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The model of counterion-induced metallacarborane aggregation in water was verified on sodium bis(1,2-dicarbollide), one of the most studied examples of metallacarboranes. This case study introduces a novel approach in anionic boron cluster self-assembly that resembles the behavior of polyoxometalate nanoions. The key prerequisite of the counterion binding to the aggregates is the strongly uneven charge distribution of cobalt bis(dicarbollide) clusters. Simultaneously, the size of the counterion should fit into the void between the metallacarborane clusters within the aggregate. In consequence, compact pentamers with two bound sodium cations via Coulombic-driven assembly are formed. This behavior cannot be explained solely by the hydrophobic effect─the paradigm of metallacarborane aggregation in the current literature. The pentamers can merge into bigger nanostructures via the second aggregation mechanism that sheds light on the complex behavior of metallacarboranes in water. The proof-of-concept of counterion-induced metallacarborane aggregation and the proposed total description of the self-assembly via the two-process model are results of calorimetry modeling, quantum chemistry calculations, and molecular dynamics simulations.

Uređajem za statičko raspršenje svjetlosti Mastersizer3000 određivane su brzine agregacije čestica SiO2 (S-1000, S-200 i S-80) i TiO2 (T-250, T-50-70 i T-15). Mjerenja su provedena za dvije pH vrijednosti pH (pH = 3 i pH = 7) i pri dvije ionske jakosti (Ic = 0,1 mol dm–3 i Ic = 0,001 mol dm–3 ). Ispitano je kako pH i ionska jakost utječu na brzinu agregacije navedenih čestica. Iz raspodjele veličine čestica po volumenu izračunate su ovisnosti brojčane koncentracije čestica o vremenu i određene konstante brzine agregacije. Za čestice SiO2 (S200) izmjereno je statičko raspršenje svjetlosti pri pH = 4,5 za niz ionskih jakosti te je određena kritična koagulacijska koncentracija koja iznosi cccc = 0,0326 mol dm–3 . Uređajem ZetasizerPro metodom dinamičkog raspršenja svjetlosti za čestice SiO2 (S-1000, S-200 i S-80) i TiO2 (T250, T-50-70 i T-15) određene su ovisnosti veličine čestica o pH, a metodom elektroforeze određene su izoelektrične točke. Aggregation rates of SiO2 (S-1000, S-200 and S-80) and TiO2 (T-250, T-50-70 and T-15) particles were determined using static light scattrering device Mastersizer3000. Measurements were performed for two pH values (pH = 3 and pH = 7) and at two ionic strengths ((Ic = 0,1 mol dm–3 i Ic = 0,001 mol dm–3 ). It was investigated how pH and ionic strength affect the speed of aggregation of the mentioned particles. The numerical concentration of particles in time are calculated from the prarticle size distribution by volume and used to determine aggregation rate constant. Static light scattering was measured for SiO2 (S-200) particles at pH = 4,5 for a range of ionic strengths and the critical coagulation concentration was determined, which is cccc = 0,0326 mol dm–3 . The ZetasizerPro device was used to detemine the dependence of particle size on pH using using the dynamic light scattering method and isoelectric points using the electrophoresis method for SiO2 (S-1000, S-200 and S-80) and TiO2 (T-250, T-50-70 and T15) particles.

Nevrodegenerativne bolezni so v današnjem času vedno večji problem zdravstva in družbe. Simptomi nevrodegenerativnih bolezni so posledica progresivne izgube nevronov centralnega ali perifernega živčnega sistema. Ena glavnih značilnosti nevrodegeneracije je agregacija endogenih proteinov v netopna inkluzijska telesca. Primer takšnega proteina je TAR (angl. transactivation response) DNA-vezavni protein 43 (TDP-43). V normalnih celicah se TDP-43 nahaja predvsem v jedru in ima pomembno vlogo pri regulaciji genov, procesiranju in prerazporejanju RNA. V patoloških pogojih pride do cepitve, hiperfosforilacije in ubikvitinacije TDP-43, kar vodi v akumulacijo in agregacijo TDP-43 v citoplazmi. Še eden izmed kritičnih proteinov pri nevrodegeneraciji je šaperon endoplazemskega retikuluma (ER) sigma receptor 1 (Sig-1R). Sig-1R se nahaja na s holesterolom bogatih predelih membrane ER, lipidnih raftih. Disregulacijo TDP-43 se lahko neposredno poveže z agregacijo Sig-1R. Šaperon Sig-1R se v preživelih motoričnih nevronih pacientov s sporadično amiotrofično lateralno sklerozo akumulira in bi lahko imel nevroprotektivno vlogo. V magistrskem delu smo se osredotočili na in vitro agregacijo TDP-43 in vpliv proteina Sig-1R na potek agregacije TDP-43. V celicah E. coli BL21[DE3] smo izrazili proteina TDP-43-MBP in Sig-1R brez ΔN-končnega transmembranskega dela. S pomočjo afinitetne kromatografije in kromatografije z ločevanjem po velikosti smo izolirali zadostno količino obeh proteinov v topni obliki. V nadaljevanju smo izvedli agregacijske in interakcijske teste. Zanimala nas je agregacija TDP-43 in vpliv Sig-1R na agregacijo TDP-43. Kot inhibitor agregacije TDP-43 smo uporabili tRNA, za katere je znano, da prispevajo k topnosti TDP-43, za ΔNSig-1R pa smo hipotezo o inhibiciji agregacije testirali. Agregacijske teste smo izvedli z merjenjem motnosti raztopine pri valovni dolžini absorbance 395 nm. Dodatek tRNA je zmanjšal oziroma preprečil agregacijo TDP-43. Za ΔNSig-1R nismo uspeli dokazati, da preprečuje agregacijo TDP-43. V nadaljevanju smo izvedli interakcijske teste z metodo interferometrije z biološkimi plastmi (BLI). Tako med TDP-43 in tRNA kot tudi ΔNSig-1R je prišlo do šibke interakcije, vendar bi morali zaradi tehničnih napak v delu eksperiment ponoviti. Neurodegenerative diseases affect millions of people worldwide. The symptoms of neurodegeneration are caused by the progressive loss of neurons in the central or peripheral nervous system. Protein aggregation into insoluble inclusion bodies is one of the main hallmarks of neurodegeneration. TAR (Trans Activation Response) DNA-binding protein 43 (TDP-43) is one of the critical proteins in neurodegeneration. In normal cells, TDP-43 is mainly present in the nucleus and plays an important role in RNA regulation. Under pathological conditions, cleavage, hyperphosphorylation and ubiquitination of TDP-43 can occur. These posttranslational modifications lead to cytoplasmic accumulation and aggregation of TDP-43. Another critical protein in neurodegeneration is the chaperone at the endoplasmic reticulum (ER) Sigma receptor 1 (Sig-1R). Dysregulation of TDP-43 can be directly caused by Sig-1R aggregation. ER chaperone Sig-1R accumulates in motor neurons of ALS patients and may have neuroprotective functions. In our work we focused on the in vitro aggregation of TDP-43 and the influence of the Sig-1R protein on the course of TDP-43 aggregation. We expressed TDP-43_MBP and Sig-1R protein lacking N-terminal transmembrane region (ΔNSig-1R) in E.coli BL21[DE3] cells. In the process of protein purification with affinity chromatography and size-exclusion chromatography we isolated enough soluble TDP-43-MBP and ΔNSig-1R for further aggregation and interaction experiments. To analyze TDP-43 aggregation, we utilized spectrofotometric turbidity measurements (optical density at 395 nm). Aggregation was followed in the presence of a previously known aggregation inhibitor tRNA and a hypothetical inhibitor ΔNSig-1R. Addition of tRNA decreased or prevented aggregation of TDP-43. Addition of ΔNSig-1R did not prevent TDP-43 aggregation. We utilized bio-layer interferometry (BLI) for interaction analysis of TDP-43 with tRNA and ΔNSig-1R. There was a weak interaction between TDP-43 and tRNA as well as ΔNSig-1R, but due to technical errors during our work, the experiment should be repeated.

Promjena potrošnje električne energije tijekom dana, tjedna, godine, zahtjeva od elektroenergetskog sustava da prilagodi proizvodnju što je više moguće da bude jednaka potrošnji. Jedan od načina koji bi riješio taj problem jest skladištenje energije. To rješenje nije najekonomičnije zbog velikih gubitaka u transformaciji energije iz jednog oblika u drugi kao i velika investicija ako se radi o stacionarnim spremnicima električne energije. Povećani broj EV-a omogućuje iskorištavanje baterijskog spremnika koji se nalazi u vozilima za pohranu električne energije za vrijeme punjenja na punionicama ili kućnom punjaču. Agregiranje EV-a je naziv za djelatnost koja omogućava vlasnicima EV sudjelovanje na tržištu električne energije uz obostrani interes. Sudjelovanjem na tržištu agregator i vlasnik EV ostvaruju financijsku dobit u iznosu koja zavisi o određenim parametrima. Ovaj rad istražuje na temelju predloženog optimizacijskog modela koji parametri i u kojem iznosu djeluju na ukupni profit ostvaren tijekom dana za vrijeme agregiranja punionice ili EV Change in electricity consumption during the day, week, year, requires the power system to adjust production as much as possible to be equal to consumption. One way to solve this problem is to store energy. This solution is not the most economical, due to large losses in the transformation of energy from one form to another as well as a large investment if it is a stationary electricity storage. The increased number of electric vehicles allows the use of the battery tank located in electricity storage vehicles during charging at a charging station or home charger. Aggregation of electric vehicles is the name for an activity that allows owners of EV to participate in the electricity market with mutual interest. By participating in the market, the aggregator and owner of EV make a financial profit in the amount that depends on certain parameters. This paper investigates, based on the optimization model which parameters and in what amount affect the total profit made during the day during the aggregation of charging stations or EV.

Delo obravnava fizikalno optimizacijo pogojev za čiščenje proteinov in povezanih metod za iskanje optimalnih pogojev za minimizacijo agregacije. Metodološko delo temelji na kombinaciji eksperimentov in modelske analize eksperimentov, pri čemer sta glavni uporabljeni eksperimentalni metodi gelska izključitvena kromatografija in intrinzična triptofanska fluorescenca. Pripravili smo več formulacij z različnimi monoklonskimi protitelesi pri različnih pH, ionskih močeh in proteinskih koncentracijah. Z meritvijo triptofanske fluorescence smo opredelili vpliv posameznih dejavnikov na konformacijsko stabilnost proteinov v formulacijah. Opazili smo temeljne razlike v obnašanju monoklonskih teles, ki kažejo na različne mehanizme denaturacije in agregacije. Z gelsko izključitveno kromatografijo smo določili delež agregatov v formulacijah po izpostavljenosti nizkemu pH. S kombinacijo meritev smo sklepali o mehanizmih agregacije monoklonskih protiteles in o vlogi konformacijske stabilnosti v začetnih korakih agregacije. Ugotovili smo, da premik k višjim valovnim dolžinam izsevane svetlobe ob izpostavljenosti nizkemu pH kaže na ireverzibilno, k nižjim pa na reverzibilno agregacijo proteinov v formulacijah. Delo je prispevek k izboljšanju procesa čiščenja terapevtskih proteinov, ki je nujen za proizvajanje pacientu varnih zdravil. This master’s thesis explores the physical optimisation of protein purification conditions and related methods for finding optimal conditions to minimize aggregation. The methodological work is based on a combination of experiments and model analysis of the experiments, with the main experimental methods used being the size-exclusion chromatography and intrinsic tryptophan fluorescence. Several formulations were prepared with different monoclonal antibodies at different pH, ionic strengths and protein concentrations. By measuring the intrinsic fluorescence of tryptophan residues, we determined the influence of individual factors on the conformational stability of proteins in formulations. We observed fundamental differences in the behavior of the monoclonal antibodies, which imply different denaturation and aggregation mechanisms. The presence of aggregates in the formulations after exposure to low pH was determined by size-exclusion chromatography. With the combination of measurements from both methods we determined the aggregation mechanisms of the monoclonal antibodies and the role of conformational stability in the initial steps of aggregation. We found that a shift toward higher emission wavelengths upon exposure to low pH indicates irreversible, whereas a shift toward lower emission wavelengths indicates reversible protein aggregation. This work is a contribution towards the improvement of therapeutic protein purification process, which is necessary for a safe use of biopharmaceuticals.

In the last couple of years electricity market landscape has significantly changed. The rising share of distributed generation and consumption increases price volatility and causes additional intermittencies to the grid. Thus, the grid must become more flexible. This paper refers to the role of aggregators as flexibility providers in the new smart grids. Its main objective was to develop an actual aggregator model which aggregates commercial and industrial consumers for providing active demand response. The idea behind it was to identify possibilities and potential challenges for aggregators on partially closed electricity markets. For this purpose, an implementation of an aggregator business model on the market in Croatia was analyzed. The paper consists of several parts. In the first part the principle of operation and value proposition of an aggregator business model are defined. The second part refers to regulatory framework and current market design. In the third part the necessary hardware and software solutions for developing the aggregator model are determined and total investment costs are calculated. The last part specifies the properties of the implemented model and analyses its cost-effectiveness. From the analysis, it can be concluded that, when implementing an aggregator on an electricity market which is not completely open, several aspects should be discussed. The main issue emerges from the current market design and the lack of incentives for prosumers to join the aggregator. A new aggregator business model requires separating the balancing responsibility, setting up a compensating financial transfer mechanism and slightly changing the regulatory framework. However, the aggregator has a positive impact on the market and its implementation is cost effective for the prosumers and for the investors. V zadnjih nekaj letih se trgi z električno energijo bistveno spreminjajo. Naraščajoči delež razpršenih virov povečuje nestanovitnost cen in povzroča dodatne nestabilnosti v omrežju, zato se pojavlja potreba po razvoju pametnih omrežij. Pametno omrežje je nov, bolj prilagodljiv sistem, ki integrira več različnih distribuiranih enot in prilagaja njihovo proizvodnjo in odjem. Ta sistem prenaša ne samo energijo, ampak tudi informacije. Vsi njegovi elementi medsebojno komunicirajo in izmenjujejo podatke, kako bi se lažje prilagodili nepričakovanim spremembam. Agregirani sistemi obnovljivih virov energije so produkti pametnega omrežja. Trend decentralizacije, dekarbonizacije in digitalizacije postavlja agregatorje v center sodobnega elektroenergetskega sistema. Njihova vloga kot aktivni udeleženci trga električne energije je bistvena za integracijo razpršenih virov v omrežje in za aktivno sodelovanje odjemalcev na trgu. Z združevanjem več majhnih, fleksibilnih odjemalcev in proizvajalcev, agregatorji povečujejo izkoristek obnovljive energije in omogočajo vstop majhnih odjemalcev na trg. Razvoj in dostopnost agregatorjev sta močno odvisna od tehničnih pogojev, kot so pametni števci in napredne komunikacijske tehnologije. Še bolj odvisna sta od dizajna trga in zakonodaje, ki morata biti oblikovana tako, da motivirata in spodbujata odjemalce k pridružitvi trgu. Namen tega magistrskega dela je analizirati vpliv agregatorjev na trg električne energije s pomočjo poslovnega agregatorskega modela na Hrvaškem. Vloga agregatorjev na sodobnih trgih električne energije je določena skozi agregiranje več majhnih odjemalcev, ki so pripravljeni prilagajati svoj odjem. Magistrsko delo je sestavljeno iz več delov. V prvem delu je razložen princip delovanja agregatorskih poslovnih modelov. Drugi del se nanaša na trenutno zakonodajo v Evropski Uniji in na Hrvaškem. Tretji del določa potrebno strojno in programsko opremo za implementacijo agregatorja. Zadnji del se nanaša na parametre implementiranega modela in na njegovo ekonomsko upravičenost. Agregatorji so posredniki med končnim odjemalcem in trgom električne energije. Razvijajo rešitve za komercialne ali stanovanjske odjemalce, s katerimi izboljšujejo njihovo energetsko učinkovitost in prispevajo k povečanju fleksibilnosti obstoječega omrežja. Osnovni model agregatorja deluje tako, da integrira več majhnih proizvajalcev in/ali odjemalcev s prilagodljivo proizvodnjo/ odjemom. Cilj agregatorja je ustvariti dobiček s prodajo servisnih storitev prenosnemu ali distributivnemu sistem operaterju. Vsak odjemalec deluje v okvirju bilančne skupine. Upravitelji bilančnih skupin so odgovorni za oskrbo z električno energijo in za korekcijo nestabilnosti, ki jo skupina s svojim delovanjem povzroči v omrežju. Bilančna skupina ali zasebni člani bilančne skupine lahko prevzamejo dodatno vlogo na trgu in postanejo agregatorji. Poleg tega obstaja tudi neodvisni agregator, ki ni povezan z dobaviteljem ali z bilančno skupino. V katero smer se bo agregatorski model razvijal je močno odvisno od zakonodaje in želenih funkcij agregatorja. Pri implementaciji agregatorja podjetje, ki namerava ustvariti agregacijo, zbere neobhodne podatke in ustvari mrežo partnerstev. Agregator in partnerji določijo poslovne pogoje in vzpostavijo sistem. Glavni vir prihodkov podjetje ustvari s prodajo energije na trgu. Ko je osnovni koncept agregatorja postavljen, se ta agregator implementira na trg. Kot prej omenjeno, je bil pri razvoju agregatorskega modela upoštevan koncept trga v Republiki Hrvaški. Enako kot vsi evropski trgi je tudi hrvaški trg sestavljen iz več bilančnih skupin. Vsak član na trgu mora delovati preko bilančne skupine kot navadni član ali kot upravitelj skupine. Vsi upravitelji bilančnih skupin so dolžni podpisati pogodbo s prenosnim operaterjem (HOPS), vsi člani bilančnih skupin pa morajo podpisati člansko pogodbo z bilančno skupino. Vsaka bilančna skupina predlaga organizatorju trga (HROTE) napoved proizvodnje/porabe en dan vnaprej. Po preverjanju skladnosti podatkov HROTE pogodbene načrte pošlje HOPS-u, ki preveri tehnične izvedljivosti predloženih količin. Odgovoren za določanje in zagotavljanje tehničnih pogojev prenosa energije in doseganje ravnotežja v sistemu je HOPS. Po fizični dobavi električne energije mora HOPS ponudnikom bilančnih storitev plačati vsako aktivacijo in še zaračunati tistim, ki so s svojo aktivacijo povzročili neravnovesje. Posledično temu je HOPS tudi odgovoren za ustvarjanje in zagotavljanje rezerv za primarno, sekundarno in terciarno regulacijo frekvence. V primeru zaznane nestabilnosti mora prenosni sistem aktivirati svoje rezerve in zagotoviti ustrezno bilančno energijo za ohranjevanje konstantne frekvence. Za primarno in sekundarno frekvenčno regulacijo so odgovorne male turbine in elektrarne v lasti hrvaškega distribucijskega sistema. Terciarna frekvenčna regulacija, ki se aktivira 15 minut po nastanku neravnovesja, je cilj implementiranega agregatorskega modela. Agregator bo v vsakem času pripravljen ponujati ročno rezervo za povrnitev frekvence HOPS-u. Za implementacijo agregatorskega modela je potrebna ustrezna strojna in programska oprema za zajem in analizo podatkov. Potrebni so krmilniki, senzorji in pametni števci. Potreben je tudi SCADA sistem za akvizicijo podatkov ter programska rešitev za analizo in napoved porabe in proizvodnje. Oprema, ki jo je treba inštalirati na strani odjemalcev, je odvisna od diagrama porabe in od že obstoječe opreme na lokaciji. Na začetku so pametni števci in krmilniki z dodatnimi I/O moduli za pošiljanje podatkov SCADA sistemu zahtevani pri vsakem porabniku, neodvisno od tipa in funkcije porabnika. Za meritev je predvidena strojniška oprema iz Schneider Electrica, na podlagi katere so izračunani investicijski stroški. Za zbiranje podatkov je predvidena GEO SCADA programska oprema. Napredna analiza podatkov in napoved porabe in proizvodnje se izvede s pomočjo avtonomne AutoGrid platforme, ki se lahko uporablja za zmogljive podatkovne analize. AutoGrid je paket rešitev z veliko podatkovno in infrastrukturno plastjo, ki izkorišča fleksibilnost odjemalcev, da bi zagotovila prilagodljivost in fleksibilnost omrežja. Platforma je sestavljena iz več zasebnih modulov. Čeprav ima vsak od teh modulov svoje lastnosti, gre navsezadnje za upravljanje, optimizacijo, združevanje in dispečiranje fleksibilnosti. Najpomembnejši elementi agregatorja so odjemalci. Agregatorski model bo sestavljen izključno iz odjemalcev, ki so pripravljeni svoj odjem spreminjati skozi čas. Pri tej analizi je bilo upoštevanih deset odjemalcev, ki pripadajo komercialnem in industrijskem sektorju. Stanovanjski odjemalci niso bili upoštevani zaradi pomanjkljivosti inštaliranih pametnih števcev in zaradi raznolikosti in nepredvidljivosti diagrama porabe. Komercialni in industrijski sektor imata različen diagram porabe. Diagram porabe komercialnega sektorja po navadi začne rahlo naraščati zjutraj, konico doseže v popoldanskem času, potem pa poraba začne zopet rahlo upadati. Odvisno od tega, ali se gre za dvoizmensko ali enoizmensko delo, imajo industrijski odjemalci različne diagrame porabe. Najlažji za modeliranje so dvoizmenski industrijski pogoni, ker imajo precej konstantno porabo. V tem magistrskem delu je bilo izbranih šest fleksibilnih objektov iz komercialnega sektorja, kot so: - hoteli, - data centri in - tržni center. Glavni produkti za prilagajanje odjema pri komercialnem sektorju pridejo iz sistemov za prezračevanje in klimatizacijo, ki se lahko regulirajo. Pametne naprave in avtomatizirani procesi nudijo dodatne prilagoditvene možnosti in rezerve. Pri industrijskem sektorju je bilo izbranih pet različnih industrijskih pogonov s prilagodljivim odjemom. Eden izmed najbolj zanimivih je pogon za proizvodnje jekla, pri katerem se s pomočjo regulacije hitrosti valjanja lahko vpliva na porabo. Izbrani so pogoni za proizvodnjo: - kamna, - jekla in - stekla. Agregator z odjemalci podpiše dvostransko pogodbo in plačuje njihovo razpoložljivost in energijo, ki jo dejansko zagotovijo z aktivacijo. V začetni fazi so odjemalci dolžni zagotoviti potrebne podatke o porabi in razpoložljivosti ter podroben opis svojih dejavnosti in naprav. Vhodni podatki se uporabljajo za optimizacijo in pridobitev optimalne rešitve za dispečiranje. Energija za uravnoteževanje se prodaja prenosnemu sistemu (HOPS-u) kot standardiziran produkt za ročno rezervo za povrnitev frekvence. Standardiziran produkt mora imeti točno določene parametre, kot so period aktivacije, period deaktivacije, velikost in period razpoložljivosti. Prodajna cena je določena na trgu izravnalnih storitev. V skladu z uredbo Evropske Unije je minimalna količina, ki se lahko prodaja kot produkt za terciarno regulacijo, 1 MW. V inicialni fazi projekta je agregator zasnovan tako, da zagotavlja samo pozitivno fleksibilnost omrežju, oziroma zmanjša svojo porabo v primeru pomanjkanja energije in rezerve gredo v sistem. Na koncu magistrskega dela je bila analizirana še ekonomska upravičenost implementiranega modela. Pri tej analizi s pomočjo metode neto sedanje vrednosti (NSV), metodo na podlagi donosnosti (ISD) in metodo dobe vračanja (DV) je izračunan neto denarni tok, akumulirani denarni tok in povračilna doba investicije. Pozitivna NSV in ISD kažeta, da je opisan agregatorski model samozadosten in izplačljiv. Iz te analize lahko zaključimo, da se pogoji za razvoj agregatorjev na evropskem trgu počasi spreminjajo. Decentralizirana proizvodnja in liberalizacija trga močno spodbujata razvoj agregacijske proizvodnje. V začetni fazi našega projekta so bili stroški in razpoložljiva zmogljivost agregatorja natančno določeni. Obnašanje agregatorja na trgu ni bilo možno popolnoma pravilno simulirati zaradi omejitev razpoložljivih informacij o prejšnjih dogodkih na trgu. Naslednje faze vključujejo podpisovanje pogodb s porabniki ter namestitev pametnih števcev in krmilnikov za zajem podatkov na objektih. Dolgoročen cilj je vključiti čim več odjemalcev in ponujati simetrične in nesimetrične izdelke na trgu, primerne ne samo za ročno, ampak tudi za avtomatsko regulacijo frekvence. Čeprav zakonodaja in regulativa še nista dokončana, bo uvedba tega modela sprožila nove prilagoditve trga in močno spodbudila razpršeno proizvodnjo energije in nadaljnji razvoj pametnih omrežij.

Monoclonal antibodies are the fastest growing class of therapeutics. However, aggregation limits their shelf life and can lead to adverse immune responses. Assessment and optimization of the long-term antibody stability are therefore key challenges in the biologic drug development. Here, we present a platform based on the analysis of temperature-dependent aggregation data that can dramatically shorten the assessment of the long-term aggregation stability and thus accelerate the optimization of antibody formulations. For a set of antibodies used in the therapeutic areas from oncology to rheumatology and osteoporosis, we obtain an accurate prediction of aggregate fractions for up to three years using the data obtained on a much shorter time scale. Significantly, the strategy combining kinetic and thermodynamic analysis not only contributes to a better understanding of the molecular mechanisms of antibody aggregation but has already proven to be very effective in the development and production of biological therapeutics.

The colloidal stability of sulfate (SL) and polyimidazolium-modified sulfate (SL-IP-2) latex particles was studied in an ionic liquid (IL) of ethylammonium nitrate (EAN) and its water mixtures. Aggregation rates were found to vary systematically as a function of the IL-to-water ratio. Repulsive electrostatic interactions between particles dominated at low IL concentrations, while they were significantly screened at intermediate IL concentrations, leading to destabilization of the dispersions. When the IL concentration was further increased, the aggregation of latex particles slowed down due to the increased viscosity and finally, a striking stabilization was observed in the IL-rich regime close to the pure IL solvent. The latter stabilization is due to the formation of IL layers at the interface between particles and IL, which induce repulsive oscillatory forces. The presence of the added salt in the system affected differently the structure of the interfaces around SL and SL-IP-2 particles. The sign of the charge and the composition of the particle surfaces were found to be the most important parameters affecting the colloidal stability. The nature of the counterions also plays an important role in the interfacial properties due to their influence on the structure of the IL surface layers. No evidence was observed for the presence of long-range electrostatic interactions between the particles in pure ILs. The results indicate that the presence of even low concentrations of water and salt in the system (as undesirable impurities) can strongly alter the interfacial structure and thus, the aggregation mechanism in particle IL dispersions.

Namen naloge je bil iz hipertermofilne arheje Aeropyrum pernix K1 izolirati majhne organske molekule (termolite) ter preučiti njihov vpliv na agregacijo/fibrilacijo inzulina. Začeli smo z izolacijo termolitov z metodo HPLC ter nadaljevali s preučevanjem agregacije inzulinskih verig pri različnih pogojih. Iz arheje smo izolirali dve majhni organski molekuli, vendar ju nismo uspeli identificirati. Agregacijo inzulinskih verig smo spremljali z emisijsko fluorescenčno spektrometrijo z barvilom tioflavinom T (ThT). Veriga B je hitreje fibrilirala v nevtralnem kot v kislem območju, veriga A je tvorila fibrile samo pri pH 2,0. Fibrilacijo verige B smo potrdili s cirkularnim dikroizmom z določitvijo za fibrile značilne β-strukture. Proces fibrilacije sta najbolj zavirala manoza in N-acetilglukozamin, slabše je učinkovala glukoza, glicerol je preprečil nastanek fibril pri visoki koncentraciji, mio-inozitol ni imel vpliva. Z določitvijo sekundarne strukture polipeptidov ob dodatku molekul smo potrdili oziroma ovrgli nastanek fibril. Z nativno poliakrilamidno gelsko elektroforezo smo ugotovili, da nekatere molekule inzulinskih verig ostanejo po fibrilaciji v monomerni obliki in se ne vgradijo v fibrile. Glede na molekulsko sidranje se nobena izmed testiranih majhnih organskih molekul ne veže močno na verigo B. V nalogi smo uspešno postavili metodo za izolacijo termolitov, ugotovili katere molekule vplivajo na fibrilacijo inzulinskih verig, vendar nismo uspeli potrditi/ovreči delovne hipoteze glede vpliva termolitov na agregacijo inzulina. The purpose of this study was to isolate small organic compounds (thermolytes) from hyperthermophilic archaeon Aeropyrum pernix K1 and research their effect on insulin aggregation/fibrillation. We started our research by isolating thermolytes with HPLC and continued to study the aggregation of insulin chains at different conditions. We isolated two small organic compounds from the archaeon, but we were unable to identify them. Aggregation of insulin chains was firstly studied using emission fluorescent spectroscopy with the dye thioflavin T (ThT). Chain B formed more fibrils at neutral than at acidic pH, while chain A fibrillated solely at pH 2.0. Measurements of circular dichroism after the fibrillation showed the formation of β-structure by chain B, which is typical for fibrils. Mannose and N-acetylglucosamine were the best inhibitors of fibrillation, glucose had small effect, glycerol only affected fibrillation at high concentration, myo-inositol did not inhibit fibrillation at all. By determining the secondary structure of polypeptides with added compounds, the formation of fibrils was confirmed or refuted. Native polyacrylamide gel electrophoresis indicated that during fibrillation some molecules of insulin chains remained in monomeric form and were not incorporated into fibrils. The molecular docking data exhibited that tested small organic compounds bind weakly to chain B. In this thesis we successfully set a method for thermolyte isolation, found out which molecules affect insulin chains fibrillation, but unfortunately, we were not able to confirm or refute the hypothesis about the effect of thermolytes on insulin aggregation.

Infrardeča spektroskopija sodi med manj pogoste metode za preučevanje konformacijskih sprememb proteinov. Kljub temu metoda nudi nekatere prednosti, kot so meritve majhnih količin vzorcev v različnih agregatnih stanjih. Pridobimo lahko informacije o medmolekulskih interakcijah, še posebej pa je metoda občutljiva pri karakterizaciji vodikovih vezi, ki sodijo med nekatere izmed glavnih sil, ki stabilizirajo strukturo proteinov. Proces denaturacije proteinov je pogosto sklopljen še z njihovo agregacijo. V magistrskem delu smo spremljali spremembe v sekundarni strukturi treh modelnih proteinov pri njihovi temperaturni in kemični denaturaciji z natrijevim dodecilsulfatom. Konformacijske spremembe smo zaznali z uporabo diferenčne spektroskopije. Z integriranjem vrhov v diferenčnih spektrih smo določili temperaturo denaturacije proteinov ter spremembe deležev elementov sekundarnih struktur. Pokazali smo, da lahko s FTIR zaznamo spremembo v temperaturi denaturacijskega prehoda ob prisotnosti majhne količine denaturanta. Z metodo dekompozicije in prileganja modelnih spektralnih trakov smo uspešno napovedali deleže sekundarnih struktur v nativnem stanju in iz površin modelnih trakov izračunali temperaturno odvisnost deleža α-vijačnic in β-agregatov za hemoglobin. Agregacija je pri hemoglobinu in konalbuminu nastopila v celotnem koncentracijskem območju preiskanim s FTIR spektroskopijo. Prisotnost dodecilsulfata je vodila do počasne agregacije proteinov. Pri visokih koncentracijah denaturanta je bila karakterizacija spektrov nemogoča zaradi prenasičenja signalov v spektru. Inzulin pri nizkih vrednostih pH raztopin ob temperaturnih meritvah ni kazal bistvenih sprememb v strukturi poleg slabenja vodikovih vezi. Ta podatek se sklada s predhodnimi rezultati, ki pokažejo rast fibril šele po začetni lag fazi. Infrared spectroscopy is an unusual method for studying conformational changes of proteins. Nevertheless, the method offers some advantages, such as the possibility to measure small amounts of samples in different states. We can obtain information about intermolecular interactions, and it is particularly sensitive in characterizing hydrogen bonds, which are among the leading forces that stabilize proteins. The process of denaturation is very often coupled with protein aggregation. In this work, we followed the changes in the secondary structure of three model proteins under temperature and chemical denaturation conditions with sodium dodecyl sulfate. We used difference spectroscopy to detect conformational changes of the proteins. By integrating peaks in difference spectra, we predicted denaturation temperatures of proteins and changes in the amount of secondary structures. We showed that we can detect changes in transition temperatures with FTIR when small amounts of denaturant are present. Using decomposition and peak fitting, we successfully predicted the amount of secondary structures in the native form and calculated the temperature dependence of the secondary structure content of α-helices and β-aggregates for hemoglobin. Hemoglobin and conalbumin experienced aggregation throughout the working concentration range of FTIR spectroscopy. The presence of dodecyl sulfate resulted in slow aggregation of the proteins, which we detected with differential spectroscopy. Characterization of spectra with high concentrations of dodecyl sulfate was not possible due to saturation of the protein spectra. Insulin at pH 3 showed no significant changes in secondary structure besides the weakening of hydrogen bonds at higher temperature measurements. These data are consistent with previous studies of insulin showing that fibrillation begins only after an initial lag phase.

The aggregation propensity of monoclonal antibodies can be modified by adding different cosolutes into the solution. A simple coarse-grained model in the combination with the thermodynamic perturbation theory was used to predict cluster distribution and viscosity of the solutions of IgG4 monoclonal anibody in the presence of L-Arginine Hydrochloride. The data were analysed using binding polynomial to describe the binding of cosolute (Arginine) to the antibody molecule. The results show that by binding to the antibody molecule the cosolute occupies some of the binding sites of the antibody, and in this way reduces the amount of binding sites available to other antibody molecules. The aggregation propensity of the antibody molecules is therefore reduced.

Monoklonska protitelesa so beljakovinske molekule, ki so nagnjene k agregaciji, kar za biološko zdravilo ni sprejemljivo, saj lahko poveča imunogenost končnega produkta. Dolgoročno stabilnost proteina ocenimo z merjenjem proteinske agregacije v vzorcih, ki so izpostavljeni povišanim temperaturam in kasnejšo ekstrapolacijo podatkov z uporabo različnih modelov. Glede na različne mehanizme nastajanja agregatov, kinetiko agregacije ter adhezije delcev na površino vial lahko pri dejanskih dolgoročnih pogojih shranjevanja pride do višje koncentracije delcev v raztopini, ki je ni mogoče napovedati na tak način. V nalogi smo preučevali vpliv površine viale, temperature shranjevanja in prisotnosti soli na kinetiko procesa agregacije na več velikostnih skalah agregatov za dve različni monoklonski protitelesi. Pri obeh smo določili, da so pri koncentracijah, značilnih za biološka zdravila, omejujoč korak hitrosti agregacije bimolekularni trki. Hitrost agregacije lahko pri poljubni temperaturi in koncentraciji napovemo le za enega od testiranih monoklonskih protiteles, medtem ko pri drugem dodaten neznan proces povzroča odstopanje dejanskih rezultatov od napovedi. To predstavlja povečano tveganje pri izbiri formulacije na podlagi presejalnih testov pri povišani temperaturi in nizki koncentraciji, ki pa ga lahko zmanjšamo z dodatnimi temperaturnimi in koncentracijskimi pogoji testiranja. Monoclonal antibodies are protein molecules that are prone to aggregation, which is not acceptable for biopharmaceuticals, since it can increase the immunogenicity of the final product. Long-term protein stability can be assessed by measuring protein aggregation in samples exposed to elevated temperatures and subsequent extrapolation of data using different models. Due to different mechanisms of aggregate formation, aggregation kinetics and particle adhesion to the vial surface, higher particle concentrations in solution may occur under actual long-term storage conditions, which cannot be predicted from accelerated studies. We studied the effect of vial surface, temperature and presence of salt on the kinetics of aggregation process on multiple size ranges of aggregates for two monoclonal antibodies. Bimolecular collisions were found to be the limiting step of aggregation at concentrations specific to biologics. The rate of aggregation can be predicted at any temperature or concentration for only one of the tested monoclonal antibodies, while the other is likely to undergo an additional unknown process, which causes a deviation of prediction from the actual results. This poses an increased risk when selecting a formulation based on screening tests at elevated temperatures and low concentrations, which can be mitigated by additional temperature and concentration conditions.

Uporaba proteinov predstavlja nov način zdravljenja bolezni ter s tem postaja pomembna veja sodobne farmacije. V primerjavi s klasičnimi zdravili, so gradniki biofarmacevtskih učinkovin precej drugačni. Biološka zdravila imajo kompleksno 3D strukturo z visoko molekulsko maso. Proteinske molekule se v primeru neustreznih pogojev utegnejo sprijeti v večje skupke podobnih velikosti (agregate). Z namenom zagotavljanja kakovosti terapevtskih proteinov se čedalje večjo pozornost namenja spremljanju lastnosti in stabilnosti proteinov ter sami interakciji med njimi. Ena izmed temeljnih lastnosti raztopin proteinov je njihova viskoznost. Merjenje viskoznosti v zgodnjih fazah razvoja zdravilne učinkovine je vedno bolj zanimivo tudi z vidika vse natančnejšega napovedovanja makroskopskega vpliva reverzibilnega povezovanja proteinskih molekul končnega produkta. Poznavanje natančnih vrednosti viskoznosti različnih formulacij prispeva k razumevanju in izboljševanju matematičnih modelov agregacijskih poti. Delo opisuje uspešno implementacijo analitske metode za določanje viskoznosti raztopine proteinov na napravi VROC Initium, ki glede na predhodne metode prinaša številne prednosti. Gre za avtomatski viskozimeter, ki temelji na uporabi mikrokanala in je prilagojen na mikrotitrske plošče ali viale. Meritve so v primerjavi s predhodno uporabljeno reometrijo hitrejše, natančnejše ter porabijo manjše količine materiala. V delu je prihranek proteinskega materiala prikazan na primeru iskanja ustreznih ekscipientov za zmanjševanje viskoznosti formulacije. Poleg tega so bili uporabljeni novi, manjši vsebniki, zato je bila znanstveno potrjena tudi ustreznost membran za filtracije in dializo. Nova analitska metoda je bila uporabljena še za potrditev korelacij med viskoznostjo raztopin proteina visokih koncentracij in interakcijami med proteini pomerjenimi pri nizkih koncentracijah. Rezultati meritev viskoznosti treh različnih razvojnih proteinov ustrezno korelirajo z meritvami dinamičnega interakcijskega parametra (kD). Umetno tvorjeni večji agregati mikrokanala niso mašili. Nova analitska metoda je tako uspešno nadomestila prej uporabljeno reometrijo. Recombinant therapeutic proteins are an advanced way of treating various life-threatening conditions and thus becoming an important branch of modern pharmacy. Compared to conventional drugs, the building blocks of biopharmaceuticals are quite different. Biological drugs have a complex 3D structure with high molecular weight. When they are exposed to different stress conditions, protein molecules may adhere to larger clusters of similar size (aggregates). In order to ensure quality, increasing emphasis is placed on monitoring the properties and stability of proteins together with interactions between them. One of the fundamental properties of protein solutions is their viscosity. Measurement of viscosity in the early stages of drug development is also becoming increasingly interesting for progressively accurate prediction of the macroscopic impact of final product reversible self-association. Knowing the exact viscosity values of different formulations contributes to understanding and improving mathematical models of the aggregation process. The thesis describes a successful implementation of an analytical method for determining the viscosity on a VROC Initium device. It is an automatic viscometer based on the use of a microchannel and adapted to microtiter plates or vials. Measurements are fast, very accurate and consume small amounts of liquid material. Viscometer VROC enabled many experiments that were not possible before or simply not to such an extent. The saving of protein material is shown in the case of examining suitable viscosity reducing additives (VRA). Reduced sample volume needed for measurement enabled the use of smaller plastic containers. The suitability of their membranes for filtration and dialysis was scientifically confirmed. Furthermore, a new analytical method was used to confirm the correlations between the viscosity of high-concentration protein solutions and the interactions between proteins measured at low concentrations. The results of viscosity measurements of three different proteins correlate accordingly with the measurements of the dynamic interaction parameter (kD). Artificially generated larger aggregates haven't clogged the microchannel. The method successfully replaced the previously used rheometry.

Preučevali smo agregacijo konkanavalina A z uporabo infrardeče spektroskopije. Konkanavalin A je protein s pretežno sekundarno strukturo beta, ki ima zelo zanimivo vrsto agregacije. V kislem mediju se z nespremenjeno strukturo agregira v amorfne agregate, medtem ko v bazičnem okolju tvori amiloidne fibrile. Zato smo ga uporabili za testiranje zaviranja tvorbe amiloidov, ki so glavni povzročitelji tako imenovanih konformacijskih bolezni, kot sta npr. Parkinsonova in Alzheimerjeva bolezen. Tvorbo agregatov in amiloidov smo pospešili z dvigovanjem temperature. Za zaviranje smo uporabili dva znana zaviralca urejenih proteinskih agregatov, homotavrin in resveratrol. Izračunali smo sigmoidne krivulje ter določili in spremljali temperature prehoda iz raztopine v agregate, z in brez inhibitorja. Z metodo diferenčne spektroskopije smo spremljali majhne strukturne spremembe, ki nastanejo kot posledica zunanjih sprememb. Analizni spekter je izračunan kot razlika med dvema posnetima spektroma ob na primer različnih temperaturah. Zato nam ponuja metoda vpogled le v vrhove, ki niso skupni obema izhodnima spektroma. Temperatura prehoda ni naraščala, tako kot smo to pričakovali z naraščanjem koncentracije inhibitorja. Kjer se izkaže, da se je dvigovala, to ovržemo z negotovostjo meritev. Možnih razlogov za to je več, kriva je lahko napačna izbira koncentracijskih razmerij, nasičena raztopina, ali pa moja napaka pri mešanju raztopin. Največ težav pri raztapljanju smo imeli pri resveratrolu, zato mogoče ni bil najboljša izbira za izbrana koncentracijska razmerja. We investigated the aggregation of Concanavalin A using infrared spectroscopy. Concanavalin A is a protein with a predominantly $beta$-structure and has a very interesting way of aggregation. In acidic medium it aggregates in amorphous aggregates, whereas in alkaline medium it forms amyloid fibrils. Therefore, we have used it for testing retarding amyloids, which are the main cause of conformation diseases, such as Parkinson and Alzheimer disease. The growth of aggregates and amyloids was accelerated with the increase in temperature. We used two well-known inhibitors to slow down settled protein aggregates, which are homotavrine and resveratrol. We calculate sigmoid curves from structural changes taken from the analysis of amide I band and monitored the temperature of the transition from solution to aggregates, with and without inhibitors. Using the method of differential spectroscopy, we were able to accompany small structural changes, which are caused by temperature changes. The analysis spectrum is calculated as the difference between two spectra recorded at different temperatures. For this reason, this method allows us to look into spectral bands that are not the same for both spectra. The temperature of the transition did not rise, as we had expected, with the increase in inhibitor concentration. Where it shows the increase, it correlates with the measurement error. There can be many reasons for this, it could be a wrong choice of concentration ratio, a saturated solution or my error with the solutions. Most of the problems with solving occurred with resveratrol, maybe it was not the best choice for the chosen concentration ratios.

Research in protein aggregation and other degradation routes is of major importance in recent years, influencing the quality and consistency of biopharmaceutical drugs used to treat various life-threatening conditions. In this work we study the open soft-matter physics questions behind such processes that affect developability, safety and efficacy of aqueous solutions of therapeutic proteins. The focus lies on processes of irreversible aggregation and reversible self-association, which can be most influenced by the physical properties of the solution. Physico-chemical interactions reflected in colloidal and conformational stability are evaluated with dynamic light scattering (DLS) and induced protein denaturation. Thermal denaturation is measured via differential scanning calorimetry (DSC). Guanidium hydrochloride and urea are used as chemical denaturants and the ensuing unfolding is measured via tryptophan fluorescent response. The physical effect of excipients such as salts and sugars on protein stability is evaluated. Irreversible aggregation is accelerated mostly with elevated temperature and measured with size exclusion chromatography (SEC), resonant mass measurement (RMM) and micro-flow imaging (MFI). A Smoluchowski type system of differential equations, describing the kinetics of binary aggregation on multiple size scales, is numerically solved in parallel, with the model output compared with experimental data, explaining several phenomena specific to protein aggregation. Adhesion of proteins and protein particles in the micrometer size range to surfaces of containers is characterised with atomic force (AFM) and optical microscopy. We identify several different regimes of particle formation, adhesion to surfaces, and accumulation in bulk solution, shown to have a major impact on determination of particle formation propensity based on elevated temperature studies. We evaluate the discrepancies with mean-field Smoluchowski type models in this non-colloidal regime. Reversible self-association resulting in increased solution viscosity is measured with microfluidic rheometry. The factors contributing to viscosity of protein solutions are analysed and evaluated. A novel interaction mechanism contributing to viscosity is proposed. Finally, this work is a contribution towards advanced protein formulation development with special emphasis on development of biopharmaceuticals. V zadnjih letih narašča pomembnost raziskav procesov kot so agregacija in degradacija proteinov, ki vplivajo na kvaliteto bioloških zdravil za zdravljenje različnih bolezenskih stanj. V tezi raziskujemo tematike fizike mehke snovi v povezavi s temi procesi, od katerih zavisi razvoj, varnost in učinkovitost vodnih raztopin terapevtskih proteinov. Osredotočamo se predvsem na procese nereverzibilne agregacje in reverzibilnega povezovanja proteinskih molekul, na katere lahko v veliki meri vplivamo s fizikalnimi lastnostmi raztopin. Fizikalno-kemijske interakcije, ki vplivajo na koloidno in konformacijsko stabilnost, ovrednotimo z dinamičnim sipanjem svetlobe (DLS) in denaturacijo. Termično denaturacijo merimo z diferencialno kalorimetrijo (DSC). Kot denaturante za kemično denaturacijo uporabljamo gvanidinijev klorid in ureo, merimo pa jo prek fluorestenčnega odziva triptofanov. Ovrednotimo fizikalni vpliv pomožnih snovi v zdravilu (ekscipientov), kot so soli in sladkorji. Tvorbo nereverzibilnih agregatov pospešimo s povišano temperaturo in merimo z izključitveno kromatografijo (SEC), resonančnim merjenjem mase (RMM) in pretočno mikroskopijo (MFI). Vzporedno z eksperimentom numerično rešimo sistem enačb po vzoru Smoluchowskega, ki opisujejo kinetiko binarne agregacije na večih velikostnih skalah, in primerjamo dobljene rezultate z eksperimentom. Tako pojasnimo osnovne pojave, povezane z agregacijo proteinov. Z mikroskopom na atomsko silo (AFM) in optično mikroskopijo raziskujemo adhezijo proteinov in proteinskih delcev na površine vsebnikov. Opišemo več režimov tvorbe delcev v raztopini in njihovega prijemanja na površine, ki imajo pomemben vpliv na interpretacijo rezultatov meritev vsebnosti delcev po študijah pri povišani temperaturi. Komentiramo tudi odstopanje rezultatov od teoretičnih napovedi modela Smoluchowskega zaradi posedanja in adhezije. Reverzibilno povezovanje proteinskih molekul, katerega makroskopski vpliv se kaže v povišani viskoznosti, merimo z mikrofluidno reometrijo. Razločimo in analiziramo različne faktorje, ki vplivajo na viskoznost. Predlagamo nov mehanizem, ki povezuje povišanje viskoznosti z elektrostatskimi interakcijami. To delo prispeva k napredku razvoja proteinskih formulacij, s posebnim poudarkom na razvoju bioloških zdravil.

Diplomska naloga opisuje postopek vizualizacije velikih količin časovnih vektorskih meritvenih podatkov. Podatke pretvorimo s časovno-frekvenčno transformacijo, da jih lahko analiziramo in zaznamo različne pojave. Težave nastanejo, kadar želimo prikazati vse zajete podatke. Cilj diplomske naloge je zmanjšati količino pretvorjenih podatkov za hiter prikaz in ohraniti njihove lastnosti. Podatke reduciramo z agregacijo podatkov. Agregacija z iskanjem maksimalne vrednosti najbolj ohrani lastnosti pretvorjenih podatkov. Z opisano metodo smo zasnovali večnivojski krožni medpomnilnik, ki reducira podatke po obeh dimenzijah. Vsak naslednji nivo medpomnilnika hrani reducirane podatke, s katerimi lahko prikažemo daljši časovni interval od prejšnjega nivoja medpomnilnika. Vizualizacija ob spremembi prikazanega časovnega intervala preklopi na ustrezen nivo medpomnilnika, s katerega prikazuje podatke. Vizualizacija je bila implementirana s pomočjo DirectX 11, pri katerem se vse transformacije izvajajo v senčilnikih. Podpira različne poglede, manipulacije podatkov in splošne uporabniške interakcije, kot so premikanje po časovni osi, rotacija in premik pogleda ter odčitavanje vrednosti pod kazalcem. This diploma thesis describes visualisation of large sets of measurement time-based vector data. The data is transformed from time to frequency domain so it can be analysed and detect various phenomena. The problem we are facing is how to display all of the data at once. The goal is to reduce the size of the transformed data for a fast visualization while preserving its properties. We reduce the data with data aggregation. The properties of the transformed data are preserved the most by using the aggregation with maximum value. With the described aggregation method a multi level circular buffer is designed, which reduces the data in both dimensions. Each next level of our buffer can display a longer time interval of reduced data than the previous one. The visualisation switches the displayed buffer level when the length of the time interval changes. The visualisation is implemented with DirectX 11, where all transformations are done in shaders. It supports different views, data manipulation and basic user interactions like changing the time interval, rotation and translation of camera view and reading the value under the cursor.

Maģistra darba literatūras apskatā apkopota vispārīga informācija par amiloīda-beta peptīdiem, to agregāciju un tās pētīšanas metodēm. Maģistra darbā eksperimentālajā daļā izstrādātas metodes fluorēto aminoskābju inkorporēšanai amiloīda-beta 42 peptīdā, un veikti fluorēto peptīdu KMR eksperimenti., Master thesis’ literature review outlines common knowledge on amyloid-beta peptides and their aggregation, and methods to study it. Methods for the incorporation of fluorinated amino acids were developed, and NMR experiments for fluorinated peptides were done.

Diplomsko delo obravnava problem razpršenosti podatkov na spletu in določa način, kako združiti ter prikazati podatke na enem mestu. Pri obravnavi problema so bili za temo izbrani avtomobilski oglasi, ki jih lahko v Sloveniji iščemo po različnih spletnih straneh. Z delom sem želel združiti podatke in jih prikazati na lep in pregleden način ter izboljšati samo uporabniško izkušnjo. Pri izdelavi dela sem uporabil slapovni model. Najprej sem naredil analizo in nato načrtoval, kako bom postavil sistem. Odločil sem se, da bom sistem razdelil na dva dela, in sicer na zaledni ter čelni del aplikacije. Zaledni sistem naj bi stregel podatke preko REST aplikacijskega programskega vmesnika, medtem ko bi jih čelni del prikazoval. Podatke iz drugih spletnih strani sem pridobival s knjižnico Scrapy, ki je zaledni sistem napolnila s podatki in jih periodično osveževala. Čelni del je implementiran v obliki enostranske aplikacije in za avtentikacijo uporablja JWT. Rezultat diplomskega dela je sistem, ki pridobiva, obdeluje in hrani podatke ter spletišče z videzom ``material design'', ki uporabnikom prikazuje ter omogoča vnos oglasov. The diploma thesis deals with the problem of online data scatterness and how to combine and display data in one place. When addressing this issue the car ads were selected as the topic, which we can search through on different sites in Slovenia. I wanted to aggregate the data and present these data in a attractive and transparent way and to improve user experience in this thesis. For developing the diploma I selected the waterfall development model. I followed the phases in the cycle. First I performed the analysis and then planned how to set up the system. I decided to split the system into two parts, on the back end and front end of the application. The back end is supposed to serve the data through the REST application programming interface while the front end is supposed only to display data. I obtained the data from other websites with the Scrapy library, which filled the back end system with data and kept it fresh. The front end is implemented as a one-page application and uses JWT for authentication. The result of the thesis is a system that collects, processes and stores data and a web site with a material design layout which displays ads and allows users to enter ads.

Complex three-dimensional structure of therapeutic proteins and their tendency for aggregate formation, is a challenge for biopharmaceutical companies, since such aggregates can evoke an immune response upon administration, cause blood vessel occlusion and lead to loss of effective monomeric proteins. Polysorbates (PS) are surface active ingredients, which are commonly supplemented to biopharmaceutical formulations with intention of preventing aggregation. They are heterogenous mixtures of a PEGylated sorbitan core with one or multiple esterified fatty acids. Although PS molecular mechanism of action is well investigated, it is to this date still not clear how different quality grades compare in stabilizing proteins. Moreover, impact of (partly) degraded PS on protein stability is not fully understood. In this thesis, a monoclonal antibody (IgG) was subjected to pumping, stirring and dropping stress, which mimic applied mechanical stresses to a proteinaceous drug product during manufacturing, transportation and administration. Protein was formulated with two PS 80 quality grades, one compliant with Ph.Eur., USP and JP, and the other in line with stricter ChP. Furthermore, the antibody was supplemented with (partly) hydrolyzed PS 80. LC-CAD was performed to confirm successful degradation and to assess PS 80 concentrations used in this study. Surfactant functionality was assessed by measuring the extent of protein aggregation. Particles in nano- and micro-meter size range were characterized by using particle characterization techniques, such as micro-flow imaging (MFI), backgrounded membrane imaging (BMI) and dynamic light scattering (DLS). Additionally, turbidity and soluble protein aggregates were analyzed by using UV spectroscopy and ultra-high performance size exclusion chromatography (UP-SEC), respectively. We noticed a concentration range, where PS 80 is effective. No discrepancy in functionality was observed for two quality grades. Fully degraded PS 80 does not possess any stabilizing properties. When a portion (more than ~10 %) of neat PS is degraded, this results in poorer protection of the antibody under mechanical stress. Similarly, presence of extra degradants at the same intact PS 80 concentration was found to be destabilizing. More studies should be carried out with focus on development of repeatable stress methods and examination of oxidized PS functionality. Kompleksna tridimenzionalna zgradba terapevtskih proteinov in njihova nagnjenost k agregaciji so velik izziv za biofarmacevtska podjetja, saj lahko proteinski agregati vzbudijo imunske reakcije, prekinejo pretok manjših žil in vodijo k zmanjšanju količine proteinskih monomerov, ki so odgovorni za terapevtski učinek. Proteinski agregati so skupki proteinskih monomerov, ki se lahko razlikujejo v velikosti, strukturi in reverzibilnosti. Njihov nastanek lahko sproži nihanje temperature, neugoden pH, prisotnost oksidativnih radikalov ali pa mehanski stres, kot je na primer tresenje, mešanje, strižne sile ali padec na trdno podlago. Znanih je več mehanizmov tvorbe proteinskih agregatov. Eden najpomembnejših je agregacija na mejnih površinah, kjer pride do konformacijske spremembe proteina zaradi adsorpcije na medfazo (npr. zrak-tekočina ali trdna snov-tekočina). Spremembe v strukturi proteina (sekundarne, terciarne, kvarterne zgradbe) vodijo do odkrivanja hidrofobnih področij, ki so bolj dovzetna za interakcije kot površina nativne konformacije proteina. Deformirana struktura proteina služi kot povod za agregacijo na medfazi. Agregacija na medfazi je še posebej močno izražena v kombinaciji z mehanskim stresom, ki prekine adsorbiran film proteina na medfazi in proteinske agregate prenese v raztopino, kjer le-ti sprožijo nadaljnjo agregacijo. Polisorbati so površinsko aktivne snovi, ki so pogosto prisotne v formulacijah s proteini z namenom preprečevanja agregacije. So heterogene zmesi mono- ali poli-estrov polioksietiliranega sorbitana z višjimi maščobnimi kislinami. Številne študije opisujejo mehanizem njihovega delovanja v smislu preprečevanja agregacije. Adsorpcija polisorbatov na mejne površine in s tem kompetitivno zaviranje adsorpcije proteina na le-te je glavna pot preprečevanja agregacije. Poleg tega sta v literaturi opisana še dva mehanizma. Prvi predpostavlja vezavo polisorbatov na hidrofobna področja proteina. Na ta način pride do zmanjšanja interakcij med samimi proteini ter med proteinom in medfazo. Drugi pa predpostavlja, da je delovanje polisorbatov podobno molekularnim šaperonom, ki proteinu olajšajo in omogočijo spremembo konformacije nazaj v nativno. Čeprav je mehanizem delovanja polisorbatov dobro raziskan, še vedno ni popolnoma jasno, kako različne stopnje kakovosti, ki ustrezajo specifikacijam iz različnih farmakopej, vplivajo na stabilnost proteinskih formulacij. Vpliv razgradnih produktov polisorbatov na stabilnost proteinov prav tako ni dobro raziskan. Znano pa je, da so polisorbati nagnjeni k razgradnji, najpogosteje po oksidativni poti zaradi faktorjev kot so UV svetloba, prisotnost reaktivnih kisikovih zvrsti in kontaminacije s prehodnimi kovinami (Fe, Cu). Druga možna pot razgradnje je hidroliza, ki v farmacevtsko relevantnih pogojih poteče zaradi encimov, ki izhajajo iz celic, uporabljenih v proizvodnem procesu in so prisotni zaradi nezadostnega čiščenja proteina. V magistrskem delu je bilo monoklonsko protitelo IgG izpostavljeno mehanskemu stresu treh različnih izvorov. Prvi mehanski stres je bil povzročen s črpanjem raztopine v sistemu dveh injekcij, ki sta bili povezani s kapilaro. Za vzbuditev drugega mehanskega stresa smo uporabili mešalnik s 96 individualnimi mešalnimi lopaticami. Zadnji mehanski stres smo povzročili s prostim padom viale na trdno podlago. Le prva metoda mehanskega stresa je bila ustrezna za primerjavo vpliva različnih koncentracij, stopenj kakovosti in razgradnih produktov polisorbata 80 na stabilnost monoklonskega protitelesa, kar je bil tudi namen te magistrske naloge. Uporabljenemu proteinu smo dodali polisorbat 80 dveh različnih stopenj kakovosti. Prva stopnja kakovosti je ustrezala specifikacijam evropske, ameriške in japonske farmakopeje, druga pa strožjim specifikacijam kitajske farmakopeje. Modelnemu proteinu smo dodali tudi (delno) razgrajen polisorbat 80, ki je bil pripravljen z bazično katalizirano hidrolizo. Uspešnost razgradnje polisorbata 80 je bila ovrednotena s tekočinsko kromatografijo, sklopljeno z detektorjem CAD (LC-CAD). Vpliv površinsko aktivne snovi na stabilizacijo proteina je bil analiziran z merjenjem stopnje njegove agregacije. Delci v nano- in mikro-metrskem velikostnem razredu so bili proučeni z metodami, kot so pretočna mikroskopija (MFI), slikovna obdelava z membrano v ozadju (BMI) in dinamičnim sipanjem laserske svetlobe (DLS). Poleg tega smo motnost raztopine določali z UV-spektroskopijo, topne agregate pa z velikostno izključitveno kromatografijo ultra visoke ločljivosti (UP-SEC). Z metodo črpanja smo določili območje koncentracij polisorbata 80, kjer je le-ta učinkovit pri preprečevanju agregacije proteina. Za stabilizacijo proteina je bila potrebna koncentracija 0.004 % (m/V) polisorbata 80. Ugotovili smo tudi, da je uporaba koncentracij polisorbata 80 pod kritično micelarno koncentracijo (< 0.0017 % m/V) lahko neugodna. Za te raztopine smo namreč po stresu pri nekaterih ponovitvah določili večjo število agregatov v primerjavi z raztopino brez dodatka polisorbata. Z uporabo iste metode za vzbuditev mehanskega stresa smo primerjali tudi delovanje polisorbata 80 dveh različnih stopenj kakovosti pri treh izbranih koncentracijah. Polisorbat 80 je protein stabiliziral v podobni meri pri obeh stopnjah kakovosti in vseh treh preizkušenih koncentracijah. Za metodo s črpanjem je bila ugotovljena velika deviacija med posameznimi ponovitvami, zato predlagamo ponovitev poskusov s polisorbatom 80 različnih stopenj kakovosti s ponovljivo metodo mehanskega stresa. Literatura prikazuje, da polisorbat 80, ki ustreza kitajski farmakopeji oksidira hitreje kot polisrbat 80 druge uporabljene kakovosti. Iz tega vidika bi bilo potrebno izvesti nadaljnje poskuse z uporabo oksidiranega polisorbata 80 in ugotavljanjem njegove aktivnosti. Pri analizi hidroliziranega polisorbata 80 smo ugotovili, da je prišlo do popolne hidrolize, saj je bil signal mono- in poli-estrov polisorbata 80 zanemarljiv v primerjavi z nerazgrajeno obliko. Dodatni signali v kromatogramu razgrajenega polisorbata 80 so najverjetneje pripadali prostim maščobnim kislinam. To domnevo smo potrdili z MFI, ki je zaznala veliko število majhnih delcev, ter z BMI. Proste maščobne kisline so lipofilne, zato jih BMI membrana prepušča, kar je bilo v literaturi opaženo že za kapljice silikonskega olja. Vpliv razgrajenega polisorbata 80 na stabilnost proteina smo preizkušali z mehanskim stresom, povzročenim s črpanjem. Ugotovili smo, da popolnoma razgrajen polisorbat 80 ne prepreči agregacije, saj smo z MFI in BMI določili zelo veliko število nastalih agregatov. Pomanjkanje učinkovitosti polisorbata 80 lahko pripišemo izgubi amfifilnega značaja. V nadaljevanju smo razgrajenemu polisorbatu 80 pri nespremenjeni celokupni koncentraciji dodajali večji delež nerazgrajenega polisorbata. Šele pri 90 % deležu nerazgrajenega polisorbata 80 je bilo število delcev enako kot pri 100 % nerazgrajeni obliki. Iz tega lahko razberemo, da imamo lahko le ~10 % razgrajenega polisorbata 80 za želen stabilizacijski učinek. V naslednjem poskusu smo stalni koncentraciji nerazgrajenega polisorbata 80 dodajali različno količino razgradnih produktov. Večja količina dodanih razgradnih produktov je povzročila nastanek večjega števila proteinskih agregatov. To pomeni, da prisotnost večje koncentracije razgradnih produktov (prostih maščobnih kislin) negativno vpliva na stabilnost monoklonskega protitelesa. Na podlagi naših rezultatov in obstoječe literature predlagamo, da je koncentracijo polisorbata 80 potrebno izbrati zelo skrbno. Prenizka koncentracija je lahko neučinkovita ali celo negativno vpliva na stabilnost proteina. V primeru previsoke koncentracije pa le-ta sodeč po literaturi lahko povzroči hitrejšo razgradnjo/agregacijo proteina. S časom pa pri večjih koncentracijah tvegamo tudi prisotnost večjega števila razgradnih produktov, ki imajo prav tako neugoden vpliv na stabilnost proteinskih formulacij.

Uvod: Priprema koncentrata trombocita (KT) iz jedinice cele krvi je metoda uslovljena velikim brojem faktora koji pojedinačno ili udruženo mogu imati značajan uticaj na kvalitet konačnog proizvoda. Kvalitet koncentrata trombocita određen je kriterijumima definisanim u Vodiču za pripremu, korišćenje i obezbeđenje kvaliteta Evropskog komiteta za transfuziju krvi Saveta Evrope. Cilj istraživanja je bio ispitati kvalitet KT dobijenih izdvajanjem iz “buffy coat”-a (BC) i izdvojiti faktore koji doprinose poboljšanju kvaliteta i standardizaciji KT. Materijal i metoda rada: Ispitivanje je obuhvatilo ukupno 80 KT izdvojenih iz (BC) prema standardnoj proceduri pripreme u Zavodu za transfuziju krvi u Nišu. Kvalitet KT je određivan na osnovu zapremine produkta i laboratorijskih testiranja prvog (KT1), trećeg (KT3) i petog dana skladištenja (KT5): hematoloških analiza, gasnih analiza (pO2, pCO2), pH, sterilnosti i agregacije trombocita izazvane dodavanjem 3,2; 6,4 i 9,6 μg kolagena/ mL (impedantna agregometrija). Rezultati: Postoji statistički značajno manji broj trombocita, pO2, pCO2 i pH u KT3 i KT5 (p

Background: Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple, or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immune response in organism. Methods: Doctoral dissertation includes proposed detection and characterization of aggregated as well as other modified forms of monoclonal antibodies (mAb) by using selected chromatographic and spectrometric methods. Additionally, the selected mAb’s aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells’ (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immune response of peripheral blood mononuclear cell (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMC to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. Results and conclusion: All developed and proposed analytical methods and immunological in vitro DC and PBMC assays could be used as a platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products, which contain monoclonal antibodies as active pharmaceutical ingredients. Ozadje: Razvoj novih rekombinantnih biotehnoloških izdelkov se je močno razširil v sodobno farmacijo in medicino. Ker so biološke rekombinantne molekule občutljive in kompleksne beljakovine, je njihova funkcija močno odvisna od njihove strukture. Poleg njihove učinkovitosti lahko biološka zdravila povzročijo neželene učinke, kot je imunogenost. Zato je odkrivanje in karakterizacija strukturnih variant beljakovin bistvenega pomena pri razvoju in nadzoru kakovosti terapevtskih proteinov, ki bi lahko sprožili imunogeni odziv v organizmu. Metode: Doktorska disertacija vključuje predlagano detekcijo in karakterizacijo agregiranih in drugih modificiranih oblik monoklonskih protiteles (mAb) z uporabo izbranih kromatografskih in spektrometričnih metod. Poleg tega smo vzorce agregatov in modificiranih strukturnih variant monoklonskih protiteles izpostavili testu z nezrelimi dendritičnimi celicami (DC), ki izhajajo iz monocitov, za spremljanje aktiviranih celic DC in ugotavljanje potencialne imunogenosti strukturnih variant monoklonskega protitelesa. Ovrednotili smo tudi potencialni prirojeni imunogenski odziv kulture mononuklearnih celic periferne krvi (PBMC) v stiku z agregati mAb, in sicer z merjenjem vnetnega citokinskega odziva pri zgodnji izpostavitvi PBMC-jev različnim strukturnim oblikam monoklonskega protitelesa in z določanjem učinka agregatov mAb na proliferacijo dolgoživeče kulture PBMC-jev. Rezultati in sklep: Vse razvite in predlagane analitske metode in imunološka in vitro testiranja s celicami DC in PBMC bi se lahko uporabljala kot platforma za komplementarno analitsko karakterizacijo in določitev potenciala za imunogenost za vse biofarmacevtske izdelke, ki vsebujejo monoklonska protitelesa kot aktivne farmacevtske sestavine.

V delu je opisan postopek konstruiranja naprave za zbiranje in kontrolo škatlic v farmacevtski industriji s pomočjo programskega paketa SolidWorks. Na kratko je opisano področje delovanja naprave, čemur sledi naročnikov zahtevnik. V delu so prikazani preračuni za najpomembnejše elemente naprave, ki so podkrepljeni s tabelami in grafi, na podlagi katerih smo le-te tudi izbrali. Nekaj besed je namenjenih tudi zaščitnim elementom, senzorjem in elektromotorjem. Na koncu dela so opisani še postopki izdelave tehniške dokumentacije, sestava naprave in njeno testiranje. The master's thesis deals with the process of constructing a device for collecting and checking packaging boxes in the pharmaceutical industry using the SolidWorks software. The scope of functioning of the device is briefly described, followed by the customer's request. The calculations are presented in a section for the most important elements of the device, which are supported by the tables and graphs based on which they were selected. A few words are also intended for protective elements, sensors and electric motors. In the last section of the thesis, the procedures for the production of technical documentation, the composition of the device and its testing are described.

Magistrsko delo obravnavna agregacijo koloidov v vodnih raztopinah pod vplivom različnih interakcijskih parskih potencialov kot efektivnega modela za agregacijo proteinov. Opisan je razvoj numerične metode za modeliranje kinetike in dinamike agregacije na osnovi programske opreme KAPSEL in rezultati simulacij, ki smo jih izvedli s to opremo. Postopek simulacije temelji na numeričnem reševanju Navier-Stokesove enačbe z metodo gladkega profila, ki mu je v vsakem koraku dodan naključen termični šum in vpilv interakcijske sile med delci. Simulirali smo agregacijo koloidov velikosti od 1 nm do 1000 nm, med katerimi deluje privlačna van der Waalsova interakcija ($1/r^{6}$), Londonove disperzijske sile med makroskopskimi telesi ($1/r$) in hidrofobna interakcija ($e^{-r/d}$), za različne številske gostote sistemov in različna razmerja moči interakcij v primerjavi s termičnim šumom. Kot osrednji rezultat študija agregacije ugotovimo, da sta kinetika in dinamika agregacije pogojeni s termodinamskim pogojem za nastanek agregacije, ki ga pogojuje razmerje med močjo interakcije in termičnim šumom ($epsilon/k_bT$), manj pa s samo obliko potencialov in njihovim dosegom. Ko je razmerje med močjo interakcije in termičnim šumom večje ali enko okvirno 2, opazimo stabilen proces agregacije. Pri zviševanju tega razmerja ostane kinetika agregacije kvalitativno nespremenjena. Prav tako je bila kinetika agregacije efektivno kvalitativno neodvisna od gostote sistema. Kinetia agregacije je tako osrednje odvisna od gibljivosti molekul v raztopini, ki je neposredno povezana s temperaturo in izpolnjenim termodinamskim pogojem za nastanek agregatov. The master thesis focuses on aggregation of colloidal solutions with different interactional pair potentials, as an effective coarse grain approximation for aggregation of proteins. We use numerical based method for the simulation of kinetics and dynamics of colloidal aggregation based on open-source program KAPSEL. The simulation solves Navier-Stokes equation for fluid flow using smooth profile method with addition of a random thermal noise and influence of interaction forces between the colloidal particles. We simulate aggregation of colloids in size range of 1 nm-1000 nm which interact with either van der Waals ($1/r^{6}$), London dispersion force between macroscopic objects ($1/r$) or hydrophobic interaction ($e^{-r/d}$). Also we explore the role of different particle density and different ratios of interactions strengths against thermal noise. As the main result of the study of aggregation we have observed, that the kinetics and dynamics of colloidal aggregation depend primarily on the ratio between the strength of inter-particle interaction and thermal noise ($epsilon/k_bT$) and less on the exact form and range of interactional potentials. When the ratio ($epsilon/k_bT$) is greater or equals roughly 2, stable process of aggregation is observed. By increasing this ration, the kinetics of aggregation qualitatively does not change and is qualitatively similar also for different particle densities of the system tested. Finally we show that aggregation of colloids in solutions depends primarily on their ability to explore space –i.e. effective Brownian motion-being directly connected with the temperature and fulfilment of thermodynamical condition for emergence of aggregates.

Bakterijski biofilmi predstavljajo velik problem v medicini in živilski industriji. Bakterijski biofilm je vir kontaminacije v prehranski verigi, problem predstavlja tudi kot mehanska ovira, hkrati pa je zelo težko odstranljiv. Agregacijska sposobnost in površinska hidrofobnost bakterij sta ključna dejavnika za nastanek biofilma. Agregacija mikroorganizmov je ključna za nastanek mikrokolonij, površinska hidrofobnost bakterij pa je v povezana z bakterijsko adhezivnostjo. To je virulentna lastnost bakterij. Pri nalogi smo uporabili različne seve Campylobacter jejuni. Ta bakterija je pogosta povzročiteljica zoonoze, bolezni, ki se prenaša posredno ali neposredno iz živali na ljudi. Pri poskusih smo primerjali različne seve in njihove kombinacije s testi agregacije s pomočjo merjenja optične gostote in izračuna agregacije in ko-agregacije. Stopnjo adhezivnosti na polistirenski površini pa smo ocenili s pomočjo primerjave dveh postopkov - s kristalvijoličnim barvilom ter s štetjem kolonij na trdnih gojiščih. Rezultati so pokazali, da je bakterijska agregacija pri bakteriji C. jejuni sevnospecifična lastnost. Ko-agregacija sevov se razlikuje od sposobnosti agregacije posameznih sevov. Naši rezultati pa niso potrdili, da je sposobnost agregacije in ko-agregacije v korelaciji s sposobnostjo adhezivnosti in ko-adhezivnosti. Bacterial biofilms are a big problem in medicine and food industry. A bacterial urce of contamination in a food chain, it presents a problem as a mechanical obstacle and it is very difficult to remove. Aggregational capability of bacteria and bacterial surface hydrophobicity are key factors for the formation of biofilm. Microbiological aggregation is necessary for microcolony formation, while bacterial surface hydrophobicity is related to bacterial adhesiveness. This is a virulent trait of bacteria. We used different strains of Campylobacter jejuni. This bacteria is a common cause of zoonoses, a disease which spreads directly or indirectly from animals to people. In the experiments, we compared different strains and their combinations in a test of aggregation with measuring optical density and a later calculation of aggregation and co-aggregation. The level of bacterial adhesiveness on a polystyrene surface was evaluated with using Kristal violet staining and plate counting. Results showed that bacterial aggregation of C. jejuni is a strain-specific trait. Bacterial co-aggregation is different from the capability of bacterial aggregation of individual strains. Our results did not confirm that the capability of bacterial aggregation and co-aggregation is in correlation with the capability of bacterial adhesiveness and co-adhesiveness.

Prethodna istraživanja pokazala su da BPC 157 sprječava nastanak okluzivnog tromba i ubrzava razgradnju već stvorenog ugruška nakon formiranja anastomoze aorte, a da, s druge strane, smanjuje vrijeme krvarenja kod štakora tretiranih varfarinom, heparinom i aspirinom. Temeljem toga, nametnula se potreba određivanja njegova utjecaja na agregaciju trombocita i viskoelastična svojstva krvnog ugruška. Za ispitivanje eventualne uključenosti NO sustava u djelovanje BPC 157 korišten je selektivni inhibitor topljive gvanilil ciklaze, ODQ. Životinje (n=60) su najprije podijeljene u skupine ovisno o tome koji antiagregacijski lijek primaju (1.aspirin, 2.klopidogrel, 3.cilostazol), a te skupine su tada nasumično podijeljene u četiri podskupine koje su primale a) fiziološku otopinu, b) BPC 157, c) ODQ, d) BPC 157 + ODQ. Mjerenja su izvršena impendancijskom agregometrijom s 4 različita agonista (ADP, AA, AA/PGE1, kolagen), te rotacijskom tromboelastometrijom s 3 različita agonista (preko vanjskog i unutarnjeg puta zgrušavanja, te bez doprinosa trombocita). Iz rezultata dobivenih usporedbom podskupina a i b, te b i d unutar svake skupine možemo zaključiti da BPC 157 oporavlja inhibiranu agregaciju trombocita, ali da nema utjecaja na viskoelastična svojstva krvnog ugruška u ispitivanih štakora. As a natural extension of previous research that confirmed the role of BPC 157 in the prevention of obstructive thrombus formation and rapid destruction of an already formed one after aortic anastomosis, but also shortening of the bleeding time in rats treated with anticoagulants and aspirin, there was a necessity to determine how BPC 157 influences platelet aggregation and viscoelastic properties of the blood clot. To assess its relation to NO system, sCG selective inhibitor (ODQ) was used. Rats (n=60) were divided into groups depending on the antiaggregatory drug they were treated with (1.aspirin, 2.clopidogrel, 3.cilostazol). Groups were further divided into four subgroups treated with a) normal saline, b) BPC 157, c) ODQ, d) BPC 157 + ODQ. Impendance aggregomery measurements with four agonists (ADP, AA, AA/PGE1 and collagen), and also rotational thromboelastometric measurements with 3 agonists (for initiating external and internal coagulation pathway and without platelet contribution) were performed. Based on the results obtained by comparing subgroups a versus b, and b versus d in each group, we can conclude that BPC 157 rescues inhibited platelet aggregation, but it has no effect on viscoelastic properties of the blood clot.

Odgovor bakterija na uticaj razliĉitih faktora sredine koja ih okruţuje, kao i njihova meĊusobna komunikacija, zavisi od procesa koji se odvijaju na površini bakterijskih ćelija. S obzirom na brojne mogućnosti primene bakterija roda Lactobacillus, kako u prehrambenoj industriji, tako i u medicini (u prevenciji i/ili leĉenju bolesti), potrebno je što bolje okarakterisati komponente koje ĉine površinski sloj ovih bakterija. Agregacija bakterija, determinisana faktorima lociranim na površini ćelija moţe se smatrati poţeljnom osobinom pri izboru probiotika, s obzirom da ima vaţnu ulogu u meĊubakterijskoj interakciji, u interakciji sa epitelijalnim ćelijama gastrointenstinalnog trakta i doprinosi sposobnosti bakterija da se adsorbuju na razliĉite površine, odnosno sposobnost agregacije omogućava uspešnu kolonizaciju staništa. Kolekcija laktobacila Laboratorije za molekularnu mikrobiologiju je pretraţena na prisustvo sojeva koji poseduju sposobnost agregacije (Agg+), nakon ĉega je za dalji rad odabrano jedanaest sojeva: BGSJ2-8, BGGR2-68, BGGR2-82, BGDP1-84, BGDP9-38, BGNJ1-3, BGNJ1-61, BGNJ1-64, BGNJ1-70, BGZLS30-6 i BGAR75. Odabrani Agg+ sojevi se na osnovu stepena agregacije i tipa formiranih agregata mogu podeliti u tri grupe: oni koji brzo, srednje i sporo agregiraju ili koji formiraju krupne, srednje i sitne agregate, pri ĉemu postoji direktna korelacija izmeĊu brzine agregacije i tipa agregata. Analizom sposobnosti vezivanja odabranih Agg+ sojeva laktobacila za komponente ekstracelularnog matriksa (ECM) zapaţen je širok dijapazon sposobnosti vezivanja za kolagen - od intenzivnog afiniteta vezivanja do odsustva sposobnosti vezivanja za matriks. Pritom su pojedini sojevi, BGGR2- 68 i BGGR2-82, ispoljili sposobnost vezivanja za plastiku (formiranje biofilma). Analizom plazmidnog sastava Agg+ sojeva konstatovano je prisustvo velikih plazmida u svim sojevima, a ĉišćenjem plazmida iz sojeva BGNJ1-64 i BGSJ2-8 potvrĊena je plazmidna lokacija gena odgovornih za ekspresiju agregacionog fenotipa. Nakon konstrukcije i analize plazmidnih biblioteka sojeva BGNJ1-64 i BGSJ2-8, izolovan je konstrukt pALb35 (koji nosi SacI fragment veliĉine 11,4 kb poreklom iz plazmida pNJ1 soja BGNJ1-64) koji dovodi do ponovne pojave autoagregacionog fenotipa transformisanog heterologog domaćina BGKP1-20 (Agg-)... Response of bacteria to different environmental factors as well as their communication primarily depends on the processes that occur on their cell surface. Due to the continuously increasing number of application possibilities of lactobacilli, both in the food industry and in medicine (in the prevention and/or treatment of the disease), there is a need for a more comprehensive characterization of the components that form the surface layer of these bacteria. Aggregation ability of bacteria, determined by factors located on the cell surface could be considered as a desirable trait in the selection of probiotics. Since it plays an important role in the interaction between bacteria, in interaction with the epithelial cells of the gastrointestinal tract plus contributing to the bacterial capability to adhere to different surfaces, which is an important feature enabling them to successfully colonize diverse habitats. Lactobacilli collection of Laboratory for molecular microbiology was screened for the presence of strains possessing the aggregation ability (Agg+) and for the further work eleven strains have been selected: BGSJ2-8, BGGR2-68, BGGR2-82, BGDP1-84, BGDP9-38, BGNJ1-3, BGNJ1-61, BGNJ1-64, BGNJ1-70, BGZLS30-6 and BGAR75 which, according to the level of aggregation and the appearance of the aggregates formed, could be clasified into three groups: the fast, the medium and the slow aggregating ones or into the groups of those forming large, medium or small aggregates wherein a direct correlation between the aggregation kinetics and the type of the formed aggregates was found. By analyzing the binding ability of the selected Agg+ strains of lactobacilli to the components of extracellular matrix (ECM) a wide range of binding capacity to collagen was observed, varying from the intensive to the complete lack of the binding capacity to the matrix. Two strains (BGGR2-68 and BGGR2-82) showed the ability to bind even to plastics (form biofilms). By analyzing the plasmid composition of Agg+ strains, the presence of large plasmids was detected in all strains. The plasmid curing experiments confirmed the plasmid location of the gene(s) responsible for the expression of aggregation phenotype in strains BGNJ1-64 and BGSJ2-8. After the construction and analysis of BGNJ1-64 and BGSJ2-8 strains plasmid libraries, selected construct pALb35 (which carries the SacI fragment of 11.4 kb. derived from plasmid pNJ1 from strain BGNJ1-64) was able to reconstitute the autoagreggation ability of the transformed heterologous host BGKP1-20 (Agg-)...

Ekspertni sustavi primjenjuju se u različitim djelatnostima, s ciljem donošenja odluka na inteligentan i transparentan način. U poslovnim djelatnostima ekspertni sustavi usmjereni su k donošenju operativnih i ekonomskih odluka. U zaštiti okoliša, uvažavajući posebnosti različitih poslovnih djelatnosti, u izboru odluka koriste se i ostali aspekti odlučivanja, koji osim ekonomsko-operativnih razloga, vrednuju i druge razloge izbora odluka. Neizrazita logika je logički i matematički aparat, koji se preporuča za primjenu u takvim ekspertnim sustavima. U radu se prikazuju neke značajke i pravila konstrukcije ekspertnih sustava za primjenu u zaštiti okoliša u rudarstvu, a koji primjenjuju neizrazitu logiku., Expert systems are used in various activities with the main goal of decision-making in intelligent and transparent way. In business decision-making, such systems are directed to pure operational and economical decisions. In environmental protection they have to accept the specificity of business sector for which they have been intended, but also use environmental reasons and evaluate them differently than economic. Fuzzy logic is logical and mathematical mean recommended for such expert systems. Some characteristics and rules for construction of systems based on fuzzy logic are provided in the work.

Darbs ir veltīts lēmumu pieņemšanai, balstoties uz ekspertu novērtējumiem un lietojot priekšrocību attiecību agregāciju. Katra eksperta viedoklim atbilstoša priekšrocību attiecību matrica ir ģenerēta uz ranžēšanas vektora pamata. Priekšrocību attiecību matricu modificētā agregācija ļauj samazināt neobjektīva eksperta ietekmi uz ekspertu grupas lēmuma pieņemšanu. Modificētā agregācija izmanto līdzības pakāpes un ļauj palielināt līdzību starp ekspertu sniegtiem novērtējumiem., This bachelor thesis is devoted to decision making based on the experts evaluations and aggragation of preference relations. Each of expert’s evaluation preference relation is generated using ranking method. The modification of aggregation of preference relation matrices allows to reduce biased expert effect on the expert group decision making. The proposed modificated aggregation uses similarity degrees and allows us to increase similarity between the expert’s evaluations.

U ovom radu proučava se razvoj strategije, razvoj globalizacije, globalnih strategija kao i primjena strategija adaptacije (A), agregacije (A) i arbitraže (A) u globalnom poduzeću. Razvojem globalizacije, rastom međunarodne trgovine te sve većim brojem međunarodnih, multinacionalnih i globalnih kompanija, između poduzeća, tržišta i zemalja nisu nestale kulturalne (C), administrativne (A), geografske (G) i ekonomske udaljenosti (E) koje se promatraju CAGE obrascem. Osim spomenutih udaljenosti globalna poduzeća poslujući u međunarodnoj okolini susreću se s brzim i učestalim promjenama, te je zbog toga važno proučavanje okoline poduzeća. Kako bi poduzeća na najbolji način organizirala svoje međunarodno poslovanje, u određenim slučajevima primjenjuju strategiju adaptacije, a kako bi se prilagodila spomenutim razlikama ; agregacije kako bi se prevladale spomenute razlike ; te strategiju arbitraže kako bi se spomenute razlike iskoristile. Nakon analize teorijskog okvira spomenutih strategija, te proučavanja njihove primjene na primjeru poduzeća, može se zaključiti da primjenom odgovarajuće „A“ strategije globalna poduzeća mogu uspješno poslovati na stranim tržištima.

Knjiga se bavi samim počecima odlučivanja kako u psihologiji tako i u matematici, s naglaskom na matematiku, a redoslijed iznošenja materijala donekle slijedi povijesni razvoj misli. Središnje mjesto među aksiomima teorije očekivane korisnosti zauzima aksiom nezavisnosti, koji na stanovit način definira racionalnost i racionalnog donositelja odluke. Od novijih neutilitarističkih pristupa spomenuta je Saatyjeva metoda svojstvenog vektora i detaljno je razrađena matematička osnova za njeno razumijevanje (Perronov teorem). Teorija potencijala, razvijena od strane autora knjige, također spada u neformalne metode koje se ne zasnivaju na nekom aksiomatskom sustavu. U Dodatku su opisane neke praktične procedure koje preciziraju pojedine faze odlučivanja. Dan je popis i opis i drugih metoda koje nisu spominjane u knjizi, a sve su ilustrirane na jednom primjeru.

Probiotici predstavljaju žive mikroorganizme koji, nakon unošenja u gastrointestinalni (GI) trakt, ostvaruju pozitivne efekte na zdravlje domaćina. Reakcija domaćina na unos probiotika u velikoj meri zavisi od sposobnosti bliske interakcije bakterija sa epitelnim ćelijama GI mukoze. S druge strane, s obzirom na nepoznatu antigenu prirodu unetih bakterija, kod imunokompromitovanog domaćina je prisutan rizik od imunske reakcije crevnog tkiva što može da dovede do narušavanja integriteta mukozne barijere i hronične inflamacije u GI traktu. Cilj istraživanja bio je ispitivanje efekta soja Lactobacillus fermentum BGHI14, alohtonog za GI trakt pacova, nakon peroralnog unosa u zdrave pacove i u pacove sa kolitisom indukovanim TNBS-om (trinitrobenzensulfonat). Metodološki, u eksperimentima su praćeni: telesna masa pacova i ekspresija iRNK za parametre inflamacije (IL-1β, TNFα, IL-17F), pokazatelje ćelijskog stresa (HSP70) i parametre uključene u održavanje integriteta mukoze (TJP1); zatim su rađene histološke i biohemijske analize i analiza sastava crevne mikroflore. Rezultati istraživanja su pokazali imunostimulišuće delovanje soja BGHI14 na nivou tkiva kolona nakon unosa u GI trakt zdravih pacova u prvih 16 dana peroralnog tretmana pacova. Iako se, histološki, sa produžetkom tretmana pacova sojem BGHI14 tokom narednih 12 dana, imunska reakcija u tkivu kolona povukla, nivoi iRNK za proinflamatorne IL-1β i TNFα citokine ostali su povišeni što ukazuje na rizik od nekontrolisane inflamacije u slučaju naknadnog oštećenja epitelne barijere kolona. U istraživanju je pokazano da je nakon imunostimulacije tkiva kolona sojem BGHI14, u slučaju indukcije kolitisa, ali uz nastavak tretmana sojem BGHI14 došlo do indukcije transkripcije iRNK za HSP70 protein, kako u ranoj (48 h) tako i u kasnoj (7 dana) akutnoj fazi kolitisa. Povećanje transkripcije Hsp70 gena, sudeći po histološkim i inflamatornim pokazateljima, kao i promenama telesnih masa pacova tokom eksperimenta, nije bilo praćeno pozitivnim delovanjem soja. S druge strane, prestanak tretmana sojem BGHI14 nakon indukcije kolitisa doveo je do manje izraženog pada telesnih masa životinja u poređenju sa kontrolnim pacovima sa kolitisom, iako bez pratećeg smanjenja inflamatorne reakcije u tkivu kolona, bar u kasnoj akutnoj fazi bolesti (sedam dana od indukcije)... Probiotics are life microorganisms that, after administration in gastrointestinal (GI) tract, excert positive effects on host’s health. Host reaction to probiotic administration largely depands on ability of bacteria to closely interact with epithelial cells of GI mucosa. On the other hand, considering the unknown antigenic nature of administered bacteria, in immune-compromised host there is a risk of immune reaction of gut tissue which could lead to disturbance of mucosal barrier integrity and chronic inflammation in GI tract. The aim of the study was to examine the effects of the strain Lactobacillus fermentum BGHI14, which is alochtonous to GI tract of rats, after peroral administration in healthy rats and in rats with TNBS (trinitrobenzenesulfonate)-induced colitis. Methodologically, following parameters were examined in experiments: body masses of rats and expression of mRNA of inflammatory cytokines (IL-1β, TNFα, IL-17F), as well as markers of cellular stress (HSP70) and barrier integrity maintenanace (TJP1); next histological and biochemical analyses and microflora profiling were performed. Results of the study indicated immune-stimulating effects of BGHI14 in colon tissue after administration in GI tract of healthy rats in first 16 days of peroral treatment of rats. Although, histologically, with prolongation of BGHI14 treatment of rats for next 12 days, immune reaction in colon tissue had retreated, mRNA levels of proinflammatory IL-1β and TNFα cytokines remained elevated which points to the risk of uncontrolled inflammation in the case of subsequent colon epithelial barrier damage. In research it was shown that after immunostimulation of colon tissue with BGHI14, in the case of colitis induction, but with sequel of treatment with bacteria, transcription of Hsp70 gene was induced, in early (48 h) as well as late (7 days) acute phase of colitis. The increase of Hsp70 gene transcription, according to histological and inflammatory markers, as well as body mass changes during the experiment, was not followed by positive action of the strain. On the other hand, the cessation of treatment with BGHI14 after colitis induction had led to less pronounced body mass decrease compared to control colitic rats, but without concomitant reduction in inflammatory scores in colon tissue, at least in late acute phase of disease (seven days from induction)...

Trombociti imaju važnu ulogu u patogenezi kardiovaskularnih i malignih bolesti. Uticaj biološki aktivnih sastojaka namirnica na aktivaciju trombocita i njihovu heterotipsku agregaciju predstavlja jedan od potencijalnih mehanizama njihove uloge u prevenciji hroničnih bolesti i promociji zdravlja. Rezultati interventnih dijetarnih studija potvrđuju uticaj polifenola na funkciju trombocita. Protočna citometrija predstavlja jednu od najoptimalnijih metoda ispitivanja uticaja dijetarnih sastojaka na trombocite. Osnovni cilj doktorske disertacije je ispitivanje uticaja biljaka koje sadrže polifenole na aktivaciju trombocita i njihovu agregaciju sa leukocitima, u in vitro eksperimentalnim uslovima, kao i nakon konzumacije namirnica biljnog porekla, koje predstavljaju dijetarni izvor polifenola. Primenom protočne citometrije pokazano je da u in vitro eksperimentalnim uslovima svi ispitivani ekstrakti (ekstrakt koprive, mirođije, šarplaninskog čaja, kelja, nara, japanske jabuke i aronije) pokazuju efekte su na neki od analiziranih parametara aktivacije trombocita i njihove agregacije sa monocitima i neutrofilima, kod zdravih ispitanika ili ispitanika sa metaboličkim sindromom. Na najveći broj parametara delovali su ekstrakti kelja i mirođije, a u tom smislu najmanje izraženo delovanje pokazao je ekstrakt nara. Ispitivanjem delovanja metabolita pokazano je da svi ispitivani metaboliti: kvercetin-3-glukuronid, 3’-metil-kvercetin-3-glukuronid, kvercetin-3’-sulfat i sulforafan-cistein-glicin pokazuju efekte na neki od parametara aktivacije trombocita i njihove agregacije sa monocitima i neutrofilima, kod zdravih ispitanika ili ispitanika sa metaboličkim sindromom. Sulforafan-cistein-glicin delovao je na veći broj parametara u odnosu na metabolite kvercetina. Delovanje 3’-metil-kvercetin-3-glukuronida bilo je izraženije u odnosu na delovanje ostalih metabolita kvercetina. Nakon ispitivanja delovanja jednokratne konzumacije namirnica koje predstavljaju dijetarni izvor polifenola pokazano je da konzumacija soka od aronije kod zdravih ispitanika dovodi do smanjenja aktivacije trombocita i to smanjenjem procenta Pselektin- pozitivnih trombocita u bazalnim uslovima, kao i smanjenjem procenta i gustine ovog adhezionog receptora nakon ex vivo delovanja adenozin-difosfata i arahidonske kiseline kao agonista... Platelets have an important role in the pathogenesis of the cardiovascular and malignant diseases. The impact of bioactive dietary compounds on platelet activation and their aggregation with leukocytes is one of the potential mechanisms of their role in health promotion and the prevention of chronic diseases. The results of dietary intervention studies confirm the effect of polyphenols on platelet function. Flow cytometry is considered to be one of the most advantageous methods in investigation of dietary compounds effects on platelets. The main aim of this study was the evaluation of potential effects of polyphenol - rich plants on platelets activation and their aggregation with leukocytes, in in vitro experimental setting and after the consumption of plant foods, as dietary sources of polyphenols. All investigated plant extracts (extracts of nettle, dill, mountain tea, kale, pomegranate, persimmon and chokeberry) affected some of the analyzed parameters of platelets activation and their aggregation with monocytes and neutrophils, in healthy subjects or subjects with metabolic syndrome The most effective were kale and dill extracts, influencing most of the parameters evaluated by flow cytometry, while the least effective was pomegranate extract. All investigated metabolites: quercetin - 3 - glucuronide, 3' - methyl - quercetin - 3 - glucuronide, quercetin - 3' - sulfate and sulforaphane - cysteine - glycine have shown the effects on some parameters of platelet activation and their aggregation with monocytes and neutrophils, in healthy subjects or subjects with metabolic syndrome. The most effective was sulforaphane - cysteine - glycine, influencing most of the analyzed parameters . 3' - methyl - quercetin - 3 - glucuronide showed more pronounced effects compared to the effects of other metabolites of quercetin. Dietary intervention studies investigating the effects of single consumption of plant-based foods as dietary sources of polyphenols have shown that consumption of chokeberry juice beneficially influenced platelet activation status by reducing the percentage of P - selectin - positive platelets under basal conditions, as well as reducing he percentage and density of P - selectin after ex vivo agonistic action of adenosine diphosphate and arachidonic acid...

Cilj ovog rada bio je karakterizacija autohtonih bakterijskih sojeva Enterococcus faecium A7 i Lactobacillus fermentum A8, izoliranih iz hrvatskog tradcionalnog svježeg sira, kao potencijalnih probiotika. Bakterijski sojevi Ec. faecium A7 i Lb. fermentum A8 preživljavaju visoke koncentracije vodikovih iona (pH 2, 0) i otporni su na djelovanje pepsina u simuliranom sekretu želuca. Neosjetljivi su prema djelovanju enzima gušterače i pokazuju otpornost prema djelovanju žučnih soli (3, 0 mg/ml). Bakterijski soj Ec. faecium A7 je osjetljiv na sve ispitane antibiotike (bacitracin, benzilpenicilin, eritromicin, kloramfenikol, rifampicin, streptomicin, tetraciklin, vankomicin), a soj Lb. fermentum A8 je rezistentan samo na vankomicin. Oba ispitana bakterijska soja pokazuju visok postotak autoagreagacije te mogućnost koagregacije s drugim bakterijama mliječne kiseline dok je soj Lb. fermentum A8 koagregirao i s ispitanim test-mikroorganizmima. Visoki postotak adhezije stanica na ksilen upućuje na njihovu hidrofobnost, a afinitet svih ispitivanih sojeva za kloroform, na elektron-donorska svojstva površine bakterijskih stanica. Svi sojevi pokazuju slabu aktivnost hidrolaze žučnih soli jer su dekonjugirali cca 12% taurokolata dodanog u hranjivu podlogu. Ispitivanjem asimilacije različitih prebiotičkih supstrata, tijekom uzgoja u hranjivim podlogama s prebioticima laktulozom i manitolom, ustanovljen je najveći rast ispitivanih sojeva, kao i najveća promjena pH vrijednosti uslijed nastajanja mliječne kiseline.

Cilj ovog rada bio je ispitati funkcionalna svojstva bakterijskih sojeva izoliranih iz probiotičkih pripravaka prisutnih na hrvatskom tržištu. Tijekom izlaganja simuliranim uvjetima gastrointestinalnog trakta broj živih stanica svih ispitanih sojeva, osim kod soja Bifidobacterium sp. 3, se održao iznad 106 CFU/ml, pri čemu je najbolje preživljavanje pokazao soj Lactobacillus sp. 1 čiji se broj živih stanica smanjio za samo 1, 1 log jedinica. Svi su ispitani sojevi pokazali autoagregacijska svojstva, pri čemu je autoagregacija soja Lactobacillus sp. 8a iznosila 70%, autoagregacija sojeva Lactobacillus sp. 1, Lactobacillus sp. 4, Bifidobacterium sp. 5a i Bifidobacterium sp. 8b oko 50%, a soja Bifidobacterium sp. 3 oko 20%. Bakterijski sojevi Lactobacillus sp. 1, Lactobacillus sp. 8a i Bifidobacterium sp. 8b su koagregirali u najvećem postotku s test-mikroorganizmom Escherichia coli 3014 (cca 40%), a sojevi Lactobacillus sp. 1 i Lactobacillus sp. 8a, i s test-mikroorganizmom Salmonella enterica serovar Typhimurium FP1. Visoki postotak adhezije stanica sojeva Bifidobacterium sp. 3, Lactobacillus sp. 8a i Bifidobacterium sp. 8b na heksan upućuje na njihovu hidrofobnost, a afinitet svih ispitivanih sojeva za kloroform, na elektron-donorska svojstva površine bakterijskih stanica. Svi sojevi pokazuju slabu aktivnost hidrolaze žučnih soli jer su dekonjugirali svega 10-15% taurokolata dodanog u hranjivu podlogu.