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Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies. Nasl z nasl. zaslona. Opis vira z dne 31. 3. 2023. Bibliografija. Abstract.

Peptidi so na trgu kozmetičnih izdelkov prisotni že skoraj 50 let. V tem času so se nekateri peptidi uveljavili kot učinkovite aktivne sestavine za široko področje kožnih problematik. Peptide sestavlja do 100 aminokislin, ki so med seboj povezane s peptidno vezjo. Nekateri peptidi niso sposobni prehajati rožene plasti in so tako odlični tvorilci filmov, okluzivi in humektanti. V to skupino spadajo tudi hidrolizirani proteini. Nekateri peptidi pa prodrejo globlje in preko različnih mehanizmov delovanja izboljšajo stanje kože. Slednje delimo na tri skupine, in sicer signalne, prenašalne in inhibitorne peptide. V diplomskem delu smo preiskovali kozmetične izdelke, ki vsebujejo peptide in so dostopni na slovenskem trgu. Osredotočili smo se na izdelke, ki se prodajajo preko spleta. Skupaj smo zbrali 107 izdelkov, in si za vsakega izpisali blagovno znamko, linijo, ime, tehnološko vrsto, mesto uporabe, ceno, količino, sestavine in oglaševanje peptidov. Izločili smo 2 izdelka, ki nista ustrezala kriterijem. Pri raziskovanju smo se osredotočili na več različnih vidikov. Izdelki, ki smo jih analizirali so bili najpogosteje namenjeni uporabi na obrazu. Največkrat smo izdelke zasledili v obliki kreme in seruma. Poleg izdelkov za nego smo zasledili proteine tudi v dekorativni kozmetiki. Skoraj polovica izdelkov, glede na postavljene kriterije, spada v visok cenovni razred. Izdelki največkrat vsebujejo kombinacijo 2 do 3 peptidov. Med najbolj zastopanimi peptidi so bili palmitoil tetrapeptid-7, palmitoil tripeptid-1, acetil heksapeptid-8, karnozin in hidrolizirani proteini. Naštete peptide smo podrobneje opisali in povzeli študije, ki so potrdile njihovo delovanje. Za konec smo se osredotočili še na nekatere neskladnosti, ki so se pojavile med raziskovanjem. Peptides have been present in the cosmetics market for almost 50 years. During this time, some peptides have established themselves as effective active ingredients for a wide range of skin problems. Peptides are composed of up to 100 amino acids linked together by a peptide bond. Some peptides are unable to cross the stratum corneum and are therefore excellent film formers, occlusive agents and humectants. Hydrolysed proteins also fall under this category. However, some peptides penetrate deeper and improve skin condition through different mechanisms of action. The latter are divided into three groups, namely signal peptides, carrier peptides and inhibitory peptides. In our thesis we investigated cosmetic products containing peptides that are available on the Slovenian market. We focused on products sold online. In total, we collected 107 products, and for each of them we wrote down the brand, line, name, technological type, place of use, price, quantity, ingredients and advertising of the peptides. We also collected ingredient lists and labelled all peptides on them. After consideration, we eliminated 2 products that did not meet the criteria. We focused our research on several different aspects. The products we found were most often intended for use on the face. The products were mainly in the form of creams and serums. In addition to skincare products, we also found decorative cosmetics. Almost half of the products belong to the high price range according to the criteria we set. The products mostly contain a combination of 2 to 3 peptides. The most represented peptides were palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, acetyl hexapeptide-8, carnosine and hydrolysed proteins. We have described these peptides in more detail and summarised the studies that have confirmed their efficiency. Finally, we have focused on some of the non-compliances that have arisen during the course of our research.

Uvod: Katestatin je biokativni peptid uključen u patofiziologiju brojnih upalnih bolesti. Adropin je sekretorni protein koji prvenstveno modulira metaboličku homeostazu i funkciju endotela. Postoji sve više dokaza koji upućuju na povezanost adropina s različitim upalnim stanjima. No, kontrolirani su podatci o njegovoj razini i mogućoj povezanosti s metaboličkim procesima u reumatoidnom artritisu (RA) nedostatni. Unatoč nedvojbenoj uključenosti katestatina u upalne procese nema studija koje su istraživale povezanost njegove razine s RA-om. Poznato je da se mediteranski način prehrane smatra optimalnim za bolesnike s RA-om zbog blagotvornoga učinka na zglobove i kardiometaboličke poremećaje te protuupalnoga učinka. Ciljevi istraživanja: Ciljevi su ove studije bili utvrditi postoji li razlika u serumskim razinama katestatina i adropina te MDSS zbira u ispitanika s RA-om u odnosu na zdrave ispitanike kontrolne skupine odgovarajuće dobi i spola. Nadalje, istraživana je povezanost serumskih razina katestatina i adropina s pokazateljima kronične upale, aktivnošću i trajanjem RA-a, funkcionalnom onesposobljenošću bolesnika, parametrima metabolizma glukoze te ostalim kliničkim, biokemijskim i laboratorijskim značajkama. Dodatni je cilj nam bio utvrditi povezanost MDSS zbira sa serumskim razinama katestatina i adropina, kao i s aktivnošću RA-a i funkcionalnošću ispitanika s RA-om. Ispitanici i metode: U ovo presječno istraživanje ukupno je bilo uključeno 160 ispitanika, odnosno 80 ispitanika s RA-om i 80 odgovarajućih ispitanika kontrolne skupine za studiju s katestatinom te 70 bolesnika s RA-om i 70 zdravih ispitanika podudarnih dobnih i spolnih značajki za studiju s adropinom. Bolesnicima uključenim u istraživanje s katestatinom dodatno je bio izračunat MDSS zbir. Svim ispitanicima s RA-om određeni su DAS28 i HAQ zbir. Svim je sudionicima u istraživanju, uz uzimanje anamnestičkih podataka, obavljen i detaljan fizikalni pregled, antropometrijska mjerenja te uzorkovanje krvi za laboratorijsku analizu. Serumske razine katestatina i adropina određene su ELISA metodom, dok su ostali laboratorijski parametri određivani standardnim laboratorijskim procedurama. Istraživanje je provedeno u Zavodu za reumatologiju, alergologiju i kliničku imunologiju Kliničkog bolničkog centra Split u periodu od 30. listopada 2020. do 30. lipnja 2021. godine. Rezultati: Serumska razina katestatina bila je značajno viša u bolesnika s RA-om u odnosu na kontrolnu skupinu ispitanika (10,53 ± 3,90 vs. 5,24 ± 2,37 ng/mL, P < 0,001). Nadalje, u skupini ispitanika s RA-om utvrđena je statistički značajna pozitivna korelacija između katestatina i dobi (r = 0,418, P < 0,001) te DAS28 (r = 0,469, P < 0,001) i HAQ zbira (r = 0,483, P < 0,001)). Također, pokazana je značajna korelacija između serumske razine katestatina i trajanja RA-a (r = 0,583, P < 0,001). Multiplom linearnom regresijskom analizom, prilagođenom za zbunjujuće varijable (dob, spol, ITM i HAQ zbroj), pokazano je da je razina katestatina zadržala statistički značajnu i pozitivnu korelaciju s trajanjem RA-a (β ± SE, 0,13 ± 0,04, P = 0,002) i DAS28 zbirom (0,94 ± 0,45, P = 0,039), sa serumskom razinom katestatina kao ovisnom varijablom. Serumska razina adropina bila je značajno niža u bolesnika s RA-om u odnosu na kontrolnu skupinu ispitanika (2,85 ± 0,91 vs. 4,02 ± 0,99 ng/mL, P < 0,001). U skupini bolesnika s RA-om, serumske razine adropina bile su u značajnoj negativnoj korelaciji s ukupnim kolesterolom (r = -0,172, P = 0,043), HbA1c (r = -0,406, P < 0,001), glukozom natašte (r = -0,377, P < 0,001) i HOMA-IR (r = -0,315, P = 0,008). Multipla linearna regresijska analiza, nakon prilagodbe zbunjujućih čimbenika, pokazala je da su serumske razine adropina zadržale značajnu povezanost s razinama glukoze natašte (β ± SE, -0,450 ± 0,140, P = 0,002) i vrijednošću HbA1c (-0,528 ± 0,223, P = 0,021). Nije pronađena statistički značajna razlika između ukupnoga MDSS zbira između 80 bolesnika s RA-om i kontrolne skupine ispitanika (7,57 ± 4,11 vs. 7,29 ± 3,61, P = 0,661). Također, u skupini bolesnika s RA-om nije utvrđena značajna korelacija MDSS zbira sa serumskim razinama katestatina (r = -0,059, P = 0, 621) i adropina (r = 0,012, P = 0,917), kao ni s DAS28 (r = 0,031, P = 0,993) i HAQ zbrojem (r = 0,222, P = 0,062). Zaključak: Rezultati dobiveni ovim istraživanjima impliciraju moguću povezanost katestatina sa složenom patofiziologijom RA-a i aktivnošću bolesti te potencijalni utjecaj adropina na metaboličku homeostazu u RA-u. Nadalje, pokazano je kako nepridržavanje mediteranske prehrane može imati negativan sveukupni učinak na aktivnost RA-a, funkcionalnost bolesnika, ali i smanjenu kvalitetu života. Za konačnu evaluaciju i integraciju dobivenih rezultata potrebne su daljnje veće multicentrične longitudinalne studije praćenja., Background: Catestatin (CST) is an important peptide that influences various inflammatory diseases. Adropin is a secretory protein that mainly modulates metabolic homeostasis and endothelial function. There is growing evidence supporting association of adropin with various inflammatory diseases. However, controlled data of possible connection among serum adropin levels and metabolic processes in rheumatoid arthritis (RA) are insufficient. Regardless the incontestable involvement of CST in inflammatory disorders, there have been no published studies that explored the association of serum CST levels with RA. The Mediterranean diet (MD) is considered optimal for patients with RA due to its beneficial effect on joints and cardiometabolic disorders and anti-inflammatory effect. Aims of the study: The aims of this study were to determine difference in serum CST and adropin levels, as well as difference in MDSS score between patients with RA and healthy control subjects matched by age and sex. Furthermore, we assessed the relationship between serum CST and adropin levels and chronic inflammation, duration and activity of RA, functional disability of patients with RA and parameters of glucose metabolism, as well as other clinical, biochemical and anthropometric parameters. An additional goal was to determine the association of the MDSS score with CST and adropin levels and RA activity and functionality of RA patients. Participants and Methods: A total of 160 subjects were included in this cross-sectional study. Study of serum CST levels was conducted on 80 patients with RA and 80 healthy control subjects matched by age and sex. 70 RA patients and 70 healthy controls, matched by age and sex, were included in the study of serum adropin levels. Additionally, the MDSS score was determined to participants involved in the study of serum CST levels. Measurement of disease activity, using DAS28 and functional disability, using HAQ, were measured only in the patient group. Detailed medical history, a physical examination, anthropometric measurements and laboratory analysis of all the participants were obtained and performed. Serum CST and adropin levels were determined by ELISA, while other biochemical analyses were performed using standard laboratory procedures. The study was conducted at the Department of Rheumatology, Allergology and Clinical Immunology, University Hospital of Split, over a period from October 30, 2020 to June 30, 2021. Results: Serum CST levels were significantly higher in RA patients than in the control group (10.53 ± 3.90 vs. 5.24 ± 2.37 ng/mL, P < 0.001). In RA patients, there was a statistically significant correlation between CST and patient age (r = 0.418, P < 0.001) and both DAS28 (r = 0.469, P < 0.001) and HAQ scores (r = 0.483, P < 0.001). There was a statistically significant correlation between serum CST levels and RA duration (r = 0.583, P < 0.001). Multiple linear regression analysis showed that serum CST levels retained a significant association with RA duration (β ± SE, 0.13 ± 0.04, P = 0.002) and DAS28 score (0.94 ± 0.45, P = 0.039) after model adjustment for age, body mass index (BMI) and HAQ score, with serum CST levels as a dependent variable. Serum adropin levels were significantly lower in RA patients than in the control group (2.85 ± 0,91 vs. 4.02 ± 0.99 ng/mL, P < 0.001). In the RA group, serum adropin levels had a significant negative correlation with total cholesterol (r = -0.172, P = 0.043), HbA1c (r = -0.406, P < 0.001), fasting glucose (r = -0.377, P < 0.001) and HOMA-IR (r = -0.315, P = 0.008). Multiple linear regression analysis showed that serum adropin levels retained a significant association with levels of fasting glucose (β ± SE, -0.450 ± 0.140, P = 0.002) and HbA1c (-0.528 ± 0.223, P = 0.021) after model adjustments. There were no statistically significant difference in MDSS score between the RA group and the control group (7.57 ± 4.11 vs.7.29 ± 3.61, P = 0.661). We did not find a significant correlation of MDSS score with serum CST (r = -0.059, P = 0.621) and adropin levels (r = 0.012, P = 0.917) and neither with DAS28 (r = 0.031, P = 0.993) and HAQ score (r = 0.222, P = 0.062). Conclusions: These findings imply that CST is possibly associated with RA complex pathophysiology and disease activity, while adropin could have an impact on metabolic homeostasis in RA. Furthermore, it was shown that non-adherence to the MD can have a negative overall impact on RA activity, patient functionality, and reduced quality of life. Future larger multicentric longitudinal follow-up studies are necessary in order to establish and integrate our results.

Peptidomimetici u odnosu na prirodne peptide formiraju rigidniju strukturu i daleko su konformacijski i proteolitički stabilniji, što im omogućuje primjenu u dizajnu i sintezi lijekova i biološki aktivnih spo-jeva. „Zaključavanje“ peptidomimetika u stabilnu bioaktivnu konformaciju alfa-uzvojnice, beta-ploče ili okreta smanjuje mogućnost interakcija s neželjenim receptorima u ljudskom organizmu umanjujući tako vjerojatnost nuspojava koje se nerijetko javljaju kod peptidnih lijekova. Budući da peptidomimetici preko svojih „hot-spot“ regija stupaju u interakcije s receptorima zahvaljujući komplementarnosti nji-hovih 3D struktura, njihov se dizajn zasniva na oponašanju sekundarnih strukturnih elemenata prisutnih u „hot-spot“ regijama. Poznati začetnik okretâ, metil 1'-aminoferocen-1-karboksilat, u ovom je završ-nom radu primjenom EDC/ HOBt metode kopuliran s homo- i/ili heterokiralnim Pro-Ala dipeptidima, a struktura nastalih biokonjugata 3-6 potvrđena je IR- i NMR-spektroskopijom. Compared to the natural peptide, peptidomimetics form a rigid structure and are much more stable in terms of conformational flexibility and proteolytic activity, which allows their use in the design and synthesis of drugs and biologically active compounds. "Locking" peptidomimetics into a stable bioac-tive conformation of alpha-helix, beta-sheet or turn reduces the possibility of interaction with undesired re-ceptors in the human body, thereby reducing the likelihood of side effects that are common with peptide drugs. Since peptidomimetics interact with receptors through their "hot-spot" regions thanks to the com-plementarity of their 3D structure, their design is based on mimicking the secondary structural elements present in the "hot-spot" regions. The well-described turn inducer, methyl 1'-aminoferrocene-1-carbox-ylate, was coupled with homo- and/or heterochiral Pro-Ala dipeptides using the EDC/ HOBt method, and the structure of the resulting bioconjugates 3-6 was determined by IR and NMR spectroscopy.

SPPS (angl. Solid-Phase Peptide Synthesis, slo. sinteza peptida na trdnem nosilcu) je dokaj enostavna in zanesljiva metoda za sintezo peptidov in manjših proteinov. Ti v naravi predstavljajo zelo pomembne molekule, saj opravljajo mnogo življenjsko pomembnih funkcij. Pri SPPS največkrat uporabljamo Fmoc skupino, kot začasno zaščito na N-koncu modificirane aminokisline. Zaščititi moramo tudi vse ostale funkcionalne skupine aminokislin, ki bi lahko reagirale tekom sinteze, tako da gradimo le eno glavno polipeptidno verigo. Pripravili smo dva peptida – enega manjšega, tako s šaržno sintezo na koloni, s kontinuirnim pretokom in brez trajnih zaščitnih skupin. Po uspešni pripravi krajšega peptida (12 aminokislin) smo se lotili sinteze nekoliko daljšega peptida (26 aminokislin). Po protokolu smo najprej na nosilec vezali prvo modificirano aminokislino, spirali z DMF, odstranili Fmoc skupino s piperidinom v DMF ter zopet spirali. Te cikle smo ponavljali, dokler nismo v verigo vključili vseh predvidenih aminokislin. Peptid smo gradili od C- proti N-koncu. Peptide smo nato očistili na reverzno fazni kromatografski koloni, ter jih analizirali s HPLC in HRMS. SPPS (Solid-Phase Peptide Synthesis) is a fairly simple and reliable method for the synthesis of peptides and smaller proteins. These are very important molecules in nature as they perform many vital functions. The Fmoc group is most commonly used as a temporary protection at the N-terminus of a modified amino acid during SPPS. However, to control the synthesis and to build only one main polypeptide chain, all other functional groups that could react during synthesis also need to be protected. We prepared 2 peptides - one smaller by manual synthesis on a column (a.k.a. batchwise method), as well as with continuous flow and without permanent protecting groups. After successful preparation of the smaller peptide (12 amino acids) we attempted the synthesis of a slightly longer peptide (26 amino acids). Following the established protocol, the first modified amino acid was first coupled to the solid support, washed with DMF, the Fmoc group was removed with piperidine in DMF, and washed again. These cycles were repeated until all the required amino acids were included into the chain. The peptide was constructed from the C- to N-terminus. The peptides were then purified on a reverse-phase chromatographic column and analysed by HPLC and HRMS.

Preiskovana je bila derivatizacija 6-aminokaprojske kisline, s pripajanjem izolevcinskega estra na Cbz-zaščiteno 6-aminokaprojsko kislino. Peptid je bil sintetiziran iz izhodnih spojin z uporabo različnih pripajalnih reagentov - CDI, EDC in EEDQ. Kot najboljši reagent glede na čistoto produkta se je iskazal EDC najslabši za to reakcijo pa je bil EEDQ. Dobljeni peptid je bil nato odščiten na N-koncu, aminska skupina pa acilirana s 4-pentinojsko kislino z uporabo EDC reagenta. Sinteza je bila uspešna, kar je bilo razvidno iz NMR spektrov končne spojine. Končni derivat 6-aminokaprojske kisline je N-konec elektrofilne sonde, katere preostanek je del diplomskih del drugih kolegov. Sinteza te elektrofilne sonde ne sovpada v obseg tega diplomskega dela. The derivatization of 6-aminocaproic acid was investigated by coupling the isoleucine ester with Cbz-protected 6-aminocaproic acid. The peptide was synthesized from the starting compounds using various coupling reagents - CDI, EDC and EEDQ. EDC proved to be the best reagent, yielding the purest product the worst for this reaction, however, was EEDQ. To the resulting peptide, 4-pentynoic acid was additionally attached using the EDC reagent. The synthesis was successful as confirmed by the NMR spectra of the final compound. The final derivative of 6-aminocaproic acid is the N-terminus of the electrophilic probe, in which the remainder of the probe is a part of the thesis of other colleagues. The synthesis of this electrophilic probe does not fall within the scope of this task.

Cilj diplomskega dela je bila sinteza peptidov z uporabo dveh različnih metod. Oba načina sta temeljila na principu SPPS (ang. Solid-phase peptide synthesis) in uporabi Fmoc metode. Pri prvem načinu smo za sintezo uporabili inštrument na osnovi pretočne SPPS, ki je omogočal hitro kemijsko sintezo peptidov. Drugi način sinteze je potekal z uporabo zbiralne vakuumske komore, za izvedbo sinteze so bili potrebni daljši časi. Uspešno smo sintetizirali tri peptide, ki so vsebovali 17 in 26 aminokislin, od katerih sta bila dva enaka, toda sintetizirana po drugačni strategiji, enkrat s pretočno SPPS, drugič ročno z uporabo zbiralne vakuumske komore. S pretočno SPPS smo peptide sintetizirali v bistveno krajšem času, kot z uporabo zbiralne vakuumske komore, vendar so bili ti bistveno manj čisti. The aim of this diploma thesis was the synthesis of peptides using two different methods. Both methods were based on the SPPS (Solid-phase peptide synthesis) principle and used the Fmoc protected amino acids. In the first method, an instrument based on flow SPPS was used for the synthesis, which enables rapid chemical synthesis of peptides. Another method of synthesis was a much slower standard SPPS method using a collection vacuum chamber. We successfully synthesized three peptides containing 17 and 26 amino acids, two of which were identical, but synthesized using a different strategy, once with flow SPPS, the second time manually using a collection vacuum chamber. With flow SPPS, peptides were synthesized in a significantly shorter time, than using a collection vacuum chamber, but these were significantly less clean.

Within this thesis, solid-state reaction medium and mechanochemical methodologies were explored to unveil weather nucleobases can self-assemble and amino acids can oligomerize under these conditions. X-ray diffraction and differential scanning calorimetry analysis showed that 2,6-diaminopurine (D) selectively self-assembles with uracil (U) and thymine (T), while adenine (A) self-assembles with hypoxanthine (H). Crystal structures of D-U cocrystal hydrate, D-T cocrystal hydrate, D-T cocrystal anhydrate, A-H cocrystal hydrate, and A-H cocrystal anhydrate were determined from powder X-ray diffraction and single-crystal X-ray diffraction data. Ion-pairing high performance liquid chromatography and mass spectrometry analysis showed that glycine and alanine undergo oligomerization by mechanical activation. Based on the results, it can be concluded that minerals as additives, time, mechanical loading, and temperature are all important parameters for the mechanochemical prebiotic peptide synthesis. In summary, solid-state reactivity of nucleobases and amino acids might have been an important factor in their prebiotic chemical evolution. U ovoj doktorskoj disertaciji koristili smo čvrsto stanje kao reakcijski medij i mehanokemijske metodologije da bismo istražili dolazi li do prepoznavanja nukleobaza i oligomerizacije aminokiselina u tim uvjetima. Analiza difrakcijom rentgenskog zračenja i diferencijalnom pretražnom kalorimetrijom pokazala je selektivno molekulsko prepoznavanje 2,6- diaminopurina (D) s uracilom (U) i timinom (T), kao i adenina (A) s hipoksantinom (H). Riješene su kristalne strukture D-U kokristalnog hidrata, D-T kokristalnog hidrata, D-T kokristalnog anhidrata, A-H kokristalnog hidrata te A-H kokristalnog anhidrata iz podataka difrakcije rentgenskog zračenja. Analiza tekućinskom kromatografijom visoke djelotvornosti i spektrometrijom mase pokazala je da glicin i alanin oligomeriziraju u peptide uslijed mehanokemijske aktivacije. Rezultati su pokazali da su minerali kao aditivi, vrijeme, prenošenje mehaničke energije, i temperatura ključni parametri za mehanokemijsku prebiotičku sintezu peptida. Zaključeno je da je reaktivnost nukleobaza i aminokiselina u čvrstom stanju mogla biti važan faktor u njihovoj prebiotičkoj kemijskoj evoluciji.

Peptidi i proteini su prirodni spojevi koji imaju ključnu ulogu u nizu imunoloških i neuroloških procesa. Djeluju kao neurotransmiteri, hormoni, antigeni i antibiotici, a poremećaji u njihovoj funkciji mogu dovesti do razvoja kroničnih ili infektivnih oboljenja. Unatoč tome, njihova upotreba kao terapeutika ograničena je lošom stabilnošću i bioraspoloživošću. Napredak u strukturnoj optimizaciji, formulaciji i proizvodnji zaslužan je za sve veći broj peptida koji ulaze u klinička ispitivanja i terapijsku primjenu. Nadalje, potreba za kontrolom funkcije peptida dovela je do razvoja peptidomimetika, spojeva koji oponašaju sekundarne strukture proteina, a time i njihove funkcije. U pripravi takvih spojeva važnu ulogu imaju neprirodne aminokiseline. Cilj istraživanja u okviru ovog diplomskog rada je sinteza tripeptida s ugrađenom neprirodnom C-glikozilnom α-aminokiselinom. Za dobivanje C-glikozilne jedinice korištena je dijastereoselektivna Passerinijeva reakcija. Kao izvor C-glikozilne jedinice korišteni su izopropilidenima zaštićeni aldehidi dobiveni iz odgovarajućih ugljikohidrata (sorboze i fruktoze). U Passerinijevoj reakciji korišteni su aminokiselinski, odnosno konvertibilni izocijanid koji se vrlo lako ukloni hidrolizom u baznim uvjetima s ciljem dobivanja derivata α-hidroksi C-glikozilnih kiselina. Post-kondenzacijskim modifikacijama α-hidroksi-derivat je preveden u aminokiselinu, koja je ugrađena u dipeptidni i tripeptidni slijed. Peptides and proteins are natural compounds that play a key role in a number of immune and neurological processes. They act as neurotransmitters, hormones, antigens and antibiotics, and disorders in their function can lead to the development of chronic or infectious diseases. Nevertheless, their use as therapeutics is limited by poor stability and bioavailability. Advances in structural optimization, formulation, and manufacturing are responsible for the growing number of peptides entering clinical trials and therapeutic applications. Furthermore, the need to control peptide function has led to the development of peptidomimetics, compounds that mimic the secondary structures of proteins and thus their functions. Unnatural amino acids play an important role in the synthesis of peptidomimetics. The objective of the research proposed in this master thesis is the synthesis of tripeptides with incorporated unnatural C-glycosyl α-amino acid. Diastereoselective Passerini reactions were used to obtain the C-glycosyl unit. Isopropylidene-protected aldehydes derived from the carbohydrates (sorbose and fructose) were used as the source of the C-glycosyl unit. In the Passerini reaction, amino acid or convertible isocyanide was used, which is very easily removed by the hydrolysis under basic conditions in order to obtain α-hydroxy C-glycosyl acid derivatives. By post-condensation modifications the α-hydroxy derivative was converted to an amino acid, which was incorporated into the dipeptide and tripeptide sequences.

The sheer size and vast chemical space (i.e., diverse repertoire and spatial distribution of functional groups) underlie peptides' ability to engage in specific interactions with targets of various structures. However, the inherent flexibility of the peptide chain negatively affects binding affinity and metabolic stability, thereby severely limiting the use of peptides as medicines. Imposing conformational constraints to the peptide chain offers to solve these problems but typically requires laborious structure optimization. Alternatively, libraries of constrained peptides with randomized modules can be screened for specific functions. Here, we present the properties of conformationally constrained peptides and review rigidification chemistries/strategies, as well as synthetic and enzymatic methods of producing macrocyclic peptides. Furthermore, we discuss the in vitro molecular evolution methods for the development of constrained peptides with pre-defined functions. Finally, we briefly present applications of selected constrained peptides to illustrate their exceptional properties as drug candidates, molecular recognition probes, and minimalist catalysts.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is a multiprotein complex which forms within cells in response to various microbial and self-derived triggers. Mutations in the gene encoding NLRP3 cause rare cryopyrin-associated periodic syndromes (CAPS) and growing evidence links NLRP3 inflammasome to common diseases such as Alzheimer´s disease. In order to modulate different stages of NLRP3 inflammasome assembly nine peptides whose sequences correspond to segments of inflammasome components NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) were selected. Five peptides inhibited IL-1β release, caspase-1 activation and ASC oligomerization in response to soluble and particulate NLRP3 triggers. Modulatory peptides also attenuated IL-1β maturation induced by constitutive CAPS-associated NLRP3 mutants. Peptide corresponding to H2-H3 segment of ASC pyrin domain selectively inhibited NLRP3 inflammasome by binding to NLRP3 pyrin domain in the micromolar range. The peptide had no effect on AIM2 and NLRC4 inflammasomes as well as NF-κB pathway. The peptide effectively dampened neutrophil infiltration in the silica-induced peritonitis and when equipped with Antennapedia or Angiopep-2 motifs crossed the blood-brain barrier in a mouse model. Our study demonstrates that peptides represent an important tool for targeting multiprotein inflammatory complexes and can serve as the basis for the development of novel anti-inflammatory strategies for neurodegeneration.

Desnosučne aminokisline so v majhnih količinah prisotne tako v sintetičnih kot tudi v nekaterih naravnih peptidih in proteinih. Določevanje vezavnih mest desnosučnih aminokislin je pomembno za podrobno analizo in razumevanje teh peptidov in proteinov. V diplomskem delu sem predstavil metode za analizo proteinov, ki vsebujejo desnosučne aminokisline. Vse metode za določevanje vezavnih mest desnosučnih aminokislin temeljijo na masni spektrometriji, ki je ključna tehnika za analizo zgradbe proteinov. Cilj eksperimentalnega dela je bil razviti metodo za ločbo proteinov, ki vsebujejo desnosučne aminokisline. Metodo sem razvijal na dveh tandemskih masnih spektrometrih, ki sta bila sklopljena s tekočinskima kromatografoma. D-amino acids are found in small quantities both in natural and synthetic peptides and proteins. Determination of their bonding sites is important for detailed analysis and understanding of these peptides and proteins. In my diploma thesis I presented methods for analysis of proteins that consist of D-amino acids. All methods for determination of bonding sites of D-amino acids are based on mass spectrometry, which is the key technique for analysing the structure of proteins. The goal of the experimental part was to develop a method to separate proteins consisting of D-amino acids. The method was developed with two tandem mass spectrometers, which were coupled with liquid chromatography instruments.

Peptidi, predvsem speti in ciklični, so v zadnjem času vzbudili veliko zanimanja na področju razvoja biološko aktivnih učinkovin. Njihova prednost je majhna velikost, zmožnost prehajanja v celico, hkrati pa tudi visoka selektivnost vezave na tarčno molekulo ter zmožnost vplivanja na interakcije med proteini. Nove tehnologije na področju sintezne biologije omogočajo produkcijo velikih zbirk peptidnih molekul oz. peptidnih knjižnic ter hitro in učinkovito izbiranje peptidov z najboljšo afiniteto vezave na tarčo. Podjetje PeptiDream, katerega tehnologija je podrobneje opisana v tem diplomskem delu, je izpopolnilo pristope pridobivanja in izboljšave pripetih peptidov, njihovi produkti pa vstopajo v klinične študije. Peptides, especially constrained and cyclic peptides, are receiving increasing attention in drug discovery. Their advantages are small size, intracellular activity, as well as high selectivity of binding to the target molecule, and the ability to effect interactions between proteins. New synthetic biology technologies enable the production of large constrained peptide libraries and the rapid and efficient selection of peptides with high target binding affinity. The company PeptiDream, whose technology is described in this diploma work, has already established an effective technology for obtaining and improving constrained peptides, and a number of products are already entering clinical trials.

Izrazite trepalnice veljajo za znak lepote, zato ni presenetljivo, da obstaja na trgu veliko različnih kozmetičnih izdelkov, katerih namen je polepšati videz zdravih trepalnic. Za najbolj pogosto uporabljen kozmetični izdelek za dekorativo oči velja maskara, ki začasno ustvari videz gostejših in podaljšanih trepalnic. Trajnejše rezultate pa lahko dosežemo z ostalimi izdelki, kot so na primer serumi za rast trepalnic. Namen slednjih je povečati dolžino, pigmentacijo, ukrivljenost in debelino trepalnice, kar z eno besedo imenujemo trihomegalija. V diplomskem delu smo proučevali sestavo serumov za rast trepalnic, pri čemer smo v nabor nabor vključili 38 izdelkov. Osredotočili smo se na kozmetično aktivne sestavine, ki smo jih razdelili po skupinah glede na kemijsko sestavo in vrednotili njihovo pojavnost v izbranih izdelkih. Za vsako sestavino je opisan mehanizem delovanja na rast trepalnic. Zanimali so nas tudi podatki o ceni, oglaševanju in ovojnini, ki odločilno vplivajo na odločitev pri nakupu izdelka. Kozmetično aktivne sestavine smo razdelili v pet skupin, in sicer si po pogostosti uporabe v serumih sledijo v naslednjem zaporedju: vitamini, peptidi, aminokisline, analogi prostaglandinov in ostale sestavine. Navadno je v enem serumu prisotnih več različnih kozmetično aktivnih sestavin. Mehanizem delovanja se med skupinami razlikuje. Pri vitaminih in aminokislinah ne gre za direkten vpliv na rast trepalnice, ampak vplivajo na strukturo trepalnice in omogočajo normalno rast. Peptidi imajo več različnih mehanizmov delovanja odvisno od peptida, najpogosteje povečajo prekrvavitev in proliferacijo keratinocitov v mešičku trepalnice. Analogi prostaglandinov imajo značilen mehanizem delovanja, in sicer trihomegalijo trepalnic dosežejo z vezavo na prostanoidni receptor na celicah mešička, ki povzroči podaljšanje anagene faze in spodbujanje prehoda mešička trepalnice iz telogene faze v anageno fazo. Poleg tega analogi prostagladinov veljajo za edine kozmetične aktivne sestavine, ki imajo s kliničnimi študijami podprto delovanje na rast trepalnic. Pri vrednotenju cene izdelkov smo opazili, da je ta odvisna od vsebnosti analogov prostaglandinov, ki prispevajo k višji ceni. Vsi pregledani serumi se nahajajo v tubularnih vsebnikih, ki omogočajo nanos na veko. Prominent eyelashes are considered as a sign of beauty, consequently contributing to many diverse cosmetic products on the market intended to beautify the look of healthy eyelashes. The most commonly used cosmetic product for eye decoration is mascara, which temporarily creates the appearance of thicker and longer lashes. More lasting results can be achieved using other products, including eyelash growth serums that increase the length, pigmentation, curvature and thickness of the eyelashes, namely trichomegaly. Within the diploma thesis, the composition of 38 eyelash growth serums was studied. Special emphasis was given towards cosmetically active ingredients, divided into groups according to chemical composition, and their occurence in selected products. For each component, the mechanism of action on eyelash growth is described. Price, advertising and packaging was also evaluated having a decisive role when purchasing a product. Cosmetically active ingredients are divided into five groups, according to the frequency of use in serums as follows: vitamins, peptides, amino acids, prostaglandin analogues and other ingredients. Usually, several different cosmetically active ingredients are present in one serum. The mechanism of action varies between groups. Vitamins and amino acids do not have a direct effect on eyelash growth, but rather affect the structure of the eyelash and allow normal growth. Peptides have several different mechanisms of action depending on the peptide, most commonly increasing blood flow and keratinocyte proliferation in the eyelash follicle. Prostaglandin analogues have a specific mechanism of action. Trichomegaly of eyelashes is achieved by binding prostaglandin analogues to the prostanoid receptor on follicular cells, which causes prolongation of the anagen phase and stimulates the transition of the eyelash follicle from the telogen phase to the anagen phase. In addition, prostaglandin analogues are considered the only cosmetic active ingredients that have a clinical study-supported effect on eyelash growth. The price of products depends on the content of prostaglandin analogues, contributing to the higher price. All examined serums are found in tubular containers that allow application to the eyelid.

Monoklonska protitelesa omogočajo specifično vezavo antigena, zaradi česar jih uporabljamo v širokem naboru aplikacij. Monoklonska protitelesa praviloma izoliramo in prečistimo z afinitetno kromatografijo na osnovi proteina A, ki ima kljub visoki selektivnosti vezave številne pomanjkljivosti. Zaradi tega razvijajo alternativne ligande, ki so stabilnejši in cenejši. V magistrskem delu smo na osnovi izhodiščnega peptida, pridobljenega v pretekli raziskavi, in rezultatov obogatitve v presejalnem testu zasnovali nove linearne in ciklične peptidne različice peptidnih ligandov, ki bi bili zmožni močnejše vezave na regijo Fc IgG v primerjavi z izhodiščnim peptidom. Zasnovane peptide smo predstavili na bakteriofagu v monovaletnem formatu in primerjalno ovrednotili njihove vezavne afinitete s testi ELISA. Rezultati s cikličnimi različicami peptida so bili nekonsistentni, zato smo ciklične različice opustili. Linarne različice peptidov smo podvrgli nizu testov ELISA, s katerimi smo postopno izločali zasnovane peptide. Za najuspešnejšega smo določili peptid GSYWYNVWF. Izbiro peptida smo potrdili z enosmerno analizo variance ANOVA, ki je pokazala na statistično značilne razlike v afiniteti vezave med izbranim peptidom in ostalimi različicami. Izvedli smo tudi bioinformacijsko analizo zasnovanih peptidov z izračunom teoretične izoelektrične točke, indeksa GRAVY in indeksa nestabilnosti, s katerimi smo podali možno razlago vzrokov za vezavne razlike med peptidi. Monoclonal antibodies allow specific antigen binding, which is why they are used in a wide range of applications. Monoclonal antibodies are currently isolated and purified mainly by using protein A-based affinity chromatography, which, despite its high binding selectivity, has a number of disadvantages. As a result, efforts are being made to develop alternative ligands that are more cost-efficient and stable. In this master's thesis, we designed new linear and cyclic peptide variants based on the starting peptide obtained in previous research and the results of enrichment during library screening. The goal was to identify peptide ligands with improved binding affinity for the Fc IgG region. The designed peptides were presented on a bacteriophage and their binding affinities were comparatively evaluated with ELISA assays. The results of cyclic variants were contradictory, so cyclic variants were not pursued further. The linear variants were subjected to a series of ELISA assays during which the peptides were gradually excluded. The GSYWYNVWF peptide was determined to have optimal binding affinity. Peptide selection was confirmed by one-way ANOVA assay, which showed statistically significant differences in binding affinity between the selected peptide and other variants. We performed a bioinformatical analysis (i.e., calculation of theoretical isoelectric point, GRAVY and instability indices), which gave a possible explanation of the reasons for the binding differences among the peptides.

I biomateriali per applicazioni di ingegneria dei tessuti devono soddisfare diversi requisiti, come sicurezza, biocompatibilità e caratteristiche meccaniche appropriate. Il processo di sviluppo di questi biomateriali comprende diversi approcci scientifici, che vanno dall' in-silico all'in-vivo. L'ottimizzazione in silico delle caratteristiche dei biomateriali sta attirando un'attenzione sempre maggiore. Infatti, il miglioramento di questo approccio consentirà di ridurre i costi aggiuntivi nel processo di sviluppo dei biomateriali, a causa di caratterizzazioni sperimentali non necessarie. Secondo questo punto di vista, in questa tesi viene presentato un approccio di dinamica molecolare per la caratterizzazione dei biomateriali. Più in dettaglio, gli scaffold degli idrogel di peptidi autoassemblanti (SAP) sono stati studiati su nanoscala e microscala, al fine di chiarire le loro relazioni intrinseche struttura-proprietà-funzione. Le dinamiche molecolari atomistiche a grana grossa (CG-MD) sono state utilizzate per studiare i meccanismi di auto-assemblamento che portano alla formazione di scaffold peptidici. A causa della mancanza di informazioni strutturali cruciali nelle simulazioni CG-MD, l'innovativa suite software, denominata Morphoscanner, è stata impiegata per la classificazione dei modelli di aggregazione conformazionale dei SAP. Quindi, le proprietà meccaniche e i meccanismi di rottura delle nanostrutture SAP sono stati studiati attraverso le simulazioni MD vincolate. Queste evidenze hanno portato allo sviluppo di un approccio CG-MD che mira a chiarire la complessa interazione tra membrane cellulari e nanofibrille SAP. In particolare, le simulazioni MARTINI CG-MD sono state utilizzate per comprendere gli effetti della nanofibrille peptidiche sulla dinamica dei domini lipidici nelle membrane neurali. Tali risultati aprono nuove dimensioni nel campo della biomateriomica, consentendo di comprendere ed eventualmente controllare i complessi fenomeni che influenzano le proprietà meccaniche e la biocompatibilità dei biomateriali a base peptidica per applicazioni di ingegneria tissutale. Biomaterials for tissue engineering applications have to comply with several requirements, such as safety, biocompatibility and appropriate mechanical features. The development process of these biomaterials encompasses several scientific approaches, ranging from in-silico to in-vivo. The in-silico optimization of biomaterials features is attracting even larger attention. Indeed, the improvement of this approach will allow to reduce additional costs int the biomaterials development process, due to unnecessary experimental characterizations. According to this point-of-view, in this thesis is presented a molecular dynamics approach for biomaterial characterization. More in details, self-assembling peptides (SAPs) hydrogels scaffolds have been investigated at the nano-scale and micro-scale, to elucidate their intrinsic structure-property-function relationships. The atomistic and coarse-grained molecular dynamics (CG-MD) have been used for the elucidation of self-assembling pathways of peptide-based scaffolds. Due to the lack of crucial structural information in CG-MD simulations, the innovative software suite, dubbed Morphoscanner, has been employed for the elucidation of conformational aggregation patterns of SAPs. Then, the mechanical properties and failure mechanisms of SAPs nanostructures have been investigated through the steered MD simulations. These evidences led the development of a CG-MD approach aiming to elucidate the complex interplay between cell membranes and SAPs nanofibrils. In particular, MARTINI CG-MD simulations have been used for understanding the effects of SAPS nanofibril on dynamics of lipid domains in neural membranes. Such achievements open up new dimensions in the field of biomateriomics, allowing to understand and eventually orchestrate the complex phenomena which affect the mechanical properties and biocompatibility of SAPs biomaterials for tissue engineering applications.

U okviru ove doktorske disertacije provedena je sinteza i konformacijska analiza peptidnih konjugata aminoferocena [Boc–(AA)n–NH–Fc, Fc = ferocenil, AA = D(L)–Pro, D(L)–Ala, Boc (tert–butoksikarbonil), Aib (α–aminoizobutirična kiselina), n = 2–4] kao i dinuklearnih ferocenskih derivata [Fc–CO–(AA)2–NH–Fc, AA = D(L)–Pro, D(L)–Ala] kako bi se istražio utjecaj nabiranja aminokiselinskog slijeda na kirooptička svojstva organometalnog kromofora. Konformacijskom analizom (IR–spektroskopija, spektroskopija NMR i CD, kristalografska analiza, računalno modeliranje) utvrđeno je da su pripravljeni spojevi, u otopini i u čvrstom stanju, stabilizirani unutarmolekulskim vodikovim vezama koje podržavaju okrete i uzvojnice slične onima u prirodnim peptidima. Kombiniranim teorijskim i eksperimentalnim pristupom pokazano je kako kiralno uređenje peptidnog segmenta inducira Cottonov efekt u blizini UV/Vis apsorpcijskog maksimuma ferocena čiji je predznak u korelaciji s diedarskim kutom koji opisuje rotaciju oko veze između ferocenskog kromofora i peptidnog segmenta. In this PhD thesis peptide derivatives of aminoferrocene [Boc–(AA)n–NH–Fc, Fc = ferrocenyl, AA = D(L)–Pro, D(L)–Ala, Aib, Boc (tert–butoxycarbonyl), Aib (α– aminoisobutyric acid), n = 2–4] as well as the dinuclear ferrocene derivatives [Fc–CO– (AA)2–NH–Fc, AA = D(L)–Pro, D(L)–Ala] were synthesized. The novel compounds were subjected to conformational analysis in order to investigate the influence of folding of amino acid sequence on the chiroptical properties of organometallic chromophore. Conformational analysis (IR, NMR, and CD spectroscopy, crystallographic analysis, computational modeling) of the prepared compounds revealed that they are stabilized by intramolecular hydrogen bonds, that support turns and helices similar to those in natural peptides, in solution as well as in solid state. The combined theoretical and experimental approach has shown that the chiral arrangement of the peptide segment induces Cotton effect near the UV/Vis absorption maximum of ferrocene, whose sign is in correlation with dihedral angle which describes rotation around ferrocene–peptide linkage.

Izotermna titracijska kalorimetrija je kalorimetrijska metoda koja služi za određivanje termodinamičkih parametara interakcija u otopini mjerenjem promjene topline tijekom kemijske reakcije. Aminopeptidaza dipeptidil-peptidaza III je enzim s bitnom funkcijom u brojnim fiziološkim procesima u organizmu, poput obrane od oksidativnog stresa i nastanka nekih tumora, a također je i eksperimentalno dokazana njena uloga u završnim fazama razgradnje bjelančevina. U ovom radu je prikazano praćenje vezanja nekih peptida na ovaj enzim metodom izotermne titracijske kalorimetrije, te je promatrano njihovo potencijalno inhibitorsko djelovanje. Zaključak je da krupni arilni supstituent pridonosi inhibitorskom efektu. Dipeptidil-2-naftilamidni supstrati te oligopeptidi spadaju u skupinu dobrih supstrata za ovaj enzim. Isothermal titration calorimetry is a calorimetric method that is used to determine relevant thermodynamic parameters of certain interactions in solution, by measuring heat changes during chemical reactions. Aminopeptidase dipeptidyl-peptidase III is an enzyme carrying important functions in numerous physiological processes in organism. For instance, the enzyme takes part in defense mechanisms regarding oxidative stress as well as in mechanisms that prevent occurrence of certain tumors. Its role in final stages of protein degradation is also experimentally proven. This thesis discusses monitoring of binding of certain peptides on the above mentioned enzyme as well as impacts of binding, such as peptide’s potential inhibitory action. Both processes are monitored through isothermal titration calorimetry. It is concluded that big aryl substituents contribute to the inhibitory effect. Furthermore, dipeptidyl-2-naphthylamides and oligopeptides belong to the group of good substrates for the enzyme discussed in this thesis.

U ovom završnom radu opisani su priprava i konformacijska svojstva ferocenskog dipeptida 1 (Boc-L-Pro-Fca-OMe) koji se pripravlja kopulacijom ferocenske aminokiseline (Fca, I) s L-Pro primjenom standardnog HOBt/EDC postupka. Da bi se ispitao utjecaj uvođenja prolina na tvorbu intramolekulskih vodikovih veza (IHB), te posljedično i na konformacijski prostor koji ferocenski dipeptid zauzima u otopini, provedena je spektroskopska analiza (IR-, NMR- i CD-spektroskopija). Kristalografskom analizom ispitana je mogućnost tvorbe IHB u čvrstom stanju. Pokazalo se da u čvrstom stanju, kao i u otopini dolazi do tvorbe IHB veze koja rezultira 7-članim prstenom, odnosno γ-okretom. The research conducted in this work includes the preparation and structural characterization of ferrocene dipeptide 1 (Boc-L-Pro-Fca-OMe), by copulation ferrocene amino acid (Fca, I) and L-Pro using standard HOBt/EDC method. To examine the effect of proline introduction on formation of intramolecular hydrogen bond (IHB) in solution, the detailed conformational analysis (IR, NMR- and CD-spectroscopy) in solution was performed. Crystallographic structure of dipeptide 2 in solid state was determined to explore possibility of IHB formation. It has been shown that the formation of intramolecular hydrogen bonding in solid state occurs, forming 7-membered ring, i.e. γ-turn. Same pattern is retained in solution state.

Nemetilirane molekule CpG-ODN aktivirajo prirojen imunski odziv z vezavo na receptor TLR9. Aktivacija prirojenega imunskega odziva pripelje do posledične aktivacije pridobljenega imunskega odziva in tvorbe imunskega spomina. V magistrskem delu smo pripravili polipeptidno ogrodje GFP:P3:P6:P10, na katerega smo v nadaljevanju želeli povezati aktivatorje receptorja TLR9 preko homolognih peptidov, ki tvorijo ovite vijačnice. Aktivatorje receptorja TLR9 smo kovalentno povezali s peptidom, ki tvori ovito vijačnico, pri čemer je izkoristek reakcije bil prenizek za nadaljnjo uporabo. Poleg tega smo za namen enostavnega čiščenja konjugata pripravili nosilec afinitetne kromatografije z magnetnimi kroglicami, ki je deloval na podlagi komplementarnosti molekul DNA. Po prvotno uspešni vezavi komplementarne molekule ODN na nosilec in spiranju le-te z nosilca, je kapaciteta nosilca izrazito padla. Unmethylated CpG-ODN molecules activate an innate immune response by binding to TLR9 receptor. The activation of the innate immune response results in the activation of adaptive immune response and thus also creating immune memory. In this MSc thesis we produced polypeptide GFP:P3:P6:P10 which acts as a scaffold for binding TLR9 receptor activators via coiled coil peptides. We have covalently bound activators of TLR9 receptors to the coiled coil peptides. The reaction had low yield and thus the conjugate was not suitable for further experiments. Furthermore we designed an affinity chromatography on the principle of complementary DNA molecules with magnetic beads for an easy and efficient cleaning of the produced conjugate. After the first successful binding of complement ODN molecule to the affinity carrier, the capacity of the carrier was significantly reduced.

Med je naravni kompleksni produkt čebel, ki nastane s procesiranjem nektarja ali mane. Sestavljen je iz vode, različnih sladkorjev, proteinov, organskih kislin, anorganskih mineralov in kovinskih kationov v sledovih. Prav slednji bi lahko služili za okoljski monitoring, saj lahko z določanjem njihove koncentracije v medu iz različnih geografskih območij spremljamo onesnaženost okolja. V medu se v manjših količinah nahajajo tudi proteini in peptidi, ki s svojim specifičnim biološkim delovanjem prispevajo k farmakološkim aktivnostim medu. Peptidi s protimikrobnim delovanjem so predvsem čebeljega izvora, torej izločki njihovih hipofaringealnih žlez, ki presnavljajo surovi med. Da bi lahko vzpostavili uporabne modele za določevanje botaničnega izvora medu in okoljskega monitoringa, smo med različnega botaničnega in geografskega porekla analizirali z optično emisijsko spektroskopijo z induktivno sklopljeno plazmo (ICP-OES). Na podlagi pridobljenih rezultatov smo skušali najti povezavo med vsebnostjo kovin in okoljskim onesnaženjem. Izkazalo se je, da obstaja povezava med vsebnostjo svinca in aluminija glede na vsebnost antimikrobnih peptidov v medu. Za preučevanje peptidov v medu je pomembna njihova dobra ločba in vizualizacija. Osredotočili smo se na peptide manjše od 10 kDa, izmed katerih jih ima veliko tudi biološke učinke. Optimizirali smo nov protokol za poliakrilamidno gelsko elektroforezo z dodatkom natrijevega dodecilsulfata (NaDS-PAGE). Klasična metoda na osnovi glicinskega pufrskega sistema ni primerna, zato smo uporabili tricinski pufer. Poleg tega barvanje z Coomasssie Brillliant Blue barvilom ni primerno za vizualizacijo peptidov nizkih koncentracij zaradi prenizke občutljivosti, zato smo uporabili in optimizirali barvanje s srebrovim barvilom. Tako smo uspešno ločili peptidni lisi velikosti 5 in 7 kDa. Na osnovi literature sklepamo, da peptidno liso velikosti 5 kDa predstavljata defenzin-1 in apisimin. Kateri proteini naj bi sestavljali peptidno liso velikosti 7 kDa iz literature ni bilo moč ugotoviti, saj kolikor je nam znano, omenjena proteinska lisa v medu še ni bila opažena in predstavlja pomembno novost našega raziskovalnega dela. Eksperimentalne rezultate smo kvantificirali v programu QuantityOne (Bio-Rad, ZDA) in pri tem kot pozitivno kontrolo uporabili matični mleček, saj vsebuje visoko vsebnost defenzina-1 in apisimina. Koncentracijo peptidnih lis smo podali kot ekvivalent ustrezne proteinske lise v znani količini matičnega mlečka. Rezultati so pokazali, da v referenčnem matičnem mlečku najdemo proteinsko liso 5 kDa, ne pa tudi 7 kDa. Nasprotno je v medu proteinska lisa 5 kDa prisotna v zgolj v sledovih, več pa je proteinske lise 7 kDa. Obe se v medu pojavljata neodvisno od njegovega geografskega in botaničnega izvora, saj med posameznimi kategorijami nismo določili statistično značilnih razlik. Honey is a complex natural bee product, produced by processing of nectar and excretions of insects. It consists of water, various sugars, proteins, organic acids, minerals and trace metals. The latter could be utilized for environmental monitoring, as environmental pollution can be monitored by determining the concentration of heavy metals in honey. Smaller quantities of peptides are also present in honey, which through their antimicrobial and anti-inflammatory action contribute to the pharmacological activity of honey. They are mostly peptides of animal origin, particularly excrements of bee hypopharyngeal glands, which process the raw honey. In order to establish useful models for determining the botanical source of honey and its usefulness for environmental monitoring, different honey samples were analysed with optical inductive coupled plasma emission spectrometry (ICP-OES). With the use of statistical analyses, we determined the variance between the concentrations of different elements in terms of geographical and botanical origin. Based on the results obtained, we tried to find a link between the content of heavy metals and environmental pollution. The content of various elements, especially heavy metals, can serve not only as a botanical and authenticy marker, but also as an indicator for environmental monitoring. To study peptides in honey, their separation and visualization are important. We focused on peptides below the size of 10 kDa and optimized a new protocol for SDS-PAGE since the classic glycine-based buffer system is not suitable. With tricine-SDS-PAGE we successfully separated two bands of peptides with apparent molecular masses of 5 kDa and 7 kDa and visualized them with silver staining. The peptide band of 5 kDa likely corresponds to defensin-1 and/or apisimin according to literature. Because the detection limit of Coomassie Brilliant Blue staining is insufficient for visualization of peptides with low concentrations, we also optimized the staining process using staining with silver nitrate. The results were quantified using QuantityOne software (Bio-Rad, ZDA) and royal jelly as control, since it is rich in protein and contains a high content of defensin-1. The concentrations of separated peptides were calculated by referencing royal jelly with a known concentration of defensin-1 peptide. The results showed the presence of the 5 kDa peptide band in the royal jelly sample, but no 7 kDa peptide band. In honey, the 7 kDa band was stronger than the 5 kDa peptide band, which was present only in small amounts. Both peptide bands were present in samples independently of botanic or geographic origin with no statistically significant differences between them.

Peptidi so zaporedja aminokislin. če imajo strukturni motiv obvite vijačnice, lahko predvidimo njihovo povezovanje in ustvarimo večje strukture. Za sestavljanje stabilne periodične mreže uporabljamo verige peptidov, ki jih v matematičnem modelu predstavimo kot graf verižne strukture. Tega vlagamo v ravninsko mrežo, da zagotovimo željeno obliko. Relacije med členi odločajo o tem, ali se bodo ti sami sestavili v mrežo. Matrika relacij je zaradi uporabe ortogonalne množice členov permutacijska. če upoštevamo pogoje iz narave, stabilnosti in oblike mreže, lahko možnosti še dodatno omejimo. Predlagamo omejitev območja iskanja periodične mreže ter preverjanja enoličnosti. Predstavimo algoritem z lenim vlaganjem, kjer mrežo gradimo s postopnim povezovanjem členov, ki jih vlagamo, ko jim za to preostane le ena možnost. Peptides are sequences of amino acids. In case of their coiled coil structural motif we can predict what bonds will form and create larger structures. We use chains made of peptides for assembly of stable periodic nets. We represent peptides as a chain structure graph in our mathematical model, which we embed into a planar net to guarantee the proper form. Relations between links given in a relation matrix decide whether or not links will self-assemble into a net. Because of the use of orthogonal link sets, relation matrices are permutation matrices. We can further limit relation matrices by applying several conditions derived from nature, stability and net shapes. For the search of periodic nets and uniqueness check we propose area restrictions. The presented lazy embedding algorithm connects one pair of links in a step and embeds them only if there is only one embedding option left.

Strukture proteina u domeni bioinformatike su dosta jednostavne strukture koje se sastoje od svega par atributa. Ako ih razdijelimo enzimom tripsinom, dobivamo peptide, koji imaju svoju jedinstvenu masu I prisutni su u više različitih proteina. Takve strukture lako je organizirati kada je u pitanju organizacija baze podataka. Naime, pregled tih podataka je spor zbog velike količine podataka od nekoliko milijardi zapisa peptida. Ovaj rad istražuje I implementira brzi pregled baze podataka peptida, pri čemu je pažnja posvećena indeksiranju, sažimanju, fizičkoj organizaciji, replikaciji i fragmentaciji. Kroz razne tehnologije, rad istražuje slučajeve relacijske, key-value i distribuirane relacijske baze podataka uz popratne rezultate i konačni izvještaj. Protein structures in the domain of bioinformatics are quite simple structures that consist of just a few attributes. If we divide them by the trypsin enzyme, we get peptides, which have their own unique mass. Different peptides are present in different proteins. When it comes to database organization, such structures are easy to organize. However, browsing this data is quite slow because of the large amount of data that goes up to several billion records. This project investigates and implements a solution for fast peptide database browser, with special attention paid on indexing, compression, physical organization, replication and fragmentation. Through various technologies, investigation is based on exploring the case of relational, key-value and distributed relational database with accompanying results and final reports.

Jednostavni konjugati aminoferocena i peptida mogu tvoriti uređene sekundarne strukturne elemente, a pritom homo- odnosno heterokiralnost aminokiselinske sekvencije uvjetuje tvorbu β- odnosno γ-okreta. U ovom radu opisana je priprava i konformacijska analiza homokiralnih bis-ferocenskih derivata VI [Fc-CO- L-Pro-L-Ala-NH-Fc; (8) i Fc-CO-D-Pro-D-Ala-NH-Fc; (9)]. Iscrpna spektroskopska analiza (CD-, IR-, 1H- i 2D NMR), u kombinaciji s molekulskim modeliranjem i kristalografskom analizom, pokazala je kako se u najstabilnijim konformacijama 8 i 9 javljaju, vodikovim vezama podržani, 10-člani prstenovi (β-okreti). Cottonovi efekti opaženi u blizini UV/Vis apsorpcijskog maksimuma ferocena u CD-spektrima 8 i 9 proizlaze iz prijenosa kiralne informacije iz lokalne sekundarne strukture do akiralnog ferocenskog kromofora. Simple conjugates of aminoferocene and peptides are able to form ordered secondary structural elements, while homo- or heterocirality of the amino acid sequence determines the formation of β- or γ-turns. This thesis describes the synthesis and conformational analysis of homochiral bis-ferrocene derivatives VI [Fc-CO-L-Pro-L-Ala-NH-Fc; (8) and Fc-CO-D-Pro-D-Ala-NH-Fc; (9)]. Thorough spectroscopic analysis (CD-, IR-, 1H- and 2D NMR), in combination with molecular modelling and X-ray diffraction, revealed the presence of 10-membered rings (β-turns), supported by hydrogen bonds, in the most stable conformations of 8 and 9. Cotton effects observed near the UV/Vis absorption maximum of ferrocene in CD spectra of 8 and 9 originate from the transfer of chiral information from the local secondary structure to the achiral ferrocene chromophore.

Cilj istraživanja: Glavni ciljevi ovog istraživanja su bili usporediti serumske koncentracije katestatina i lipidih parametara u bolesnika s opstrukcijskom apnejom tijekom spavanja (OSA) i kontrolnih ispitanika te utvrditi moguću povezanost katestatina s polisomnografskim i lipidnim parametrima u populaciji OSA bolesnika. Ispitanici i metode: Ovo presječno kliničko istraživanje provedeno je u Centru za Medicinu spavanja Kliničkog bolničkog centra Split i Medicinskog fakulteta u Splitu tijekom razdoblja od prosinca 2016. do veljače 2018. godine, a uključilo je ukupno 75 odraslih novodijagnosticiranih bolesnika s OSA- om i 50 kontrolnih ispitanika usklađenih prema dobi, spolu i indeksu tjelesne mase. OSA je dijagnosticirana kontinuiranom cjelonoćnom laboratorijskom polisomnografijom (Alice 5LE, Philips Respironics, Eindhoven, Nizozemska) prema važećim praktičnim smjernicama. Svim ispitanicima uzeta je detaljna anamneza te izvršen fizikalni pregled i antropometrijsko mjerenje. Venski uzorci krvi su uzeti natašte za analizu lipidnih parametara i određivanje serumskih koncentracija katestatina metodom dvostrukog enzim-imunoadsorpcijskog testa humanog katestatina (Phoenix Pharmaceuticals, Burlingame, CA, SAD), prema uputstvima proizvođača. Rezultati: Serumske koncentracije katestatina su značajno više u OSA bolesnika u odnosu na kontrolnu skupinu (2, 99 ± 0, 14 vs. 1, 37 ± 0, 13 ng/mL ; P

Jednostavni konjugati aminoferocena i peptida mogu tvoriti uređene sekundarne strukturne elemente, a pritom homo- odnosno heterokiralnost aminokiselinske sekvencije uvjetuje tvorbu beta- odnosno gama-okreta. U ovom radu opisana je priprava i konformacijska analiza homokiralnih bis-ferocenskih derivata VI (Fc-CO- L-Pro-L-Ala-NH-Fc ; (8) i Fc-CO-D-Pro-D-Ala-NH-Fc ; (9)). Iscrpna spektroskopska analiza (CD-, IR-, 1H- i 2D NMR), u kombinaciji s molekulskim modeliranjem i kristalografskom analizom, pokazala je kako se u najstabilnijim konformacijama 8 i 9 javljaju, vodikovim vezama podržani, 10-člani prstenovi (beta-okreti). Cottonovi efekti opaženi u blizini UV/Vis apsorpcijskog maksimuma ferocena u CD-spektrima 8 i 9 proizlaze iz prijenosa kiralne informacije iz lokalne sekundarne strukture do akiralnog ferocenskog kromofora.

Cilj istraživanja: Glavni ciljevi ovog istraživanja su bili usporediti serumske koncentracije katestatina i lipidih parametara u bolesnika s opstrukcijskom apnejom tijekom spavanja (OSA) i kontrolnih ispitanika te utvrditi moguću povezanost katestatina s polisomnografskim i lipidnim parametrima u populaciji OSA bolesnika. Ispitanici i metode: Ovo presječno kliničko istraživanje provedeno je u Centru za Medicinu spavanja Kliničkog bolničkog centra Split i Medicinskog fakulteta u Splitu tijekom razdoblja od prosinca 2016. do veljače 2018. godine, a uključilo je ukupno 75 odraslih novodijagnosticiranih bolesnika s OSA-om i 50 kontrolnih ispitanika usklađenih prema dobi, spolu i indeksu tjelesne mase. OSA je dijagnosticirana kontinuiranom cjelonoćnom laboratorijskom polisomnografijom (Alice 5LE, Philips Respironics, Eindhoven, Nizozemska) prema važećim praktičnim smjernicama. Svim ispitanicima uzeta je detaljna anamneza te izvršen fizikalni pregled i antropometrijsko mjerenje. Venski uzorci krvi su uzeti natašte za analizu lipidnih parametara i određivanje serumskih koncentracija katestatina metodom dvostrukog enzim-imunoadsorpcijskog testa humanog katestatina (Phoenix Pharmaceuticals, Burlingame, CA, SAD), prema uputstvima proizvođača. Rezultati: Serumske koncentracije katestatina su značajno više u OSA bolesnika u odnosu na kontrolnu skupinu (2,99 ± 0,14 vs. 1,37 ± 0,13 ng/mL; P, Objectives: The aims of the study were to compare serum catestatin concentrations between patients with obstructive sleep apnea (OSA) and control subjects and to determine potential associations of catestatin with polysomnographic and lipid parameters in OSA population. Patients and methods: This cross-sectional study was conducted at the Split Sleep Medicine Center from December 2016 to February 2018 and included a total of 75 adult newly-diagnosed patients with OSA and 50 control subjects that were body mass index/age/sex-matched. OSA was diagnosed by full-night in-laboratory polysomnography (Alice 5LE, Philips Respironics, Eindhoven, Netherlands) according to established guidelines. A detailed medical history interview along with physical examination and anthropometric measuring were undertaken in each enrolled subject. Venous blood samples were taken from fasting subjects and were used to determine serum catestatin concentrations and lipid profile parameters. Serum catestatin concentrations were determined by an enzyme-linked immunosorbent assay (ELISA) method by using a commercial kit for human catestatin (Phoenix Pharmaceuticals, Burlingame, CA, USA) according to manufacturer's instructions. Results: Serum catestatin concentrations are significantly higher in OSA patients than in control group (2.99 ± 0.14 vs. 1.37 ± 0.13 ng/mL; P

Forward osmosis (FO) membranes have gained interest in several disciplines for the rejection and concentration of various molecules. One application area for FO membranes that is becoming increasingly popular is the use of the membranes to concentrate or dilute high value compound solutions such as pharmaceuticals. It is crucial in such settings to control the transport over the membrane to avoid losses of valuable compounds, but little is known about the rejection and transport mechanisms of larger biomolecules with often flexible conformations. In this study, transport of two chemically similar peptides with molecular weight (Mw) of 375 and 692 Da across a thin film composite Aquaporin Inside™ Membrane (AIM) FO membrane was investigated. Despite the relative large size, both peptides were able to permeate the dense active layer of the AIM membrane and the transport mechanism was determined to be diffusion-based. Interestingly, the membrane permeability increased 3.65 times for the 692 Da peptide (1.39 × 10−12 m2·s−1) compared to the 375 Da peptide (0.38 × 10−12 m2·s−1). This increase thus occurs for an 85% increase in Mw but only for a 34% increase in peptide radius of gyration (Rg) as determined from molecular dynamics (MD) simulations. This suggests that Rg is a strong influencing factor for membrane permeability. Thus, an increased Rg reflects the larger peptide chains ability to sample a larger conformational space when interacting with the nanostructured active layer increasing the likelihood for permeation.

In an effort to understand the vast complexity of the underlying processes within a cell at a molecular level, the first step lies in revealing the fundamental physical and chemical properties, as well as the structure, of biopolymers (proteins and DNA). With the development of modern experimental techniques it has become possible to study these large molecules under well-defined conditions in the gas phase, by closely inspecting their interactions with energetic photons and electrons. In this Thesis, we present the experimental setups for the action spectroscopy of peptides, proteins and nucleotides, as well as the hydrated complexes (hydrated nucleotides), in the gas phase. We present the details and the operation of the two experimental setups based on coupling the linear quadrupole ion trap with: (1) a VUV or a soft X-ray synchrotron beamline and (2) a focusing electron gun. In the case (1), the existing experimental setup consisting of a commercial quadrupole ion trap mass spectrometer (LTQ XL from Thermo Scientific), equipped with an electrospray ion source, was coupled to the VUV beamline DESIRS and the soft X-ray beamline PLEIADES at the synchrotron SOLEIL (France). The setups were used to study the photo-induced ionization/fragmentation of trapped biopolymers and nanosolvated species. The results obtained with this setups include VUV action spectroscopy of protonated Leucine-Enkephalin peptide (both a monomer and a dimer) and a nanosolvated nucleotide Adenosine monophosphate (AMP), in (5-15) eV photon energy range. The inner-shell action spectroscopy in the soft X-ray energy range (around C and N K-edge), was performed for multiply charged precursor of Ubiquitin protein. The photo-dissociation and photo-fragmentation ion yields for several fragment ions from all above mentioned macromolecules were extracted and the obtained spectral features were discussed considering relevant photon-induced processes. In the case (2), new experimental setup was developed by coupling the same LTQ XL ion trap with a focusing electron gun, in order to perform an electron activation tandem mass spectrometry, as well as an electron-impact action spectroscopy of trapped biopolymer ions. The ion optic simulations using SIMION program were performed in order to investigate the propagation of the electron beam in the RF+DC ion trap. Tests measurements for electron-induced fragmentation of Substance P, Melittin and Ubiquitin are presented for the impact energy of 300 eV. Finally, we present the electron-impact inner-shell action spectroscopy of the multiply charged Ubiquitin protein, in the vicinity of C K-edge energies of (280-300) eV. The electron-impact results are compared with the soft X-ray photon-impact action spectroscopy results obtained for the same target. U nastojanju da se razume ogromna složenost procesa u okviru ćelije na molekulskom nivou, prvi korak je otkrivanje fundamentalnih fizičko-hemijskih osobina, kao i strukture biopolimera (proteina i DNK). Razvojem savremenih eksperimentalnih tehnika omogućeno je proučavanje velikih biološki relevantnih molekula pod jasno definisanim uslovima u gasnoj fazi, izučavanjem njihovih interakcija sa fotonima i elektronima velikih energija. U ovom radu su predstavljene eksperimentalne postavke za akcionu spektroskopiju peptida, proteina, nukleotida, kao i nanosolvatisanih kompleksa (hidratisani nukleotidi) u gasnoj fazi. Prikazani su detalji i princip rada dve eksperimentalne postavke zasnovane na povezivanju linearne kvadrupolne jonske zamke sa: (1) sinhrotronskim fotonskim mlazom (VUV i meki X-zraci) i (2) fokusirajućim elektronskim topom. U slucaju (1), postojeća eksperimentalna aparatura koja sadrži linearnu kvadrupolnu jonsku zamku u okviru komercijalnog masenog spektrometra (LTQ XL od firme Thermo Scientific) povezana je sa VUV mlaznom linijom DESIRS i mlaznom linijom za meke X-zrake PLEIADES na sinhrotronu SOLEIL (Francuska). Aparatura je upotrebljena za izučavanje foto-indukovanih procesa jonizacije i fragmentacije zarobljenih jona biopolimera i nanosolvatisanih čestica. Rezultati dobijeni na ovoj aparaturi uključuju VUV akcionu spektroskopiju protonisanog leucin-enkefalin peptida (monomer i dimer), kao i nanosolvatisanog nukleotida adenosin monofosfata (AMP), u opsegu energija fotona od (5-15) eV. Akciona spektroskopija unutrašnje ljuske koristeći meke X-zrake (u oblasti C i N K-ljuske), urađena je za višestruko naelektrisani prekursor proteina ubikuitin. Za sve gore pomenute makromolekule, izučavani su absorpcioni spektri za neke od dobijenih jonskih produkata, pri čemu su analizirane uočene spektralne karakteristike dobijene u pomenutim fotonski indukovanim procesima. U slucaju (2) razvijen je novi eksperimentalni sistem zasnovan na povezivanju iste LTQ XL jonske zamke sa elektronskim topim sa mlazom elektrona srednjih energija, koja omogućava tandem masenu spektrometriju i elektronski indukovanu akcionu spektroskopiju zarobljenih jona biopolimera. Korišćenjem programa SIMION, urađene su simulacije sa ciljem ispitivanja transmisije elektrona kroz jonsku zamku sa RF+DC potencijalima. Inicijalni testovi aparature uradjeni su fragmentacijom peptida supstance P i melitin, kao i ubikuitin proteina pri energijama elektrona u oblasti oko 300 eV. Na kraju, prikazani su rezultati elektronski-indukovane akcione spektroskopije višestruko naelektrisaniog jona ubikuitin proteina, u oblasti energija oko C K-ljuske (280-300) eV. Ovi rezultati su upoređeni sa rezultatima dobijenim za istu metu pri fotonskiindukovanim (X-zraci) procesima iz iste oblasti energija.

Peptidni kozmeceutici su relativno nove tvari koje se koriste u liječenju raznih stanja kože. Tri su glavne skupine peptidnih kozmeceutika: peptidi koji moduliraju signalne puteve, peptidi koji moduliraju neurotransmitore i peptidi koji moduliraju prijenosne nosače. Iako njihove prednosti trenutno nisu strogo ispitane, dokazi o njihovoj upotrebi u stalnom su porastu. Postoji značajan broj potencijalno aktivnih peptidnih tvari koje se mogu koristiti u proizvodima za njegu kože, posebno uzimajući u obzir brojne peptidne aktivne tvari koje su kao sirovina dostupne za izradu kozmetičkih proizvoda. Omjer cijene i učinkovitosti kozmetičkog proizvoda ključan je čimbenik njegovog uspješnog oblikovanja. Sukladno tome ograničavajući čimbenik korištenja pojedinog peptida pri oblikovanju kozmetičkog proizvoda je primjeren odnos isplativosti i potrebnog udjela peptida u kozmetičkom proizvodu te reproducibilnog učinaka takvog peptidnog kozmeceutika. Peptide cosmeceuticals are one of the new, popular options to treat different skin conditions. There are three main categories of cosmeceutical peptides: signal peptides, neurotransmitter-affecting peptides and carrier peptides. Although their benefits currently may not be as rigorously tested, the evidence to support their use is growing. The potential for bioactive peptide ingredients in skin care products is enormous, especially when you consider the diverse assortment of molecules available to the cosmetic formulator today. The cost-benefit ratio is key in this area, and the balance of economics, peptide concentration and, reproducible, stable, deliverable bioactivity, will be the key driver for the success of each peptide on an individual basis.

The objective of this study is to identify fish protein markers for detecting multiple species based on a comparative proteomic approach that relies on fragments with identical sequences. The possibilities and challenges of the use of peptides obtained from carp (Cyprinus carpio) and herring (Clupea harengus) proteins are discussed. A bioinformatic analysis was followed by an LC-MS/MS experiment to identify markers predicting the presence of fish allergenic proteins. Selected myosin peptides were found in carp protein hydrolysates with known sequences and in herring protein hydrolysates with unknown sequences. The results obtained for carp and herring proteins myosin and parvalbumin indicate that proteins with unknown sequences can be identified by peptide markers. Such markers can be designed by disregarding the principle that peptides should be unique (present in one sequence). The challenge is to determine a group of proteins that can be detected by peptide identification., Svrha je ovoga rada bila identificirati markere koji se mogu upotrijebiti za istodobno određivanje proteina iz nekoliko vrsta riba, i to usporedbom proteoma i pronalaskom identičnih sekvencija. Razmotrena je mogućnost identifikacije fragmenata proteina u uzorcima šarana (Cyprinus carpio) i haringe (Clupea harengus), te su ispitani nedostaci metode. Markeri za proteinske alergene pronađeni su u ribama pomoću bioinformatičke analize, nakon koje je provedeno ispitivanje u vezanom sustavu tekućinske kromatografije i tandemne spektrometrije masa (LC-MS/MS). Odabrani peptidi koji sačinjavaju strukturu miozina pronađeni su u hidrolizatima proteina šarana poznatih sekvencija i hidrolizatima proteina haringe nepoznatih sekvencija. Rezultati dobiveni analizom miozina i parvalbumina iz šarana i haringe pokazuju da se pomoću peptidnih markera mogu odrediti proteini nepoznatih sekvencija. Takvi se markeri mogu pripremiti zanemarujući princip da peptidi moraju biti jedinstveni (prisutni u samo jednoj sekvenciji). U daljnjem bi istraživanju trebalo odrediti skupinu proteina koja se može identificirati ovom metodom.

CILJ ISTRAŽIVANJA Cilj predloženog specijalističkog rada je pregledno opisati i kritički raspraviti nove spoznaje povezane s degenerativnim promjenama fibroblasta i kolagena kao ključnog mehanizma kronološkog i fotostarenja kože s naglaskom na kozmeceutike i dermatokozmetičke pripravke koji se koriste ili ispituju u svrhu preveniranja i/ili usporavanja tih degenerativnih promjena. MATERIJALI I METODE Pretraživanje literature obavljeno je elektroničkim putem, a pretražena je baza podataka PubMed. Pri pretraživanju literature traženi su odgovori na specifična pitanja vezana za problematiku ovog specijalističkog rada. Literatura je pretraživana od općih prema specijaliziranim člancima pri čemu su odabrani članci relevantni za problematiku ovog specijalističkog rada. REZULTATI Propadanje kolagenskog matriksa dermisa kože karakteristična je osobina kronološki ostarjele kože, a ta je promjena ubrzana kod fotooštećene kože. U organizaciji i održavanju kolagenskog matriksa ključnu ulogu igra mehanička napetost fibroblasta odgovornih za sintezu kolagena. Smanjenje mehaničke napetosti koje se javlja starenjem kritična je pokretačka sila molekularnih promjena u podlozi starenja kože. Iako postoji mnoštvo kozmeceutika za koje se tvrdi da poboljšavaju izgled i funkciju ostarjele i/ili fotooštećene kože samo je mali broj njih primjereno ispitan te određen mehanizam njihovog djelovanja. ZAKLJUČAK Retinoidi su zasad najučinkovitiji u sprječavanju degenerativnih promjena dermisa, a vrlo važnu skupinu predstavljaju i peptidi koji stimuliraju aktivnost fibroblasta te potiču proizvodnju kolagena. Učinak topikalno primjenjenih kozmeceutika koji ublažavaju i/ili sprječavaju degenerativne promjene dermisa ograničen je njihovom permeacijom do mjesta učinka. Pristup za poboljšanje bioraspoloživosti kozmeceutika u dermisu ovisi o fizičkoj kemijskim svojstvima kozmeceutika te može uključivati: (i) modifikaciju same molekule kozmeceutika s ciljem poboljšanja lipofilnosti i/ili stabilnosti, (ii) dodatak pospješivača apsorpcije u formulaciju, (iii) uklapanje,kozmeceutika u nosače nanometarskih dimenzija. OBJECTIVES The objective of this thesis is a review and critical discussion of new pieces of knowledge about fibroblast and collagen degenerative changes as a key mechanism of chronological ageing and photoageing. Special emphasis was given to the cosmeceuticals and formulations which are being used or tested for prevention and/or slowing down of such degenerative changes. MATERIALS AND METHODS Literature search was conducted electronically with PubMed being the database searched. During the research of the literature the answers to the specific questions related to the problems of this thesis were searched. The literature was researched from the general articles to the specialized articles and the articles relevant for this thesis were selected. RESULTS A major feature of aged skin is fragmentation of the dermal collagen matrix and this fragmetation is acceleraated by sunlight. The collagen network is organized and maintained by dynamic mechanical tension provided by the fibroblasts responsible for its production. Age related reduction of mechanical tension is a critical driving force for molecular changes that underlie the appearance of aged skin. Although there exists a multitude of cosmeceuticals that are claimed to improve appearance of chronologically/photoaged skin only few have been rigorously evaluated and their mechanism of action elucidated. CONCLUSION Amongst various antiaging agents retinoids are the most promising agents that are available for the treatment of aging dermis. Emerging agents are also peptides that stimulate fibroblast activity and induce collagen production. However the efficacy of topically applied cosmeceuticals is restricted by their permeation to the site of their action. The approach to improve the dermal bioavailability of cosmeceuticals depends on their physicochemical properties of and may include: (i) modification of the molecule in order to improve its lipophilicity and/or stability (ii) incorporation of absorbtion enhancers in the formulation (iii) and the encapsulation of cosmeceutical into different nanocarriers.

Recently, we described cyclopeptide opioid agonists containing the D-Trp-Phe sequence. To expand the scope of this atypical pharmacophore, we tested the activity profiles of the linear peptides Ac-Xaa-Phe-Yaa (Xaa = L/D-Trp, D-His/Lys/ Arg; Yaa = H, GlyNH2). Ac-D-Trp-PheNH2 appeared to be the minimal binding sequence, while Ac-D-Trp-Phe-GlyNH2 emerged as the first noncationizable short peptide (partial) agonist with high μ-opioid receptor affinity and selectivity. Conformational analysis suggested that 5 adopts in solution a β-turn conformation.

The myocardial response to mechanical stretch (Frank-Starling law) is an important physiological cardiac determinant. Modulated by many endogenous substances, it is impaired in the presence of cardiovascular pathologies and during senescence. Catestatin (CST: hCgA352-372), a 21-amino-acid derivate of Chromogranin A (CgA), displays hypotensive/vasodilatory properties and counteracts excessive systemic and/or intra-cardiac excitatory stimuli (e.g., catecholamines and endothelin-1). CST, produced also by the myocardium, affects the heart by modulating inotropy, lusitropy and the coronary tone through a Nitric Oxide (NO)-dependent mechanism. This study evaluated the putative influence elicited by CST on the Frank-Starling response of normotensive Wistar Kyoto (WKY) and hypertensive (SHR) hearts by using isolated and Langendorff perfused cardiac preparations. Functional changes were evaluated on aged (18-month-old) WKY rats and SHR which mimic human chronic heart failure (HF). Comparison to WKY rats, SHR showed a reduced Frank-Starling response. In both rat strains, CST administration improved myocardial mechanical response to increased end-diastolic pressures. This effect was mediated by EE/IP3K/NOS/NO/cGMP/PKG, as revealed by specific inhibitors. CST-dependent positive Frank-Starling response is paralleled by an increment in protein S-Nitrosylation, AKT/eNOS/nNOS and PLN phosphorylations. Our data suggested CST as a NO dependent physiological modulator of the stretch-induced intrinsic regulation of the heart. This may be of particular importance in the aged hypertrophic heart, whose function is impaired because of a reduced systolic performance accompanied by delayed relaxation and increased diastolic stiffness. Scuola di Dottorato Life Sciences, Indirizzo: Biologia animale, Ciclo XXVIII, a.a. 2015-2016 Università della Calabria

U sirevima se tijekom zrenja odvijaju kompleksne mikrobiološke i biokemijske promjene koje imaju značajan utjecaj na formiranje organoleptičkih karakteristika gotovog proizvoda. Proteolitički enzimi razgrađuju kazein do većih i manjih peptida te slobodnih aminokiselina. Količina i omjeri pojedinih aminokiselina i topljivih peptida znatno utječu na teksturu i organoleptička svojstva sira. Razgradnjom aminokiselina u siru nastaju alkoholi, aldehidi, esteri, kiseline i sumporni spojevi koji formiraju specifične arome raznih vrsta sireva. U siru je identificirano više od 200 različitih hlapljivih komponenti. Okus sira je koncentriran u frakciji topljivoj u vodi (peptidi, aminokiseline, organske kiseline i amini), dok je aroma uglavnom koncentrirana u hlapljivoj frakciji (organske kiseline, aldehidi, amini, esteri). Mnogi sirevi sadrže iste ili slične komponente, ali u različitim koncentracijama i odnosima. Posebnosti arome sira ne zasnivaju se samo na jednom specifičnom spoju već na kombinaciji različitih spojeva nastalih tijekom zrenja. Djelovanjem na proteolitičke procese može se ubrzati i modificirati tehnološki proces proizvodnje sira, te ih se stoga intenzivno proučava., Complex microbiological and biochemical changes take place during cheese ripening process, having a significant impact on the formation of organoleptic characteristics of the final product. Proteolytic enzymes degraded casein to larger and smaller peptides and free amino acids. The quantity and the ratios of particular amino acids and soluble peptides significantly influence the texture and organoleptic properties of cheese. The products of amino acid degradation in cheese are alcohols, aldehydes, esters, acids and sulphur compounds, which form specific aromas of various cheese types. More than 200 different volatile components have been identified in cheese. Cheese flavour is concentrated in the watersoluble fraction (peptides, amino acids, organic acids and amines), while aroma is mainly concentrated in the volatile fraction (organic acids, aldehydes, amines, esters). Many cheeses contain the same or similar components, but in different concentrations and ratios. Specific characteristics of cheese aroma are not based only on one specific compound, but on combination of different compounds produced in the maturing process. The technological process of cheese production can be accelerated and modified by influencing proteolytic processes, which are therefore the subject of intense research.

A systematic study of the non-radiative deactivation mechanisms of the three photoexcited N-acetylphenylalanylamide conformers was conducted in order to disclose the experimentally observed conformational dependent lifetime of phenyl vibrationless 1* excited state. The all-atom trajectory surface hopping non-adiabatic molecular dynamics simulations, based on linear response time dependent density functional theory, were utilized for blind screening of relaxation pathways and revealed a number of excitation transfer mechanisms from 1* to the 1n* excited states localized on each of the two amide groups. Possible pathways were further refined by obtaining conical intersection barrier energies from corresponding reaction paths constructed at the coupled cluster (CC2) level of theory. Finally, from semiclassical consideration of conical intersection accessibility with only the nuclear zero point vibrational energy and from the increased rigidity of the second amide group towards distorsion upon its methylation, it was concluded that the classically accessible part of conical intersection seam for population transfer to the 1n* state of the second amide group is the largest for the conformer with the shortest 1* state lifetime. Provedeno je sustavno istraživanje neradijativnih deaktivacijskih mehanizama odgovornih za eksperimentalno opaženu konformacijsku ovisnost vremena života vibracijski osnovnog pobuđenog fenilnog 1* elektronskog stanja u tri konformera N-acetilfenilalanilamida. Simulacije metodom neadijabatske molekulske dinamike, s preskokom među plohama elektronske potencijalne energije dobivenih vremenski ovisnom teorijom funkcionala gustoće, ukazale su na nekoliko mehanizama prijenosa ekscitacije iz 1* u 1n* stanja locirana na pojedinim amidnim grupama. Pronađeni mehanizmi potom su utočnjeni pripadajućim vrijednostima energijama barijera koničnih presjecišta dobivenih ih reakcijskih puteva izračunatim na razini teorije spregnutih grozdova (CC2). Konačno, iz poluklasičnog razmatranja dostupnosti koničnog presjecišta samo na temelju nuklearne vibracijske energije nulte točke te iz povećanja rigidnosti druge peptidne skupine naspram njene deformacije usljed metilacije, određeno je kako klasično dostupan dio šava koničnog presjecišta za prijenos populacije u 1n* stanje druge peptidne skupine je najveći u konformeru s najkraćim vremenom života pobuđenog 1* stanja.

The ubiquitin-proteasome pathway (UPP) influences essential cellular functions including cell growt, differetiation, apoptosis, signal transduction, antigen processind and inflammatory responses. Themain proteolytic component of the ubiquitin-proteasome pathway (UPP)is the 26S proteasome, responsible for the turnover of many cellular proteins and represents an attractive target for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases.Natural and synthetic proteasome inhibitors having different chemical structures and potency have been discovered.In my PhD I worked on the design, synthesis and biological activity of molecules bearing pseudopeptidiche farmacoforiche different units at the C-terminus, as potential substrates for the interaction with the catalytic enzyme threonine, can inhibit specifically and selectively multicatalytic complex.Based on the data obtained from other series of inhibitors developed in our research group, which provided interesting biological results, I have designed and prepared based molecules peptides bearing new units farmacoforiche C-terminal, represented by groups isoxazolin vinyl-ester and dichloro- naftochinonici. Another series of derivatives instead, presents the methyl ester of trans-trans-muconic which pharmacophore linked to the side chain of a lysine residue common to all pseudo-tripeptides of the series.The new compounds were tested on the enzyme isolated based on their ability to inhibit three different catalytic activities of the proteasome: some of these, such as derivatives dichloro-naftochinonici, have shown a good ability of inhibition of the site post-acidic β1 and site chyme-tryptic β5.

Using molecular graph theory we studied the binding of NSFRY-NH2 and 12 related pentapeptide amides to Cu(II) as a model system for atrial natriuretic factor (ANF) peptide interactions with copper. Linear regression models based on the valence connectivity index of the 3rd order (3χv) reproduced experimental stability constants (log β) for 1N, 2N, 3N, and 4N coordinated complexes with the standard error of 0.30-0.39 log β units. We developed separate models for seven tyrosinic (N=28) and five non-tyrosinic peptides (N=20), and a common model for both kinds of peptides (N=48) with an indicator (dummy) variable. The results indicate additional aromatic stabilisation in 4N complexes due to metal cation-π interactions with tyrosine but not with the phenylalanine residue. We have also amended the log K and log K* values to correct miscalculations published by Janicka-Klos et al. in 2013., Služeći se teorijom molekularnih grafova istraživali smo vezivanje NSFRY-NH2 i 12 srodnih pentapetidnih amida za Cu(II) kao modelnog sustava za interakciju bakra s peptidnom molekulom atrijalnog natriuretičkog faktora (ANF). Modeli linarne regresije temeljeni na valencijskom indeksu povezanosti trećega reda (3χv) reproducirali su eksperimentalnu konstantu stabilnosti (log β) za komplekse koordinacije 1N, 2N, 3N i 4N sa standardnom pogreškom u rasponu od 0,30 do 0,39 log β jedinica. Razvili smo odvojene modele za sedam tirozinskih (N=28) i pet netirozinskih (N=20) peptida te skupni model s indikatorskom varijablom za obje vrste peptida (N=48). Rezultati upućuju na dodatnu aromatsku stabilizaciju u kompleksima vrste 4N zbog interakcija kationa s π-orbitalama tirozinskog ostatka, ali ne i fenilalaninskoga. Ispravili smo i pogrešne vrijednosti log K i log K* nastale omaškom u radu Anne Janicka-Klos i sur. 2013.