Improvement of methods for enrichment and analysis of N-glycosylation of membrane proteins

Analiza glikanskog dijela membranskih glikoproteina postala je važan analitički izazov zbog njihove izuzetne važnosti u međustaničnom prepoznavanju te interakcijama pojedinih molekula s receptorima na staničnoj površini. Brojni pokušaji izolacije membranskih glikoproteina naišli su na niz prepreka s obzirom na nedovoljnu ponovljivost metoda ili pak onečišćenje citosolnim glikoproteinima. Stoga je cilj ovog rada bio potvrditi učinkovitost i ponovljivost metode izolacije membranskih proteina s Triton X-114 i pročišćavanja organskim otapalima u svrhu analize njihove N-glikozilacije. Sama analiza N-glikana provedena je HILIC-UPLC kromatografijom. Korištene su stanice iz THP stanične linije, ali je ponovljivost potvrđena i na stanicama ljudskog mozga, leukocitima i lizosomima. U metodama za pročišćavanje izoliranih membranskih proteina korištena su različita organska otapala pomoću kojih je dobiven precipitat proteina. Organska otapala koja su upotrijebljena bila su aceton, kombinacija aceton/metanol i kloroform/metanol, gdje se posljednja kombinacija pokazala najučinkovitijom, iako sve tri metode daju kromatograme jednakog glikanskog profila. Također je pokazano da početna količina stanica utječe na intenzitet kromatografskih vršaka. Radi potvrde prisutnosti glikanskih struktura u dobivenim uzorcima, sakupljeni su pojedini kromatografski vršci koji su sekvencirani razgradnjom kombinacijom egzoglikozidaza koje otkidaju pojedine monosaharide s nereduktivnih krajeva glikana, a pokazuju određeni stupanj specifičnosti prema kidanju pojedinih veza. Na taj način uspješno su dokazane i identificirane pojedine glikanske strukture. Važnost ove studije je u napretku izolacije membranskih proteina čime je omogućen bolji uvid u njihovu N-glikozilaciju koja je, osim u dijagnostici i prognostici bolesti, danas važna i biofarmaceutskoj industriji zbog toga što glikozilacija ima učinak na fizikalno-kemijska svojstva proteina što se odražava na njihovu učinkovitost i sigurnost.Analysis of glycan part of membrane glycoproteins has become an important analytical challenge because of its high importance in intercellular recognition and interactions of molecules with receptors on the cell surface. Numerous attempts to isolate membrane glycoproteins have encountered a number of obstacles due to the lack of reproducibility of the method or pollution with cytosolic glycoprotein. Therefore, the aim of this study was to confirm the efficacy and reproducibility of the method of isolation of membrane proteins with Triton X-114 and its purification with organic solvents for the purpose of analyzing the N-glycosylation. The analysis of N-glycans was performed with HILIC-UPLC chromatography. Used cells were from THP cell lines, but the repeatability was confirmed with human brain cells, white blood cells and lysosomes. In the method for purifying the isolated membrane proteins are used various organic solvents with which we got a protein precipitate. Organic solvents which have been used are acetone, combinations of acetone/methanol and chloroform/methanol, where the last combination came as most effective, although all three methods gave chromatograms with the same glycan profile. It was also found that the initial amount of cells affects the intensity of the chromatographic peak. To confirm the presence of glycan structure in the obtained samples, individual chromatographic peaks were collected and sequenced with combination of exoglycosidases that are tearing certain non-reducing ends of the monosaccharides from glycans. Thus were successfully proven and identified some glycan structures. The importance of this study is progress in isolation of membrane proteins thus providing a better insight into their N-glycosylation that is, except for the diagnosis and prognosis of disease, today very important in biopharmaceutical industry because glycosylation has an effect on the physical and chemical properties of the protein which is reflected in their effectiveness and safety

Improvement of methods for enrichment and analysis of N-glycosylation of membrane proteins

Analiza glikanskog dijela membranskih glikoproteina postala je važan analitički izazov zbog njihove izuzetne važnosti u međustaničnom prepoznavanju te interakcijama pojedinih molekula s receptorima na staničnoj površini. Brojni pokušaji izolacije membranskih glikoproteina naišli su na niz prepreka s obzirom na nedovoljnu ponovljivost metoda ili pak onečišćenje citosolnim glikoproteinima. Stoga je cilj ovog rada bio potvrditi učinkovitost i ponovljivost metode izolacije membranskih proteina s Triton X-114 i pročišćavanja organskim otapalima u svrhu analize njihove N-glikozilacije. Sama analiza N-glikana provedena je HILIC-UPLC kromatografijom. Korištene su stanice iz THP stanične linije, ali je ponovljivost potvrđena i na stanicama ljudskog mozga, leukocitima i lizosomima. U metodama za pročišćavanje izoliranih membranskih proteina korištena su različita organska otapala pomoću kojih je dobiven precipitat proteina. Organska otapala koja su upotrijebljena bila su aceton, kombinacija aceton/metanol i kloroform/metanol, gdje se posljednja kombinacija pokazala najučinkovitijom, iako sve tri metode daju kromatograme jednakog glikanskog profila. Također je pokazano da početna količina stanica utječe na intenzitet kromatografskih vršaka. Radi potvrde prisutnosti glikanskih struktura u dobivenim uzorcima, sakupljeni su pojedini kromatografski vršci koji su sekvencirani razgradnjom kombinacijom egzoglikozidaza koje otkidaju pojedine monosaharide s nereduktivnih krajeva glikana, a pokazuju određeni stupanj specifičnosti prema kidanju pojedinih veza. Na taj način uspješno su dokazane i identificirane pojedine glikanske strukture. Važnost ove studije je u napretku izolacije membranskih proteina čime je omogućen bolji uvid u njihovu N-glikozilaciju koja je, osim u dijagnostici i prognostici bolesti, danas važna i biofarmaceutskoj industriji zbog toga što glikozilacija ima učinak na fizikalno-kemijska svojstva proteina što se odražava na njihovu učinkovitost i sigurnost.Analysis of glycan part of membrane glycoproteins has become an important analytical challenge because of its high importance in intercellular recognition and interactions of molecules with receptors on the cell surface. Numerous attempts to isolate membrane glycoproteins have encountered a number of obstacles due to the lack of reproducibility of the method or pollution with cytosolic glycoprotein. Therefore, the aim of this study was to confirm the efficacy and reproducibility of the method of isolation of membrane proteins with Triton X-114 and its purification with organic solvents for the purpose of analyzing the N-glycosylation. The analysis of N-glycans was performed with HILIC-UPLC chromatography. Used cells were from THP cell lines, but the repeatability was confirmed with human brain cells, white blood cells and lysosomes. In the method for purifying the isolated membrane proteins are used various organic solvents with which we got a protein precipitate. Organic solvents which have been used are acetone, combinations of acetone/methanol and chloroform/methanol, where the last combination came as most effective, although all three methods gave chromatograms with the same glycan profile. It was also found that the initial amount of cells affects the intensity of the chromatographic peak. To confirm the presence of glycan structure in the obtained samples, individual chromatographic peaks were collected and sequenced with combination of exoglycosidases that are tearing certain non-reducing ends of the monosaccharides from glycans. Thus were successfully proven and identified some glycan structures. The importance of this study is progress in isolation of membrane proteins thus providing a better insight into their N-glycosylation that is, except for the diagnosis and prognosis of disease, today very important in biopharmaceutical industry because glycosylation has an effect on the physical and chemical properties of the protein which is reflected in their effectiveness and safety

Individual and situational factors of work efficiency of people with the status of a disabled person

V preteklosti je veliko raziskovalcev preučevalo odnos med delovno učinkovitostjo in mnogoterimi različnimi dejavniki. V pričujoči nalogi smo pozornost namenili odnosu, ki ga oblikuje delovna učinkovitost z izbranimi psihološkimi koncepti na populaciji zaposlenih invalidnih oseb. Uporabljeni so bili vprašalniki o delovni učinkovitosti, vprašalnik delovnih zahtev in resursov, vprašanje o delovnem zadovoljstvu in vprašalnik spoprijemanja s stresom. V raziskavi je sodelovalo 137 oseb, ki imajo status invalida in so zaposlene na prostem trgu dela, v invalidskem podjetju ali v zaposlitvenem centru. Rezultati so pokazali, da delovno učinkovitost najbolje napovedujejo raznolikost delovnih nalog in opora sodelavcev kot koncepta delovnih zahtev in resursov ter usmerjenost na problem, manj uporabne strategije in novejša dimenzija uporabe substanc in humorja kot strategije spoprijemanja s stresom. Pokazale so se tudi signifikantne razlike med skupinami glede na vrsto zaposlitve in delovne zahteve ter resurse. Do statistično pomembnih razlik je prišlo na dimenziji opora sodelavcev in poddimenziji instrumentalna opora sodelavcev med zaposlenimi v zaposlitvenem centru in zaposlenimi v invalidskem podjetju ter zaposlenimi v zaposlitvenem centru ter na prostem trgu dela. Do statistično pomembnih razlik je prišlo še med zaposlenimi glede na vrsto zdravstvenih težav in samooceno lastnih omejitev. Posamezniki, ki imajo psihofizične poškodbe in/ali bolezni, vrednotijo svoje težave višje oz. jih pri delu ovirajo bolj kot tiste, ki imajo bodisi samo fizične poškodbe in/ali bolezni bodisi samo psihične poškodbe in/ali bolezni. Ugotovitve tako nakazujejo, da je socialna opora na delovnem mestu bistvena sestavina uspešnega kadra, še posebej, ko govorimo o ranljivih skupinah. Nedvomno je vsaka invalidna oseba individuum zase, zato je edino pravilno, da je deležna individualne obravnave, pri posploševanju rezultatov moramo biti tako skrajno previdni. Kljub temu pa dobljeni podatki dajejo neke grobe smernice za nadgradnjo nadaljnjega dela z zaposlenimi invalidi, da bo zadovoljstvo s sodelovanjem prisotno, tako na strani delodajalca kot na strani zaposlenega invalida.In the past, many researchers studied the relations between work efficiency and multiple factors. In this study, we focused on the relation formed by working efficiency with selected psychological concepts on the sample of employed persons with disabilities. Questionnaires on work efficiency, work requirements and resources questionnaire, question of work satisfaction and questionnaire for coping with stress were used. The survey involved 137 persons with the status of a disabled person and employed in the open labor market, in a disabled company or in a job center. The results have shown that work efficiency is best predicted by the diversity of work tasks and the support of employees presented as concepts of work requirements and resources, and problem focus. A less useful strategy, and a newer dimension would be the use of substances and humor as a strategy for coping with stress. Significant differences between groups according to the type of employment, work requirements and resources have also been shown. Statistically significant differences occurred at the dimension of employee support and sub-dimension of the instrumental support of employees among employees in the employment center and the disabled company, as well as among employees in the employment center and on the ones on the open labor market. Statistically significant differences occurred among employees and their range of health problems and self-esteem of their own limitations. Individuals with psychophysical injuries and/or illness evaluate their problems higherthey hinder them at work more than those who have either physical injuries and/or illnesses, or only psychological injuries and/or illnesses. The findings thus indicate that social support at the workplace is an essential component of a successful working group, especially when it comes to more vulnerable groups. Undoubtedly, every disabled person is an individual for himself, so it is only right that he is receiving individual treatment, and thus when summarizing the results, we must be extremely careful. Nevertheless, the obtained data provides some rough guidelines for the upgrading of continued work with disabled people, so that the satisfaction with the collaboration will be present both on the side of the employer and on the side of the employed disabled person

ANALYSIS OF DAYLIGHT IN A BUSINESS BUILDING

V tem diplomskem delu je izvedena analiza naravne osvetljenosti poslovne stavbe zgrajene v leseni montažni gradnji. Predstavljeni so principi gradnje z lesom, energetski razredi stavb, svetloba in njene osnovne fizikalne lastnosti, vpliv geografske lege in orientacije na zasnovo nizkoenergijskega objekta ter analiza naravne osvetlitve poslovne stavbe. Diplomsko delo predstavlja nadgradnjo projekta zasnovanega v okviru študentske skupine za delavnico »Energijsko učinkovite lesene javne stavbe Destrnik«. Projekt delavnice je v diplomskem delu optimiziran z manjšimi spremembami. Analiza osvetlitve je izvedena s programom Velux Daylight Visualizer.In this diploma there has been done an analysis of natural light in a business building with a timber frame construction. We have presented, principles of building with wood, energy classes of buildings, light and its physical properties, the impact of geographical location and orientation on the design of a low-energy building and an analysis of natural lighting in commercial buildings. The diploma represents an upgraded project, conceived in the context of student groups for the workshop "Energy-efficient wooden public buildings Destrnik." The project workshop in this diploma is optimized with minor modifications. Exposure Analysis was conducted using the program Velux Daylight Visualizer

The role of an internal auditor in fighting against money laundering

Delo diplomskega projekta obravnava problem pranja denarja in njegove (večkrat) dolgoročne posledice za družbo in gospodarstvo. Denar, ki izvira iz vseh vrst kaznivih dejanj, imenujemo umazan denar. Storilci tovrstnih kaznivih dejanj morajo, da bi lahko ušli organom pregona, prikriti vse sledi za pravim izvorom tega denarja. Ker so tehnike pranja denarja različne in se vedno znova spreminjajo ter pojavljajo nove, morajo podjetja vzpostaviti nek notranji nadzor, ki bo nadzoroval tudi to področje tveganj. Tukaj se pojavi potreba po notranjem nadzoru in zelo znani obliki notranjega nadzora, notranji reviziji. Notranji revizor je tisti, ki revidira področja v podjetju, ki so najbolj izpostavljena tveganjem. Notranji revizor je tudi zaščitnik vrednosti organizacije, saj odkriva pomanjkljivosti in o tem obvešča upravo. Pomembno vlogo pri odkrivanju in zmanjševanju prevar predstavlja prav notranja revizija. Zaradi vloge, ki jo v organizacijah opravlja, je večkrat nezaželena in vzbuja nezaupanje. Organizacije pa se morajo zavedati, da je vzpostavitev notranjega revidiranja pomembna preventiva. Prihrani jim lahko ogromne stroške, ki jih povzročijo prevare v podjetjihv nekaterih primerih vodijo celo do propada podjetij.The bachelor’s thesis project discusses the problem of money laundering and its (multiple) long-term consequences on society and the economy. Money that comes from all kinds of crimes is called dirty money. The perpetrators of such crimes must conceal all traces of the true origin of this type of money to escape law enforcement. Because of different, constantly changing, and new techniques of money laundering, companies need to establish a type of internal control that will also control this area of risk. This is where the need arises for internal control and the well-known type of internal control, internal audit. An internal audit is the one that audits the most at-risk areas in the company. An internal auditor is also the protector of the organization\u27s values because he discovers deficiencies and informs the management of them. Internal audit plays an important role in detecting and reducing fraud. Therefore, it is often undesirable and arouses distrust. However, organizations need to be aware of the importance of establishing internal auditing as a preventive measure. It can save them huge costs caused by corporate fraud which, in some cases, even leads to corporate collapse

Clinical and Structural Features of Sperm Head Vacuoles in Men Included in the In Vitro Fertilization Programme

The human sperm head vacuoles and their role in male infertility are still poorly understood. The aim of this study was to identify the clinical and ultrastructural features of human sperm head vacuoles in men included in the in vitro fertilization programme: men with normal (normozoospermia) and impaired sperm morphology (teratozoospermia). The sperm samples were observed under 6000-time magnification using motile sperm organelle morphology examination (MSOME). The proportion of sperm with head vacuoles was evaluated and related to the outcome of in vitro fertilization. The sperm of men with impaired sperm morphology was characterized by a higher proportion of sperm head vacuoles. The sperm head vacuoles were related to impaired semen quality (sperm concentration, motility, and morphology) but were not influenced by male factors (semen volume, height, age, weight, or body mass index). Moreover, sperm head vacuoles were related to impaired fertilization rate merely after classical in vitro fertilization (IVF), while there was no relation to pregnancy. In a subgroup of men, the sperm was fixed and observed by transmission electron microscopy (TEM). The ultrastructural study revealed that sperm head vacuoles are large nuclear indentations of various sizes and positions, packed with membranous material organized in membrane whorls (MW)

An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse

International audienceHemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions that stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling and outline its role with respect to α6β4 integrin recycling. We find that α6β4 integrin chains localize to the plasma membrane, caveolae, and ADP-ribosylation factor-6+ (Arf6+) endocytic compartments. Based on fluorescence recovery after photobleaching and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 but neither caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, α6β4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1, and Cavin1. We propose that mechanically induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover

Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo

International audienceTumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs’ hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo

Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells

International audienc