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Pahorjeva in Devetakova Nekropola: raziskovanje tesnobe, krivde in sramu v medznakovnem prevodu
No full textPrispevek raziskuje, kako so v Devetakovi stripovski priredbi Pahorjeve Nekropole zastopana občutja tesnobe, krivde in sramu, pri čemer je odnos med deloma razumljen kot medznakovni prevod. Pri obravnavi izbranih prizorov iz romana v stripu in ustreznih odlomkov iz romana se prispevek v veliki meri opira na smernice za analizo slikovnega gradiva, ki so se vzpostavile pri raziskovanju večkodnih besedil. Pri tem stopajo v ospredje posamezni elementi Devetakove vizualne govorice, kot so izbira barv, igra svetlobe in senc, pomenljive praznine, ponavljajoči se motivi in obrazna mimika, prek katerih se zrcalita vzdušje romana in čustveni horizont protagonista. Prispevek želi poudariti vlogo interpretacije pri medznakovnem prevajanju ter dialoško naravo odnosa med izvirnikom in njegovo stripovsko priredbo.Through the lens of intersemiotic translation, this article examines the representation of anxiety, guilt, and shame in Jurij Devetak’s adaptation of Boris Pahor’s Nekropola (Necropolis) into a graphic novel. In observing selected scenes from the graphic novel and the corresponding excerpts from Pahor’s novel, the article draws on established guidelines for analyzing visual elements in multimodal texts. Devetak’s choice of color, his play of light and shadow, and the recurring themes and facial expressions are particularly revealing in this respect, mirroring the novel’s atmosphere and the protagonist’s emotional horizon. The article highlights the role of interpretation in intersemiotic translation and the dialogic nature of the relationship between the original and its visual adaptation
Comparison of tooth nuclear DNA preservation between cementum and other dental tissues
No full textFor the genetic identification of skeletal remains, teeth can be used since they contain well-preserved DNA, which is present in various dental tissues, including dental pulp, dentin, and cementum. In forensics, the standard extraction method utilizes the whole tooth and destructive grinding. In our previous study, a highly effective non-destructive method was introduced to extract DNA from tooth cementum, bypassing whole tooth destruction. An analysis was performed on sixty-two canines obtained from adult skeletons from two archaeological cemeteries. After tooth cementum DNA extraction, teeth were stored in the freezer, and later used for destructive DNA extraction, grinding the entire tooth left after non-destructive extraction. The purpose of this study was to compare the preservation of DNA in tooth cementum and other tooth tissues. To extract the DNA from the stored teeth, they were pulverized, and DNA was obtained using the full demineralization method. Real-time PCR was employed to evaluate the quality and quantity of DNA, followed by STR typing. The amount of DNA, degradation rate, and success of STR typing were compared between DNA retrieved from tooth cementum and DNA extracted from the other tooth tissues. The results showed no successful STR typing in most stored teeth, and only five of them produced full or almost full profiles. Comparison to tooth root cementum, where 46 out of 62 teeth generated highly informative STR profiles, indicates that in the aged canines analysed, DNA preservation is strongest in tooth root cementum, while other tooth tissues generally yield little or no recoverable DNA. In five teeth that generated informative STR profiles, we assume that not all the tooth root cementum was decalcified using a non-destructive extraction method, and after grinding the whole tooth, some cementum tissue remained, contributing to successful genetic typing. In this study, the importance of tooth root cementum in archaeological samples is emphasized while exploring the forensic relevance of various tooth tissues. It highlights the notably poorer DNA preservation in dental pulp and dentin compared to cementum in the context of aged teeth
Microstructured magnetoactive elastomers for switchable wettability
No full textWe demonstrate the control of wettability of non-structured and microstructured magnetoactive elastomers (MAEs) by magnetic field. The synthesized composite materials have a concentration of carbonyl iron particles of 75 wt.% (≈27 vol.%) and three different stiffnesses of the elastomer matrix. A new method of fabrication of MAE coatings on plastic substrates is presented, which allows one to enhance the response of the apparent contact angle to the magnetic field by exposing the particle-enriched side of MAEs to water. A magnetic field is not applied during crosslinking. The highest variation of the contact angle from (113 ± 1)° in zero field up to (156 ± 2)° at about 400 mT is achieved in the MAE sample with the softest matrix. Several lamellar and pillared MAE structures are fabricated by laser micromachining. The lateral dimension of surface structures is about 50 µm and the depth varies between 3 µm and 60 µm. A systematic investigation of the effects of parameters of laser processing (laser power and the number of passages of the laser beam) on the wetting behavior of these structures in the absence and presence of a magnetic field is performed. In particular, strong anisotropy of the wetting behavior of lamellar structures is observed. The results are qualitatively discussed in the framework of the Wenzel and Cassie–Baxter models. Finally, directions of further research on magnetically controlled wettability of microstructured MAE surfaces are outlined. The obtained results may be useful for the development of magnetically controlled smart surfaces for droplet-based microfluidics
E-mentoring in higher education: a case study of the Faculty of Doba
No full textMagistrska naloga obravnava e-mentorstvo kot specifično obliko mentoriranja v
visokošolskem prostoru, s posebnim poudarkom na Doba Fakulteti v Mariboru. V
teoretičnem delu so predstavljeni različni modeli mentorstva, njihove značilnosti in ključni
elementi, kot so zaupanje, sprejemanje in prilagodljivost, ki pridobijo nove dimenzije v
digitalnem okolju. Analizirane so prednosti in izzivi e-mentorstva, vključno z vprašanji
tehnične podpore, vloge e-mentorja, institucionalnega okvira ter dinamike odnosa med
študentom, e-mentorjem in visokošolskim učiteljem. Empirični del raziskave se osredotoča
na razumevanje vloge e-mentorjev na Doba Fakulteti, ki delujejo kot povezovalni člen med
študenti in visokošolskimi učitelji ter izvajajo pedagoško, tehnično in motivacijsko podporo.
Kvalitativno zasnovana raziskava, ki temelji na intervjujih s študenti, visokošolskimi učitelji,
e-mentorji in vodstvom Doba fakultete v Mariboru, se osredotoča na vprašanja, kako se v
multipartitnem mentorskem odnosu določajo, porazdeljujejo in razmejujejo vloge, naloge in
odgovornostikako študenti dojemajo in doživljajo razmejitev vlog, nalog in odgovornosti
med visokošolskih učiteljem in e-mentorjemkatere prednosti, izzive in elemente
učinkovitega e-mentorstva razpoznavajokakšna institucionalna podpora je na voljo
različnim udeležencem v mentorskem procesu ter kako vsi deležniki ocenjujejo te obstoječe
institucionalne podporne mehanizme v skladu z elementi učinkovitega e-mentorstva?
Ugotovitve kažejo, da so vloge učitelja, e-mentorja in institucije formalno jasno razmejene,
vendar njihova dejanska razmejitev in učinkovitost temelji na stalni komunikaciji, refleksiji
in prilagajanju. Študenti razliko med učiteljem in e-mentorjem oblikujejo skozi izkušnjo, pri
čemer e-mentorja dojemajo kot socialno in motivacijsko podporo, učitelja pa kot
akademskega vodjo. Učinkovitost e-mentorstva se kaže v kombinaciji strukturne jasnosti,
procesne odzivnosti in empatične prisotnosti e-mentorja. Institucionalna podpora je ključna, a
mora biti refleksivna in vključujoča, da omogoča trajnostni razvoj e-mentorstva kot
sestavnega dela organizacijske kulture.The master\u27s thesis deals with e-mentoring as a specific form of mentoring in higher
education, with a special focus on the Faculty of Economics in Maribor. The theoretical part
presents various models of mentoring, their characteristics, and key elements such as trust,
acceptance, and flexibility, which take on new dimensions in the digital environment. The
advantages and challenges of e-mentoring are analyzed, including issues of technical support,
the role of the e-mentor, the institutional framework, and the dynamics of the relationship
between the student, e-mentor, and higher education teacher. The empirical part of the
research focuses on understanding the role of e-mentors at Doba Faculty, who act as a link
between students and university teachers and provide pedagogical, technical, and
motivational support.
The qualitative research, based on interviews with students, university teachers, e-mentors,
and the management of the Doba Faculty in Maribor, focuses on the following questions how
roles, tasks, and responsibilities are determined, distributed, and delimited in a multipartite
mentoring relationshiphow students perceive and experience the demarcation of roles, tasks,
and responsibilities between university teachers and e-mentorswhat advantages, challenges,
and elements of effective e-mentoring they recognizewhat institutional support is available
to the various participants in the mentoring process and how all stakeholders assess these
existing institutional support mechanisms in accordance with the elements of effective
e-mentoring?
The findings show that the roles of the teacher, e-mentor, and institution are formally clearly
defined, but their actual demarcation and effectiveness are based on constant communication,
reflection, and adaptation. Students shape the difference between the teacher and the
e-mentor through experience, perceiving the e-mentor as social and motivational support and
the teacher as an academic leader. The effectiveness of e-mentoring is demonstrated by a
combination of structural clarity, process responsiveness, and the empathetic presence of the
e-mentor. Institutional support is crucial, but it must be reflective and inclusive to enable the
sustainable development of e-mentoring as an integral part of organizational culture
Impact of Karst Phenomena on Construction Technology of Beka Tunnel (T2) on Second Railway Track between Divača and Koper
No full textGradnja predorov v kraškem okolju predstavlja izjemno zahteven inženirski izziv zaradi izrazite heterogenosti kamninskega masiva, nepredvidljivega pojavljanja kraških votlin ter kompleksnih hidrogeoloških razmer. Uspešna izvedba takšnih projektov zahteva stalno in celovito spremljanje geoloških, geofizikalnih in geotehničnih razmer ter sposobnost hitrega prilagajanja tehnologije gradnje ob odkritju nepredvidenih kraških pojavov. Ključnega pomena je pravočasno in usklajeno delovanje vseh vpletenih strok – geologov, krasoslovcev, hidrogeologov, geotehnikov, nadzora in izvajalca – saj le interdisciplinaren pristop omogoča varno, časovno učinkovito in okolijsko sprejemljivo gradnjo. Ob odkritju kraškega pojava se tehnologija gradnje praviloma spremeni: izkop se lahko začasno ustavi ali omeji, uvedejo se dodatni raziskovalni in zaščitni ukrepi, prilagodi se način podpiranja ter izvedejo obvodi za ohranitev naravnega režima pretakanja podzemne vode. Posebna pozornost je namenjena preprečevanju dviga hidrostatskih tlakov, zaščiti kraških vodonosnikov ter ohranjanju občutljivega kraškega okolja. Hkrati je za zmanjševanje časovnih zastojev in dodatnih stroškov nujna ustrezna organizacija gradbišča, ki omogoča nadaljevanje del na drugih izkopnih fazah ali napadnih točkah, kadar je napredovanje v kaloti začasno omejeno. Magistrska naloga obravnava navedena izhodišča gradnje predorov v kraškem okolju na primeru gradnje Drugega tira železniške proge Divača–Koper, s poudarkom na predoru Beka in odkritju kraškega pojava 2TDK-025. Na konkretnem primeru je prikazan vpliv kraških pojavov na spremembo tehnologije gradnje, potek odločanja o primerni vrsti sanacijskih del, izvedba začasnih in trajnih sanacijskih ukrepov. V nalogi je poudarjen pomen pravočasne detekcije kraškega in ustreznega tehničnega odziva projektanta, inženirja in izvajalca. Naloga potrjuje, da je gradnja predorov v kraškem okolju lahko uspešna le ob stalnem nadzoru, prilagodljivi tehnologiji gradnje ter doslednem upoštevanju naravovarstvenih in hidrogeoloških zahtev.Tunnel construction in karst environment represents an exceptionally demanding engineering challenge due to the pronounced heterogeneity of the rock mass, the unpredictable occurrence of karst cavities, and complex hydrogeological conditions. The successful execution of such projects requires continuous and comprehensive monitoring of geological, geophysical, and geotechnical conditions, as well as the ability to promptly adapt construction technology when unforeseen karst phenomena are encountered. Timely and coordinated cooperation among all involved disciplines—geologists, karstologists, hydrogeologists, geotechnical engineers, supervisory staff, and contractors—is essential, as only an interdisciplinary approach can ensure safe, time-efficient, and environmentally acceptable construction. When a karst feature is discovered, the construction technology typically needs to be modified: excavation may be temporarily halted or limited, additional investigation and protective measures are introduced, the support system is adapted, and bypass systems are implemented to preserve the natural regime of groundwater flow. Particular attention is given to preventing the increase in hydrostatic pressures, protecting karst aquifers, and preserving the sensitive karst environment. At the same time, minimizing delays and additional costs requires appropriate site organization, enabling construction activities to continue in other excavation phases or working faces when advancement in the tunnel crown is temporarily restricted. This master’s thesis examines principles of tunnel construction in karst environment through the case study of the Second Track railway project Divača–Koper, with a focus on the Beka Tunnel and the discovery of the karst feature 2TDK-025. The case study demonstrates the impact of karst phenomena on changes in construction technology, the decision-making process for the choice on remedial measures the implementation of temporary and permanent remediation measures. It is highlited in the thesis the importance of timely detection and an appropriate technical response of designer, enginere and contractor. The thesis confirms that successful tunnel construction in karst environments is only achievable through continuous monitoring, adaptable construction technology, and strict compliance with nature conservation and hydrogeological requirements
General vicar Giovanni Antonio Ricci and his Status dioecesis Goritiensis (1788–1789)
No full textLjubljanski nadškof Leopold baron Brigido se je zavedal, da upravlja izje-mno veliko in zahtevno nadškofijo in metropolijo. Zahodni del nove cerkvene pokrajine je bil vse od imenovanja Franca Filipa grofa Inzaghija za gradiškega škofa leta 1788 do njegove umestitve konec aprila 1789 brez rezidencialnega škofa, zato se je nadškof metropolit odločil ukrepati. Za navedeno kratko obdo-bje je imenoval metropolitanskega generalnega vikarja v duhovnih zadevah, ki je prevzel škofovska opravila na zahodu metropolije. Na to mesto je imenoval »domačina« iz Krmina, ljubljanskega stolnega kanonika Janeza Antona Riccija, ki se je pri delu izjemno izkazal, za kar si je pozneje prislužil pohvalo samega dunajskega dvora.Novoimenovani generalni vikar se je temeljito lotil dela in v soglasju s škofom Inzaghijem v kratkem obdobju uspel izdelati t. i. Status dioecesis Go-ritiensis, tj. popis vseh 20 dekanij generalnega vikariata po štirih sklopih, in sicer: 1. po duhovščini, 2. po župnijah in lokalnih kaplanijah, 3. po župnijskih in kuratnih cerkvah ter 4. po beneficijih. Odgovore je dobil od 533 duhovnikov. Celotno gradivo so na ordinariatu prepisali v 20 rokopisnih knjig, ki se danes hranijo v treh škatlah v Nadškofijskem arhivu v Gorici. Obstaja sicer še ena škatla »Riccijevega« gradiva v nevezanem stanju, ki pa v pričujoči monografiji ni obdelano oziroma objavljeno.Gradivo je bilo do sedaj poznano le redkim zgodovinarjem, si pa zaradi bogate vsebine zagotovo zasluži objavo in širše poznavanje. Z njo tako skušamo narediti korak naprej v raziskavah zgodovine goriške nadškofije, gradiške ško-fije in tudi goriško-gradiške škofije, saj je turbulentno obdobje cerkvene zgodo-vine tega ozemlja med letoma 1784 in 1830 slabo raziskano in si zasluži večjo pozornost raziskovalcev, ne samo zgodovinarjev.Gradivo, ki ga je zbral generalni vikar Ricci, nam podaja uvid v vrsto za-nimivosti. Nekatere tabele so izpolnjene v celoti, nekatere vsebujejo le osnovne podatke. Prva tabela z 20 vprašanji se je nanašala na duhovščino. Vsak klerik se je moral predstaviti z imenom, priimkom in funkcijo. Navesti je moral kraj iz-vora, mizni naslov, starost in leto prejema mašniškega posvečenja, kraj in vrsto študija, čas delovanja v dušnem pastirstvu, službena mesta in morebitne bene-ficije, ki jih je imel v lasti. Ostala vprašanja so se dotikala sposobnosti govorni-štva, branja primernih knjig in posledično znanja, navad, umnosti, obnašanja, fizičnih moči, vrlin, napak in pomanjkljivosti, sprejemanja pri vernikih, more-bitnih opominov itd. Za konec je moraj vsak podati tudi svoje finančno stanje.
Druga tabela se je nanašala na župnije in lokalne kaplanije in je vsebova-la 12 vprašanj. Navesti je bilo treba ime župnije ali lokalne kaplanije, ime žu-pnika ali lokalnega kaplana in sodelavcev (kaplanov ali ekspozitov). Sledila so vprašanja o redovnem življenju v župniji: o številu in vrsti samostanov, imena redovnikov oziroma redovnic skupaj z njihovo starostjo. Ostala vprašanja so se dotikala ustanovnih maš, župnijskega patrona in odvetnika, vasi in sosesk, ba-bic, prestopkov, šolstva, oddaljenosti posameznih vasi in lege župnije ter števila vernikov. Tretja tabela z 11 vprašanji se je posvečala cerkvam. Odgovoriti je bilo tre-ba na vprašanja o imenu župnijske cerkve, njenem patrociniju in posvetitvi, o posameznih oltarjih, podružničnih cerkvah in ekspoziturah, bratovščinah, oratorijih in križevem potu, odpustkih in oltarnih privilegijih, odvetnikih, cer-kvenih obračunih, ključarjih, cerkovnikih in njihovih dohodkih ter finančnem stanju cerkva.Zadnja tabela z 10 vprašanji pa se je dotikala beneficijev. Podati je bilo treba ime in kraj beneficija, navesti ustanovitelja in čas nastanka, imetnika be-neficija, njegove obveznosti ter vrsto beneficija, patrona in odvetnika beneficija ter podrejenost župniku, beneficiatov dom, vrsto šol, njihovo financiranje in vzdrževanje ter dohodke beneficija.Za zgodovino obravnavanega obdobja in ozemlja je prav gotovo zanimiva vsaka posamezna tabela in tudi prav vsak odgovor na zastavljena vprašanja. Prav vsak, ki se zanima za zgodovino tega časa ali območja, lahko v objavljenem viru najde kako uporabno navedbo. In s tem je namen objave dosežen.The Ljubljana Archbishop Michael Leopold Freiherr von Brigido was acutely aware of the fact that he was managing a vast and demanding archdi-ocese and metropolis. From the appointment of Franz Philipp von Inzaghi to Gradisca d’Isonzo in 1788 until his installation at the end of April 1789, the western part of the new metropolis was without a residential bishop, which led the archbishop to his decision to act. For this short period, he appointed a metropolitan general vicar in spiritual matters who assumed bishop’s duties in the west of the metropolis. For this position, he appointed “a local” from Cor-mons, the Ljubljana cathedral canon Giovanni Antonio Ricci, who performed his work with distinction, for which he later received a commendation from none other than the Viennese court.The newly appointed general vicar embarked on his duties vigorously and in agreement with Bishop Inzaghi managed, in a very short time, to prepare the so-called Status dioecesis Goritiensis, i.e. a census of all 20 deaneries of the gen-eral vicarage in four parts, according to: 1. clergy, 2. parishes and local curacies, 3. parish and curacy churches, and 4. benefices. He received answers from 533 priests. At the ordinariate, the entire material was transcribed into 20 manu-script books, which are today kept in three boxes in the Archdiocesan Archives in Gorizia. In fact, there exists another box of “Ricci’s” material that is unboundhowever, this material is not discussed or published in this monograph.Until today, the material has been known to only a handful of histori-anshowever, due to its valuable content it certainly deserves an independent publication and wider awareness. Hence, this publication strives to take a step forward in the research of the history of the Gorizia Archdiocese, Diocese of Gradisca d’Isonzo, and also of the Gorizia-Gradisca d’Isonzo Diocese, since the turbulent period of church history of this territory between 1784 and 1830 is poorly studied and deserves greater attention of researchers, not only historians.The material collected by General Vicar Ricci offers an insight into a se-ries of interesting facts. Some of the tables are filled in completely, others only include basic data. The first table, with 20 questions, pertained to clergy. Each cleric had to introduce himself with his name, surname, and function. He had to state his place of origin, titulus mensae, age, and the year he received his Rite of Ordination, the place and type of studies, the period spent working in pastoral care, posts performed, and potential benefices held. Other questions touched upon his ability in elocution, reading of appropriate books, and consequently the knowledge, habits, intelligence, behaviour, physical strength, virtues, flaws, and deficiencies, the manner of acceptance with the believers, possible repri-mands, etc. At the end, everyone had to present his financial situation.The second table inquired about parishes and local chaplaincies and in-cluded 12 questions. The name of the parish or local chaplaincy and the name of the priest or local chaplain and their associates (chaplains or expositi) had to be written. This was followed by questions regarding monastic life in the parish: the number and types of monasteries and convents in the parish and names of priests or nuns together with their age. Other questions pertained to foundation masses, the parish patron and lawyer, villages and neighbourhoods, midwives, offences, schools, distance to individual villages, the position of the parish, and the number of believers.The third table, including 11 questions, was dedicated to churches. The name of the parish church, its patronage and consecration, individual altars, filial churches and expositurae, brotherhoods, oratories and Way of the Cross, indulgences and altar privileges, lawyers, church accounts, caretakers, sextons and their income, and the financial state of churches had to be given.The last table, with 10 questions, pertained to benefices. The name and place of the benefice had to be given together with its founder and time of foun-dation, the holder of the benefice, his duties and the type of the benefice, the patron and lawyer of the benefice, as well as its subordination to a priest, the benefice’s home, the type of schools, their financing and maintenance, and ben-efice’s income.Each and every one of these tables as well as every answer to the given questions are certainly very interesting for the history of the discussed period and territory. Every person interested in the history of this era or area can find some useful information in the published source. This, in its own right, achieves the purpose of the publication
Construction of plasmids with hsa-miR-20b-5p targets and their validation with luciferase assay in mammalian cells
No full textMolekule mikro RNA postajajo vedno bolj zanimive za raziskovanje, saj se vpletajo v mnogo fizioloških in patoloških procesov v telesu ter tudi v procese kostne prenove. Porušena kostna prenova vodi v različne bolezni, med drugim tudi v osteoporozo. V predhodnih raziskavah na Katedri za klinično biokemijo so ugotovili spremenjeno izražanje hsa-miR-20b-5p v povezavi z zdravljenjem osteoporoze. Namen naloge je bil poiskati in eksperimentalno potrditi tarčne gene te mikro RNA. Na podlagi bioinformatične analize smo izbrali gena FZD3 in BMPR2. Zasnovali smo oligonukleotide, ki so vsebovali vezavno mesto za hsa-miR-20b-5p na osnovi zaporedij nukleotidov v informacijski RNA tarčnih genov. Poleg tarčnih zaporedij smo zasnovali tudi zaporedje za pozitivno kontrolo, ki je bila komplementarno zaporedje nukleotidov mikro RNA. Sledila je optimizacija procesa vnosa oligonukleotidov v plazmid pmirGLO. Celoten proces je obsegal restrikcijo plazmida, agarozno gelsko elektroforezo, ligacijo oligonukleotidov v razrezan plazmid in transformacijo celic Top10 s tem plazmidom. Pripravljene plazmide smo v procesu transfekcije vnesli v dve sesalski celični liniji (HOS in HeLa). Na teh celicah smo nato izvedli dvojni luciferazni test. Izmerili smo kresničkino in Renilla luciferazno aktivnost. Ti dve vrednosti smo med sabo delili, da smo podatke normalizirali, nato pa smo jih s pomočjo statističnega programa SPSS analizirali. Ugotovili smo, da se je hsa-miR-20b-5p v celični liniji HeLa vezala na zaporedji za FZD3 in BMPR2, s čimer smo potrdili njihovo direktno interakcijo. V celični liniji HOS ni bilo statistično pomembnih razlik med nobenim parom vzorcev, vzrok temu je bila najbrž slabša transfekcija, ki bi jo bilo v prihodnje treba optimizirati. Dokazana vezava hsa-miR-20b-5p na ta dva gena bi lahko bila v prihodnje uporabna osnova za razvoj novih terapevtskih možnosti zdravljenja bolezenskih stanj, kjer je cilj zmanjšanje tvorbe kostnine (npr. kostni rak), po drugi strani bi lahko to vezavo poizkušali zavirati, s čimer bi dobili nove terapevtske možnosti pri zdravljenju osteoporoze.MicroRNA molecules are becoming increasingly interesting for research, as they are involved in numerous physiological and pathological processes in the body, including bone remodelling. Disrupted bone remodelling leads to various diseases, including osteoporosis. Previous studies conducted at the Department of Clinical Biochemistry identified altered expression of hsa-miR-20b-5p in connection with osteoporosis treatment. The aim of this thesis was to identify and experimentally confirm the target genes of this microRNA. Based on bioinformatic analysis, we selected genes FZD3 and BMPR2. We designed oligonucleotides containing the binding site for hsa-miR-20b-5p, based on the nucleotide sequences in the mRNA of the target genes. In addition to the target sequences, we also designed a sequence for a positive control, which was a complementary sequence to the microRNA. This was followed by an optimization of the process for inserting the oligonucleotides into the pmirGLO plasmid. The entire process included plasmid restriction, agarose gel electrophoresis, ligation of the oligonucleotides into the digested plasmid, and transformation of Top10 cells with the recombinant plasmid. The prepared plasmids were then transfected into two mammalian cell lines (HOS and HeLa). A dual-luciferase reporter assay was performed on these cells. We measured firefly and Renilla luciferase activity. These values were normalized by calculating their ratio, and the data were subsequently analyzed using the statistical software SPSS. We found that hsa-miR-20b-5p bound to the sequences of FZD3 and BMPR2 in the HeLa cell line, confirming their direct interaction. In the HOS cell line, there were no statistically significant differences between any of the sample pairs, most likely due to inefficient transfection, which would need to be optimized in future experiments. The confirmed binding of hsa-miR-20b-5p to these two genes could serve as a basis for developing new therapeutic strategies for conditions where suppression of bone formation is desired (e.g., bone cancer). Conversely, inhibiting this interaction might provide novel therapeutic options for treating osteoporosis
Development of methods to increase the yield of extracellular heterologous proteins expressed in Streptomyces rimosus
No full textNamen doktorske naloge je razvoj novih metod za detekcijo in povečanje koncentracije heterolognih zunajceličnih proteinov pri bakteriji S. rimosus oz. preizkusiti več različnih pristopov ter ovrednotiti njihovo uporabnost. V začetnem delu smo poenostavili dosedanje načine detekcije zunajceličnih proteinov z uporabo različnih fluorescentnih proteinov. Z analizo ekso-proteoma smo pridobili nove informacije o najbolj zastopanih zunajceličnih proteinih, te informacije smo nato uporabili pri dizajnu ekspresijskih kaset. Z dodatkom poročevalskega sistema smo optimizirali molekularno orodje CRISPR-Cas9 in sam postopek izvajanja delecij s tem orodjem v bakteriji S. rimosus. Razvit sistem Cas9_SD_GusA je uporaben tudi pri inženiringu drugih bakterij, kjer CRISPR orodje ne deluje dobro. Zasnovali in testirali smo tudi sistem usmerjene evolucije na osnovi eritromicinske esteraze EreB, ki omogoča povečevanje količine tarčnega proteina. Z optimiziranim sistemom CRISPR smo izvedli delecije večjih odsekov genoma S. rimosus, in ustvarili metabolno očiščen sev S. rimosus ATCC Δotc Δ145kb, ki ne proizvaja nobenega od glavnih metabolitov te bakterije (rimocidini, oksitetraciklin) in je zato teoretično bolj primeren za industrijsko proizvodnjo heterolognih proteinov in drugih produktov. V konstruiranem sevu smo nato testirali delovanje verzij fuzijskega proteina mCherry-kaminizin in ovrednotili delovanje tega poročevalskega sistema za proizvodnjo industrijsko relevantne proteaze Kaminizin. Rezultati opravljenega dela kažejo prednosti in omejitve vsakega izmed testiranih pristopov ter ustvarjajo podlago za nadaljnji razvoja gostiteljskega organizma S. rimosus za produkcijo heterolognih proteinov in drugih produktov.The aim of the doctoral thesis was to develop new methods for the detection and increase of the concentration of heterologous extracellular proteins in the bacterium Streptomyces rimosus, and to test several different approaches and evaluate their usefulness. In the initial part, we simplified the existing methods of detecting extracellular proteins by using various fluorescent proteins. Through exoproteome analysis, we obtained new information about the most abundant extracellular proteins, which we then used in the design of expression cassettes. By adding a reporter system, we optimized the CRISPR-Cas9 molecular tool and the procedure for performing deletions with this tool in the bacterium S. rimosus. The developed Cas9_SD_GusA system is also useful for engineering other bacteria where the CRISPR tool does not work well. We also designed and tested a directed evolution system based on erythromycin esterase EreB, which enables the increase of the target protein amount. With the optimized CRISPR system, we performed deletions of larger genome segments of S. rimosus and created a metabolically purified strain of S. rimosus ATCC Δotc Δ145kb, which does not produce any of the main metabolites of this bacterium (rimocidins, oxytetracycline) and is therefore theoretically more suitable for the industrial production of heterologous proteins and other products. In the constructed strain, we then tested the functionality of versions of the fusion protein mCherry-kaminizin and evaluated the performance of this reporter system for the production of the industrially relevant protease Kaminizin. The results of the work performed demonstrate the advantages and limitations of each of the tested approaches and provide a basis for further development of the host organism S. rimosus for the production of heterologous proteins and other products
Review and comparison of the incidence and effects of per- and polyfluoroalkyl compounds
No full textPer- in polifluoroalkilne spojine z okrajšavo PFAS so spojine, ki so in se zaradi svojih edinstvenih lastnosti veliko uporabljajo v industriji in v potrošnih izdelkih. Prisotne so v sintezi polimerov, v gasilnih penah in kot dodatki za izboljšavo lastnosti ovojnin za živila, ličil in tekstila. Zaradi njihove obstojnosti in dolgega razpolovnega časa predstavljajo veliko tveganje za zdravje ljudi. Regulatorne institucije preučujejo učinke teh kemikalij na okolje ter na ljudi in so že ukinile uporabo perfluorooktansulfonske kisline (PFOS) in perfluorooktankarboksilne kisline (PFOA), vendar pa tudi veliko drugih PFAS lahko povzroči škodljive učinke. Namen te magistrske naloge je bil pregledati pojavnosti PFAS v naravnem okolju po svetu in ugotoviti, katere PFAS povzročajo največ škodljivih učinkov na ljudi in živali. Osredotočili smo se na neželene učinke ugotovljene v in vivo in in vitro raziskavah. Za zbiranje člankov smo uporabili sistematični literaturni pregled dveh podatkovnih baz PubMed in Science Direct. Med pridobljenimi članki pa smo informacije, ki so bile primerne za literaturni pregled, izbrali v skladu z metodologijo PRISMA. Potencialni viri izpostavljenosti spojinam, ki smo jih raziskali, so izdelki za potrošnike, prah in zrak notranjih prostorov, vode in sedimenti ter odpadne vode in blato iz čistilnih naprav. Po vseh celinah so raziskave pokazale prisotnost PFAS v notranjih prostorih, zemlji in vodi. V prahu in zraku so pogosto prisotne PFOS in PFOA, pri čemer je raven PFAS višja v urbaniziranih območjih. V Evropi in Severni Ameriki se najpogosteje pojavljajo PFOA in PFOS, medtem ko je v Aziji bolj opazen premik k alternativnim kratkoverižnim PFAS (npr. 6:2 fluorotelomersulfonat (6:2 FTS), kalijeva sol 6:2 fluorotelomer sulfonata (F-53B)). Območja, kjer se uporabljajo vodne filmotvorne gasilne pene z okrajšavo AFFF, kažejo najvišje stopnje onesnaženosti. Potrošniški izdelki z najvišjo vsebnostjo PFAS pa so voski za smuči. V pregledu raziskav pojavnosti pri ljudeh smo iz zbrane literature pridobili predvsem podatke o vsebnosti PFAS v krvi populacij. Najpogosteje detektirane spojine v krvi ljudi so bile PFOA, PFOS, perfluoroheksansulfonska kislina (PFHxS) in perfluorononanojska kislina (PFNA). Poklicno izpostavljeni ljudje po vsem svetu imajo višje ravni PFAS v primerjavi s splošno populacijo. V pregledanih in vitro raziskavah so predvsem PFOA, PFOS, PFHxS, PFNA in PFDA pokazale učinke na estrogenske in androgene receptorje ter na ščitnični hormonski sistem. Pregledane in vivo raziskave na živalih kažejo predvsem vpliv na jetra, endokrini sistem in razvoj potomcev. V preglednih raziskavah humanega biomonitoringa smo odkrili neenotne rezultate. Vendar pa se je pokazal nezanemarljiv vpliv na ščitnične hormone, prenatalni razvoj, dojenje ter na plodnost
in spolne hormone. Prav tako pa bodo potrebne dodatne raziskave za pojasnitev njihovega vpliva na imunski sistem ter na nevrološki razvoj predvsem pri otrocih.Per- and polyfluoroalkyl compounds, abbreviated PFAS, are compounds that are widely used in industry and consumer products due to their unique properties. They are present in polymer synthesis, in fire-fighting foams and as additives to improve the properties of food packaging, make-up and textiles. Their persistence and long half-life pose a significant risk to human health. Regulatory institutions are studying the effects of these chemicals on the environment and on humans and have already phased out the use of perfluorooctane sulfonic acid (PFOS) and perfluorooctanecarboxylic acid (PFOA), but many other PFASs can also cause harmful effects. The aim of this master thesis was to review the occurrence of PFASs in the natural environment worldwide and to identify which PFASs cause the most adverse effects on humans and animals. We focused on adverse effects identified in in vivo and in vitro studies. A systematic literature review of two databases, PubMed and Science Direct, was used to collect the articles. The information suitable for the review was selected from the retrieved articles according to the PRISMA methodology. Potential sources of exposure to the compounds we have investigated include consumer products, dust and indoor air, water and sediment, and wastewater and sewage sludge. Across continents, surveys have shown the presence of PFASs in indoor air, soil and water. PFOS and PFOA are commonly present in dust and air, with higher levels in urbanised areas. In Europe and North America, PFOA and PFOS are the most commonly found, while in Asia there is a more marked shift towards alternative short-chain PFASs (e.g. 6:2 fluorotelomer sulfonate (6:2 FTS), potassium salt of 6:2 fluorotelomer sulfonate (F-53B)). Areas where aqueous film-forming firefighting foams, abbreviated AFFF, are used show the highest levels of contamination. The consumer products with the highest PFAS content are ski waxes. In our review of human incidence studies, we have mainly extracted data on PFAS levels in the blood of populations from the literature. The most frequently detected compounds in human blood were PFOA, PFOS, perfluorohexanesulfonic acid (PFHxS) and perfluorononanoic acid (PFNA). Occupationally exposed people around the world have higher levels of PFAS compared to the general population. In the in vitro studies reviewed, PFOA, PFOS, PFHxS, PFNA and PFDA in particular have shown effects on estrogen and androgen receptors and on the thyroid hormone system. The in vivo animal studies reviewed show mainly effects on the liver, the endocrine system and the development of offspring. In the human biomonitoring studies reviewed, inconsistent results were found. But a non-negligible impact on thyroid hormones, prenatal development, lactation and on fertility and sex hormones has been
demonstrated. Many questions remain about the impact on the immune system and on neurodevelopment, particularly in children