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In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

S1.1. Checking of N protein purity Recombinant N protein purity was checked after imidazole removal and buffer exchange by SDS PAGE (Figure 6.). For comparison, commercial high-purity HSA was also analyzed. S1.2. Identification of N protein Tandem mass spectrometry identification of proteins in an in-gel digested band of N protein (Figure S1, lane 3), confirmed the identity of N protein with high scores and peptide coverage (Fig. S2.). S2. Purification of polyclonal antibodies from mice and rabbit sera For the development of an ELISA test specific for the detection of SARS-CoV-2 N protein, recombinantly produced N protein was used for the immunization of mice and rabbits. Sera obtained from rabbits and mice were then tested for titer and specificity (Figure S3 and Figure 1). To determine the titer of polyclonal sera required to detect N protein in samples, we use wells coated with N protein and serial dilution of sera pools from different animals. After multiple washing steps, we detected the binding of rabbit and mice antibodies using secondary biotinylated antibodies and streptavidin-alkaline phosphatase chimaera or secondary antibodies with previously coupled alkaline phosphatase, where the amount of enzymes’ substrate converted to the product was measured as an increase in absorbance at 405 nm. As shown in Figure S3A, unpurified sera pools from both animals showed very high titers and expected logarithmic decrease of signal with dilution. Based on the obtained data titer for unpurified sera was determined to be X. The same trend was observed for pools purified using AS precipitation and rabbit sera purified using protein A affinity chromatography (Figure S3B and S3C). As shown in Figure S3D, clear bands from antibodies could be observed in both full and purified samples. Western blot analysis showed only one protein band on mass around 40 kDa, a Accession number / Protein Name Score Coverage (%) Unique peptides P0DTC9|NCAP_SARS2 Nucleoprotein OS=Severe acute res

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

Novi korona virus (SARS CoV-2) koji se pojavio u Vuhanu 2019. godine pripada grupi jednolančanih RNK virusa [1]. Predstavlja novi infektivni agens za humanu populaciju i veoma je brzo detektovan u velikom broju zemalja. Uzročnik je respiratornih infekcija koje mogu da budu praćene i veoma teškom kliničkom slikom. Brzo širenje, odsustvo imuniteta na ovaj virus i odsustvo pouzdanih testova za detekciju virusa u trenutku izbijanja pandemije su bolest izazvanu ovim virusom brzo pretvorili u zdravstveni i društveni problem najvišeg prioriteta na globalnom nivou. Iako su najveće biotehnološke kompanije ubrzano počele sa razvojem i masovnom proizvodnjom dijagnostičkih testova i vakcina, njihova dostupnost u trenucima najveće potražnje je i dalje nedovoljna, a cene istih su limitirajući faktor za bolju kontrolu bolesti i širenja pandemije [2]. Razvoj sopstvenih i održiva proizvodnja testova i vakcina za COVID-19 su od velikog društvenog značaja. Važan preduslov za održivu proizvodnju testova je dostupnost rekombinantnih antigena virusa i mogućnost proizvodnje istih na velikoj skali za potrebe proizvodnje domaćih testova. Ovim tehničkim rešenjem se opisuje dobijanje dva ključna antigena novog korona virusa rekombinantnom tehnologijom i njihova primena u serološkom ELISA testu koji proizvodi Institut za primenu nuklearne energije, INEP, kao i za dobijanje reagenasa za detekciju antigena novog korona virusa (specifičnih antitela). U prvoj fazi, optimizovane su sekvence proteina koje su podigle osetljivost postojećih seroloških testova. Inovativnost našeg pristupa se ogleda i u razrađenim eksperimentalnim protokolima za dobijanje rekombinantnih proteina nukleokapsida na velikoj skali, kao i u solubilnoj formi, što olakšava postupak prečišćavanja. Izbor fragmenta nukleokapsida koji se heterologo eksprimira u solubilnoj formi, a specifično detektuje antitela i generiše jak imuni odgovor tokom imunizacije životinja (imunogenost) na osnovu pregleda poznatih epitopskih sekvenci je k

Plastic materials have become indispensable in the modern world, with their extensive use resulting in their environmental accumulation. A promising solution for overcoming this ecological threat may be found in recombinantly produced plastic-degrading enzymes. Due to the complexity of the heterogeneous catalysis occuring during enzymatic PET hydrolysis, quantifying and comparing activities of such enzymes is rendered difficult. Here, we have assessed various assays documented in existing literature, employing different preparations of the purified recombinant Ideonella sakaiensis PETase mutant W159H/S238F (expressed from commercial plasmid Addgene #112203). The investigated methods were as follows: p-nitrophenyl acetate (pNA) hydrolysis assay, bis-(2-hydrohxyethyl)-terephthalate (BHET) agar and PET agar diffusion assays, and UV absorbance monitoring after PET particle and PET bottle cut-out hydrolysis. Additionally, we introduced an indirect colorimetric assay using the indicator pyrocatechol violet (PCV). Our work reveals many advantages and problems for each of the tested methods. The pNA hydrolysis assay is the quickest, but many substances which are usually present in enzyme buffers and solutions tend to hydrolyse this compound (e.g. imidazole). It is also unspecific due to hydrolysis by other esterase enzymes. The BHET diffusion assay offers a great tool for activity comparison and estimation, with greater enzyme specificity. However, it is slow and accurate activity quantification is difficult. PET hydrolysis was conducted on in-house prepared PET particles with UV spectrophotometric measurement or by a diffusion assay. Due to the measuring wavelength (240 nm), the importance of proper blanking is critical, but accurate results can still be obtained. The sensitivity of the diffusion assay is much lower in comparison to the similar BHET assay. We also report on a modification of the phenol red indirect colorimetric assay using PCV as the indicator and PET part

Polyethylene terephthalate (PET) is a widely used plastic material. Due to its convenient physicochemical properties, it has become irreplaceable in many scientific, industrial, medical and everyday uses, leading to an accumulation of this material in the environment and initiating many ecological problems, especially in marine ecosystems. One of the solutions for overcoming this ecological threat may be found in recombinantly produced PET degrading enzymes. The genes encoding proteins with prominent PET hydrolyzing activity (PETases) that have been successfully produced in Escherichia coli are commercially available (Addgene #112203 and #162667). These genes encode Ideonella sakaiensis PETase mutant W159H/S238F, and the fusion of the wild-type enzyme to MHETase (I. sakaiensis mono-(2-hydroxyethyl)terephthalic acid hydrolyzing enzyme). Initially, we have done sequence alignment by ClustalW of the sequences corresponding to the entries available in the PAZy database (pazy.eu/doku.php) that contains information on many PET-degrading enzymes. We have identified amino acid substitutions that might be of interest for mutation towards improving the PET hydrolytic activity of IsPETase: at position W159 substitutions into H, I and L and at position S238 substitutions into F, T, Y, W, L and G. Since we are aiming to produce all of the abovementioned (double) mutants, we used different bioinformatic tools to predict the expression solubility of the mutated enzymes. To evaluate the accuracy of the available tools we have tested the expression levels and solubility of IsPETase W159H/S238F and IsPETase-MHETase fusion in E. coli. The IsPETase W159H/S238F protein was expressed fully soluble only at 20 oC, whereas the larger (~92 kDa) IsPETase-MHETase fusion protein was insoluble in all tested conditions. NetSolP (services.healthtech.dtu.dk/services/NetSolP-1.0/) gave the most accurate solubility predictions for the tested proteins and we used it for prediction of the solubility of

Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the difference

Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja za suzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostima ELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studije je bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih seruma za rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjam apsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testa proizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima i ze evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumi visokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog za ovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adherirani na dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli ite koncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu za kvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jim poliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe uje spektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikaciju N-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototip ELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ 10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekciju N-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela. Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u za kvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma., The accurate diagnosis of people with suspected infection with the SARS-CoV-2 is essential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can be detected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens in biological fluids in ELISA or similar techniques using antibodies developed in animals. The aim of the study was the establishment of a quantitative polyclonal sera-based test for routine measurement of the concentration of SARS CoV-2 nucleocapsid protein using absorbance measurement in a standard 96-well microtiter plate. For the purposes of the test development, recombinant N protein was produced and used for the production of mice and rabbit antisera. Produced antisera were purified and titer was determined. High-affinity polyclonal N-protein specific antisera were used for N-protein specific ELISA test development. The test is based on mice polyclonal sera adhered to microtiter plate bottom for the capture of the N protein from the specimen. Various concentrations of the recombinant N-protein were used to generate a standard curve for protein quantification. The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera and anti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement. We have successfully developed the prototype ELISA for the quantification of N-protein with the detection limit being in the range of ng/mL. The average LOD value for the prototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was 10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for the detection of N-protein with affinity and specificity similar to, or better than commercial antibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence for quantification of the N-protein in protein-rich samples, similar to human sera.

Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom., Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.

Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom., Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.

Late embryogenesis abundant (LEA) proteins are induced in cellular dehydration, such as freezing, drought, or desiccation. They can be involved in antioxidative defense, ion sequestration, and structural stabilization of both membranes and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation 1. The genome of the model plant Arabidopsis thaliana contains 51 genes encoding LEA proteins2. The majority of these LEA proteins (35%) belongs to Pfam LEA_4 (PF02987) family. In silico analysis suggested that these proteins are highly hydrophilic proteins with significant intrinsically disordered protein (IDP) properties. In order to evaluate structural properties and possible functions of LEA_4 protein family under different water content, a representative AtLEA25 protein (At2g42560, 635 aa), naturally located in the cytoplasm of seeds3 was obtained in Escherichia coli by recombinant DNA technology. Although this technology has been traditionally used to over-express and purify various globular proteins, numerous reports have shown that the IDPs, due to their structural plasicity are naturally highly susceptible to proteolytic cleavage. To conduct structural and functional studies we developed a robust method to produce highly purified (>95% pure) AtLEA25 with no detectable amount of protein breakdown products.

By considering regulations defined by laws, plans and decisions on different levels related to urban development of Belgrade riverside, specific conditions of privatization of land, changes in legislation and insufficiently defined planning procedures have been recognized, which reflect the informal practice of land use development. The example of such coastal areas can be found along the Sava and Danube riverside, including the illegal appropriation of the aquatory, which are regarded in this research as a specific practice of creating a unique cultural landscape. This practice reflects a spontaneous urban development of the riverside through the formal and informal establishment of temporary and permanent housing, numerous commercial and recreational activities in different scale along the 200km of Belgrade riverbank. The shaping of the cultural landscape of Belgrade riverside will be analyzed through the comparison of the advantages and disadvantages of different approaches to the ongoing riverside urbanization, which manifest the complex linkage between formal institutions, and members of the local community. The importance of closer examination of cultural, economic, environmental and spatial aspects of this process is in the recognition of indigenous building regulations and relations to the natural resources. The main goal of the research is to understand different levels on which this informal practice persists and to outline the possibilities for the improvement of the current planning of Belgrade riverbank through their institutionalisation.

This paper aims to open up a discussion about relations between former Yugoslavia's socialism and practice resulting from self-managing system established in early 1950s. Although this system was applied through a top-down approach, it implied, at least allegedly, coordination, integration and democratic harmonisation of particular interests with common and general ones on local level. The paper will briefly review the history and concept of socialist ideology and consider the impact that it had on institutional arrangements evolution and planning practice in Serbia. It will then touch on the role of ideology for urban planning process at the local level, understanding self-managing planning principles, their benefits, role and significance in planning practice.

Тема истраживања су односи Кнежевине Србије са Бугарском егзархијом у формативном периоду њеног постојања. Хронолошки оквир је време од формирања Бугарске егзархије 1870. до завршетка Берлинског конгреса 1878. Објашњено је какве су промене у Православљу наступиле стварањем Бугарске егзархије. Указано је какве су ставове о томе имале помесне цркве. Дати су подаци о органима и мрежи епархија Бугарске егзархије. Указано је на различите видове српске подршке бугарским циљевима. Она је показивана школовањем бугарских богослова и стипендирањем студената, али и неучестовањем Православне цркве у Кнежевини Србији на Сабору 1872, на коме је проглашено да је Егзархија у стању шизме. Од проглашавања шизме односи Кнежевине Србије са Бугарском егзархијом одржавани су преко црквених општина, епархијских митрополита и српског дипломатског агента у Цариграду. Део рада посвећен је дефинисању различитих струја у Бугарској егзархији, и њиховом односу према српским интересима. Закључено је да активност струје окупљене око егзархијског Мешовитог савета због недостатка информација од српске владе није одмах препозната као непријатељска. По окончању Велике источне кризе 1878. део територије Бугарске егзархије ушао у састав Србије. Свештенство и верници приступили су Православној цркви у Кнежевини Србији, и тако изашли из тешког стања шизме. Од 1878, односи два народа настављени су на међудржавном нивоу., The topic of this research is the relations between the Principality of Serbia and the Bulgarian Exarchate during the formative years of its existence. The chronological framework extends from the institution of the Bulgarian Exarchate in 1870 up to the conclusion of the Congress of Berlin in 1878. An explanation is provided of what changes happened within Orthodoxy with the establishment of the Bulgarian Exarchate, and what was the stance of the local churches to this event. Data is also provided about the administrative organs and networks of eparchies of the Bulgarian Exarchate. Various forms of support provided by Serbia to Bulgarian goals are indicated as well. This support not only took the form of providing education to Bulgarian theologians and providing scholarships to students, but also in the fact that the Orthodox Church in the Principality of Serbia refused to participate in the Council of 1872 which proclaimed that the Exarchate was in schism. Since the proclamation of schism, the relations between the Principality of Serbia and the Bulgarian Exarchate were maintained through ecclesiastical communities, eparchial metropolitans and the Serbian diplomatic agent in Constantinople. Part of the work is dedicated to defining the various currents within the Bulgarian Exarchate, and their stance towards Serbian interests. The conclusion is that the activity of the current gathered around Mixed Council of the Exarchate was not immediately recognized as inimical due to a lack of information on the side of the Serbian government. After the conclusion of the Great Eastern Crisis in 1878, part of the territory of the Bulgarian Exarchate was incorporated into Serbia. The clergy and laity joined the Orthodox Church in the Principality of Serbia, thereby leaving the difficult state of schism. After 1878, the relations of the two peoples were continued on an international level.

The aim of the present study is the comparison of ambient pollution in working environment during infectious medical waste treatment in two relevant health care institutions in the Republic of Serbia - The Blood Transfusion Institute of Serbia and The Clinical Centre of Serbia by different sterilization methods (sterilizer-convertor and autoclave, respectively). Monitoring and analysis of the following chemical compounds were performed in both institutions: water vapor, carbon dioxide, carbon monoxide, hydrochloric acid, methane, ethane, propane, hexane, formaldehyde, benzene, toluene, m-xylene, phenol, acrolein, ethanol, ethyl acetate, ethylamine, ethylene oxide, methanol, dimethyl disulphide, dimethyl sulphide, ethyl mercaptan, methyl mercaptan, freon 11 (trichlorofluoromethane), carbonyl sulphide and hydrogen chloride. The determination of vapor-phase concentrations was done by extractive Fourier transform infrared spectroscopy. Monitoring of ambient pollution during infectious medical waste sterilization has shown that mercaptans, acrolein, formaldehyde, dimethyl sulphide, and ethylene oxide are emitted in both health institutions, in concentrations which are not permitted by regulations, while increased concentrations of hydrogen chloride and phenol were found in the Clinical Centre of Serbia. A comparison of ambient pollution with two different sterilization methods has shown that higher concentrations of pollutants are emitted at higher temperatures. Considering the fact that mentioned compounds exhibit high toxicity, hence represent the risk to air quality in working and living environment, i.e. represents a risk to human health it is necessary to revise and improve the existing treatment methods.

The aim of this study was to identify short digestion-resistant peptides (SDRPs) released by pepsin digestion of the whole cow's milk and examine their IgE reactivity and allergenicity. Raw milk was subjected to simulated gastric digestion. SDRPs were fractionated from the digests and identified by MS. Milk SDRPs were evaluated for aggregability, propensity to compete for IgE binding with individual milk allergens, and ability to bind IgG4 from allergic and milk-tolerant individuals. The majority of milk SDRPs originated from caseins (97% of peptides) and overlapped with the known IgE epitopes of cow's milk allergens. SDRPs competed with milk proteins for binding to human IgE and readily formed aggregates. The average peptide length was 10.6 +/- 3.5 amino acids. The ability to provoke allergenic in vivo responses was confirmed by skin-prick testing (SPT) in five milk-allergic subjects. This was attributed to the peptide ability to aggregate into non-covalent complexes. SDRPs are able to induce response in SPT, but only in 50% of the sera SDRPs were able to inhibit IgG4 binding to caseins. Hence, SDRPs corresponding to the mainly continuous epitopes of milk proteins induce allergenic in vivo responses in milk-allergic subjects due to aggregation.

A simple organic-phase synthesis process was used to produce bare NiFe2O4 and ZnFe2O4 and core-shell ZnFe2O4@NiFe2O4 ferrite nanoparticles. X-ray powder diffractograms for all investigated powders show characteristic peaks of a spinel cubic structure without a secondary phase. Transmission electron microscopy (TEM) indicated the presence of nanoparticles that are smaller than 20 nm. The release of divalent ions (Ni2+ and Zn2+) from synthesized nanoparticles that were dispersed in saline solution, phosphate-buffered saline (PBS) and human serum, as determined by the inductively coupled plasma mass spectrometry (ICP-MS) method, was lower than 2 wt %. These results demonstrate the stability of the investigated nanoparticles in biologically relevant media and exclude the toxicity of Ni2+ and Zn2+ due to metal ion release, thereby opening a broad range of (bio)medical applications.

Figure S1-S3: Figure S1: Digestion of BLG at pH 1.2, 2.5 and 4.0; Figure S2: Digestion of ALA at pH 1.2, 2.5 and 4.0; Figure S3: MALDI spectra of peptides used in the study Table S1. IgE levels of patients used in the study determined by ImmunoCAP Methods: 1.1 Detection of ALA and BLG by immunoblot; 1.2 Mass spectrometry analysis; 1.3 Size-exclusion chromatography; 1.4 IgG4-binding properties of peptides obtained by digestion; 1.5 Digestion of purified ALA and BLG at different pH; 1.6 MALDI-TOF MS.

A simple organic-phase synthesis process was used to produce bare NiFe2O4 and ZnFe2O4 and core-shell ZnFe2O4@NiFe2O4 ferrite nanoparticles. X-ray powder diffractograms for all investigated powders show characteristic peaks of a spinel cubic structure without a secondary phase. Transmission electron microscopy (TEM) indicated the presence of nanoparticles that are smaller than 20 nm. The release of divalent ions (Ni2+ and Zn2+) from synthesized nanoparticles that were dispersed in saline solution, phosphate-buffered saline (PBS) and human serum, as determined by the inductively coupled plasma mass spectrometry (ICP-MS) method, was lower than 2 wt %. These results demonstrate the stability of the investigated nanoparticles in biologically relevant media and exclude the toxicity of Ni2+ and Zn2+ due to metal ion release, thereby opening a broad range of (bio)medical applications.

Alergija na hranu je reakcija preosetljivosti imunskog sistema na hranu koja dovodi dostvaranja IgE antitela. Ne postoji efikasan tretman i izbegavanje namirnica koje izazivajusimptome je jedini način za sprečavanje ozbiljnih posledica. Cilj ove teze je izučavanjeposttranslacionih i hemijskih modifikacija (PTM i HM) i interakcija alergena hrane sabiološki aktivnim polifenolom biljnog porekla.Alergija na crveno meso je novootkriveni tip odložene reakcije, sa PTM galaktozil-α-1,3-galaktozom (α-Gal) kao epitopom. Ispitani su podložnost model proteina - tiroglobulina(TG) digestiji pepsinom, prisustvo α-Gal na rezultujućim peptidima i pretpostavljenomesto vezivanja α-Gal za TG. Pokazano je da je α-Gal celim tokom simulirane želudačnedigestije vezan za nastale peptide TG, koji zadržavaju sposobnost da vežu IgE.Alergija na kikiriki je među najopasnijim zbog učestalosti, postojanosti i ozbiljnostisimptoma. Postoje razlike u alergenosti između sirovog i pečenog kikirikija, koje mogupoticati od razlika u modifikacijama alergena. U ovoj disertaciji je proučena razlika umodifikacijama glavnih alergena iz sirovog i pečenog kikirikija. Detektovano je 27modifikacija, od kojih 4 isključivo na proteinima pečenog kikirikija. Pokazane su razlikeu profilima modifikacija proteina između ekstrakata, koje mogu uticati na alergenost.2S albumini kikirikija su zbog kompaktne strukture otporni na delovanje digestivnihenzima, što se povezuje sa njihovom alergenošću. Ispitane su interakcije između njih iepigalokatehin-3-galata (EGCG), katehina zelenog čaja sa imunomodulatornimsvojstvima koji pospešuje digestiju proteina pepsinom. Vezivanje EGCG-a za alergeneizaziva promene u njihovoj strukturi (entalpijski povoljan proces), sa konstantamavezivanja reda veličine 104 M-1., Food allergy is an immune system reaction to certain foods that results in IgE antibodygeneration. Efficient treatment does not exist, so avoidance of trigger foods is the onlyway to prevent serious consequences. The aim of this thesis was to study posttranslationaland chemical modifications (PTM and CM) of food allergens and their interactions withbiologically active plant polyphenol.Red meat allergy is a newly discovered type of allergy with delayed symptoms and aPTM - galactose-α-1,3-galactose (α-Gal) as an epitope. Digestibility of a model protein -thyroglobulin (TG) by pepsin and presence of α-Gal on resulting peptides was studied,and the position of α-Gal containing glycan on TG was proposed. It was shown thatthroughout simulated gastric digestion α-Gal remains bound to IgE reactive TG peptides.Peanut allergy is among most dangerous types of food allergies due to prevalence,persistence and symptom severity. Differences in allergenicity between raw and roastedpeanuts were previously shown and might stem from differences in protein modifications.Differences in major allergen modifications between raw and roasted peanut werestudied. Twenty-seven modifications were detected, 4 of which exclusively on allergensfrom roasted peanuts. Differences in modification profiling were found between the twoprotein preparations that can affect their allergenicity.Peanut 2S albumins are compact digestion-resistant proteins, which attributes to theirallergenicity. Their interactions with epigallocatechin-3-gallate (EGCG), animmunomodulatory green tea catechin that facilitates pepsin digestion of proteins werestudied. The results show that binding of EGCG to these allergens induces conformationalchanges, and is enthalpy favorable with binding constants of 104 M-1.

INFOGEST metoda predstavlja standardizovani protokol za in vitro simulacijudigestije kompletne hrane, zasnovanom na fiziološki relevantnim uslovima. Predmetrada ove disertacije je ispitivanje digestibilnosti alergena kikirikija iz celog zrnaprimenom INFOGEST metode, kao i karakterizacija njihovih fragmenata otpornih naproteolizu.Za odstranjivanje lipida primenjena je metoda taloženja proteina, koja se pokazala kaosuperiornija u odnosu ekstrakciju lipida organskim rastvaračem, usled manjegkvalitativnog i kvantitativnog gubitka proteina.U ovoj tezi je pokazano da termički tretmani kikirikija, pored matriksa hrane, dodatnootežavaju oslobađanje proteina iz zrna, što čini glavne alergene kikirikija Ara h 1, Ara h2 Ara h 3 i Ara h 6 nedostupnijim za pepsinsku hidrolizu. Oslobađanje proteinakikirikija, kao i digestibilnost, u gastričnoj fazi se pokazala znatno izraženijom, uodnosu na intestinalnu fazu, s tim da je digestija kod pečenog kikirikija otežana uodnosu na sirovi. Nakon oralno-gastrične digestije celog zrna sirovog kikirikija, glavnialergeni kikirikija u velikoj meri ostaju intaktni, a njihovi peptidi otporni na digestijuzadržavaju alergeni kapacitet. Pokazano je da većina Ara h 2 i Ara h 6 alergena ostajerezistentna na digestiju. Ara h 1 i Ara h 3 kaskadno podležu pepsinolizi, do fragmenatakoji i dalje zadržavaju IgE vezujući potencijal. Mali peptidi koji potiču od Ara h 2alergena, su se pokazali kao najpotentniji inhibitori vezivanja IgE iz seruma pacijenataalergičnih na kikiriki, u odnosu na male Ara h 1 i Ara h 3 peptide.U ovoj disertaciji je pokazana izuzetno važna uloga efekata matriksa hrane, kao i njenetermičke obrade, na digestiju proteina hrane, koji mogu povećati stabilnost alergenahrane tokom digestije, i time omogućiti zadržavanje potencijala aktivacije alergijskereakcije nakon oralno-gastrične faze digestije., INFOGEST method is standardized protocol for in vitro simulation of complete fooddigestion, based on physiologicaly relevant conditions. The objective of thisdissertation was to investigate digestibility of peanut allergens from whole peanutkernel by INFOGEST method, as well as to characterize their fragments resistant toproteolysis.For delipidation, protein precipitation approach was applied, showing to be superior incomparison to delipidation by organic solevent, due to lower qualitative andquantitative protein loss.In this thesis it was shown that peanut thermal processing, in addition to effect of foodmatix, further complicates the extractability and digestibility of proteins from the grain,making peanut allergens Ara h 1, 2, 3 and 6, less accessible for pepsin hydrolysis.Extractability and digestibility of peanut proteins in the gastric phase have shown to besignificantly more pronounced, in comparison to intestinal phase, and roasted peanutdigestion was impaired compared to the raw. It was shown that after oral and gastricdigestion of whole raw peanut grains peanut allergens largely remain intact, and theirdigestion resistant peptides retain allergenic capacity. The most Ara h 2 and Ara h 6allergens have been shown to remain resistant to digestion. Ara h 1 and Ara h 3undergo pepsinolysis with cascade pattern to consequently smaller peptide fragmentswith retained IgE binding capacity. Small peptides from Ara h 2 allergens were themost potent inhibitors of IgE binding from sera of peanut allergic patients, compared tosmall peptides from Ara h 1 and Ara h 3.This thesis points to the great importance of the effects of food matrix, as well as foodthermal processing, on protein digestibility, which can create additional stability offood allergens during digestion, and thus enable retaining of their potential for thesensitization or triggering of allergic reactions.

Majarova reakcija (MR) je spontana reakcija izmeĊu karbonilne i amino grupe iod velikog je znaĉaja u hemiji hrane. Proteini modifikovani u MR imaju poboljšanetehno-funkcionalne osobine i nalaze primenu u prehrambenoj industriji. TakoĊe,strukture Majarovih proizvoda modifikovanih proteina imaju brojne efekte naalergenost proteina hrane.Predmet rada ove disertacije bilo je ispitivanje efekata MR indukovaneultrazvukom ili sušenjem raspršivanjem na fiziĉko-hemijske i tehno-funkcionalneosobine kravlje surutke i kamiljeg mleka, kao i efekata intenzivnog glikovanja u MR naimunološke osobine glavnog alergena kravljeg mleka, β-laktoglobulina (BLG).Kombinovana primena ultrazvuka i makromolekulskog nagomilavanja jeznaĉajnije ubrzala MR i glikovanje proteina nego sam ultrazvuĉni tretman.Makromolekulsko nagomilavanje, preko ubrzavanja MR, povećava oksidativnepromene na proteinima i formiranje struktura sliĉnih amiloidima. Visoko glikovaniproteini surutke su pokazali poboljšanu rastvorljivost u širokom pH i temperaturnomopsegu, kao i povećanu antioksidativnu moć.U kamiljem mleku u prahu sušenom raspršivanjem više temperature (230 °C –250 °C) su rezultirale većim stepenom MR u odnosu na niže temperature sušenja (190°C – 210 °C). Poboljšanje tehno-funkcionalnih osobina proteina kamiljeg mleka uprahu, kao što su antioksidativna moć i rastvorljivost, je pozitivno koreliralo sastepenom MR.Glikovanje BLG u MR, dovelo je do njegovog smanjenog transporta kroz modelsistem intestinalne barijere, povećanog preuzimanja od strane dendritskih ćelija,smanjenog luĉenja citokina u mešovitoj kulturi dendritskih ćelija sa CD4+ T-ćelijama, ido smanjene aktivacije bazofila, ukazujući da modifikovanje BLG u MR znaĉajnomenja njegovu sudbinu u procesima koji odreĊuju alergenost proteina mleka., Maillard reaction (MR) is a spontaneous reaction between carbonil and aminogroup, and it is of high importance in food chemistry. Proteins modified in MR possessimproved techno-functional properties and they have application in food industry. MRproducts of food proteins exert numerous effects on food proteins allergenicity.The subject of this doctoral dissertation was to examine the effects of MRinduced by ultrasound or spray-drying on phisyco-chemical and techno-functionalproperties of cow whey proteins and camel milk proteins, as well as the effects of MRon immunologic properties of major cow milk allergen, β-lactoglobulin (BLG).Combined application of ultrasound and macromolecular crowding enhancedMR to a greater extent than ultrasound treatment alone. Macromolecular crowdingindirectly, by enhancing MR, intensified oxidative modifications of whey proteins andformation of amyloid-like structures. Highly glycated proteins exhibited improvedsolubility in wide pH range, thermal stability and antioxidative power.Upon spray-drying treatment of camel milk, higher temperatures (230 °C – 250°C) induced higher degree of MR compared to lower drying temperatures (190 °C – 210°C). Improvement of techno-functional properties of camel milk proteins, such asantioxidative power and solubility, strongly correlated with the degree of MR.Intensive glycation of BLG in MR reduced its transport through the modelsystem of intestinal barrier, increased uptake by dendritic cells and decreased cytokineproduction in dendritic cells/CD4+ T-cells coculture, as well as reduced basophilactivation, indicating that modification of BLG in MR alters its fate in processescrucially involved in allergenicity of milk proteins.

Alergija na hranu je reakcija preosetljivosti imunskog sistema na hranu koja dovodi do stvaranja IgE antitela. Ne postoji efikasan tretman i izbegavanje namirnica koje izazivaju simptome je jedini način za sprečavanje ozbiljnih posledica. Cilj ove teze je izučavanje posttranslacionih i hemijskih modifikacija (PTM i HM) i interakcija alergena hrane sa biološki aktivnim polifenolom biljnog porekla. Alergija na crveno meso je novootkriveni tip odložene reakcije, sa PTM galaktozil-α-1,3- galaktozom (α-Gal) kao epitopom. Ispitani su podložnost model proteina - tiroglobulina (TG) digestiji pepsinom, prisustvo α-Gal na rezultujućim peptidima i pretpostavljeno mesto vezivanja α-Gal za TG. Pokazano je da je α-Gal celim tokom simulirane želudačne digestije vezan za nastale peptide TG, koji zadržavaju sposobnost da vežu IgE. Alergija na kikiriki je među najopasnijim zbog učestalosti, postojanosti i ozbiljnosti simptoma. Postoje razlike u alergenosti između sirovog i pečenog kikirikija, koje mogu poticati od razlika u modifikacijama alergena. U ovoj disertaciji je proučena razlika u modifikacijama glavnih alergena iz sirovog i pečenog kikirikija. Detektovano je 27 modifikacija, od kojih 4 isključivo na proteinima pečenog kikirikija. Pokazane su razlike u profilima modifikacija proteina između ekstrakata, koje mogu uticati na alergenost. 2S albumini kikirikija su zbog kompaktne strukture otporni na delovanje digestivnih enzima, što se povezuje sa njihovom alergenošću. Ispitane su interakcije između njih i epigalokatehin-3-galata (EGCG), katehina zelenog čaja sa imunomodulatornim svojstvima koji pospešuje digestiju proteina pepsinom. Vezivanje EGCG-a za alergene izaziva promene u njihovoj strukturi (entalpijski povoljan proces), sa konstantama vezivanja reda veličine 104 M-1., Food allergy is an immune system reaction to certain foods that results in IgE antibody generation. Efficient treatment does not exist, so avoidance of trigger foods is the only way to prevent serious consequences. The aim of this thesis was to study posttranslational and chemical modifications (PTM and CM) of food allergens and their interactions with biologically active plant polyphenol. Red meat allergy is a newly discovered type of allergy with delayed symptoms and a PTM - galactose-α-1,3-galactose (α-Gal) as an epitope. Digestibility of a model protein - thyroglobulin (TG) by pepsin and presence of α-Gal on resulting peptides was studied, and the position of α-Gal containing glycan on TG was proposed. It was shown that throughout simulated gastric digestion α-Gal remains bound to IgE reactive TG peptides. Peanut allergy is among most dangerous types of food allergies due to prevalence, persistence and symptom severity. Differences in allergenicity between raw and roasted peanuts were previously shown and might stem from differences in protein modifications. Differences in major allergen modifications between raw and roasted peanut were studied. Twenty-seven modifications were detected, 4 of which exclusively on allergens from roasted peanuts. Differences in modification profiling were found between the two protein preparations that can affect their allergenicity. Peanut 2S albumins are compact digestion-resistant proteins, which attributes to their allergenicity. Their interactions with epigallocatechin-3-gallate (EGCG), an immunomodulatory green tea catechin that facilitates pepsin digestion of proteins were studied. The results show that binding of EGCG to these allergens induces conformational changes, and is enthalpy favorable with binding constants of 104 M-1.

Majarova reakcija (MR) je spontana reakcija izmeĊu karbonilne i amino grupe i od velikog je znaĉaja u hemiji hrane. Proteini modifikovani u MR imaju poboljšane tehno-funkcionalne osobine i nalaze primenu u prehrambenoj industriji. TakoĊe, strukture Majarovih proizvoda modifikovanih proteina imaju brojne efekte na alergenost proteina hrane. Predmet rada ove disertacije bilo je ispitivanje efekata MR indukovane ultrazvukom ili sušenjem raspršivanjem na fiziĉko-hemijske i tehno-funkcionalne osobine kravlje surutke i kamiljeg mleka, kao i efekata intenzivnog glikovanja u MR na imunološke osobine glavnog alergena kravljeg mleka, β-laktoglobulina (BLG). Kombinovana primena ultrazvuka i makromolekulskog nagomilavanja je znaĉajnije ubrzala MR i glikovanje proteina nego sam ultrazvuĉni tretman. Makromolekulsko nagomilavanje, preko ubrzavanja MR, povećava oksidativne promene na proteinima i formiranje struktura sliĉnih amiloidima. Visoko glikovani proteini surutke su pokazali poboljšanu rastvorljivost u širokom pH i temperaturnom opsegu, kao i povećanu antioksidativnu moć. U kamiljem mleku u prahu sušenom raspršivanjem više temperature (230 °C – 250 °C) su rezultirale većim stepenom MR u odnosu na niže temperature sušenja (190 °C – 210 °C). Poboljšanje tehno-funkcionalnih osobina proteina kamiljeg mleka u prahu, kao što su antioksidativna moć i rastvorljivost, je pozitivno koreliralo sa stepenom MR. Glikovanje BLG u MR, dovelo je do njegovog smanjenog transporta kroz model sistem intestinalne barijere, povećanog preuzimanja od strane dendritskih ćelija, smanjenog luĉenja citokina u mešovitoj kulturi dendritskih ćelija sa CD4+ T-ćelijama, i do smanjene aktivacije bazofila, ukazujući da modifikovanje BLG u MR znaĉajno menja njegovu sudbinu u procesima koji odreĊuju alergenost proteina mleka., Maillard reaction (MR) is a spontaneous reaction between carbonil and amino group, and it is of high importance in food chemistry. Proteins modified in MR possess improved techno-functional properties and they have application in food industry. MR products of food proteins exert numerous effects on food proteins allergenicity. The subject of this doctoral dissertation was to examine the effects of MR induced by ultrasound or spray-drying on phisyco-chemical and techno-functional properties of cow whey proteins and camel milk proteins, as well as the effects of MR on immunologic properties of major cow milk allergen, β-lactoglobulin (BLG). Combined application of ultrasound and macromolecular crowding enhanced MR to a greater extent than ultrasound treatment alone. Macromolecular crowding indirectly, by enhancing MR, intensified oxidative modifications of whey proteins and formation of amyloid-like structures. Highly glycated proteins exhibited improved solubility in wide pH range, thermal stability and antioxidative power. Upon spray-drying treatment of camel milk, higher temperatures (230 °C – 250 °C) induced higher degree of MR compared to lower drying temperatures (190 °C – 210 °C). Improvement of techno-functional properties of camel milk proteins, such as antioxidative power and solubility, strongly correlated with the degree of MR. Intensive glycation of BLG in MR reduced its transport through the model system of intestinal barrier, increased uptake by dendritic cells and decreased cytokine production in dendritic cells/CD4+ T-cells coculture, as well as reduced basophil activation, indicating that modification of BLG in MR alters its fate in processes crucially involved in allergenicity of milk proteins.

INFOGEST metoda predstavlja standardizovani protokol za in vitro simulaciju digestije kompletne hrane, zasnovanom na fiziološki relevantnim uslovima. Predmet rada ove disertacije je ispitivanje digestibilnosti alergena kikirikija iz celog zrna primenom INFOGEST metode, kao i karakterizacija njihovih fragmenata otpornih na proteolizu. Za odstranjivanje lipida primenjena je metoda taloženja proteina, koja se pokazala kao superiornija u odnosu ekstrakciju lipida organskim rastvaračem, usled manjeg kvalitativnog i kvantitativnog gubitka proteina. U ovoj tezi je pokazano da termički tretmani kikirikija, pored matriksa hrane, dodatno otežavaju oslobađanje proteina iz zrna, što čini glavne alergene kikirikija Ara h 1, Ara h 2 Ara h 3 i Ara h 6 nedostupnijim za pepsinsku hidrolizu. Oslobađanje proteina kikirikija, kao i digestibilnost, u gastričnoj fazi se pokazala znatno izraženijom, u odnosu na intestinalnu fazu, s tim da je digestija kod pečenog kikirikija otežana u odnosu na sirovi. Nakon oralno-gastrične digestije celog zrna sirovog kikirikija, glavni alergeni kikirikija u velikoj meri ostaju intaktni, a njihovi peptidi otporni na digestiju zadržavaju alergeni kapacitet. Pokazano je da većina Ara h 2 i Ara h 6 alergena ostaje rezistentna na digestiju. Ara h 1 i Ara h 3 kaskadno podležu pepsinolizi, do fragmenata koji i dalje zadržavaju IgE vezujući potencijal. Mali peptidi koji potiču od Ara h 2 alergena, su se pokazali kao najpotentniji inhibitori vezivanja IgE iz seruma pacijenata alergičnih na kikiriki, u odnosu na male Ara h 1 i Ara h 3 peptide. U ovoj disertaciji je pokazana izuzetno važna uloga efekata matriksa hrane, kao i njene termičke obrade, na digestiju proteina hrane, koji mogu povećati stabilnost alergena hrane tokom digestije, i time omogućiti zadržavanje potencijala aktivacije alergijske reakcije nakon oralno-gastrične faze digestije., INFOGEST method is standardized protocol for in vitro simulation of complete food digestion, based on physiologicaly relevant conditions. The objective of this dissertation was to investigate digestibility of peanut allergens from whole peanut kernel by INFOGEST method, as well as to characterize their fragments resistant to proteolysis. For delipidation, protein precipitation approach was applied, showing to be superior in comparison to delipidation by organic solevent, due to lower qualitative and quantitative protein loss. In this thesis it was shown that peanut thermal processing, in addition to effect of food matix, further complicates the extractability and digestibility of proteins from the grain, making peanut allergens Ara h 1, 2, 3 and 6, less accessible for pepsin hydrolysis. Extractability and digestibility of peanut proteins in the gastric phase have shown to be significantly more pronounced, in comparison to intestinal phase, and roasted peanut digestion was impaired compared to the raw. It was shown that after oral and gastric digestion of whole raw peanut grains peanut allergens largely remain intact, and their digestion resistant peptides retain allergenic capacity. The most Ara h 2 and Ara h 6 allergens have been shown to remain resistant to digestion. Ara h 1 and Ara h 3 undergo pepsinolysis with cascade pattern to consequently smaller peptide fragments with retained IgE binding capacity. Small peptides from Ara h 2 allergens were the most potent inhibitors of IgE binding from sera of peanut allergic patients, compared to small peptides from Ara h 1 and Ara h 3. This thesis points to the great importance of the effects of food matrix, as well as food thermal processing, on protein digestibility, which can create additional stability of food allergens during digestion, and thus enable retaining of their potential for the sensitization or triggering of allergic reactions.

Thanks to its excellent properties, such as electrical resistivity, flexibility and resistance, ethylene-propylene-diene (EPDM) is widely used as an insulating material for electrical cables. Insulation layers of power cables, which make up 95% of electrical cables at nuclear power plants, are made of elastomers. Therefore, normal work can be seriously compromised if insulation of some cables is easily damaged or degraded. The performance of electrical cables depends on the physical integrity of the key components in the insulation layers, because micro-cracks can lead to loss of insulation characteristics. The use of polymeric materials for the production of electrical insulation is based on their favorable properties, which are related to their high stability. Oxidation as a major degradation process changes the functional characteristics of electrical insulations. The obtaining of insulating layers of current cables with satisfactory properties in an electric field were achieved by a well-chosen formulation consisting of high performance materials. In this dissertation, a research was carried out regarding the choice of polymer formulations with EPDM, testing the crosslinking of the appropriate EPDM formulation using peroxide compounds in order to slow down the degradation of insulation polymer layers of medium voltage cables. The proposed EPDM formulations were examined in terms of degradation, improvement of rheological, mechanical and electrical performances, and thermal stability. The possibility of reducing the concentration of zinc oxide in the formulation with simultaneous improved thermal and electrical stability due to the adverse impact of zinc oxide on the environment as well as the costeffectiveness of the insulation process on the cables were investigated. A new HPLC method that were developed could be used in the cable industry for routine measurements of percentage peroxide utilization in the process of making insulating polymer layers of EPDM. The

Resistance to digestion by digestive proteases represents a critical property of many food allergens. Recently, a harmonized INFOGEST protocol was proposed for solid food digestion. The protocol proposes digestion conditions suitable for all kinds of solid and liquid foods. However, peanuts, as a lipid-rich food, represent a challenge for downstream analyses of the digestome. This is particularly reflected in the methodological difficulties in analyzing proteins and peptides in the presence of lipids. Therefore, the removal of the lipids seems to be a prerequisite for the downstream analysis of digestomes of lipid-rich foods. Here, we aimed to compare the digestomes of raw and thermally treated (boiled and roasted) peanuts, resulting from the INFOGEST digestion protocol for solid food, upon defatting the digests in two different manners. The most reproducible results of peanut digests were obtained in downstream analyses on TCA/acetone defatting. Unfortunately, defatting, even with an optimized TCA/acetone procedure, leads to the loss of proteins and peptides. The results of our study reveal that different thermal treatments of peanuts affect protein extraction and gastric/gastrointestinal digestion. Roasting of peanuts seems to enhance the extraction of proteins during intestinal digestion to a notable extent. The increased intestinal digestion is a consequence of the delayed extraction of thermally treated peanut proteins, which are poorly soluble in acidic gastric digestion juice but are easily extracted when the pH of the media is raised as in the subsequent intestinal phase of the digestion. Thermal processing of peanuts impaired the gastrointestinal digestion of the peanut proteins, especially in the case of roasted samples

Structural details of changes accompanying interaction between insulin-related hormones and their binding partners are often enigmatic. Here, cross-linking/mass spectrometry could complement structural techniques and reveal details of these protein-protein interfaces. We used such approach to clarify missing structural description of the interface in human insulin-like growth factor (IGF-1): Drosophila melanogaster imaginal morphogenesis protein-late 2 protein (Imp-L2) complex which we studied previously by X-ray crystallography. We crosslinked these proteins by heterobifunctional cross-linker sulfosuccinimidyl 4,4′-azidopentanoate (Sulfo-SDA) for the subsequent mass spectrometry (MS) analysis. The MS analysis revealed IGF-1:Imp-L2 interactions which were not resolved in the crystal structure of this assembly, and they converged with X-ray results, indicating the importance of the IGF-1 N-terminus interaction with the C-terminal (185–242) part of the Imp-L2 for stability of this complex. Here, we also showed the advantage and reliability of MS approach in solving details of protein-protein interactions that are too flexible for solid state structural methods.