen homodimer z aktin-vezavno domeno na obeh koncih, katerega osrednja vloga je prečno povezovanje aktinskih filamentov v snope in mreže. Za razliko od sorodnega α-aktinina-1 je prisoten tudi v nukleoplazmi, kjer naj bi deloval kot transkripcijski ko-aktivator številnih genov, vpletenih v tumorske spremembe celic. Dosedanje raziskave so pokazale, da vezava Ca2+-iona v kalmodulinu podobno domeno α aktinina-1 sproži konformacijske spremembe v tem delu molekule, ki se po predvidevanjih prenesejo na aktin-vezavo domeno in posledično reorganizacijo aktinskega citoskeleta. Z namenom primerjati Ca2+-vezavne lastnosti ne-mišičnih α aktininov smo se osredotočili na še neokarakterizirano izoobliko 4. V okviru magistrskega dela smo pripravili proteinske kristale dimerne oblike α-aktinina-4, ki vsebuje vse domene udeležene v zgoraj opisani interakciji, ter posneli set difrakcijskih podatkov do ločljivosti 2,9 Å. Z bioinformatsko analizo, kjer smo primerjali zaporedja kalmodulinu podobnih domen različnih izooblik, smo določili tiste aminokislinske ostanke v kalmodulinu podobni domeni α aktinina-4, ki so najverjetneje ključni pri koordinaciji Ca2+ iona. Podobno kot pri α aktininu-1 je tudi pri tej izoobliki pri vezavi Ca2+-ionov aktivna le ena izmed štirih t. i. EF-dlani. Da bi to hipotezo preverili smo v kalmodulinu podobno domeno α aktinina-4 uvedli različne točkovne mutacije, mutantne oblike pa nato okarakterizirali z uporabo izotermne titracijske kalorimetrije. Potrdili smo, da je res aktivno le eno od potencialnih vezavnih mest za Ca2+, afiniteta za vezavo pa je 4-krat nižja kot pri α aktininu-1. Predvidevamo, da je opažena razlika eden izmed faktorjev, ki vplivajo na različno obnašanje teh dveh izooblik ter posledično na njuno različno preferenčno vključenost v citoskeletne strukture kot so fokalni stiki in ukrivljene membranske strukture. The activity of many cytosolic proteins depends on calcium concentration. One of the important intracellular proteins that respond to calcium ions is α-actinin-4. α actinins are cytoskeletal proteins of the spectrin family that play a crosslinking role between different cellular structures. They consist of four regions, namely an actin binding domain, a rod region with four spectrin repeats, linker region “neck” and a calmodulin-like domain. α actinins are divided into two groups according to their tissue specificity. The muscle isoforms, α-actinin-2 and -3, do not bind Ca2+-ions. On the other hand, the non-muscle isoforms, α-actinin-1 and -4, bind Ca2+-ions, which affects their structure and consequently the spatial arrangement of the actin filaments connected to them. The active form of α-actinin-4 is an antiparallel homodimer with an actin-binding domain at both ends, the central role of which is the cross-linking of actin filaments into bundles and networks. Unlike the related α-actinin-1, it is also present in the nucleoplasm, where it is thought to act as a transcription co-activator for many genes involved in tumorigenic cell changes. Research so far has shown that the binding of Ca2+-ion in the calmodulin-like domain of α-actinin-1 triggers conformational changes in this part of the molecule, which are predicted to be transferred to the actin-binding domain and the resulting reorganization of the actin cytoskeleton. To compare the Ca2+-binding properties of non-muscle α actinins, we focused on the yet uncharacterized isoform 4. As part of the master's thesis, we prepared protein crystals of the dimeric form of α-actinin-4, which contains all the domains involved in the interaction described above, and recorded a set of diffraction data up to a resolution of 2.9 Å. By bioinformatic analysis, where we compared the sequences of the calmodulin-like domains of different isoforms, we determined those amino acid residues in the calmodulin-like domain of α-actinin-4, which are most likely to be crucial in the coordination of the Ca2+-ion. Like α-actinin-1, only one of the four EF1-hands is active in binding Ca2+-ions. In order to test this hypothesis, we introduced various point mutations in the calmodulin-like domain of α-actinin-4, and the mutant forms were then characterized using isothermal titration calorimetry. We confirmed that only one of the potential binding sites for Ca2+ is indeed active, and the binding affinity is 4 times lower than that of α-actinin-1. We assume that the observed difference is one of the factors influencing the different behavior of these two isoforms and consequently, to their different preferential involvement in cytoskeletal structures such as focal junctions and curved membrane structures.

Namen: Roženični epitel je površinski roženični sloj, ki ščiti roženico pred zunanjim okoljem in je podvržen številnim mehaničnim, kemičnim, biološkim in fizikalnim dejavnikom. Nekateri izmed omenjenih dejavnikov so znani aktivatorji ali modulatorji mehanosenzitivnih kationskih kanalov, med katere prištevamo receptor prehodnega potenciala vaniloida tipa 4 (TRPV4) in Piezo1 kanale. Aktivacija mehanoreceptorjev vodi v aktivacijo znotrajceličnih poti in procesov, ki so povezani z motnjami v delovanju roženice, vnetjem, hiperalgezijo/nocicepcijo in disfunkcijo tkiva. V doktorski disertaciji smo se zato osredotočili na identifikacijo in lokalizacijo mehanosenzitivnih kationskih ionskih kanalov v celicah roženičnega epitela, ovrednotili njihov funkcionalni pomen in opredelili vlogo TRPV4 kanalov v parakrinem signaliziranju ter izločanju vnetnih in bolečinskih mediatorjev. Metode: Roženicam miši linije C57BL/6, transgene miši z izničenim genom za TRPV4 (TRPV4-/-) in transgene miši, ki smo jim gene za TRPV4 zamenjali s fluorescenčno označenimi bakterijskimi geni, ki kodirajo TRPV4 (TRPV4eGFP), smo encimsko odluščili sloj roženičnega epitela in jih uporabili za in situ poskuse. Celice roženičnega epitela, ki smo jih uporabili za in vitro poskuse, smo disociirali, nasadili na silikonske membrane ali objektna stekelca in jih gojili v inkubatorju 4–7 dni pri 37 °C. Celice roženičnega epitela smo izpostavili različnim specifičnim agonistom in antagonistom, cikličnemu vzdolžnemu mehaničnemu raztezku in ultravijoličnemu B (UVB) sevanju. S pomočjo optičnega slikanja smo ovrednotili njihov vpliv na homeostazo kalcijevih ionov (Ca2+) v različnih eksperimentalnih pogojih. Za določitev izražanja genov za izbrane proteine in njihovo lokalizacijo smo uporabili tehniki imunooznačevanja in semikvantitativne verižne reakcije s polimerazo v realnem času (qRT-PCR). Izločanje ATP iz celic roženičnega epitela po izpostavitvi različnim dražljajem smo v supernatantu kvantificirali z luciferin-luciferazno reakcijo, izločanje vnetnih molekul in citokinov pa s pomočjo kemiluminiscence. Rezultati: Celice roženičnega epitela močno izražajo TRPV4 in Piezo1 kanale. Prvi se v roženičnem epitelu pojavljajo v neenakomernem, drugi pa v bolj enakomernem vzorcu glede na sklade celic roženičnega epitela. Skladno s tem sta specifična agonista GSK101 in YODA1 povzročila povečanje koncentracije prostih znotrajceličnih kalcijevih ionov ([Ca2+]i) v celicah roženičnega epitela. Signali Ca2+ so bili zmanjšani po izpostavitvi mehanskemu hipotoničnemu stresu ob uporabi specifičnega TRPV4 antagonista (HC-06) in po izpostavitvi mehanskemu raztezku ob uporabi specifičnega Piezo1 antagonista (GsMTx4). Izpostavitev temperaturi 37 °C je v celicah roženičnega epitela vodila v od TRPV4 kanalov odvisno povečanje [Ca2+]i. Aktivaciji TRPV4 kanalov je sledilo od Ca2+ odvisno izločanje ATP, ki je bilo zavrto ob farmakološki blokadi paneksinskih hemikanalov s probenecidom. Farmakološka aktivacija proteazno aktiviranih receptorjev 2 (PAR-2) je senzibilizirala TPRV4 kanale za aktivacijo z agonistom. UVB sevanje je v celicah roženičnega epitela vodilo do reverzibilnega povečanja [Ca2+]i, učinek je bil delno zmanjšan ob farmakološki blokadi TRPV4 kanalov ali njihovi genetski odstranitvi. Povečanje [Ca2+]i ob stimulaciji z UVB sevanjem je bilo primarno posledica sprostitve Ca2+ iz znotrajceličnih rezerv, predvsem iz endoplazemskega retikuluma. Tako farmakološka aktivacija TRPV4 kanalov kot UVB sevanje sta v celicah roženičnega epitela povzročila povečano izločanje nekaterih provnetnih citokinov in vnetnih mediatorjev, ki je bilo specifično glede na dražljaj in izničeno ob farmakološki blokadi TRPV4 kanalov. Zaključki: Naši rezultati kažejo, da je pri mehanotransdukciji v celicah roženičnega epitela udeleženih več različnih mehanoreceptorjev. Eni izmed najpomembnejših mehanoreceptorjev so TRPV4 kanali, ki so aktivirani z osmotskim stresom. Preko homeostaze znotrajceličnega Ca2+ so povezani z od Ca2+ odvisnim izločanjem ATP iz celic preko paneksinskih hemikanalov in so tako vključeni v purinergično avtosignalizacijo. V celicah roženičnega epitela TRPV4 kanali niso povezani z zaznavanjem mehanskega raztezka, ki ga zaznavajo Piezo1 kanali. TRPV4 kanale aktivirata tudi povečana temperatura in UVB sevanje, senzibilizira pa jih aktivacija PAR-2 receptorjev. Na škodljivo UVB sevanje se celice roženičnega epitela odzovejo z aktivacijo TRPV4 kanalov in sprostitvijo Ca2+ iz znotrajceličnih zalog. Molekule, ki se nahajajo v solzah, lahko prispevajo k od PAR-2 receptorjev odvisni regulaciji TRPV4 kanalov in tako posredno, tudi preko TRPV4 kanalov, vplivajo na delovanje roženičnega epitela v patoloških pogojih. Na roženično okolje najverjetneje vplivajo tudi vnetne molekule, sproščene iz celic roženičnega epitela ob aktivaciji TRPV4 kanalov oziroma ob izpostavitvi UVB sevanju. Vnetne molekule imajo lahko učinek na nocicepcijo, celjenje in prozornost roženice. V splošnem ta študija opredeljuje Piezo1 kanale kot pomembne mehanoreceptorje in TRPV4 kanale kot potencialno novo tarčo pri procesih, povezanih z vnetjem očesne površine in tkivno poškodbo v celicah roženičnega epitela. Purpose: Corneal epithelium – the outermost corneal layer that protects the cornea from the environment – is subjected to various mechanical, biological, chemical, and physical stressors. Many are known activators or modulators of mechanosensitive cation channels. Mechanoreceptors' activation can lead to downstream pathways that are linked to impaired corneal physiology, inflammation, hyperalgesia/nociception, and tissue dysfunction. We localised mechanotransducing cation permeable TRPV4 and Piezo1 ion channels to corneal epithelial cells, characterized their functional properties and assessed their functions in paracrine signalling and the release of inflammatory and nociceptive transmitters. Methods: Corneal epithelia from C57BL/6, TRPV4-/- and TRPV4eGFP mice were enzymatically detached from the stroma and used for in situ experiments. In vitro, epithelial cells were dissociated, plated onto silicon membranes or cover slips, and cultured for 4-7 days. Cells were subjected to either assorted agonists and antagonists, cyclic equiaxial mechanical stretch, or UVB radiation. Optical imaging assessed the properties of Ca2+ homeostasis under different experimental conditions. Immunolabeling and qRT-PCR were performed to determine the changes in gene and protein expression, and localization. For ATP and cytokine release assays, cells were dissociated, subjected to assorted stimuli and supernatants were evaluated by luciferin-luciferase and chemiluminescence reaction, respectively. Results: Corneal epithelial cells strongly express TRPV4 and Piezo1 channels, the former being localized in a non-uniform and the latter in a uniform pattern. Accordingly, the selective agonists GSK101 and YODA1 increased [Ca2+]i in corneal epithelial cells, whereas swelling and pressure induced Ca2+ signals were averted by the selective TRPV4 (HC-06) and Piezo1 inhibitor (GsMTx4), respectively. Heating of corneal epithelium to 37°C evoked TRPV4 dependent [Ca2+]i elevations. The epithelial cells responded to TRPV4 activation with massive release of ATP that was sensitive to the pannexin 1 blocker probenecid and showed Ca2+ dependence. Pharmacological activation of the protease-activated receptor-2 (PAR-2) sensitized TRPV4 for agonist activation. UVB radiation reversibly elevated [Ca2+]i, principally reflected release of Ca2+ from the pool stored intracellularly within the endoplasmic reticulum and was modestly attenuated by TRPV4 ablation or inhibition. UVB radiation and TRPV4 activation evoked stimulus-specific release of proinflammatory cytokines and inflammatory substances, effects that were antagonized by pharmacological blockade of TRPV4. Conclusions: We found that corneal mechanotransduction involves activation of multiple force-activated channels which regulate fundamental aspects of corneal epithelial biology. A principal mechanosensor is TRPV4, which is activated by osmotic stress and contributes to [Ca2+]i homeostasis through Ca2+-dependent release of ATP, purinergic auto-feedback and hemichannel (pannexin) activation. Interestingly, TRPV4 fails to mediate stretch-evoked Ca2+ responses, which are instead mediated by another mechanochannel, Piezo1. In addition to osmotic gradients, TRPV4 channels in the corneal epithelium are activated by moderate heat, UVB radiation and sensitized by the PAR-2 receptor. Our data suggest that corneal epithelial cells respond to harmful UVB radiation with parallel and additive activation of TRPV4 channels and release of Ca2+ from intracellular stores. It is possible that substances residing in tears contribute to PAR-2-mediated TRPV4 regulation in corneal epithelial cells under pathological conditions. In addition, the corneal milieu is likely to be influenced by TRPV4- and/or UVB-dependent release of cytokines and inflammatory substances, with potential effects on transparency, wound healing, and nociception. Overall, this study identifies Piezo1 as an important corneal epithelial mechanosensor that may be triggered by acute impact and TRPV4 as a potential target of ocular surface inflammation, UVB impact and tissue damage.

Jabučni ocat je jedinstveni prirodni fermentirani proizvod bogat polifenolima, organskim kiselinama te drugim farmakološko aktivnim komponentama. Zbog blagotvornog utjecaja na metabolizam lipida i glukoze te antioksidativnog i antibakterijskog svojstva upotreba mu je sve češća. U jabučnom octu se nalaze i biološki važni metali koji su neophodni za ispravno funkcioniranje ljudskog organizma. Oni djeluju kao kofaktori u mnogim enzimima, imaju antioksidativno svojstvo, te je održavanje njihovih koncentracija u organizmu neophodno u prevenciji nastanka patoloških stanja. Sadržaj pojedinih metala u uzorcima jabučnih octi određen je metodom induktivno spregnute plazme s masenom sprektrometrijom (ICP-MS). Dodatak meda i vlakna inulina u jabučni ocat omogućio je praćenje koncentracija metala te utjecaj tih dodanih komponenti. Apple cider vinegar is a unique natural fermented product rich in polyphenols, organic acids and other pharmacologically active components. Due to its beneficial effect on lipid and glucose metabolism, as well as its antioxidant and antibacterial properties, its everyday use is becoming more frequent. Apple cider vinegar also contains biologically relevant metals that are necessary for the proper functioning of the human body. They act as cofactors for many enzymes and possess antioxidant properties. Maintaining their homeostasis in the body is necessary in the prevention of different pathological conditions. The content of individual metals in apple cider vinegar samples was determined by inductively coupled plasma mass spectrometry (ICP-MS). Addition of honey and inulin fiber to the apple cider vinegars made it possible to compare the biometals' concentrations of the „basic starter ingredient“, apple cider vinegar, and monitor changes in metal concentrations in the samples with addition of other supplements (honey and inulin). Moreover, the impact of added components on the content of individual metals was also assessed and discussed.

Exposure to actual or simulated microgravity results in alterations of renal function, fluid redistribution, and bone loss, which is coupled to a rise of urinary calcium excretion. We provided evidence that high calcium delivery to the collecting duct reduces local Aquaporin 2 (AQP2)-mediated water reabsorption under vasopressin action, thus limiting the maximal urinary concentration to reduce calcium saturation. To investigate early renal adaptation into simulated microgravity, we investigated the effects of 10 days of strict bedrest in 10 healthy volunteers. We report here that 10 days of inactivity are associated with a transient, significant decrease (day 5) in vasopressin (copeptin) paralleled by a decrease in AQP2 excretion, consistent with an increased central volume to the heart, resulting in reduced water reabsorption. Moreover, bedrest caused a significant increase in calciuria secondary to bone demineralization paralleled by a decrease in PTH. Urinary osteopontin, a glycoprotein exerting a protective effect on stone formation, was significantly reduced during bedrest. Moreover, a significant increase in adrenomedullin (day 5), a peptide with vasodepressor properties, was observed at day 5, which may contribute to the known reduced orthostatic capacity post-bedrest. We conclude that renal function is altered in simulated microgravity and is associated with an early increase in the risk of stone formation and reduced orthostatic capacity post-bedrest within a few days of inactivity.

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.

Aktivni metabolit vitamina D 1, 25(OH)₂D₃ ima mnoge važne uloge u organizmu ptica i sisavaca. U intenzivnoj proizvodnji je važno pravodobno praćenje zdravlja peradi, pri čemu hematološke i biokemijske pretrage mogu biti od iznimnog značenja. Cilj ovog istraživanja bio je utvrditi utjecaj dodavanja 1, 25(OH)₂D₃-glikozida biljnog podrijetla u hranu na ukupan broj i populacije leukocita, te koncentracije kalcija, fosfora i magnezija u krvi. Istraživanje je provedeno na 10000 nesilica kokoši teške linije Ross 308. Pokusnoj skupini nesilica u hranu je umiješan 1, 25(OH)₂D₃-glikozid biljnog podrijetla, dok je kontrolna skupina konzumirala komercijalno dostupnu hranu, sukladno preporukama za genetiku. Uzorci venske krvi uzeti su punkcijom krilne vene u epruvete s litijevim heparinom kao antikoagulansom i u epruvete s gelom. Krv je uzorkovana 14., 25. i 35. dana od početka dodavanja pripravka vitamina D u hranu. U uzorcima krvi određen je ukupan broj leukocita te su iz krvnog razmaza morfološki diferencirane populacije leukocita. Izračunat je apsolutni broj pojedinih morfoloških oblika leukocita te omjer heterofila i limfocita. U uzorcima seruma određene su koncentracije kalcija, fosfora i magnezija. U ovom istraživanju dodavanje 1, 25(OH)₂D₃-glikozida biljnog podrijetla u hranu nesilica dovelo je do smanjenja ukupnog broja heterofila, limfocita i eozinofila uz povećanje postotka limfocita te povećanje koncentracije kalcija i magnezija u krvi. Na osnovi rezultata možemo zaključiti kako suplementiranje nesilica 1, 25(OH)₂D₃-glikozidom nakon 55. tjedna starosti privremeno smanjuje omjer H/L ukazujući na manju osjetljivost na stres, te povećava koncentraciju kalcija u serumu, čime je omogućena proizvodnja kvalitetnije ljuske jajeta.

This research aimed to determine the chemical and physical properties, hygienic quality, calcium concentration, and coagulation properties from different types of milk (bovine, ovine, and caprine). A specific aim was to compare three different types of milk and to define key parameters affecting their coagulation properties. A total of 33 milk samples were collected from March to December 2021 from three different regions in the Republic of Croatia. Chemical composition was determined with Milkoscan FT3 (Foss, Denmark) and physical properties were determined with a Seven Multi pHmeter (Mettler Toledo, Switzerland) and Cryostar 1 (Funke Gerber, Germany). Hygienic quality was determined with Bactoscan FT 50 (Foss, Denmark) and Fossomatic Minor (Foss, Denmark). Calcium concentration was determined by inductively coupled plasma-mass spectrometry, ICP-MS (Shimadzu, Japan). Milk coagulation properties were determined with a lactodynamograph (MA.PE SYSTEM, Italy). The highest average casein content (g/100 g) was determined in ovine (4.76), bovine (2.77), and caprine milk (2.42). The average ionometric acidity (pH) of the bovine milk was higher (6.55) than caprine (6.40) and ovine milk (6.42). Different calcium concentrations (mg/kg) in bovine (1501.11), caprine (1387.65), and ovine milk (2271.43) were determined. The conclusion based on the results was that calcium concentration, casein content, and ionometric acidity had a significant effect (P

Biljke i dodaci prehrani su važan izvor biološki aktivnih sastavnica koje pozitivno utječu na zdravlje ljudi. Osim toga, primjena dodataka prehrani u stalnom je porastu. Budući da se indijski tamjanovac, justicija i kurkuma koriste u ublažavanju simptoma upalnih bolesti crijeva, cilj ovog rada bio je procijeniti kvalitetu dodataka prehrani obzirom na sadržaj kalcija i magnezija. Sadržaj navedenih minerala u analiziranim dodacima prehrani određen je plamenom tehnikom atomske apsorpcijske spektrometrije nakon postupka mikrovalne digestije. Nadalje, u analiziranim uzorcima ispitan je gubitak sušenjem prema propisu Američke Farmakopeje. Rezultati istraživanja upućuju na visoki sadržaj ovih minerala te se analizirani dodaci prehrani mogu smatrati dobrim izvorom minerala. Osim toga, svi analizirani dodaci prehrani osim jednog uzorka koji sadrži suhi ekstrakt papra zadovoljavaju USP zahtjeve obzirom na gubitak sušenjem.

Ionsko-selektivne elektrode su vrste potenciometrijskih senzora čiji potencijal je ovisan o jednoj ionskoj vrsti odnosno o koncentraciji određene ionske vrste u otopini. Ionsko selektivna membrana koristi se za prepoznavanje određene vrste iona, a najčešće je izrađena od stakla i polimernog materijala. Ionsko-selektivne elektrode mogu biti elektrode s kristalnom membranom, s nekristalnom membranom i specijalne ionsko-selektivne elektrode. Primjena ionsko-selektivnih elektroda je široka, a jedna od prednosti je što za provedbu mjerenja ne troše analit i ne zahtijevaju posebnu pripravu uzorka. Kalcijeva ionsko-selektivna elektroda je kombinirana elektroda koja ima polimernu membranu selektivnu za kalcijeve ione. Zadatak ovog završnog rada je bio opisati kalcijevu ionsko-selektivnu elektrodu, odrediti linearnost metode i odrediti sadržaj kalcija u uzorku soka pomoću kalcijeve ionsko-selektivne elektrode. Ion-selective electrodes are types of potentiometric sensors whose potential depends on one ion type or the concentration of a certain ion type in solution. An ion-selective membrane is used to recognize a particular type of ion and is most often made of glass and polymeric material. Ion-selective electrodes can be crystal membrane electrodes, non-crystalline membrane electrodes and special ion-selective electrodes. The application of ion-selective electrodes is wide, and one of the advantages is that they do not consume analyte to perform measurements and do not require special sample preparation. A calcium ion-selective electrode is a combined electrode that has a polymer membrane selective for calcium ions. This work aimed to describe the calcium ion-selective electrode, determine the linearity of the method and determine the calcium content in the fruit juice sample using a calcium ionselective electrode.

Glavni namen diplomskega dela je bilo preučiti, ali se ponujena hrana v domu starejših občanov sklada z referenčnimi vrednostmi za vnos makro in mikroelementov, ki so predpisane za to starostno skupino. S pomočjo kemijske analize petih izbranih elementov (natrija, kalija, kalcija, magnezija in železa) v celodnevnih obrokih smo njihove vsebnosti primerjali z referenčnimi vrednostmi za vnos teh elementov s hrano. Obravnavali smo 14 celodnevnih obrokov, ki so jih sestavljali zajtrk, kosilo, popoldanska malica in večerja. Ugotovili smo, da je v obrokih v obravnavanem domu starejših preveč natrija in soli, saj je tedenski vnos natrija in soli v obeh tednih presegal ocenjene vrednosti za minimalen vnos tega elementa. Najvišja izmerjena vrednost Na (2994 mg/dan) je skoraj za dvakrat presegla referenčno vrednost NIJZ za ta element (1500 mg/dan) (NIJZ, 2020). Vsebnosti soli v celodnevnih obrokih so presegale mejo 5 g/dan (NIJZ, 2016) v dvanajstih od štirinajstih dni, z najvišjo vrednostjo 7,6 g/dan. Za makroelement K smo ugotovili, da njegove vsebnosti v obrokih niso dosegale priporočenih vrednosti. Največja dnevna izmerjena vsebnost kalija v obrokih dveh tednov je bila 2605 mg, kar je še zmeraj veliko manj od priporočene vrednosti 4000 mg/dan, ki jo navaja NIJZ (2020). Tudi povprečna količina Ca v celodnevnih obrokih je bila nižja od priporočene vrednosti 1000 mg/dan. Največja vsebnost Ca v celodnevnem obroku je bila 903 mg, najnižja celo samo 339 mg/dan, medtem ko je priporočena vrednost 1000 mg/dan. Za Mg smo ugotovili, da izmerjene vrednosti v obrokih ne dosegajo priporočil NIJZ (2020) za moške (350 mg/dan), ter z izjemo enega dneva tudi ne priporočil za ženske (300 mg/dan). Po naših analizah so vsebnosti mikroelementa železa po večini ustrezale priporočeni vrednosti NIJZ (2020) 10 mg/dan, saj so se gibale med 10 in 19 mg/dan. The aim of the thesis was to examine whether the food offered in the elderly nursing home complies with the reference values for macro and micronutrients intake prescribed for the elderly age group. Chemical analysis of five selected elements (sodium, potassium, calcium, magnesium, and iron) was used in all-day meals, these contents were compared with the reference values for the intake of these elements with food. We discussed 14 all-day meals consisting of breakfast, lunch, afternoon snack, and dinner. We found that there was too much sodium and salt in the meals in the nursing home in question, as the weekly sodium and salt intake in both weeks exceeded the estimated values for the minimum intake of this element. The highest measured value of Na (2994 mg/day) was almost twice the reference value of the National Institute of Public Health - NIJZ (2020) for this element (1500 mg/day). The salt content in all-day meals exceeded the limit of 5 g / day (NIJZ, 2016) in 12 of 14 days, with a maximum value of 7.6 g. For the macroelement K, we found that its content in the meals did not reach the recommended levels. The highest measured potassium content in the two-week meals was 2605 mg, which is still well below the NIJZ (2020) recommended value of 4000 mg/day. The average quantity of Ca in all-day meals was lower than the recommended value of 1000 mg/day. The maximum Ca content in the whole day meal was 903 mg, the lowest even only 339 mg/day, while the recommended value was 1000 mg/day. For Mg, we found that the measured values in meals do not meet the recommendations of the NIJZ (2020) for men (350 mg/day), and except for one day also do not meet the recommendations for women (300 mg/day). According to our analysis, the iron trace element contents mainly corresponded to the recommended NIJZ (2020) value of 10 mg/day, as they ranged between 10 and 19 mg/day

Kalcij je esencijalni makronutrijent važan za normalno funkcioniranje mnogih procesa u ljudskom organizmu. Nedostatak kalcija u organizmu nadomješta se dodacima prehrani. Kao uzorci za usporedbu dviju analitičkih metoda i određivanje sadržaja kalcija poslužili su dodaci prehrani dvaju različitih proizvođača. Cilj ovog diplomskog rada bio je usporediti klasičnu – gravimetrijsku i instrumentalnu – FAAS metodu u svrhu određivanja sadržaja kalcija u dodacima prehrani. Obje metode su visoke točnosti i preciznosti. Provedenim istraživanjem uočene su prednosti određivanja koncentracije kalcija plamenom tehnikom koja se pokazala kao jednostavnija i brža metoda naročito za veći broj uzoraka. Niske dobivene RSD vrijednosti upućuju na zadovoljavajuću ponovljivost određivanja koncentracije kalcija plamenom tehnikom. Na temelju dobivenih vrijednosti LOD i LOQ te vrijednosti za točnost provedene metode, plamena tehnika se može smatrati prikladnom za određivanje kalcija u uzorcima dodataka prehrani te u usporedbi s klasičnom gravimetrijskom metodom i metodom izbora. Calcium is an essential macronutrient that is important for the normal functioning of many processes in the human body. A deficiency of calcium in the body is compensated for dietary supplements. Dietary supplements from two different manufacturers served as samples to compare the two methods of analysis and determine the calcium content. The aim of this work was to compare the classical - gravimetric and the instrumental - FAAS method for the determination of calcium content in food supplements. Both methods are characterised by high accuracy and precision. The study showed the advantages of the determination of calcium concentration by FAAS, which proved to be an easier and faster method, especially with a larger number of samples. The obtained low RSD values indicate a satisfactory repeatability of the determination of calcium concentration by the flame technique. Based on the obtained LOD and LOQ values and the values for the accuracy of the performed method, FAAS can be considered suitable for the determination of calcium in samples of food supplements and the method of choice compared to the classical gravimetric method.

The production system, especially fertilisation has an important effect on yield and quality of strawberries. In the present study, plants were fertilized with different doses of nitrogen (0–100% recommended doses), calcium chelate, as well as nano fertilizer Lithovit. Strawberry cultivar ‘Clery’ yield and quality parameters (fruit color and firmness) including nutritional indicators (total soluble solids, sugars, organic acids, phenolic and volatile compounds) were monitored. Volatiles were identified and monitored using headspace solid phase microextraction and analysed using gas chromatograph-mass spectrometry (SPME/GC-MS) and sugars, organic acids, and phenolic compounds with high performance liquid chromatography. Organic acids and phenolic compounds were detected with mass spectrometer (HPLC/MS). Both nitrogen and calcium fertilisation had altered sugars, organic acids, volatile and phenolic contents in strawberry fruits. Fertilisation with higher doses of nitrogen and calcium increased the content of unpleasant aromas aldehydes hexanal (up to 3.8-fold) and (E)-2-hexen-1- (up to 3.7-fold). The content of fruity esters was uppermost in fruits fertilised with nano-fertiliser Lithovit (up to 2.3-fold). Fertilisation with N and Ca decreased the strength of ketone and terpenoids fruity aroma. The highest content of total phenols, as well as all individual hydroxycinnamic and hydroxybenzoic acid derivatives were obtained in the nano-fertiliser Lithovit treatment. Fertilisation, especially with nitrogen, had mostly negative impact on strawberry flavour while nano-fertilization with Lithovit improved strawberry phenolic content and aroma.

CALR mutations are a revolutionary discovery and represent an important hallmark of myeloproliferative neoplasms (MPN), especially essential thrombocythemia and primary myelofibrosis. To date, several CALR mutations were identified, with only frameshift mutations linked to the diseased phenotype. It is of diagnostic and prognostic importance to properly define the type of CALR mutation and subclassify it according to its structural similarities to the classical mutations, a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2 mutation), using a statistical approximation algorithm (AGADIR). Today, the knowledge on the pathogenesis of CALR-positive MPN is expanding and several cellular mechanisms have been recognized that finally cause a clonal hematopoietic expansion. In this review, we discuss the current basis of the cellular effects of CALR mutants and the understanding of its implementation in the current diagnostic laboratorial and medical practice. Different methods of CALR detection are explained and a diagnostic algorithm is shown that aids in the approach to CALR-positive MPN. Finally, contemporary methods joining artificial intelligence in accordance with molecular-genetic biomarkers in the approach to MPN are presented.

Cilj: Cilj istraživanja je odrediti koncentracije kalcija, fosfora i njhov međusobni odnos u uzorcima mliječnih i kariotičnih zubi. Materijal i metode: Istraživanje je provedeno na zubima prikupljenim u stomatološkoj poliklinici. Prikupljeni zubi su mliječni zubi i trajni zubi zahvaćeni karijesom. Koncentracija kalcija je određena plamenom apsorpcijskom spektrometrijom (FAAS) na atomskom apsorpcijskom spektrometru AnalytikJena AG, model AAS Vario 6. Koncentracija fosfora je određena UV VIS spektrofotometrijom, na UV VIS spektrofotometru Perkin Elmer Lambda 25, double beam. Rezultati: Istraživanje je provedeno na 20 zubi: 10 mliječnih i 10 trajnih (kariotičnih) zubi. Srednja vrijednost kalcija u mliječnim zubima je 26,372 %, a srednja vrijednost kalcija u uzorcima trajnih zubi je 24,597 %. Srednja vrijednost fosfora u mliječnim zubima je 11,785 % a srednja vrijednost fosfora u trajnim zubima 14,115 %. Omjer Ca/P je 2,277 u mliječnim zubima, a u trajnim zubima omjer Ca/P je 1,748. Zaključak: Istraživanjem je utvrđeno da je koncentracija kalcija niža u uzorcima trajnih (kariotičnih) zubi, koncentracija fosfora viša u uzorcima trajnih (kariotičnih) zubi te da je omjer Ca/P niži u uzorcima trajnih (kariotičnih) zubi u odnosu na mliječne zube. Dobiveni rezultati ukazuju na potrebu za daljnim istraživanjem s obzirom da ne postoji velik broj ispitivanja ove tematike., Objectives: The aim of the research is to determine the concentrations of calcium, phosphorus and their interrelationship in samples of primary and permanent teeth affected by caries. Material and methods: The study was conducted on teeth collected at a dental clinic. Collected teeth are primary teeth and permanent teeth affected by caries. Calcium concentration was determined by flame absorption spectrometry (FAAS) on an atomic absorption spectrometer AnalytikJena AG, model AAS Vario 6. Phosphorus concentration was determined by UV VIS spectrophotometry, on a UV VIS spectrophotometer Perkin Elmer Lambda 25, double beam. Results: The study was conducted on 20 teeth: 10 deciduous and 10 permanent (karyotic) teeth. The mean value of calcium in primary teeth is 26.372%, and the mean value of calcium in samples of permanent teeth is 24.597%. The mean value of phosphorus in primary teeth is 11.785% and the mean value of phosphorus in permanent teeth is 14.115%. The Ca / P ratio is 2.277 in primary teeth and the Ca / P ratio in permanent teeth is 1.748. Conclusion: The study found that the concentration of calcium is lower in samples of permanent (karyotic) teeth, the concentration of phosphorus is higher in samples of permanent (karyotic) teeth and that the Ca / P ratio is lower in samples of permanent (karyotic) teeth compared to primary teeth. The obtained results indicate the need for further research since there is not a large number of studies on this topic.

Prehranska dopolnila so koncentrirani viri posameznih ali kombiniranih hranil ali drugih snovi s prehranskim ali fiziološkim učinkom, ki se tržijo v obliki »odmerka« (npr. tablete, kapsule, tekočine v izmerjenih odmerkih ipd.). V prehranskih dopolnilih je lahko prisotna široka paleta hranil in drugih sestavin, vključno z vitamini, minerali, aminokislinami, maščobnimi kislinami, prehranskimi vlakninami ter različnimi rastlinskimi in zeliščnimi ekstrakti. Nekatera prehranska dopolnila lahko povzročajo negativne in nepričakovane stranske učinke zaradi prekomerno zaužite količine, onesnaženj, interakcij z drugimi komponentami iz hrane, zdravil ali bodisi drugih prehranskih dopolnil. Prehranska dopolnila ne morejo nadomestiti pestre in raznovrstne prehrane. Zdravi posamezniki, ki kljub uživanju pestre in raznovrstne prehrane mislijo, da ne morejo zadostiti svojim potrebam po določenih snoveh, bodisi po makro- kot tudi mikro-hranilih, posežejo po prehranskih dopolnilih. V tem primeru lahko pride do prekomernega vnosa hranil in v skrajnih primerih tudi do toksičnih učinkov. S prehranskimi dopolnili sicer lahko zadostimo potrebam po raznih hranilih v primeru uživanja hranilno revne hrane. Industrija na področju proizvodnje in trženja prehranskih dopolnil je precej agresivna. Potrošniki kupijo prehransko dopolnilo, ki ga promovirajo kot učinkovitega in preprostega za uživanje, pogosto ne glede na to, ali sploh obstajajo dejanski znanstveno dokazani pozitivni učinki določenega prehranskega dopolnila. Možni negativni učinki uporabe prehranskih dopolnil pri trženju niso omenjeni in zato potrošnikom ostajajo neznani. Food supplements are a source of nutrients and other substances with a nutritional or physiological effect, and are sold in a form of »doses« (tablet, capsule, fluids in measured doses). Vitamins, minerals, amino acids, fatty acids, fiber and various plant and herbal extracts can be found in food supplements. The amounts of ingested food supplements will also determine their positive, negative, or unexpected effects on our body. In the case of multiple food supplement use or the use of food supplements in combination with medicines, -different interactions between them,- can lead to increased health risks or even organ damage. Food supplements can not replace a rich and healthy diet. Healthy individuals who think that even with a rich diet they can not fulfil their nutritional needs, whether macro or micro nutrient needs, reach for food supplements. This can lead to a nutritional overdose or, in the worst case, can have toxic effects. In case of nutrient poor food consumption, dietary supplements can help to meet the needs for various nutrients. The food supplement industry is quite aggressive and the consumers mostly buy what is being promoted as effective and easy to use, mostly regardless of any scientifically proven positive effects. The goal of marketing is to sell, not to provide real positive effects to the consumers. Consequently, possible negative effects of food supplements are not mentioned in marketing and- therefore remain unknown to the consumers.

Oksalati so antinutritivna komponenta hrane. So soli oksalne kisline, ki nastajajo in se akumulirajo v vakuolah posebnih rastlinskih celic, idioblastov. Oksalati se pojavljajo v rastlinah v različnih oblikah. Lahko so v obliki topnih soli, katere tvorijo z Na+, K+ in NH4+ in v obliki netopnih soli s Ca2+, Fe2+ in Mg2+. Najpogosteje jih v rastlini najdemo v obliki kalcijevih oksalatov. Kalcijevi oksalati so vidni kot kristali različnih oblik. V rastlini imajo več različnih funkcij. Če rastlina raste v okolju bogatem s kalcijem, imajo oksalati lahko funkcijo uravnavanja ravni kalcija v rastlini. Oksalati pa lahko služijo tudi kot obrambni mehanizem, saj kristali lahko predstavljajo statične ali pa aktivne obrambne strukture, ki odvračajo žival od paše. Nekatere rastline bogate z oksalati so pomembne pri razstrupljanju okolja z aluminijm in drugimi težkimi kovinami. Vsebnost oksalatov v rastlinah je lahko med 3% in tudi do 80% rastlinine suhe teže. Rastline bogate z oksalati se ponavadi v prehrani ljudi uživajo v manjših porcijah, obstajajo pa tudi kmetijske rastline, ki jih uživamo v večjih količinah, kljub temu, da vsebujejo relativno velike količine oksalatov. To sta predvsem navadna špinača (Spinacia oleracea) in rdeča pesa (Beta vulgaris var. vulgaris). Vsebnost oksalatov v hrani se lahko zniža z nekaterimi načini priprave. V nalogi so predstavljene oblike kristalov, ki jih tvorijo kalcijevi oksalati, vloga oksalatov v rastlinah ter način nastajanja, kmetijske in okrasne rastline bogate z oksalati, ter vpliv oksalatov na zdravje ljudi in živali. Oxalates are an antinutritive component of food. They are salts of oxalic acid that are formed and accumulate in the vacuoles of special plant cells, idioblasts. Oxalates occur in plants in various forms. They can appear as soluble salts, formed with Na+, K+ and NH4+, and as insoluble salts formed with Ca2+, Fe2+ and Mg2+. In plant they are most commonly found in the form of calcium oxalates. Calcium oxalates are visible as crystals of various shapes. They have several different functions in the plant. If the plant grows in a calcium-rich environment, oxalates may have the function of regulating the calcium levels in the plant. Oxalates, however, can also serve as a defense mechanism, as crystals can represent static or active defense structures that deter an animal from grazing. Some oxalate-rich plants are important in detoxifying the environment with aluminum and other heavy metals. The content of oxalates in plants can be between 3% and up to 80% of the plant's dry weight. Plants rich in oxalates are usually consumed in small portions in human consumption, but there are also crop plants that are consumed in larger quantities, despite the fact that they contain relatively large amounts of oxalates. These are mainly spinach (Spinacia oleracea) and beetroot (Beta vulgaris var. vulgaris). The oxalate content of food can be reduced by some methods of preparation. This paper presents the forms of crystals formed by calcium oxalates, the role of oxalates in plants and the method of formation, agricultural and ornamental plants rich in oxalates, and the impact of oxalates on human and animal health.

Kalcij je nepogrešljiv makroelement v našem telesu. Kostem in zobem zagotavlja trdnost, hkrati je ključni element za delovanje mišic, celično signalizacijo in sekrecijo hormonov. Pomanjkanje kalcija in posledice, ki s tem pridejo, preprečimo z uživanjem kalcijevih prehranskih dopolnil. Vsebnost kalcija v prehranskih dopolnilih lahko določamo z uporabo klasičnih ali instrumentalnih analiznih metod. Za metodo se odločimo na podlagi lastnosti vzorca ter zahtev analize. V delu sem vključila pregled lastnosti AAS, Ca-ISE, volumetrije (kompleksometrično določanje) in gravimetrije. Primerjala sem področja uporabe posamezne metode, analite, natančnost, občutljivost, selektivnost, destruktivnost, hitrost, cene ter inovativnost. Pregledala sem načine priprave vzorcev za metode in jih med seboj primerjala. Calcium is an indispensable macronutrient in our body. It ensures the strength of bones and teeth and is also a key element for muscle function, cellular signalling and hormone release. Taking calcium supplements prevents calcium deficiency and its consequences. The calcium content in food supplements can be determined using classical or instrumental analytical methods. The choice of method depends on the properties of the sample and the requirements of the analysis. In this thesis I have included an overview of the properties of AAS, Ca-ISE, volumetry - complexometric titration and gravimetry. I did a comparison of the scope of the method, the analytes, accuracy, sensitivity, selectivity, destructiveness, speed, price and innovation. I reviewed sample preparations for each method and compared them.

V magistrski nalogi smo preverjali odziv celic in protoplastov, pridobljenih iz kalusnega tkiva koruze, na dodatek acetilholina in agonistov acetilholinskih receptorjev. Razvoj kalusa smo inducirali z nacepom mladih poganjkov na gojišče Murashige-Skoog z dodatkom sintetičnega avksina 2,4-D [3 mg/L]. Odzivanje celic in protoplastov s povišanjem [Ca2+]i ob izpostavitvi 60 µM acetilholin-jodidu, nikotinu ali pilokarpin-hidrokloridu smo spremljali prek sprememb v intenziteti fluorescence celic po stimulaciji. Za zaznavo sprememb v [Ca2+]i smo uporabili fluorescenčni indikator za Ca2+ ione, Fluo-4-AM. Ne glede na uporabljeni stimulans v izbrani koncentraciji, pri protoplastih in kalusnih celicah nismo opazili odziva, tj. spremenjene [Ca2+]i, ki bi se statistično značilno razlikoval od negativne kontrole (celice in protoplasti stimulirani s hranilno raztopino). Povišanja [Ca2+]i v času stimulacije so sicer bila prisotna, a so bila najverjetneje posledica mehanskega vzdraženja ob dodatku raztopin. Rezultati torej kažejo na odsotnost acetilholinskih receptorjev pri nediferenciranih celicah koruze oziroma, v primeru njihovega morebitnega obstoja, njihovo neodzivnost na uporabljeno koncentracijo 60 µM acetilholin-jodida, nikotina ali pilokarpin-hidroklorida. Odziv celic in protoplastov koruze na ionomicin je bil neznačilen. Kot pozitivna kontrola za so bile v eksperimentu uporabljene celice HUVEC. Ob izpostavitvi celic acetilholin-jodidu, nikotinu ali pilokarpin-hidrokloridu smo opazili odzive, tj. povišanje [Ca2+]i, ki so bili statistično značilno različni od negativne kontrole (HUVEC stimulirane z zunajcelično raztopino z 10 mM glukozo). Rezultati torej potrjujejo zaznavo 60 µM acetilholin-jodida, nikotina in pilokarpin-hidroklorida ter odzivnost celic HUVEC. Sprememba [Ca2+]i po izpostavitvi HUVEC ionomicinu je bila statistično značilna. In this master thesis we checked the response of the callus-derived maize cells and protoplasts to acetylcholine and acetylcholine receptor agonists. Callus formation was induced by inoculation of young shoots of maize on Murashige-Skoog medium with addition of synthetic auxin 2,4-D [3 mg/L]. We followed the responses of cells and protoplasts to stimulation by 60 µM acetylcholine-iodide, nicotine or pilocarpine-hydrochloride, by alterations in fluorescence intensity. Fluorescent calcium indicator, Fluo-4-AM was used to follow the alterations in [Ca2+]i. We did not observe statistically significant differences between responses of cells and protoplasts to stimulants and negative control (plant cells and protoplasts stimulated by nutrient solution). Increased [Ca2+]i was observed at the time of stimulation, but probably as a consequence of mechanical stimulation when solutions were added to cells and protoplasts. Results show absence of achetilcholine receptors on the dediferentiated maize callus cells or, if expressed, their unresponsiveness to used concentration – 60 µM of acetylcholine-iodide, nicotine or pilocarpine-hydrochloride. The response to ionomycin was uncommon. HUVEC cells were used as positive control. When cells were exposed to acetylcholine-iodide, nicotine and pilocarpine-hydrochloride, statistically significant differenences from negative control (HUVEC stimulated by extracellular solution with 10 mM glucose) were observed in responses, thus increased [Ca2+]i. Results confirm sensing of acetylcholine-iodide, nicotine and pilocarpine-hydrochloride, and responsiveness of HUVEC cells. Alteration in [Ca2+]i after addition of ionomycine to HUVEC was statistically significant.

Kalcij je najzastupljeniji mineral u čovjekovom organizmu. Uloga kalcija u ljudskom organizmu je od velike važnosti za normalan metabolizam. Važna je sastavnica kostiju i zubiju, regulira kontrakciju i relaksaciju mišića, funkciju živčanog tkiva, zgrušavanje krvi, utječe na regulaciju krvnog tlaka i provođenje prijenosnih signala živčanog sustava. Eliminira se urinom i fecesom. Ukoliko unos kalcija i apsorpcija iz crijeva ne zadovoljavaju fiziološke potrebe ljudskog organizma, tada se kalcij resorbira iz kostiju, što narušava zdravlje kostiju. Kroničan deficit kalcija dovodi do smanjenja koštane mase, osteoporoze i učestale frakture kostiju. Deficit kalcija moguće je spriječiti korištenjem namirnica obogaćenih kalcijem u svakodnevnoj prehrani. Cilj ovoga rada bila je optimizacija metode plamene atomske apsorpcijske spektrofotometrije u svrhu određivanja kalcija u uzorcima žitarica. Ispitivanja su provedena na atomskom apsorpcijskom spektrofotometru Analyst 800 s deuterijskim korektorom nespecifične površine na valnoj duljini od 422,7 nm. Postojeća metoda je validirana ispitivanjem parametara validacije prema važećim smjernicama. Dobiveni rezultati su pokazali da su izmjerene mase kalcija u uzorcima usporedive s deklariranim masama proizvođača. Metoda se pokazala brzom, jeftinom, pouzdanom i pogodnom za određivanje kalcija u različitim uzorcima žitarica. Calcium is one of the most prevalent elements in the human body. As a such, it plays an important role in normal human metabolism. It is a significant part of human bones and teeth, regulates muscle contraction and relaxation, nerve tissue function, blood coagulation, affects high blood pressure regulation, and conducting nerve system transmission signals. It is eliminated by urine and feces. If calcium supply and bowel absorption are not sufficient to satisfy the physiological needs of the human body, then calcium is resorbed from bones, which impairs bones’ health. Chronic calcium deficit leads to bone mass decreasing, osteoporosis and frequent bone fractures. Calcium deficit can be stopped using calcium enriched food daily. The main purpose of this thesis was optimisation of flame atomic absorption spectroscopy due to calcium quantification in cereal samples. Tests were done using atomic absorption spectrophotometer Analyst 800 with non-specific surface deuterium corrector at the wavelength of 422,7 nm. The existing method has been validated by the validation parameter examination. Results show that measured calcium masses in samples are comparable with declared masses. The method proved to be fast, cheap, reliable and appropriate for calcium determination in different cereal samples.

Kalcij je esencijalni makronutrijent i najzastupljeniji mineral u ljudskom organizmu. Vrlo je bitan za zdravlje ljudskog organizma, rast i razvoj kostiju te održavanje njihove gustoće. Sudjeluje u prijenosu živčanih impulsa, kontrakciji i relaksaciji mišića te koagulaciji krvi aktivacijom pretvorbe protrombina u trombin. Bitan je u aktivaciji brojnih enzima te je važan za permeabilnost membrane i regulaciju krvnog tlaka. Kod nedostatka kalcija može doći do gubitka pamćenja, delirija, depresije, halucinacija i zbunjenosti, a pri značajnom manjku kalcija mogući su bolovi u mišićima, konvulzije i tetanija. Hiperkalcijemija uzrokuje depresiju živčanog sustava, refleksi su usporeni i dolazi do opstipacije i gubitka apetita. Rosuvastatin je sintetski lijek koji pripada novijoj generaciji statina, skupini lijekova za snižavanje kolesterola, a dolazi u obliku kalcijeve soli. Statini su najvažnija skupina lijekova koja se koristi za snižavanje razine lipida u krvi i jedni su od najčešće propisivanih lijekova. Selektivni su i kompetitivni inhibitori 3-hidroksi-3-metilglutaril-koenzim A (HMG-CoA) reduktaze, enzima koji pretvara HMG-CoA u mevalonat, preteči kolesterola. Koristi se u terapiji primarne hiperkolesterolemije (tipa IIa, uključujući heterozigotnu obiteljsku hiperkolesterolemiju) ili miješane dislipidemije (tipa IIb) te homozigotne obiteljske hiperkolesterolemije. Cilj ovog rada bio je optimizirati metodu za određivanje kalcija u obliku tableta rosuvastatina pomoću plamene atomske apsorpcijske spektrofotometrije. Prije samog ispitivanja uzoraka, metoda je validirana ispitivanjem parametara kao što su linearnost, preciznost, točnost te granica dokazivanja i određivanja. U ispitivanju se koristio atomski apsorpcijski spektrofotometar Analyst 800 (PerkinElmer Instruments, Norwalk, CT, SAD) s deuterijskim korektorom nespecifične površine na valnoj duljini od 422,7 nm. Metoda je prikladna za određivanje kalcija u tabletama rosuvastatina te je brza, pouzdana i relativno jeftina. Calcium is an essential macronutrient and the most abundant mineral in the human body. It is necessary for the health of the human body, growth and development of bones, and maintenance bone maintenance density. It participates in the transmission of nerve impulses, muscle contraction, and relaxation, and it enables blood clotting by activating the conversion of prothrombin to thrombin. It activates many enzymes, enhances membrane permeability, and it regulates blood pressure. Calcium deficiency can lead to memory loss, delirium, depression, hallucinations, and confusion. Significant calcium deficiency can lead to muscle aches, convulsions, and tetany. Hypercalcemia causes depression of the nervous system, slowed reflexes, obstipation, and loss of appetite. Rosuvastatin is a synthetic drug that belongs to a newer generation of statins, a group of cholesterol-lowering drugs, and comes in the form of calcium salt. Statins are the most dominant group of drugs used to lower blood lipid levels, and they are one of the most commonly prescribed drugs. They are competitive and selective inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that converts HMG-CoA to mevalonate, a precursor to cholesterol. Rosuvastatin is used to treat primary hypercholesterolaemia (type IIa, including heterozygous familial hypercholesterolaemia) or mixed dyslipidemia (type IIb) and homozygous familial hypercholesterolaemia. The aim of this study was to optimize the method for the determination of calcium quantity in the form of rosuvastatin calcium tablets, using flame atomic absorption spectroscopy. Before sample testing, the method was validated by determining linearity, precision, accuracy and detection limits and quantitation. An atomic absorption spectrophotometer Analytical 800 (PerkinElmer Instruments, Norwalk, CT, USA) with a deuterium corrector of a non-specific surface at a 422,7 nm wavelength was used in the study. This method is suitable for determining calcium in rosuvastatin tablets and is fast, reliable, and inexpensive.

Dosadašnje studije o odnosu mineralnog sastava sline i parodontnog statusa ostale su bez jasnih zaključaka, stoga je svrha ovog rada ispitati utjecaj i povezanost mineralnog sastava sline pušača i nepušača s parodontnim statusom. Uzorci nestimulirane sline prikupljeni su od muških ispitanika mlađe životne dobi koji su nepušači ili koji puše više od 20 cigareta na dan. Određen je protok sline. Koncentracije kalcija, magnezija i fosfata u slini izmjerene su induktivno spregnutom plazmom sa spektrometrijom masa. Dubina sondiranja izmjerena je za svaki zub, a mjerenje je uključivalo vrijednosti iz mezijalnog, središnjeg i distalnog dijela gingivnog sulkusa s vestibularne i oralne strane. Također, za svaki zub izmjereni su parodontološki indeksi kliničkog gubitka pričvrstka (CAL), krvarenja pri sondiranju (BOP) i prisutnost tvrdih zubnih naslaga (CA). Za određivanje značenja podataka dobivenih prikupljanjem sline i parodontološkim pregledom korištena je metoda neparametrijske deskriptivne statistike te je određen Spearmanov koeficijent korelacije. Prosječna i maksimalna vrijednost CAL-a je u statistički značajnoj pozitivnoj korelaciji s godinama pušenja (r=0.625520, r=0.630565, p

Določanje kalcijevih ionov v bioloških matricah (serum, kri, urin itd.) je možno z uporabo klasičnih in instrumentalnih metod. Pri "in vitro" študijah interakcij kalcija s trinatrijevim citratom in humanim albuminom je najprimernejša metoda potenciometrija z uporabo kalcijeve iono-selektivne elektrode (Ca/ISE). Ca/ISE predstavlja najboljšo kombinacijo odzivnosti, občutljivosti, specifičnosti in ponovljivosti za "in vitro" model kvantitativnega spremljanja sprememb koncentracij kalcijevih ionov ob konici dializnega katetra v resničnem času. V serumu je kalcij ioniziran, kompleksiran ali vezan na različne proteine, med katerimi prevladuje albumin. Nemoteno delovanje človeškega telesa je pogojeno z vzdrževanjem normalnega razmerja serumske koncentracije kalcija. Zaradi izhajanja trinatrijevega citrata iz konice katetra lahko pride do znižanja koncentracij ioniziranega kalcija v telesu. V sklopu magistrskega dela smo izvajali eksperimente pri različnih pogojih npr.: v odprtem sistemu pri sobni temperaturi, v zaprtem sistemu pri 36,5 °C, v ali ob odsotnosti zraka, ob prisotnosti albumina itd. Z izbiro ustrezne Ca/ISE in kalibracijo v izbranem koncentracijskem območju smo zagotovili točnost, natančnost in ponovljivost meritev. Pri poskusih smo zaradi dobre ponovljivosti in majhnega odstopanja izmerjenega naklona od teoretične vrednosti uporabili Mettler Toledo perfectIONTM Ca/ISE. Odzivni čas elektrode je 100 s, njen naklon v zaprtem sistemu je 29,3 ± 0,3 mV, kar je zelo blizu Nernstovega naklona. Potrdili smo vpliv temperature na odziv Ca/ISE z višanjem temperature narašča tudi naklon elektrode. Poleg sprememb koncentracij kalcijevih ionov ob izhajanju trinatrijevega citrata iz konice katetra smo merili tudi pH. Potrdili smo mehanizem vezave kalcija na albumin ter ovrednotili vpliv kislinsko-bazičnega ravnotežja – pri višjem pH se je koncentracija prostih kalcijevih ionov znižala zaradi njihove vezave na humani albumin. Determination of calcium ions in biological matrices (serum, blood, urine etc.) is possible with the use of classical and instrumental methods. With in vitro studies of interactions of calcium with trisodium citrate and human albumin, the preferred method is potentiometry with the use of calcium ion-selective electrode (Ca/ISE). Ca/ISE presents the best combination of responsiveness, sensitivity, specificity and repeatability for the in vitro model of quantitative monitoring of changes of calcium ions concentrations at the tip of the dialysis catheter in real time. In the serum, calcium is ionised, complexed or bound to different proteins, among which albumin predominates. Smooth functioning of the human body is contingent on maintaining a normal ratio of serum concentration of calcium. The leakage of trisodium citrate from the tip of the catheter can lead to reduced concentrations of ionised calcium in the body. Within this master’s thesis, experiments have been carried out under different conditions, for instance in an open system at room temperature, in a closed system at 36.5 °C, in the absence of air, in the presence of albumin etc. With the choice of an appropriate Ca/ISE and calibration in the chosen concentration range, accuracy, precision and repeatability of the measurements have been ensured. In experiments, Mettler Toledo perfectIONTM Ca/ISE has been used due to good repeatability and small deviation of the measured slope from the theoretical value. The response time of the electrode is 100 s, its slope in the closed system is 29,3 ± 0,3 mV, which is very close to the Nernst slope. The influence of temperature on the reaction of Ca/ISE has been confirmed increase in temperature results in the increase of the electrode slope. In addition to the changes of calcium ions concentrations after the leakage of trisodium citrate from the tip of the catheter, also pH has been measured. The mechanism of calcium binding to albumin has been confirmed and the influence of the acid-base balance evaluated – with a higher pH, the concentration of free calcium ions decreased due to their binding to human albumin.

Uvod: Izraz prezgodnja menopavza opredeljuje prenehanje hormonske dejavnosti jajčnikov pred 40. letom starosti. V okviru preučevanja pojava prezgodnje menopavze se v ospredje vse bolj postavljajo sociodemografski dejavniki dejavniki povezani z življenjskim slogom dejavniki, povezani z zdravjem reprodukcijskega organskega sistema dejavniki, povezani z izpostavljenostjo škodljivim zunanjim vplivom. Namen: Namen magistrskega dela je raziskati vpliv različnih dejavnikov tveganja na pojav prezgodnje menopavze ter hkrati razviti in testirati presejalni vprašalnik za ugotavljanje statusa vitamina D pri slovenskih pomenopavznih in perimenopavznih ženskah. Metode dela: Izvedena je bila presečna epidemiološka raziskava. Uporabljena je bila deskriptivna metoda dela, narejen je bil pregled literature, v empiričnem delu pa neeksperimentalna kvantitativna metodologija z uporabo anketnega vprašalnika. Vzorec je predstavljalo 81 pacientk, starih med 40. in 65. letom, ki so v obdobju od julija 2019 do septembra 2019 obiskale izbrano referenčno ambulanto v Zdravstvenem domu dr. Julija Polca v Kamniku. Zbrane podatke smo obdelali in analizirali s pomočjo programov Microsoft® Excel in SPSS, različica 21.0. Rezultati: Ob upoštevanju vključitvenih in izključitvenih meril smo v nadaljnjo analizo vključili 59 preiskovank. Povprečna starost preiskovank je 57,05 leta, povprečna starost ob pojavu menopavze pa je 50,85 leta. Povprečna vrednost vnosa vitamina D prek hrane je 1,32 µg dnevno, s prehranskimi dopolnili in/ali z zdravilom Plivit D3 pa 4,11 µg dnevno. Z raziskavo smo ugotovili, da kajenje vpliva na zgodnejši nastop menopavze (p < 0,05) ter dokazali vpliv višje stopnje izobrazbe na višji vnos kalcija z mlekom in mlečnimi izdelki (p < 0,05). V nasprotju s predhodnimi študijami z našo raziskavo nismo dokazali povezave med starostjo ob pojavu prve menstruacije in starostjo ob nastopu menopavze (p > 0,05). Ugotovili smo tudi, da večje izpostavljanje soncu ne vpliva na starost ob nastopu menopavze (p > 0,05). Razprava in zaključek: Zaradi povezanosti med življenjskim slogom in starostjo nastopa menopavze se nakazuje priložnost za preventivno delovanje z namenom krepitve reproduktivnega zdravja žensk v vseh starostnih obdobjih. Introduction: The term premature menopause defines the cessation of ovarian hormonal activity before the age of 40. In the context of the study of the onset of premature menopause, sociodemographic factors lifestyle related factors factors related to health of the reproductive organ system and factors related to exposure to harmful external influences are increasingly being highlighted. Purpose: The purpose of the thesis is to investigate the impact of various risk factors on the onset of premature menopause and at the same time develop and test a screening questionnaire to determine the status of vitamin D in Slovenian postmenopausal and perimenopausal women. Methods: A cross-sectional epidemiological survey was conducted. Used was the descriptive work method, prepared was an overview of literature and in the empirical part a non-experimental quantitative methodology was carried out using a questionnaire. The sample consisted of women aged 40-65 who visited reference clinics in the Health Center dr. Julija Polca Kamnik during July and September in 2019. The collected data were processed and analyzed using Microsoft® Excel and SPSS version 21.0. Results: Considering the inclusion and exclusion criteria, 59 subjects were included in the follow-up analysis. The mean age of the subjects was 57.05 years and the mean age at onset of menopause was 50.85 years. The average value of vitamin D intake with food was 1,32 µg daily, while with dietary supplements and/or Plivit D3 the value of vitamin D intake was 4.11 µg daily. Study showed that smoking has an effect on the onset of menopause (p < 0.05) and demonstrated the impact of higher education on higher calcium and milk intake (p < 0.05). In contrast to previous studies, our study did not demonstrate an association between age at onset of menarche and age at onset of menopause (p > 0.05). Results also showed that greater exposure to the sun does not affect the age at onset of menopause (p > 0.05). Discussion and conclusion: Due to the association between lifestyle and age of onset of menopause, the opportunity for preventive health care interventions are being suggested to improve the reproductive health of women at all ages.