POSTER MINUTE




AI in napredne



tehnologije v znanosti



Zbornik posterjev





Ljubljana, 22. oktober 2024

Zbornik posterjev





AI IN NAPREDNE TEHNOLOGIJE V ZNANOSTI



Poster minute, Ljubljana, 22. oktober 2024



Poster minute se je odvijal v okviru interdisciplinarnega strokovno-



izobraževalnega dogodka BEST dnevi znanosti (22. – 24. oktober 2024)



Kraj dogodka: Univerza v Ljubljani, Fakulteta za kemijo in kemijsko tehnologijo,



Večna pot 113, 1000 Ljubljana



Urednika:



Deni Krašna



Lara Likar



Organizacijski odbor:



Zara Bunc, Neža Bajec, Špela Blaznik, Lara Likar, Tinkara Korošec, Neja Brumec, Karin



Kunstelj, Lucija Kovaček, Deni Krašna, Manca Simončič, Nik Čadež



Grafično oblikovanje: Neža Bajec



Založnik zbornika in organizator konference:



Društvo BEST Ljubljana



Kraj in leto izida:



Ljubljana, 2024



Publikacija je izšla v elektronski obliki in je dostopna na spletnem naslovu:



https://www.best-dnevi-znanosti.si



Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v

Ljubljani

COBISS.SI-ID 218927875

ISBN 978-961-96808-2-7 (PDF)





Pravilnost odgovorov in omejitve prosto dostopnega orodja



ChatGPT v slovenščini na primerih okoljske problematike



I. Kralj Cigić, T. Balaško, N. Guzelj, L. Lengar, J. Levstek, G. Pirnat, L. Stepanova, L. Šarić, J. Štenkler, K.



Ziherl, H. Prosen*



Univerza v Ljubljani, Fakulteta za kemijo in kemijsko tehnologijo, Ljubljana, Slovenia *helena.prosen@fkkt.uni-lj.si



OZADJE



V novembru 2022 je podjetje OpenAI iz ZDA dalo v prosti medmrežni dostop prvo različico orodja ChatGPT (Sl. 1), ki je tako imenovani chatbot, torej orodje umetne inteligence (UI), ki se aktivno “pogovarja” z uporabnikom. Preko dostopa do medmrežnih virov zbira informacije, ki jih uporabniku predstavi v obliki odgovora na zastavljeno vprašanje. Omogoča pisanje daljših in krajših besedil na želeno tematiko, obseg in vsebino pa lahko uporabnik z dodatnimi vprašanji in ključnimi besedami nadalje usmerja v želeno smer. Orodje so doslej že večkrat posodobili, na voljo pa je bodisi v preprostejši, prosto dostopni, bodisi v bolj zmogljivi plačljivi različici. Pojav orodja ChatGPT je sprožil pospešen razvoj podobnih orodij UI drugih ponudnikov. Slika 1: Logo ChatGPT S pojavom ChatGPT so se razvile intenzivne razprave o koristnosti oziroma škodljivosti orodij UI. V zvezi z visokošolskim izobraževanjem je pogosto izražena zaskrbljenost, da študentje svoje pisne izdelke lahko napišejo z orodji UI in da je taka zloraba neizsledljiva. Univerza v Ljubljani se je odzvala z dokumentom ‘Priporočila Univerze v Ljubljani pri uporabi umetne inteligence’ [1], ki navaja tako primere koristne kot neustrezne oziroma nedovoljene uporabe orodij UI. V času od pojava ChatGPT so njegov način delovanja ter potencialno koristno uporabo v visokem šolstvu proučevali že v številnih raziskavah [2]. Čeprav je ob pravilni uporabi učinek na učni proces lahko pozitiven [2], pa podane informacije niso vedno pravilne [3]. Zlasti je potrebna previdnost pri citatih in navajanju virov, saj jih ChatGPT pretežno generira sam in niso resnične reference [3].



Pri predmetu Kemija okolja za ZASNOVA RAZISKAVE Vprašanje 1: Kakšen je vpliv globalne proizvodnje, predelave, dostave in študente univerzitetnega študijskega potrošnje hrane na Zemeljsko klimo? programa Kemija na Fakulteti za kemijo in kemijsko tehnologijo UL so ChatGPT se je pri poljudnem sestavku osredotočil na nekaj glavnih problematik vprašanja: emisije študenti preverjali pravilnost odgovorov in omejitve orodja

ChatGPT na primerih toplogrednih plinov, deforestacija, transport, poraba vode, odpadki ter izguba biotske raznovrstnosti. vprašanj iz okoljske problematike. Nekaj

podobnih raziskav je bilo Dotaknil se je torej skoraj vseh največjih problemov prehrambene industrije. Odgovori so bili sicer že izvedenih v angleškem jeziku, predvsem

v zvezi z informacijami o klimatskih spremembah [4,5] ali klimatski pretežno pravilni, vendar opisani precej površno, brez specifičnih podatkov. Odgovor je bil razumljiv tudi

politiki [6], pa tudi o za laike, čeprav so se pojavili tudi strokovni izrazi, npr. »anaerobna razgradnja«, ter tujke ali napačni zmožnosti orodja, da identificira pomembne

okoljske tematike, ki direktni prevodi – primer: “rastlinjaki plini” (angl. greenhouse gases) namesto “toplogredni plini”. še niso dovolj raziskane [7]. V Pri strokovnem odgovoru orodje ni podalo nič več podrobnosti kot pri poljudnem, temveč se je naši raziskavi smo se pri uporabi ChatGPT omejili na prosto

dostopno, osredotočilo izrecno na razlago ključnih besed, ki so bile dodane vprašanju. neplačljivo orodje. Vsa vprašanja, ključne besede in odgovori so bili v slovenščini. S tem smo se želeli približati izkušnji

slovenskega laičnega uporabnika. Študentje so najprej zastavili Vprašanje 2: Prednosti in slabosti uporabe električnih avtomobilov. Ali so poljudno vprašanje, zatem pa zahtevali bolj strokoven odgovor z električni avtomobili zares odgovor za čistejšo prihodnost? dodajanjem specifičnih ključnih besed. Pri obeh odgovorih pa so

nato preverili pravilnost s primerjavo z znanstveno literaturo. ChatGPT v nobenem od odgovorov ni definiral, o kateri podvrsti električnih avtomobilov razpravlja

(baterijska električna vozila, električna vozila z gorivno celico, hibridna električna vozila). Uporabil je

Vprašanje predpostavko, da je bralec seznanjen s tehnično terminologijo, kar ni nujno res. Sicer pa je odgovor 3: Kakšne so prednosti in slabosti

napisan v preprostem jeziku, primernem tudi za nezahtevne bralce. Izstopa zaokroženost besedila.

recikliranja odpadkov? V strokovnem odgovoru trditve niso bile zadostno utemeljene, temveč predstavljene kot neizpodbitna V laičnem odgovoru je ChatGPT predstavil pravilne podatke, a na dejstva. Večina besedila ni bila v protislovju z znanstvenimi dognanji, vendar tudi informativna vrednost nekoliko bolj enostaven način. Pri tem je vseeno navedel dovolj ni bila velika. V izogib posplošitvam je orodje pogosto uporabilo trditev, da se v različnih primerih izbran informacij, da si lahko bralec ustvari boljšo predstavo o recikliranju, parameter obravnava različno, ne da bi podalo osnovno delitev na različne primere. Izstopa njegovih prednostih in slabostih. fragmentirana oblika besedila in odsotnost osrednje teze. Citirani so bili članki iz uglednih znanstvenih V strokovnem sestavku je podal verodostojne, a ne poglobljene revij, toda ob pregledu je bilo ugotovljeno, da ne obstajajo. odgovore. Osredotočil se je le na prednosti recikliranja odpadkov,

navedel pa ni niti ene slabosti. Kljub zahtevi po strokovnosti članka RAZPRAVA IN ZAKLJUČKI je bilo orodje precej skopo s strokovnim izrazjem.

Skupna analiza poljudnih in strokovnih odgovorov, ki so jih študenti dobili z uporabo orodja ChatGPT v

9

8 neplačljivi slovenski različici, je omogočila identifikacijo nekaterih prednosti in slabosti orodja za 7 informiranje o okoljskih tematikah v slovenščini.

6

5

4 Prednosti so:

3 - omogoča hiter pregled ogromne količine informacij, dostopnih v medmrežnih virih;

2 - glede vsebine odgovorov je sicer zanesljiv, a precej bolj primeren za splošno (laično, nestrokovno) 1

0 uporabo, ko želi uporabnik pridobiti le osnovne informacije – Sl. 2;

JASNOST NATANČNOST GLOBINA UPORABNOST - lahko se uporablja za osnovno informiranje in kot izhodišče za nadaljnje raziskovanje, vendar ni

POLJUDNI SESTAVEK STROKOVNI SESTAVEK

Slika 2 zanesljiv kot vir pri kompleksnih in poglobljenih temah. : Primerjava poljudnega in strokovnega sestavka glede na različne

vidike – primer vprašanja 4.

Glavne slabosti:

Vprašanje 4: Kaj so prednosti in slabosti uporabe -ažurnost podatkov, iz katerih črpa (npr. v avgustu 2024 je baza podatkov segala do oktobra 2023); živega -upošteva tudi nepreverjene medmrežne vire; srebra?

Poljudni odgovor zajema bistvo problematike uporabe - uporaba napačnih slovenskih izrazov zaradi neposrednega in nepreverjenega prevajanja iz drugih živega srebra,

čeprav jezikov, zlasti angleščine; je opis prednosti in slabosti zelo poenostavljen. ChatGPT na začetku - orodje je naučeno, da prekomerno upošteva ključne besede, zato se pojavi nevarnost podajanja predstavi prednosti uporabe Hg, kjer pa že takoj opozori, da

so v primerjavi s strupenostjo Hg v resnici zanemarljive. To je zelo nepravilnih informacij, če uporabnik vztraja na izbranih zahtevah; pomemben podatek za razumevanje, zakaj se dandanes - odgovori so zelo splošni, mestoma tudi netočni, ter brez konkretnih podatkov; živo srebro umika iz izdelkov in proizvodnih procesov. - uporaba pravilnega standarda citiranja in verodostojnost naslova članka ter znanstvene revije

Strokovni sestavek ni dosegel učinkovito prikrivajo, da gre za navidezni vir, medtem ko ostajajo dejanski viri necitirani, kar pričakovane globine in natančnosti .

Pomanjkanje podrobnih informacij in primerov ter pomanjkljiva izpostavlja uporabnika orodja morebitnim posledicam kršitve pravic intelektualne lastnine.



strokovnjake. Nekatere navedbe so bile REFERENCE površinske uporaba strokovnih terminov so zmanjšali njegovo uporabnost za ali celo netočne,

kar je [1] Priporočila Univerze v Ljubljani pri uporabi umetne inteligence. Dostopno na: https://www.uni-lj.si/novice/2023-09-20-priporocila-univerze-v-ljubljani- zmanjšalo zanesljivost sestavka kot vira informacij (Sl. 2).

pri-uporabi-umetne-inteligence (dostop 21.6.2024)

[2] M. Montenegro-Rueda, J. Fernández-Cerero, J. M. Fernández-Batanero, E. López-Meneses. Computers 2023, 12, 153.

https://doi.org/10.3390/computers12080153

ZAHVALA [3] T. Day. The Professional Geographer 2023, 75, 1024-1027, DOI: 10.1080/00330124.2023.2190373 [4 ] A. Krzyżewska. Miscellanea Geographica 2024, 28 , 5-12, DOI: 10.2478/mgrsd-2023-0017

Avtorji se zahvaljujejo Javni agenciji za znanstvenoraziskovalno in inovacijsko dejavnost [5] B. Sommer, S. von Querfurth. Ambio2024, 53, 951–959, https://doi.org/10.1007/s13280-024-01997-7 Republike Slovenije ARIS (programski skupini P1-0153 in P1-0447), ki je finančno omogočila [6] F. Salekpay, J. van den Bergh, I. Savin. Ecological Economics 2024, 226, 108352, https://doi.org/10.1016/j.ecolecon.2024.108352 raziskovalno delo. [7] E. Agathokleous, C. J. Saitanis, C. Fang, Z. Yu. Science of the Total Environment 2023, 888, 164154, http://dx.doi.org/10.1016/j.scitotenv.2023.164154



Development of a continuous δ-viniferin synthesis in a microreactor using



immobilized horseradish peroxidase





Natalija Tomažina, Marko Božinovića, Francesca Annunziatab, Andrea Pintob, Polona Žnidaršič-Plazla,*





aUniversity of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-1000 Slovenia

bUniversità degli Studi di Milano, Department of Food, Environmental and Nutritional Sciences, Via Celoria 2, IT-20133

Milano, Italy



*email: polona.znidarsic@fkkt.uni-lj.si





INTRODUCTION



δ-viniferin is a resveratrol dehydrodimer, an isomer of ε-viniferin, which widely exists in grapes, knotweed, peanuts, and red wine. It was found to have biological activities, such as antiviral, anti-inflammatory, antibacterial, anticancer, and antioxidation. It possesses strong antioxidant properties, which can help protect the body against free radicals and oxidative stress. Additionally, δ-viniferin has been found to have anti-inflammatory properties, aiding in reducing inflammation in the body. Some researchers have also suggested that δ-viniferin could have the potential to fight various diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders.1,2 However, at a cost often exceeding 300 euros for just 1 milligram, the expense associated with δ-viniferin may severely restrict research efforts and its global applicability.

AIM

• to develop a cost-effective and sustainable process for synthesizing δ-viniferin from bio-derived materials using horseradish peroxidase (HRP) • optimization of crosslinked enzyme aggregates of HRP (CLEA-HRP) generation using microfluidic system3

Glutaraldehyde buffer solution

EXPERIMENTAL

Enzyme buffer solution

Optimization of CLEA-HRP generation (Figure 1)

Optimization focused on determining the optimal residence time for precipitation and crosslinking, screening various precipitation solvents, and adjusting the glutaraldehyde (GA) concentration for enzyme crosslinking at 25°C in PTFE tubes of various lenghts with 0.8 mm inner diameter. Horseradish peroxidase (HRP) was dissolved in 0.1 M potassium phosphate buffer (pH 6.0). Aqueous acetone solution Residence times in a microfluidic system obtained by changing the tube‘s lenghts: 3.77, 1.88, 0.94, 0.47 and 0.34 min

Figure 1: Scheme of a continuous CLEA-HRP production in a

HRP inlet concentration: 0.02 mg/mL microfluidic system

HRP inlet solution flow rate: 50 µL/min

Organic solvents tested for precipitation: acetone, acetonitrile, isopropanol and ethanol.

Organic solvent flow rate: 50 µL/min

Crosslinking agent: 1, 1.5, 0.5, 0.1 mM glutaraldehyde (GA) solution

Crosslinking agent flow rate: 100 µL/min

The size of CLEA-HRP was assessed through dynamic light scattering analysis (DLS).

The activity of CLEA-HRP was measured spectrophotometrically using ABTS test4- the activity of CLEA-HRP was compared to the free enzyme (recovered activity).

Figure 2: HRP-catalyzed synthesis of δ-viniferin from

Batch reaction (Figure 2)

5 resveratrol

Various amounts (80 mg, 100 mg, 120 mg) of resveratrol were dissolved in 4.621 mL of citrate buffer (pH 5.0) with 50% (v/v) of acetone. 3,79% (v/v) of HRP in Milli-Q water (1 mg/mL) was added and the mixture was stirred for 30 min at tested temperature. Subsequantly, 1.43% (v/v) of H 2O2 was added and the mixture was stirred for 1 h at tested temperature.



Temperature: 40°C



For quenching the reaction, the solution was placed in ice. The concentrations of resveratrol and δ-viniferin were analyzed using HPLC with Gemini-NX 3 µm C18 110 Å (150 × 4.60 mm) column and UV/VIS detector.





Continuous flow reaction Figure 3: Scheme of a continuous δ-viniferin production in a ( Figure 3 ): Work in progress microreactor with CLEA-HRP immobilized on the membrane surface



RESULTS DLS



The resulting CLEA-HRP exhibited an average particle radius of



Optimization of CLEA-HRP generation 150 nm ( 100



Figure 7).



Table 1: Results of testing different residence times; organic ity 80 , %



solvent: pure acetone, 1 mM GA ivct a 60





Tube length, cm ed τ, min Recovered activity, % St. dev., %er 40 ov 100 3.77 74.96 3.10 .c 20 50 1.88 85.48 4.13 Re

25 0.94 93.15 2.37 0

12.5 0.47 93.32 1.68 100% Ac 90% Ac 80% Ac 70% Ac Figure 7: Size distribution of CLEA-HRP; 90%

9 0.34 98.36 1.67 (v/v) acetone, 1 mM GA, retention time 0.34 min Figure 5 : Results of testing different acetone:buffer ratios; 1

mM GA, residence time: 0.34 min Batch reaction

Tube with a length of 9 cm and a residence time of 0.34 min was

selected for further testing due to its highest achieved recovered The final choice of organic solvent was 90% (v/v) acetone due to Temperature of reaction: 40 °C activity ( its highest recovered activity (Figure 5). 25 Table 1 ).

L 20

100 /m 15



, % 100 10 resveratrol , mg γ 80 5 δ-viniferin ity iv , % 80 0 ct 60 ity a iv 0 50 100 ct 60 ed a t, min er 40 ed ov er 40 Figure 8 : Concentrations of resveratrol and δ-viniferin .c 20 ov at 40 °C, initial mass of resveratrol: 100 mg Re .c 20 Re 0 The yield of the reaction performed in a batch process was

Acetonitrile Ethanol Isopropanol Acetone 0 45.7% after 15 min (Figure 8).

1.5 mM GA 1 mM GA 0.5 mM GA 0.1 mM GA

Figure 4: The effect of tested organic solvents as precipitation

solvents on recovered activity; 1 mM GA, residence time 0.34 min Figure 6: Results of testing different concentrations of glutaraldehyde; CONCLUSIONS



Acetone was selected among the tested organic solvents as the 0.34 min with acetone and glutaraldehyde concentrations of 90% (v/v) and 1 mM was selected as the best GA concentration for cross-linking best-tested solvent for precipitation because of the highest organic solvent: 90 vol.% acetone, residence time 0.34 min The highest recovered activity of 99% was achieved at a residence time of

achieved recovered activity ( enzyme because the highest recovered activity was retrained 1 mM, respectively. The resulting CLEA-HRP exhibited an average particle Figure 4). radius of 150 nm.

(Figure 6). The reaction was successfully performed with a yield of 45.7% after 15 min.

ACKNOWLEDGEMENT

This work was supported by Slovenian Research and Innovation Agency (ARIS) through Grants J4-4562 and P2-0191. MB was financed through Horizon Europe MSCA Doctoral Network project GreenDigiPharma (Grant 101073089), while FA was supported by National Recovery and Resilience Plan (NRRP), Mission4 Component 2 Investment 1.3 -Call for tender No. 341 of 15/03/2022 of Italian Ministry of University and by the European Union–NextGenerationEU, in the frame of the project Research and innovation network on food and nutrition Sustainability, Safety and Security (ON Foods).

REFERENCES

(1) Shang, Y.; Li, X.; Sun, T.; Zhou, J.; Zhou, H.; Chen, K. Comparative theoretical researches on the anti-oxidant activity of δ-viniferin and ε-viniferin. Journal of Molecular Structure 2021, 1245, 131062. https://doi.org/10.1016/j.molstruc.2021.131062. (2) Zwygart, A. C.; Medaglia, C.; Huber, R.; Poli, R.; Marcourt, L.; Schnée, S.; Michellod, E.; Mazel-Sanchez, B.; Constant, S.; Huang, S.; Bekliz, M.; Clément, S.; Gindro, K.; Queiroz, E. F.; Tapparel, C. Antiviral properties of trans-δ-viniferin derivatives against enveloped viruses. Biomedicine & Pharmacotherapy 2023, 163, 114825. https://doi.org/10.1016/j.biopha.2023.114825.

(3) Menegatti, T.; Lavrič, Ž.; Žnidaršič-Plazl, P. Microfluidics-based preparation of cross-linked enzyme aggregates. WO2023175002A1. (4) https://www.sigmaaldrich.com/HR/en/technical-documents/protocol/protein-biology/enzyme-activity-assay-of-peroxidase-abts-as-substrate (5) Mattio, L. M.; Dallavalle, S.; Musso, L.; Filardi, R.; Franzetti, L.; Pellegrino, L.;D’Incecco, P.; Mora, D.; Pinto, A.; Arioli, S. Antimicrobial Activity of Resveratrol-Derived Monomers and Dimers against Foodborne Pathogens. Sci. Rep. 2019, 9 (1), 1–13.





Understanding the mode of activation of plasmacytoid dendritic cells in different skin disorders



Neža Lesjak1, Jeremy Di Domizio1*

1 Department of Dermatology, CHUV University Hospital and University of Lausanne (UNIL), Lausanne, Switzerland * Corresponding authors: Jeremy.Di-Domizio@chuv.ch



BACKGROUND and HYPOTHESIS

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset which represents from 0.2 - 0.8% of human peripheral blood mononuclear cells (PBMCs). These rare circulating cells express Toll-like receptors TLR7 and TLR9 and can produce large amounts of type I interferons (IFNs) upon viral stimulation, thereby playing a critical role in linking innate and adaptive immunity (Ref 1). However, in inflammatory autoimmune diseases such as psoriasis and lupus, pDCs are abnormally activated. Conversely, in many cancers pDCs infiltrate tumors but produce less IFN-alpha, promoting immune suppression and tumor growth (Ref 2).

PDCs have shown phenotypic heterogeneity upon activation leading to two distinct populations: IFN production (IFN-pDCs) or antigen-presenting capacities (APC-pDCs). However, pDC differentiation into these two distinct types remains poorly understood.

We hypothesized that plasmacytoid dendritic cell diversification upon activation leads to two main phenotypes named IFN-pDC and APC-pDC, that can be predicted by the identification of specific transcriptional modules using scRNAseq.



• Do pDCs exclusively differentiate into one or the other phenotype

upon activation?

• Can single pDCs harbor two activation phenotypes overtime ?

• What is the transcriptional signature that predict pDC

differentiation ?

• What are the activation phenotypes of pDC inflitrating skin

diseases ?



METHODS



PRELIMINARY RESULTS



1. Verifying pDC phenotypes upon stimulation with four different agonists To determine whether plasmacytoid dendritic cells (pDCs) exhibit distinct phenotypic profiles in response to different stimuli, we stimulated freshly isolated pDCs using

Imiquimod (R837), Resiquimod (R848), CpGA, and CpGB. The cells were subsequently

a b IFN-⍺ production c IL-6 production analyzed using surface markers including IFN-α, CD40, CD80, and CD83. Our results indicate the presence of an antigen-presenting cell (APC)-like phenotype following

stimulation with CpGB (a TLR-9 agonist) and R837 (a TLR-7 agonist) (Fig. 1a). To

further investigate cytokine production, a cytometric bead array (CBA) was performed to

detect interferon signalling, utilizing antibodies against IFN-α and IL-6. Notably, we

observed that stimulation with R837 not only promoted an APC signature but also led to

the production of IFN-α. This suggests a potential role for scRNA-seq in further

elucidating the distinction between TLR agonist responses (Fig. 1b). In addition to IFN

type I production, pDCs demonstrated IL-6 expression, indicating cellular activation and

maturation (Fig. 1c).

In summary, these data demonstrate that the two

phenotypes (IFN and APC) can be obtained following

stimulation with different TLR-7/9 agonists.



2. Identifying the subsets in single cell RNA sequencing

a e





c Following single-cell RNA sequencing of pDCs stimulated for 24 hours, we employed various bioinformatic tools to identify cellular subsets.



After testing several integration methods, mutual nearest neighbor (MNN) integration was selected to produce a more suitable data transformation, particularly when visualized via UMAP, enabling clear identification of clusters corresponding to cells stimulated with



b different TLR agonists (Fig.2a). We then wanted to characterize the different clusters (Fig. 2b). We found that cluster 3 expressed APC markers; HLA-DOB, CD80, CD40, CD86, IL-15, IL-6. (Fig. 2c). Cluster 2 expressed markers of the IFN-pDC phenotype: CCL4, CCL3, TNF,



8 IRF7, GZMB and IFNA-1. Interestingly, IFNA-1 expression was nearly absent (Fig. 2d). Upon further analysis of the most representative

5

markers within MNN-derived clusters, we identified an emerging pDC subset characterized by an intermediate expression profile, clustering

4 3 1 prominent markers: TNFSF4, CEACAM1, CSRP2, and S1PR1 (Fig. 2e). These markers are well-known for their roles in mediating cellular between the two primary subsets: APC-pDCs and IFN-pDCs. This newly classified intermediate subset is defined by the expression of four

0

1

2 adhesion, immune interactions, and functions related to binding, cell proliferation, and migratory capacity.

0 4

3

umapmnn_2 5 d These findings demonstrate that the two subsets (APC-pDC and IFN-pDC) of pDCs can 0

2

4

−4 be distinguished at the single cell level, suggesting that they harbor different

−5 0 5 transcriptomes. Moreover, we observed an emerging third subset that might be an umapmnn_1

intermediate stage during pDC differentiation.



SUMMARY ONGOING RESEARCH

Activation of pDC leads to the two main phenotypes APC-pDC and IFN-pDC. • To be able to track pDC differentiation over time, we will add another timepoint at 6h.

• To confirm our results in vivo and to identify the pDC subsets that infiltrate different skin diseases, we will analyze pDC phenotypes in spatial

transcriptomics datasets previously generated by the group.



• Activation of plasmacytoid dendritic cells with TLR 7/9



? agonists leads to the two main phenotypes APC-pDC and



IFN-pDC.



• An emerging third intermediate phenotype that express



adhesion and migration markers could be the link between



the two subsets.





Ref.1 Lande, R., Gilliet, M. (2010). Plasmacytoid dendritic cells: Key players in the initiation and regulation of immune responses. Annals of NY Academy of Science 1183, 89-103. Ref.2 Conrad, C. et al. (2009). Plasmacytoid dendritic cells in the skin: To sense or not to sense nucleid acids. Seminars in Immunology 21, 101-109.





Bioconjugation and covalent binding of native proteins using



azide-alkyne cycloaddition



Nadja Suhorepeca a a a a a , Luka Ciber , Uroš Grošelj , Bogdan Štefane , Marko Novinec , Jurij Svete

a University of Ljubljana Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana



INTRODUCTION



~ Bioconjugation reactions are bioorthogonal reactions in which a covalent bond is formed between two molecules, one of which is a biological molecule or its fragment. [1] Such bioorthogonal reactions are also suitable for chemical cross-linking of proteins. [2]



~ We developed a protocol for binding NHS esters, maleimides and benzotriazolides that are functionalized with either an azide group or a cyclooctyne group to proteins (lysine or cysteine residues) and an analytical method for quantifying the binding (loading) to proteins.



~ Labeled proteins could thus undergo dimerization via strain-promoted azide–cyclooctyne [3+2] cycloaddition reaction (SPAAC), resulting in formation of a covalent 1,2,3-triazole linker. [3]



STRATEGY RESULTS



SDS-PAGE gels:

Step 1: • obtained after reaction between BSA,

Bioconjugation labeled with tags ref, 1-5, and of tags with two

complementary fluorescent probes and scanned for bioorthogonal groups

(azide and cyclooctyne group, separately) fluorescein fluorescence:

to the protein. reference 1 2 3 4 5



Step 2: ‘click‘ reaction between Used tags: fluorescent probe Spectrophotometrical determination of loading of each bioorthogonal groups and labeled protein

(CuAAC or SPAAC mechanism) 1

with fluorescein derivatives. reference



Fluorescent probes: 3 2



4 5

Average number of bound

Tag

tags per protein molecule

FPLC and SDS-PAGE reference 2.190

1 4.630

Step 3: 2 0.911

3 0.467

Determination of optimal length of a 4 0.429

linker for desired cross-linking. 5 0.611



Bifunctional reagents that act attempts at dimerization

as intermediate connectors:

• obtained after attempted dimerization

using a single intermediate

N3-N3 : bifunctional reagent

1 +

5 + Cokt-

BSA0 N-N C

3 3 okt

C Dimerization-C okt okt

[kDa]

potential

116.0 - cross-linking

66.2 -

CONCLUSION

45.0 -



We successfully prepared various NHS esters, benzotriazolides and maleimides that were functionalized • obtained after iterative addition of with either an azide group or a cyclooctyne group. We developed a protocol for binding these molecules

bifunctional reagents to determine the

to proteins and an analytical method for quantifying the loading. With known loading of labels on the

optimal linker length for desired

protein in hand, we were able to perform experiments on protein dimerization via azide–cyclooctyne

dimerization:

[3+2] cycloaddition reaction. To extend the linker, we prepared bifunctional reagents that could act as

intermediate connectors of the two protein molecules. BSA0 rd st nd 1 2 3

[kDa] potential

cross-linking

116.0 -

References: Contact: 66.2 -

[1] G. T. Hermanson: Introduction to Bioconjugation. In Bioconjugate Techniques; Elsevier, 2013; pp 1–125. suhorepec.nadja@gmail.com

[2] N. Forte, M. Livanos, E. Miranda, M. Morais, X. Yang, V. S. Rajkumar, K. A. Chester, V. Chudasama, J. R. Baker: Tuning the Hydrolytic Stability of Next 45.0 -LinkedIn: Nadja Suhorepec



Generation Maleimide Cross-Linkers Enables Access to Albumin-Antibody Fragment Conjugates and Tri-ScFvs. Bioconjugate Chem. 2018, 29, 486–492.



[3] J. Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks, F. P. J. T. Rutjes, J. C. M. Van Hest, D. J. Lefeber, P. Friedl, F. L. Van Delft: Readily BSA0 – control, unlabelled BSA



Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells. Angew. Chem. Int. Ed. 2010, 49, 9422–9425. 1st, 2nd, 3rd – number of iteration





STUDY OF THE DESORPTION OF NANOPARTICLES PREVIOUSLY





ADSORBED ON POLYETHLENE MICROPLASTICS





Tjaša Likeb1 1 1 1 , Ula Rozman , Jernej Imperl , Gabriela Kalčíková



1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000

Ljubljana, Slovenia

tjasa.likeb@gmail.com



INTRODUCTION MATERIALS & METHODS



Microplastics (1-1000 • μm ) are introduced into Nanoparticles TiO and ZnO (100 mg/L) were adsorbed on 2 the environment through various pathways, with polyethylene microplastics. wastewater being one of the primary sources. In • Desorption was studied at pH values of 6 and 8.3, with

wastewater, microplastics can adsorb various conditions including shaking at 170 rpm, and sampling at

pollutants, intervals of 3, 6, 12, and 24 hours. Nanoparticle concentrations including nanoparticles. These

nanoparticles can later desorb in the digestive were determined using ICP-MS and ICP-OES techniques.

• The desorption kinetics were analyzed using various Lagergren

tracts of organisms, potentially causing harmful



effects. pseudo-second-order models. kinetics models, including first-order, pseudo-first-order, and



100 100



INTERACTIONS 90 90 80 80



• ] ] 70 Initial concentration of adsorbed 70 [% [% 60 60 nanoparticles on microplastics: tion 50 tion 50

c(nTiO 40 2 ) = 923,7 µg/g 40

c(nZnO) = 284,2 µg/g Desorp 30 Desorp 30

20 20

• The maximum desorption for 10 10

nTiO 0 0 was reached after 6 h

(Figure 1) and for nZnO after 24 h 2 0 5 10 15 20 25 0 10 20 30 40 50

Time [h] Time [h]

(Figure 2). pH = 6 pH = 8 pH = 6 pH = 8



Figure 1: The desorption of nTiO 2 with time Figure 2: The desorption of nZnO with time



DESORPTION KINETICS



Table 1: Parameters for the pseudo-second-order kinetic model



pH [/] K 2 2 K pseudo-second-order rate constant [g/µg∙h] [g/µg∙h ] W [µg/g] W [µg/g] R [/] 2 e,cal e,exp

W calculated desorbed concentration at equilibrium e,cal

6 0,00277 666,7 718,4 0,9977 [µg/g]

TiO2

8 0,00336 666,7 704,0 0,9983 W experimentally determined desorbed e,exp

concentration at equilibrium [µg/g]

6 0,0129 28,9 31,6 0,9833

ZnO

8 0,00536 45,0 51,5 0,9754 2 R The coefficient of determination [/]



CONCLUSIONS



The study investigated the impact of pH on the desorption of nanoparticles adsorbed onto microplastics. The desorption

kinetics for both nTiO and nZnO nanoparticles were accurately modeled using the pseudo-second order Lagergren 2 model, with a coefficient of determination (R²) near unity. The findings indicated that pH did not influence the quantity of nanoparticles desorbed. However, the kinetic rate constants revealed that both nanoparticles desorbed more quickly at lower pH levels.

Izražanje in izolacija proteina



fenilalanin tRNA-sintetaze (FARS)



Špela Rapuš1,×, mag. Urša Čerček2, prof. dr. Boris Rogelj1,2,*

1: Univerza v Ljubljani, Fakulteta za kemijo in kemijsko tehnologijo, 2: Institut Jožef Stefan, Oddelek za biotehnologijo



Uvod Rezultati in diskusija



• Razširitvena mutacija G4C2 na Izražanje pri 18 ⁰C



genu c9orf72 po več Po izražanju pri 23 ⁰C mehanizmih vpliva na razvoj (ni prikazano) smo nevrodegenerativnih bolezni FARS izražali pri 18 kot sta ALS in FTD. ⁰C, da bi dobili več

• Protismerni prepis RNA se veže proteina v topni

na protein FARS, kar vodi v frakciji. Podenoto

inhibicijo aminoacilacije FARSA smo dobili

tRNAPhe in upad zastopanosti nekaj več v topni

fenilalanina v proteomu. frakciji medtem ko

• podenote FARSB v

Za boljše razumevanje načina



vezave in interakcije smo zaznali. topni frakciji nismo

protein FARS izrazili v Slika 1: a) vsi proteini barvani s Slika 2: a) vsi proteini barvani s E.coli



BL21[DE3] in izolirali. FARSA in heksahistidinski oznaki CBB, b) imunodetekcija proti CBB, b) imunodetekcija proti

FARSB in heksahistidinski oznaki



Metode Izražanje pri 18 ⁰C z dodanim MBP (maltoza vezavni protein)



• Transformacija bakterij, Fuzijski partner MBP

• izražanje rekombinantnih pomaga pri zvijanju

proteinov, proteina, da ga tako

• NaDS-PAGE, nastane več v topni

• prenos western in barvanje s frakciji. Podenoto

Coomassie Blue, FARSA smo uspeli v 90

• PCR, % izraziti v topni

• agarozna gelska elektroforeza, frakciji, FARSB pa 50 %.

• čiščenje produktov PCR,

• molekulsko kloniranje z



• CBB, b) imunodetekcija proti CBB, b) imunodetekcija proti izolacija plazmidov, FARSB in heksahistidinski oznaki FARSA in heksahistidinski oznaki Gibsonovo reakcijo, Slika 3: a) vsi proteini barvani s Slika 4: a) vsi proteini barvani s

• restrikcijska analiza in

določanje nukleotidnega Poskusna izolacija

zaporedja, Izolacijo smo izvedli z nikljevo



• izolacija proteina. afinitetno kromatografijo. V primeru





Zaključek FARSA smo največ proteina zbrali v



prvih treh eluatih (3,65 mg), v primeru





✓ FARSB pa v eluatih 2–5 (1,77 mg). Obe podenoti proteina FARS



smo uspeli izraziti v Začetni volumen bakterijske kulture je E. coli v zadostni količini bil 400 mL. Kljub uporabi inhibitorjev

proteaz in delu na ledu, je prišlo do

✓ FARS se boljše izraža pri nižji proteolitične razgradnje, kar bi lahko

temperaturi, skupaj s izboljšali z delom v hladni sobi.

fuzijskim partnerjem MBP



✓ FARS smo uspeli izolirati v Viri



manjši količini z afinitetno *korespondenčni avtor: boris.rogelj@ijs.si, × spela.rapus@gmail.com kromatografijo. [1] M. DeJesus-Hernandez, I. R. Mackenzie, B. F. Boeve, A. L. Boxer, M. Baker, N.J. Rutherford, A. M.





✓ Nicholson, N. C. A. Finch, H. Flynn, J. Adamson, et al.: Expanded GGGGCC hexanucleotide repeat in V prihodnosti bi morali noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 2011, 72, 245 – 256. optimizirati postopek izolacije [2] R. Balendra, A. M. Isaacs: C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat Rev



in izvesti študije interakcije z Neurol 2018, 14, 544–558. [3] J. P. Taylor, R. H. Brown, D. W. Cleveland: Decoding ALS: from genes to mechanism. Nature 2016, RNA 539, 197 – 206. Vloga transteritinu-podobnih proteinov v proteostazi pri C. elegans





Lena Kogoj1,2, Dr. Emmanouil Kyriakakis1, Prof. Anne Spang1 1. The Biozentrum, University of Basel, Basel, Switzerland 2. Fakulteta za kemijo in kemijsko tenologijo, Univerza v Ljubljani, Ljubljana, Slovenja



UVOD



Staranje je časovno odvisen funkcionalni upad. Znanstveniki so z raziskavami molekularnih mehanizmov staranja uspeli določiti devet znakov staranja (angl. hallmarks of aging), ki skupaj določajo fenotip staranja1. En od aspektov staranja je tudi izguba proteostaze ali homeostaze proteinov, ki ima lahko za posledico razvoj degenerativnih boleznih, kot sta Alzheimerjeva in Parkinsonova bolezen. Izguba proteostaze pomeni, da se proteini ne uspejo pravilno zviti in zato ne morejo upravljati svoje funkcije, pride pa tudi do njihove agregacije. Da se to ne bi dogajalo, je v celici prisotno kompleksno omrežje, ki zagotavlja proteastazo. Cilj raziskave je bil določiti, ali zmanjšanje izražanja transteritinu-podobnih proteinov (v nadaljevanju ttr) poveča število proteinskih agregatov v C. elegans, kar bi lahko pomenilo, da so ttr del proteastaznega omrežja2.



RAZPRAVA IN REZULTATI



Shematski prikaz poteka dela





Slika 1: Zmanjšanje izražanja genov z uporabo metode RNA-interferenca. Uporabljen je bil sev AM141, pri katerem se morebitni proteinski agregati zaradi oznake z YLP vidijo pod fluorescenčnim mikroskopom. Vključenost v proteastazno omrežje smo preverjali za ttr-5, ttr-15, ttr-16, ttr-17, ttr-25, ttr-37 in ttr-44. RNA-interferenca je bila izvedena tako, da se je vsak vzorec C. elegans hranil z E. coli z vstavljenim plazmidom za zmanjšanje izražanja gena za specifičnen ttr. Povečanje števila agregatov po zmanjšanju izražanja specifičnega ttr nakazuje na vpletenost tega ttr v proteastazno omrežje.



Rezultati RNA-interference



A) kontrola C) Slika 2: A) Mikrofotografija kontrole – brez RNA-



interference. B) Mikrofotografija po RNA-



ov interferenci za zmanjšane količino ttr-35. C) Graf

at števila agregatov v odvisnosti od specifičnega ttr,

eg čigar sinteza je bila zmanjšana z RNA-interferenco.

agr



B) ttr-35 o





evil Zmanjšanje izražanja genov za prav vse izbrane ttr Št poveča število agregatov napram kontroli v C.

elegans, kar nakazuje na to, da bi ttr lahko bili del

proteastaznega omrežja. Največja korelacija je



kontrola vidna pri ttr-35, najmanjša pa pri ttr-47.



ZAKLJUČEK IN UGOTOVITVE



Pokazali smo, da bi transteritinu-podobni proteini lahko igrali vlogo v proteastaznem omrežju, katerega naloga je preprečevanje agregacije proteinov.

Ne ve pa se še, na kakšen način bi ttr to lahko počeli – lahko bi namreč pomagali pri translaciji, zvitju proteinov (šaperoni) ali pa pri razgradnji nepravilno zvitih proteinov (proteaze / pot ubikvitin-proteasom / avtofagija)2.



REFERENCE



1. López-Otín, C.; Blasco, M. A.; Partridge, L.; Serrano, M.; Kroemer, G. The Hallmarks of Aging. Cell 2013, 153 (6), 1194–1217. https://doi.org/10.1016/j.cell.2013.05.039. 2. Hipp, M. S.; Kasturi, P.; Hartl, F. U. The Proteostasis Network and Its Decline in Ageing. Nat. Rev. Mol. Cell Biol. 2019, 20 (7), 421–435. https://doi.org/10.1038/s41580-

019-0101-y.

Milena Stojkovska Docevska



University of Ljubljana, Faculty of Chemistry and Chemical Technology



ABSTRACT INTRODUCTION



The FreeStyle 293-F cells were utilized to optimize proDPPI

expression using six different peptones, including plant-

based peptones and animal-derived Tryptone N1.

The goal of the study was to evaluate the effectiveness of

these peptones in producing the proDPPI-Twin-Strep-tag

construct from FreeStyle 293-F cells, conducted in

suspension flasks.

Results revealed that peptones derived from guar, soy, and

pea proteins improved protein production by approximately

eightfold compared to the control without added peptones.

The plant-based peptones used were G115, S146B, and P112.

Producing proteins in the right quantity and quality is a crucial need in modern times. The use of mammalian cells for protein

MATERIALS AND METHODS production has notably increased due to their ability to ensure proper protein folding, post-translational modifications, and

product assembly, all of which are vital for full biological activity. The ultimate goal of process development in animal cell culture is to increase product quality and yield while reducing cost.

EXPRESSION SYSTEM



• Cells: FreeStyle 293-F suspension cells hydrolysates, rich in amino acids, vitamins, and peptides, are gaining popularity as serum alternatives. They not only support Serum is essential for cell growth but is costly, inconsistent, and poses contamination risks. To address this, plant-based

• cell growth but also improve protein quality and reduce production costs. Overall, hydrolysates offer a promising solution to Vector : pDSG-IBA104

replace animal serum in cell culture.

• Protein: DPPI-Dipeptidyl-peptidase I

In our study, we expressed the proDPPI protein from FreeStyle 293-F cells five differet plant-based protein hydrolysates. To

MEDIA COMPOSITION enhance protein solubility and purification efficiency, we employed the Twin-Strep-tag for this experiment.

• FreeStyle 293 Expression Medium

(12338026)

• 0.5% Peptones

C-CELL PEPTONES



• Tryptone N1(#19553) of animal origin)

• C-CELL P112, (#17112) and P118, (#17118)

of pea origin

RESULTS

• G115, (#17115) of guar origin

• Protein expression detection by SDS-PAGE and Western-blot analysis S204 , (#17204) and S146B (#E0003) of soy



origin Expression Cell Mass Line Peptone 0,5% volume/ density/ A280 nm [µg/mL]* mL cells/mL

PRE-CULTURE Western blot, HRP- 6 1 Tryptone N1 25 1,25 x10 0,060 36 streptavidin

antibody 6 2 C-CELL P118 25 1,25 x10 0,140 83

Before transfection, pre- 6 3 C-CELL P112 25 1,25 x10 0,200 119

culture of cells. 6 4 C-CELL S146B 25 1,3 x10 0,245 145

5 6 C-CELL G115 25 1,3 x10 0,250 148

PROTEIN EXPRESSION 6 6 C-CELL S204 25 1,1 x10 0,160 95

Coomassie staining 6 7 Control 25 1,25 x10 0,043 25

Transfection with *The mass was calculated from the measured absorbance at 280 nm on NanoDrop

pDSG-IBA104 plasmid. and the amino acid sequence of the protein with the tags.

Feeding with 0,5% Quantification of pro-DPPI in the conditioned medium from Western-blot peptones at 48h and

96 h.

Sample collection at

120h.



SDS-PAGE AND WB

Coomassie staining;



Western blot, HRP- DISCUSSION AND CONCLUSIONS

streptavidin antibody.

A proDPPI expression study was conducted to assess the ability of various peptones to enhance protein production yields.

QUANTIFICATION Different peptones were compared to standard expression without supplementation, with the protein secreted into the

Quantification of pro culture medium. The results indicated that plant-derived peptones significantly boosted proDPPI secretion compared to the

control.

DPPI in the conditioned

medium from Western After purification via affinity chromatography, protein quantification was done using absorbance at 280 nm, and Western blot

blot by using Bio-Rad provided semi-quantitative analysis. Peptones enhanced the protein production by approximately eightfold compared to the

Image lab 6.1 control, particularly C-CELL G115, C-CELL S146B and P112 also showed more significant increase in protein production. All six

peptones dissolved easily in a preheated expression media.



ACKNOWLEDGEMENTS



We would like to extend our special thanks to Organotechnie SAS for their invaluable support and the provision of peptones, which greatly contributed to the success of this study. Your collaboration has been crucial for the research, and we sincerely appreciate your contribution.





Tissue-specific element profiles in edible seeds



Blaž Režonja1, Ela Vavpetič1, Neža Kokalj1, Aleš Kladnik1, Primož Vavpetič2, Mitja Kelemen2, Paula Pongrac1,2 1 Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia



2 Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia



paula.pongrac@bf.uni-lj.si; paula.pongrac@ijs.si





INTRODUCTION



Optimising nutritional yield of existing farmland to feed the increasing global population can be achieved through biofortification [1]. Staple foodstuff, mainly seeds, differ in their element profiles and their distribution can importantly influence the success of biofortification, food processing and diets worldwide. Elemental profiles of tissues in seeds of six nutritionally important dicots (Figure 1) were captured using Figure 1: Taxonomic relationships

micro-PIXE. A five-step workflow (Figure 2) was followed: after manual sectioning, of selected dicot seeds.

microscopy discerned tissues in seeds (Figure 3), distribution analysis depicted tissue-specific allocations of essential elements (Figure 4 disclosing results for Ca, Fe and Zn), image analysis provided their tissue-specific concentrations (Figure 5), and, ultimately, led to the estimation of nutritional potential of each seed (Table 1).



RESULTS



Figure 3: Autofluorescence microscopy of seed cross-sections under white (VIS), ultraviolet (UV) and blue (BLUE)

Figure 2: Workflow with key methods. seed coat (brown), endosperm (beige), endosperm (green), perisperm (orange) and auxiliary structures (gray) (right). excitation light (left) and microscopy-derived seed cross-section diagrams with colour-coded tissues distinguished:

The displayed seeds are caraway (A), chia (B), flax (C), poppy (D), quinoa (E) and rapeseed (F).



Figure 4: Micro-PIXE localisation maps of Ca, Fe and Zn. Values on colour scales are in

mg kg-1 dry matter. (SQRT) marks scales of square-rooted concentration values.

The displayed seeds are caraway (A), chia (B), flax (C), poppy (D), quinoa (E) and rapeseed (F).

Table 1: Calculated minimum daily intake for adult males and females with all

essential elements assumed to be fully bioavailable.



Figure 5: Whole-section and tissue-specific (embryo, endosperm and testa) element concentrations in all six seeds analysed.

Note: The concentrations in mg kg-1 are displayed on a logarithmic scale.



CONCLUSIONS

● Substantial differences in Ca, Fe and Zn concentrations between species and seed tissues were found. ● In general, whole grain concentrations follow the order: P > K > S > Ca > Cl > Fe > Zn > Mn. ● Of the studied seeds, poppy seed contained the highest concentrations of Ca (allocated to the seed coat, as in other seeds) and the highest concentrations of Fe and Zn

were measured in flax seeds (Fe allocated to the seed coat and Zn to the embryo).

● The elemental profiles of the endosperm and embryo in all studied dicot seeds were quite similar, which is in contrast to observations in monocot cereal grains [2]. ● Data on essential element distributions in different seeds and their tissues has the potential to support informed decisions in food processing and diets worldwide.





Bibliography Acknowledgements





1. P. J. White & M. R. Broadley, New Phytologist. 2009, 182, 49–84. This work was supported by the Slovenian Research and Innovation Agency (through research core funding [No. P1-0112, 2. A. Detterbeck, P. Pongrac, S. Rensch, S. Reuscher, M. Pečovnik, P. Vavpetič, P. Pelicon, S. Holzheu, U. Krämer, S. Clemens, P1-0212 and P1-0034] and project funding [J4-3091]) and the Infrastructural Centre Microscopy of Biological Samples.. New Phytologist. 2016, doi: 10.1111/nph.13987





Aleksander Kravos, Martina Mileva, and Helena Prosen University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia (aleksander.kravos@fkkt.uni-lj.si)



Simplified and fast analysis of selected pharmaceuticals with



dispersive micro solid-phase extraction (DMSPE)





40 mL sample 30 min ultrasound 30 min twitching 30 min fast shaking



1.2 mL 2-propanol bath Centrifugation



Decantation

67 mg C18

33 mg SAX Binding of the analytes onto freely dispersed sorbent in the sample and their desorption afterwards.

pH 4.2



Unveiling practical aspects of DMSPE-HPLC-UV for analysis of 4 selected pharmaceuticals



Moxifloxacin (MOX) Rosuvastatin (ROS) Simvastatin (SIM) Telmisartan (TEL)



RESULTS



1) Sorbent screening 2) Sorbent load 3) Time



250 80 120 Ext/Twi/Des (min) MOX , ROS , SIM , TEL MOX , ROS , SIM , TEL

70 30/30/15

200 30/15/30 100

60

Mix C18/strong anion 30/15/15



(%) 80 C18, hydra C18, or C8 exchanger (SAX) 50 15/15/30 ies 150 (%) (%) y ver SDB - Styrene-y 15/30/15 40 60 divinylbenzene ver ver reco 15/15/15 f o 100 reco 30 reco 15/30/30 m 40

su CRS - Chromasorb 30/30/30 20

50 W-AW DCMS 20

10



0 0 0 27+13 40+20 53+27 67+33 MOX ROS SIM TEL

sorbent load XX mg C18 + YY mg SAX

sorbent mixture (m = 60 mg), pH



The C18 and C8 sorbents (pH 7) provided the higest The higher the sorbent mass, the higher Prolonged extraction (30 min), twitching (30 min), and recoveries. However, addition of SAX and creation of mixture the recoveries. This is especially true for desorption (30 min) times were the most beneficary for C18/SAX even increased efficiency, especially at pH 4. very polar MOX. extraction efficiency. However, in general no significant

effects were observed.



4) Desorption solvent 5) Method performance



140 120 180 methanol ultrapure water

120 methanol/formic acid 160 seawater

methanol/acetonitrile 100 river water 140

100 acetonitrile/methanol/formic acid ME (seawater)

(%) acetonitrile 80 120 ME (river water) (%) 80 y acetonitrile/water (%) y 100 ect

ver acetonitrile/acetone 60 ver eff 80 60x ri reco acetonitrile/formic acid reco

40 60 mat

40

40

20 20

20

0 0 0

MOX ROS SIM TEL MOX ROS SIM TEL



Optimized method had recoveries between 50 and 80% reaching > 25 preconcentration

Acidic conditions (by adding formic acid) were found the most

factors. Recoveries obtained in seawater/river water were different compared to

important parameter in desorption regardless of the solvent.

ultrapure water suggesting the impact of the ionic strength. Repeatability was < 5%

However, acidic acetonitrile finally provided the best compromise of

RSD. Matrix effects were below 20% except for MOX (the most polar & first-eluting

recoveries compared to slightly less efficient acidic methanol.

analyte) that suffered up to 100% signal enhancement in seawater.



CONCLUSIONS



DMSPE-HPLC-UV is a promising & simple procedure with high multiplexing capacity.



It is applicable for fast extraction & preconcentration of analytes in water analysis. ŠTUDIJA DEGRADACIJE ZGODOVINSKEGA PAPIRJA



Z METODAMI POSPEŠENE RAZGRADNJE



Nik Nikolić*, Jan Ocepek‡, Ida Kraševec, Matija Strlič

Univerza v Ljubljani, Fakulteta za kemijo in kemijsko tehnologijo

*nik.nikolic18@gmail.com, ‡jan.ocepek@gmail.com



Kislinsko katalizirana hidroliza in oksidacija sta glavna procesa razgradnje papirja. Njune posledice lahko vodijo do izgube knjižne in tiskane dediščine. Med razgradnjo papirja se krajšajo polimerne celulozne verige; oksidacijski produkti povzročijo njegovo rumenenje; kisli produkti pa znižujejo pH-vrednost papirja. Spremembe papirja med njegovim staranjem, ki so pri sobnih pogojih nezaznavne, opazujemo z metodo pospešene razgradnje, pri kateri vzorce izpostavimo povečani temperaturi in relativni vlažnosti. Na podlagi ekstrapolacije rezultatov na sobne pogoje lahko proučimo dejavnike, ki pospešujejo oz. zavirajo razgradnjo in napovemo, kako se bodo lastnosti papirja spreminjale in življenjsko dobo papirja.



CILJ RAZISKAVE: Ovrednotenje vpliva temperature, relativne vlažnosti (RH), lignina in načina razgradnje na degradacijo papirja

N

AČI

I N

N



N 30 % RH AL 90 °C 80 °C 18 dni 65 % RH stopmja polimerizacije (DP) SEC 36 dni

IC vsebnost mlečne, etanojske,

N 70 °C 60 °C CIE L*a*b* oksalne kisline STEKLO VENCIO metanojske, sukcinsek &

KO 50 % RH 80 % RH pH

62 dni 183 dni

JIGE STEKLO

KN

JA

ACI vzorec

papir

IMIT



Iz sprememb stopenj polimerizacije (DP) med Pokritim vzorcem se je pH-vrednost med po- MLEČNA KISLINA

pospešeno razgradnje smo izračunali konstan- spešeno razgradnjo znižala bolj kot prostim

te razgradnje pri posameznih pogojih, iz teh pa vzorcem. Hitrosti spreminjanja pH so višje pri 60 °C + 80 % RH 70 °C + 50 % RH 80 °C + 85 % RH 90 °C + 30 % RH

nato razpolovni čas razgradnje. Predpostavili višjih temperaturah pospešene razgradnje.

smo prvi red razgradnih reakcij. Vsebnost večine kislin, ki so v vzorcu že priso-

1,2 tne, na začetku pade zaradi njihovega izhlape- ETANOJSKA

1,0

vanja (metanojska, etanojska kislina) oz. oksi- KISLINA

3 60 °C + 80 % RH 70 °C + 50 % RH 80 °C + 85 % RH 90 °C + 30 % RH dacije (mlečna kislina). Koncentracija oksalne

0,8 kisline narašča med celotno razgradnjo, predvi-

) * 10 0

1/DP 0,6 doma ker je pri pogojih razgradnje končni pro-

delež vzorcev s signifikantno razliko v končni konc. kisline

- t dukt oksidacije. Izhlapevanje/oksidacija je

delež vzorcev z nesignifikantno razliko v končni konc. kisline

/DP 0,4 izrazitejše pri prostih vzorcih (polna črta grafu Med razgradno papirja sta se najbolj spremi-(1

0,2 spodaj), akumulacija pa v pokritih (prekinjena njali barvni koordinati b* in L*. Hitrost rume-

vzorec 993

pHzač = 5,57 črta). nenja smo izračunali kot k = ln(b*)/t in s

0,0 b* razgradnje

0 50 100 150 0,50 60 °C pomočjo MLR ustarili model, ki napove kb*.

št. dni 80 % RH Hitrosti rumenenja pokritih vzorcev so višje

90 °C, 30 % RH 80 °C, 65 % RH 70 °C, 50 % RH 60 °C, 80 % RH vzorec 405

Graf 1 ] pHzač = 5,04 : Vpliv različnih eksperimentalnih pogojev na razgradnjo celuloze v od hitrosti rumenenja prostih vzorcev. k je rja

vzorcu 993. Prosti vzorci so označeni s polno črto, pokriti pa s črtkano pi 0,25 b* pa

odvisna predvsem od temperature, pri pokritih

250 [mg/g vzorcih pa tudi od RH in pri prostih vzorcih od

vzorec 993 lin

0,00 vsebnosti lignina.

pHzač = 5,57 1,50 0 50 100 150 203,9

200 nost kis 0,018 eb

vs 1,00

ni 150 130,8 d ]

št. 113,6 0,50 0,014

[ 105,0

0,5 t 100

0,00

0 50 100 150



50 36,3 mlečna kislina etanojska kislina oksalna kislina ani 27,2 24,8 Graf 3: Koncentracija kislin v vzorcu 405 med njegovo razgradnjo št. dni b* 0,010 k 16,2 ed PROSTI VZORCI

0 Končne ov 1 napovedni model: k b* = 0,0928 – 31,2 ×[K-1] 0,006 koncentracije merjenih kislin v pokritih T nap + 6,10 × 10 -6 × lig [mg g -1 ]

90 °C, 30 % RH 80 °C, 65 % RH 70 °C, 50 % RH 60 °C, 80 % RH 2 prilagojeni R = 0,8125 vzorcih smo primerjali s Studentovim testom.

prosti POKRITI VZORCI pokriti Razlika med koncentracijo etanojske kisline v

0,002 = 0,0232 napovedni model: kb* 1 -1

– 80,5 × [K ]

Graf 2: Primerjava t T 0,5 -4 + 1,32 med pokritimi in prostimi vzorci pri različnih pokritih in prostih vzorcih je signifikantna pri

× 10 × RH [%]

pogojih 2 prilagojeni R = 0,8383



Pri vseh pogojih pospešene razgradnje je de- večini vzorcev; za mlečno kislino je razlika v 0,000 0,004 0,008 0,012 0,016 0,020 0,024-0,002 0,5 gradacijski razpolovni čas t manjši pri pokri-koncentraciji signifikantna pri nižjih tempera-izmerjeni k b*

tih vzorcih, kar pomeni, da tam razgradnja po- turah. Pri ostalih kislinah razlike med končnimi Graf 4: Primerjava napovedanih in izmerjenih hitrosti razgradnje V nadaljnjih raziskavah bomo poskusili merjene

teka hitreje. Razgradnja je najhitrejša pri 90 °C, koncentracijami v prostih in pokritih vzorcev lastnosti povezati z vsebnostjo lignina.

30 % RH, najpočasnejša pa pri 60 °C, 80 % RH. niso statistično pomembne.



Delo je bilo opravljeno v okviru projekta J4-3085, ki ga financira javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost RS (ARIS). Več podrobnost o projektu dobite na spletni



strani: https://hslab.fkkt.uni-lj.si/2021/10/04/effects-of-lignin-degradation-on-paper-based-materials-in-extreme-conditions/.





Preučevanje interakcije PD-1/PD-L1 v



mikrookolju glioblastoma



Pia Mencin1,2, Metka Novak2, Barbara Breznik2



1Fakulteta za kemijo in kemijsko tehnologijo, Univerza v Ljubljani



2Nacionalni inštitut za biologijo



UVOD

§ Glioblastom je najpogostejši primarni maligni tumor osrednjega živčnega sistema, ki velja za neozdravljivega s trenutnimi

terapevtskimi pristopi [1].



§ Tumorsko mikrookolje glioblastoma je interheterogeno (med bolniki) in intraheterogeno (znotraj posameznega tumorja bolnika),

kar otežuje razvoj univerzalno učinkovite terapije [2].



§ Potencialne terapevtske tarče so imunske kontrolne točke, kot je tudi inhibitorni receptorski par: receptor programirane celične

smrti 1/ligand programirane celične smrti 1 (PD-1/PD-L1) [3].



§ Zaradi raznolikosti tumorjev med bolniki uporaba protiteles proti PD-1 in PD-L1 za zdravljenje številnih vrst raka ni uspešna pri

vseh bolnikih. Zato je ključnega pomena identiCicirati bolnike, pri katerih bi bila ta terapija učinkovita.



METODE REZULTATI



Trenutne študije kot potencialen indikator za uspešnost imunoterapije proti PD-1 in/ali PD-L1 predlagajo samo interakcijo PD-1/PD-L1. Za določitev interakcije smo izbrali metodo test ligacijske bližine (PLA), ki omogoča in situ vizualizacijo in kvantiCikacijo proteinskih interakcij na molekularnem nivoju. Uporabili smo komplet reagentov Naveni PD1/PD-L1 Atto647N, proizvajalca

Navinci. ikln

Bo



1. 2. 3. 4.





Zgornja slika prikazuje glavne stopnje metode PLA: 1. inkubacijo s primarnimi la protitelesi (Ab) proti tarčnima epitopoma, 2. inkubacijo z naveni Ab ront (sekundarna protitelesa konjugirana z enoverižnim oligonukleotidom), 3. Ko nastanek krožne DNA iz plus in minus oligov naveni Ab in povezovalnih oligov, 4. podvojevanje in detekcija.





ZAKLJUČEK





V sklopu diplomske naloge smo potrdili prisotnost interakcije PD-1/PD-L1 Prisotnost interakcij PD-1/PD-L1 na tkivni

v tumorskih biopsijah in organoidih glioblastoma ter primerljivo število rezini biopsije glioblastoma bolnika (zgoraj)

interakcij PD-1/PD-L1 v tumorskih biopsijah in organoidih glioblastoma. Pa in pripadajoča negativna kontrola (spodaj).

vendar na podlagi pridobljenih rezultatov ne moremo narediti zagotovih Na vzorcih je bil izveden PLA, inkubacija s

sklepov, saj je eksperimentalno delo zajemalo premajhno število vzorcev in Cluorescenčno označenimi protitelesi proti

so bili signali negativnih kontrol razmeroma visoki. Kljub temu rezultati označevalcu levkocitov CD45 ter barvanje

predstavljajo obetaven začetek identiCikacije metod za določanje jeder s Hoechst. Z belo puščico so označene

primernosti terapij proti PD-1 in PD-L1. Uporaba metode PLA bi bila lahko zaznane interakcije PD-1/PD-L1. Vzorci so

aplikativna tudi pri zdravljenju ostalih heterogenih tumorjev, ne le bili opazovani s Cluorescentnim invertnim

glioblastoma. mikroskopom pri 200× povečavi, merilo na

slikah je 125 μm.



REFERENCE

[1] Q. T. Ostrom, M. Price, C. Neff, G. Ciof5i, K. A. Waite, C. Kruchko, J. S. Barnholtz-Sloan: CBTRUS Statistical Report: Primary Brain and KONTAKT Other Central Nervous System Tumors Diagnosed in the United States in 2016—2020. Neuro Oncol 2023, 25, iv1–iv99. [2] piamencin@gmail.com S. DeCordova, A. Shastri, A. G. Tsolaki, H. Yasmin, L. Klein, S. K. Singh, U. Kishore: Molecular Heterogeneity and Immunosuppressive Microenvironment in Glioblastoma. Front Immunol 2020, 11, 1402.

[3] T. Yang, Z. Kong, W. Ma: PD-1/PD-L1 immune checkpoint inhibitors in glioblastoma: clinical studies, challenges and potential. Hum Vaccin Immunother 2021, 17, 546.

.



Optimization of analytical methods for photodegradation products



of PAH and phthalate esters adsorbed on microplastics



Katarina Čubej1*, Samo Bordon1, Jena Jamšek2, Oliver Bajt2, Helena Prosen1

1 Faculty of Chemistry and Chemical technology, University of Ljubljana, Ljubljana, Slovenia

2 National Institute of Biology – Marine Biology Station Piran, Piran, Slovenia

* Corresponding author: katarina.cubej@gmail.com



Microplastics (MPs) are particles smaller than 5 mm in diameter and present a global environmental pollution problem. MPs serve as

vectors for organic pollutants and therefore modify their photodegradation pathways [1]-[3].

The aim of this research was the optimization of analytical methods for photodegradation products adsorbed on MPs for two groups of

important environmental pollutants: phthalate esters and polycyclic aromatic hydrocarbons.



HPLC method Solid Phase Exctraction



Standard solutions used Optimised for both groups separately

PHTHALATES Final procedure:

phthalic acid (a) - Column:

monomethyl phthalate (b) phthalates: HLB



dimethylphthalate (d) PAH: LC-8

monobutylphthalate (g) - pH value of the sample:

dibutyl phthalate (k) original

PAH - Ion strength of the sample:

2,7-naphthalenediol (c) 38 ‰ NaCl (sea salinity)

acenaphtenequinone (e) increases SPE yield*

2-naphtol (f) - Washing step:

naphthalene (h) 5 mL MQ

acenaphtene (i) - Elution solvent:

phenanthrene (j) phthalates: 2 mL MeOH

fluoranthene (l) PAH: 2 mL ACN

HPLC-DAD chromatogram of mix of analytes with concentration *did not use it on model samples to avoid formation of adducts 10 mg/L in 10 % ACN in MQ at 270 nm



LC-MS/MS method and TOF MS analysis



The complete optimised method was applied to model aqueous samples. Identities of peaks were proposed based on fragmentation patterns and

confirmed with additional high-resolution MS analysis.



• Electrooxidation of dibutyl phthalate



ESI+





ESI-





• Photodegradation of PAH adsorbed on MP‘s particles





CONCLUSION



With the optimised method, some structures of degradation products of dibutyl phthalate and PAH were successfully determined. Reference



[1] Y. Yu, W. Y. Mo, T. Luukkonen: Adsorption Behaviour and Interaction of Organic Micropollutants with Nano and Microplastics – A Review. Sci. Total Environ. 2021, 797, 149140. [2] L. Fu, J. Li, G. Wang, Y. Luan, W. Dai: Adsorption Behavior of Organic Pollutants on Microplastics.Ecotoxicol. Environ. Saf. 2021, 217, 112207. [3] J. Huang, P. Duan, L. Tong, W. Zhang: Influence of Polystyrene Microplastics on the Volatilization, Photodegradation and Photoinduced Toxicity of Anthracene and Pyrene in Freshwater and Artificial Seawater. Sci. Total Environ. 2022, 819, 152049.





VPLIV OZNAKE FLAG NA FAZNO


SEPARACIJO TEKOČE-TEKOČE

Avtor: Žiga Koren Ključne besede: fazna separacija tekoče-tekoče, oznaka

Mentor: doc. dr. San Hadži FLAG, FPLC, UV-Vis, kloniranje s sestavljanjem in vivo



Uvod



Fazna separacija tekoče-tekoče (LLPS) je način fazne separacije, v katerem raztopine makromolekul, na primer proteinov ali nukleinskih kislin, kondenzirajo v bolj gosto tekoče fazno stanje, podobno kapljam. Bolj kondenzirana faza soobstaja z redkejšo fazo. Do LLPS pride zaradi intramolekularnih sil, kot so kation anion, dipol-dipol, π-π in kation-π interakcije. Nastanek kondenzatov je odvisen od koncentracije makromolekule ter od zunanjih dejavnikov. Odvisno od teh dveh parametrov se makromolekula lahko nahaja v eni ali v dveh fazah. LLPS je v celicah ključen za tvorbo brezmembranskih organelov, pospeševanje biokemijskih reakcij in za varovanje celic pred variacijami v koncentracijah makromolekul, saj so koncentracije znotraj faz konstantne, menjuje se le volumski delež med njimi.





Namen Metode

Tvorba kondenzatov z LLPS lahko Pripravili smo konstrukte preiskovanega vpliva na fizikalne lastnosti in na proteina (nanotelesa 55) z 0x, 1x, 2x in 3x funkcijo proteina. Sintetični epitop oznako FLAG z uporabo kloniranja s FLAG je pogosto uporabljen fuzijski sestavljanjem in vivo (IVA). Proteine smo partner. Zanimalo nas je, če dodatek izolirali s kovinsko-kelatno imobilizirano oznake FLAG vzpodbudi tvorbo LLPS, kromatografijo (IMAC), pojavu smo sledili z saj lahko fenomen predstavlja UV-Vis spektrofotometrom pri 550 nm s



potencialen vpliv na druge raziskave. hlajenjem s hitrostjo 0,1 °C/min.



Rezultati in diskusija

Rezultate izolacije rekombinantnih proteinov, pridobljenih s kloniranjem IVA, smo preverili s poliakrilamidno gelsko elektroforezo z natrijevim dodecil sulfatom. Proteini so se med seboj razlikovali v zapisu za 1x FLAG (DYKDDDDK), pričakovali smo razlike okoli 1 kDa, kar se tudi sklada s pridobljenimi rezultati. Ob hlajenju smo pojav LLPS zaznali kot porast absorbance, do katerega pride zaradi sipanja svetlobe po pojavu goste faze. Vzorec 3x FLAG je tvoril kondenzate pri temperaturi 13 °C, vzorec 2x FLAG pa pri 8 °C. Pri vzorcih 1x FLAG in 0x FLAG do pojava ni prišlo, kar ponazarja pomembnost prisotnosti intramolekularnih sil, ki jih omogoča oznaka FLAG, za tvorbo kondenzatov. Pojav smo spremljali tudi pri naraščajoči koncentraciji NaCl, pri čemer smo ugotovili, da nizke koncentracije soli zavirajo, višje pa popolnoma zatrejo LLPS.



Z eksperimenti smo pokazali, da prisotnost oznake FLAG poveča nagnjenost proteina k temu, da tvori biomolekularne kondenzate. Kondenzati vplivajo na različne biokoemijske lastnosti makromolekul, ki jih tvorijo. Lokalno koncentrirani encimi hitreje vršijo biokemijske reakcije kot tisti, ki se ne nahajajo v kondenzatih, saj so kondenzati dinamične strukture, ki omogočajo hitrejšo izmenjavo snovi. Proteini v kondenzatih lahko tudi zavirajo biokemijske reakcije, saj služijo kot fizične ovire, ali pa se zaradi LLPS narobe zvijejo, kar ovira njihovo funkcijo. Glede na uporabnost oznake FLAG pri detekciji in izolaciji proteinov, je pomembno, da se pred raziskavami, posebej pri raziskavah kinetičnih lastnosti encimov, preveri, če do pojava pride, saj lahko zaradi LLPS pride do neskladanja med rezultati in vitro in med dogajanjem in vivo.





Zaključek Reference



Uspešno smo pripravili in izrazili proteinske produkte in potrdili povezavo Z. Gao, W. Zhang, R. Chang, S. Zhang, G. Yang, G. Zhao: Liquid-Liquid



med številom ponovitev oznake FLAG in med tvorbo LLPS. V prihodnjih Phase Separation: Unraveling the Enigma of Biomolecular Condensates in

raziskavah bi bilo dobro pripraviti samostojen polipeptid FLAG in S. Alberti, A. Gladfelter, T. Mittag: Considerations and Challenges in Microbial Cells. Front. Microbiol. 2021, 12, 751880.

preveriti, če tvori LLPS. Sam pojav je viden na makroskopski ravni, kar v Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.

prihodnje omogoča tudi slikanje kondenzatov pod mikroskopom. Cell 2019, 176, 419– 434. Ugotovili smo tudi, da prisotnost soli zavira pojav, kar bi lahko W. Li, C. Jiang, E. Zhang: Advances in the phase separation-organized

membraneless organelles in cells: a narrative review. Transl Cancer Res

pripomoglo k preprečevanju pojava pri raziskavah, kjer LLPS bistveno TCR 2021, 10, 4929–4946. spremeni lastnosti obravnavanega proteina.

Modulating inflammation, oxidative stress and urothelial barrier



dysfunction: The role of taurine in an in vitro model of interstitial



cystitis



Tadeja Kuret1, Janja Bohte2, Peter Veranič1*

1 2 Institute of Cell Biology, Faculty of Medicine, University of Ljubljana Faculty of Chemistry and Chemical Technology, University of Ljubljana



INTRODUCTION & AIM RESULTS



Interstitial cystitis is a chronic inflammatory disease of the urinary bladder with Taurine downregulates oxidative stress-induced inflammatory mediators

no long-term effective treatment available to date.

The exact etiology and pathobiology of the disease remain unknown, however, Taurine significantly downregulated the mRNA expression of the inflammatory

disturbed assembly of urothelial cell tight junctions, increased urine-blood cytokine IL-6, and the chemokines CXCL-1, and CXCL-10, which were upregulated

barrier permeability, inflammation and oxidative stress have been proposed to by stimulation with GO. No significant effect of taurine was observed on the mRNA

play crucial roles. expression of IL-1β and TNF-α. The protein levels of IL-6 and IL-8 released in the

Agents that can simultaneously modulate all processes associated with supernatants of urothelial cells incubated with taurine were also significantly lower

interstitial cystitis will be of paramount importance for future therapy. compared to cells stimulated with GO. One such example is taurine (2-aminoethanesulfonic acid), the most abundant free amino acid in human. Taurine has already been shown to reduce inflammation as well as oxidative stress and improve integrity of various epithelial tissues.

Our aim was to look into the effects of taurine on inflammation, oxidative stress and blood-urine barrier function of urothelial cells in an in vitro model of interstitial cystitis.



METHODS



The in vitro model consisted of normal human

urothelial cell line SV-HUC1, stimulated with

glucose oxidase (GO; 20 mU/ml), which mimics GO (20 mU/ml) taurine (2mM)

prolonged low levels of oxidative stress. Cells



were preincubated with taurine (2 mM) for 2h Substrate qPCR



and then stimulated with/without GO for 24h. SV-HUC-1 ELISA



Untreated cells served as control. Cells were



lysed for RNA isolation and subsequent qPCR Immunofluorescence



analysis while protein levels were determined Effects of taurine on inflammatory mediators in GO-stimulated cells. a) mRNA by ELISA and immunoflourescence (IF). expression of inflammatory cytokines and chemokines, obtained by the qPCR method,



normalized to the endogenous controls ACTB and GAPDH. b) protein levels of IL-6 and IL-8, determined by ELISA. Shown are means ± SD for each group.



*p<0.05; **p<0.01; ***p< 0.001.



CONCLUSION



Taurine reverses the assembly of intercellular contacts disrupted by



Our findings suggest that taurine has the potential to mitigate inflammation, oxidative stress oxidative stress as well as maintain the integrity of the urothelial barrier, all of which are implicated in the development and progression of interstitial Taurine significantly increased the mRNA expression of E-cadherin, encoding for



cystitis. adherent junction protein and zonula occludens-1 (ZO-1), encoding tight junction



protein, which were downregulated in the presence of GO. This was subsequently confirmed also on protein levels showing altered assembly of adherens and tight junctions in the presence of GO, reversed by the addition of taurine.

Taurine upregulates catalase



Taurine pregulated the mRNA expression of the antioxidant enzyme catalase (CAT), downregulated by stimulation with GO. However, no influence of taurine on the expression of the redox sensitive transcription factor NRF-2 and the antioxidant enzymes SOD-2 and HMOX-1 were observed.





Effects of taurine on intercellular junctions in GO-stimulated cells. a) mRNA expression of CDH-1 and ZO-1, obtained by the qPCR method, normalized to the endogenous controls ACTB and GAPDH. Shown are means±SD for each group. *p<0.05; **p<0.01;



Effects of taurine on NRF2-activation pathway and antioxidant enzymes in GO- ****p<0.0001. b) Representative images showing localization and distribution of E-



stimulated cells, determined by the qPCR method, normalized to the endogenous cadherin (green) and ZO-1 (red). Nuclei are stained blue. Images were taken at the 20x

controls ACTB and GAPDH. Shown are means ± SD for each group. and 63x magnification. Scale bars: 20 µm and 5 µm. **p<0.01; ***p<0.001.

Created with BioRender Poster Builder



pri inhibiciji invazije 300.000 AVTOR: Ula Mikoš novih primerov 5 rakavih Nepričakovan preobrat celic! % na leto na svetu petletno preživetje



Namen Metode



Glioblastom je primarni možganski tumor, ki je eno najbolj • Gojenje celic NIB140 (glioblastomska celična linija agresivnih in smrtonosnih malignih obolenj. Njegova agresivnost vzpostavljena na NIB). se kaže v hitri difuzni invaziji, ki vodi do visoke stopnje ponovitve • MTT test celične viabilnosti (koncentracije 1 µg/mL, 5 µg/mL, in na koncu do slabe prognoze. 10 µg/mL in 20 µg/mL protitelesa proti CD155 (IgCD155) in CD155 (imenovan tudi kot PVR ali Necl-5) se v rakavih celicah mišjega kontrolnega protitelesa IgG izotipa 1 (mIgG1)). S tem izraža v povečani meri, kar je povezano z agresivnostjo tumorja. testom smo želeli ugotoviti ali ima protitelo toksičen učinek na Zaradi njegove vloge pri regulaciji celične adhezije in migracije je celice glioblastoma. CD155 potencialna tarča za zdravljenje glioblastoma. • 3D test invazije sferoidov v Matrigelu (tretiranje s Namen diplomskega dela je bil raziskati vlogo proteina CD155 koncentracijo 10 µg/mL). S tem testom smo simulirali invazijo pri rasti in invaziji celic glioblastoma. Zanimalo nas je, ali lahko z celic glioblastoma v 3D prostoru. Opazovali smo, kako uporabo protitelesa proti CD155 vplivamo na invazivnost celic protitelo vpliva na sposobnost celic, da se širijo v umetni glioblastoma in s tem potencialno izboljšamo možnost za zunajcelični matriks (Matrigel). zdravljenje. • Analiza invazije s programom ImageJ.



REZULTATI



IgCD155 ne vpliva na viabilnosti IgCD155 ne vpliva na invazijo celic NIB140 mIgG1 zmanjša invazijo za 30 – 40 % celic NIB140



Slika 1: Viabilnost celic NIB140 48 h po tretiranju z različnimi koncentracijami IgCD155 in Slika 3: Invazivna razdalja celic sferoidov NIB140 96 h po tretmaju z IgCD155 in mIgG1. mIgG1. Prikazane so povprečne viabilnosti in standardni odklon. Kontrolo predstavljajo celice v Prikazane so povprečne invazivne razdalje in standardni odklon. Kontrolo predstavljajo celice v dopolnjenem gojišču, brez dodatka protiteles. dopolnjenem gojišču, brez dodatka protiteles. Legenda: *** = p < 0,001 in **** = p < 0,0001.



Slika 2: Primerjava invazije celic sferoida NIB140 v času inkubacije brez dodatka protiteles. Slika 4: Invazija celic sferoida NIB140 96 h po tretmaju. A – kontrola brez protitelesa, A –sferoid v začetnem stanju, B – sferoid po 24 h inkubacije, C – sferoid po 96 h inkubacije. B – z dodatkom IgCD155, C – z dodatkom mIgG1.



Razprava



Protitelo proti CD155 ni vplivalo na invazijo celic glioblastoma, vendar ne moremo z gotovostjo trditi, da IgCD155 ne inhibira invazije celic glioblastoma, saj bi poskus morali ponoviti na večjem številu glioblastomskih celičnih linij, prav tako bi morali testirati še druge koncentracije protitelesa. Za najbolj realno sliko, kako protitelo vpliva na tumor, pa bi morali uporabiti celične modele kot so glioblastomski organoidi. Mišje kontrolno protitelo IgG izotipa 1 je zmanjšalo invazijo celic glioblastoma, kar je verjetno posledica njegove vezave na Fc receptorje in ne neposrednega toksičnega učinka protitelesa, saj smo uporabili dokazano netoksično koncentracijo protitelesa za našo celično linijo. V pregledani literaturi nismo zasledili, da bi o vplivu mIgG1 na invazijo že kdo poročal, zato je potreben podrobnejši vpogled v delovanje tega protitelesa na invazijo celic glioblastoma, predno lahko predpostavimo njegovo klinično uporabnost.



Vpliv vezave protitelesa proti CD155 na invazijo celic glioblastoma



avtor: Ula Mikoš, soavtor: dr. Metka Novak, mentor: dr. Barbara Breznik



kontakt: Barbara.Breznik@nib.si





Impact of Substrate on Optical Properties of




1-D Photonic Crystals for White LEDs



Martin Jazbec,1,2 Prasenjit P Sukul, 2 Luís F. Santos, 2 Rui M. Almeida 2



1 Faculty of Chemistry and Chemical Technology, University of Ljubljana,

Večna pot 113, 1000 Ljubljana, Slovenia

2 Instituto Superior Técnico, University of Lisboa, Av. Rovisco Pais 1,

1049-001 Lisboa, Portugal



INTRODUCTION

RESULTS

White light generation (WLG) was achieved by using 1D

photonic crystal structures (Bragg mirrors (BM) and ➢ Calibration curves presenting layer thickness as a



Fabry-Pérot function of ethanol volume microcavities (MC)) on different

substrates. Samples with tunable light emission

spectra were created by exposing them to different

laser light angles, optimizing emission characteristics.



Figure 4: Aluminosilicate sol, layer Figure 5: Titania sol, layer thickness

thickness calibration calibration



➢ Reflectivity measurements (on SiO substrate): 2

• Lower angle → red emission spectra

Figure 1: SEM image of MC structure • Higher angle → blue emission spectra





MATERIALS & METHODS

➢ Sol-gel process:

▪ High refractive index solution = Titania sol

▪ Low refractive index solution = Aluminosilicate sol with

0.3/0.5/5.0 mol. % of (Tm3+ 3+ 3+ /Er /Yb) doping materials

➢ Substrates:

▪ p-type Silicon (100) wafer, SiO2 (vitreous silica)

wafer, Gallium arsenide (GaAs)

▪ Figure 6: Reflectivity measurements of Figure 7: Reflectivity measurements of Substrate material significantly impacted the

BM structure on SiO2 substrate MC structure on SiO2 substrate

adhesion and optical properties of the deposited

layers, affecting light emission spectra.



➢ Spin Coating was used for the deposition of thin layers



➢ Characterization techniques:



▪ Ellipsometry, FTIR, SEM, Photoluminescence (PL),

Reflectivity





Figure 8: PL spectra of BM and MC Figure 9: PL spectra of BM and MC



structures on SiO2 substrate structures on Si substrate



Figure 2: Substrates before deposition; from left to right: CONCLUSION

a) SiO2 wafer, b) Silicon wafer, c) GaAs, d) MC deposition on Silicon substrate 2 SiO substrate demonstrated superior performance among

all three studied substrates, providing the most intense photoluminescence (PL) spectra and peak values at reflectivity measurements within the white light range. Lower angles of laser exposure resulted in red-shifted emission spectra, while higher angles produced more blue-shifted emissions. GaAs was excluded from further research due to poor thermal stability and reactivity with deposited layers during heat treatment.



Figure 3: Substrates after deposition; from left to right:



a) Deposited samples on different substrates, b) MC deposition on Silicon substrate,



c) BM deposition on Silica substrate Izražanje γ-enolaze in njeno uravnavanje s katepsinom X v





poškodovanih dopaminergičnih nevroblastomskih celicah SH-SY5Y





Lora Gržin, mag. farm. pod vodstvom mentorice izr. prof. dr. Anje Pišlar,

mag. farm., in somentorice asist. Selene Horvat, mag. farm.



NEVRODEGENERACIJA PRI NEVROTROFIČNA IN VITRO PROUČEVANJE

PARKINSONOVI BOLEZNI PODPORA NEVRODEGENERACIJE





NAMEN DELA METODE





1. Vzpostavitev modela diferenciacije celic SH-SY5Y



2. Vzpostavitev modela nevrodegeneracije dopaminergičnega



podtipa



3. Vrednotenje izražanja γ-enolaze in njene ko-lokalizacije s

katepsinom X

4. Vrednotenje zaščitnega vpliva zaviralca katepsina X AMS36



1. VZPOSTAVITEV MODELA 2. VREDNOTENJE IZRAŽANJA 3. VREDNOTENJE

DIFERENCIACIJE IN γ-ENOLAZE IN NJENE ZAŠČITNEGA VPLIVA

NEVRODEGENERACIJE KO- LOKALIZACIJE S ZAVIRALCA KATEPSINA

SH-SY5Y KATEPSINOM X X AMS36





SKLEPI





1. Celice SH-SY5Y lahko diferenciramo v dopaminergično podvrsto z dodatkom RA v kombinaciji s PMA, kar se



odraža v povišanem izražanju dopaminergičnih označevalcev TH in FOLR1 ter v podaljšanju celičnih izrastkov.

2. Dodatek 6-OHDA poveča delež mrtvih nediferenciranih in diferenciranih celic SH-SY5Y. Za in vitro celični model

Parkinsonove bolezni je najprimernejša 24-urna izpostavitev 6-OHDA s končno koncentracijo 50 ali 100 µM.

3. Raven izražanja aktivne oblike γ-enolaze je višja pri diferenciranih celicah, raven izražanja celokupne γ-enolaze pa je

podobna pri nediferenciranih in diferenciranih celicah. Opazna je ko-lokalizacija γ-enolaze s katepsinom X, ki je izrazitejša v diferenciranih celicah, medtem ko izpostavitev celic 6-OHDA vpliva na zmanjšano ko-lokalizacijo.

4. Aktivnost katepsina X je višja v diferenciranih celicah, pri čemer je najvišja v celicah, izpostavljenih RA in PMA.

5. Dodatek zaviralca katepsina X AMS36 ima zaščitni učinek na nevrotoksične učinke 6-OHDA. Zaviranje katepsina X v

celicah, poškodovanih s 6-OHDA, poviša raven izražanja aktivne oblike γ-enolaze.

Sheme so bile pripravljene v programu BioRender. Prva shema je prirejena po Dovonou A, Bolduc C, Soto Linan V, Gora C, Peralta Iii MR, Lévesque M. Animal models of Parkinson’s disease: bridging the gap between disease hallmarks and research questions. Transl

Neurodegener. 19. julij 2023;12(1):36. in BioRender (2021). Intrinsic Circuitry and Outputs of the Basal Ganglia. https://app.biorender.com/biorender-templates/figures/all/t-605e2f6959e85b00a6c651b7-intrinsic-circuitry-and-outputs-of-the-basal-ganglia

Created with BioRender Poster Builder

Triazolijeve soli kot napredni ligandi:



Sinteza in njihova uporabnost



POVZETEK: Sintetizirali smo 3,4-difenil-5-metil-1-(p-tolil)-1H-1,2,3-triazolijev triflat in 3,4-



difenil-5-etil-1-(p-tolil)-1H-1,2,3-triazolijev triflat. Sintezo smo začeli s 4-aminotoluenom 1, ki



smo ga pretvorili v 4-azidotoluen 2. Med njim in fenilacetilenom 3 smo izvedli z bakrom



katalizirano azid-alkin cikloadicijo (CuAAC) in dobili 1,4-disubstituiran-1,2,3-triazol 4. Tega smo



reagirali z jodonijevo soljo 5 v prisotnosti bakrovega sulfata in dobili 1,3,4-trisubstituirano 1,2,3-



triazolijevo sol 6. V zadnji stopnji smo na mesto 5, v prisotnosti reagentov n-butillitija in



ustreznega alkil jodida vezali metilno ali etilno skupino ter dobili produkta 7. Ta sta uporabljena



kot 1 13 liganda, ki ob vezavi kovinskega iona delujeta kot katalizatorja. Karakteritirali smo ju z H, C



NMR in IR spektroskopijo ter masno spektrometrijo visoke ločljivosti.



Metodologija Uporaba triazolijevih soli



Tvorba 1,2,3-triazola z CuAAC: Triazolijeve soli služijo kot prekurzorji za



sintezo N-heterocikličnih olefinov (mNHO).

o spada med “klik reakcije”, za katere je



bila leta 2022 podeljena Nobelova



nagrada;



o preprosta in hitra izvedba brez topila, v



prisotnosti katalizatorja Cu(PPh3)3Br, z



enostavnim čiščenjem (prekristalizacija); Uporabni so:



o kot katalizatorji za reakcije

o povprečen izkoristek petih ponovitev je



91 % hidroboriranja in N-metiliranja .

primarnih aminov;



Čiščenje produktov: o za reakcije z Lewisovimi kislinami,



o ekstrakcija; kisikom in aril azidi;



o prekristalizacija; o za dostop do diazoolefinov in njihovih



o kolonska kromatografija; usteznih bakrovih kompleksov.

o filtracija skozi čep silikagela.





Anastazija Rakar anastazija.rakar@gmail.com





Mentor prof. dr. Janez Košmrlj Celotno diplomsko delo: Pretvorbe elektronsko bogatih aromatov Skeniraj Skeniraj me! me!





pri alternativnih pogojih Zoja Žnidarič

Mentor: prof. dr. Marjan Jereb





Diplomsko delo Diplomsko delo zoja.znidaric@gmail.com





POVZETEK REZULTATI



Tabela 2: Pregled produktov



V svojem diplomskem delu sem testirala Strukturna formula Izkoristek [%] pretvorbo elektronsko bogatih aromatov s

karboksilnimi anhidridi in trifluoroocetno 74,93 %

kislino (TFA).

92,02 %

Reakcija je trajnostna alternativa klasični

reakciji Friedel-Craftsovega aciliranja, saj 77,14 %

omogoča sintezo brez uporabe toksičnega



in nevarnega katalizatorja AlCl . 65,55 % 3



TFA deluje kot katalizator in topilo hkrati,

prav tako pa jo lahko po reakciji ponovno 91,29 %

uporabimo in se tako izognemo

nepotrebnemu odpadku. Aciliranje sem 45,05 %



izvajala na spojinah z različno sterično 81,29 %

zahtevnimi funkcionalnimi skupinami.



Raziskovala sem tudi vpliv strukture 2,72 %

elektronsko revnejših oziroma elektronsko



bogatejših aromatov na potek aciliranja 82,56 %

aromatskega obroča.

13,38 %



METODOLOGIJA ZAKLJUČEK

Aciliranje je bilo uspešno na manj sterično zahtevnih

• spojinah. Izkoristki so bili dokaj visoki, med 50 in 90 %. Mešanje na sobni temperaturi



• Za manj uspešno se je izkazalo na spojinah, ki vsebujejo Prekinitev reakcije z dodatkom NaHCO ₃

estrsko funkcionalno skupino. Čeprav lahko estrska

• Ekstrakcija in odstranitev topila skupina, vezana preko kisika, donira elektrone, je



• Analiza z resonančni učinek omejen zaradi elektron-privlačnega H NMR spektroskopijo 1

učinka karbonilne skupine. Tako se posledično zmanjša tudi

• Čiščenje aktiviranost aromata. : prekristalizacija, kolonska

Temeljna literatura: G. Liu, B. Xu: Hydrogen bond donor solvents enabled metal and halogen-

kromatografija free Friedel– Crafts acylations with virtually no waste stream. Tetrahedron Lett. 2018, 59, 869– 872.



• Potrditev strukture s HRMS = število ekvivalentov



in IR spektroskopijo PRVI TIP REAKCIJE



Tabela 1: Primerjava TFA in AlCl 2

3



Lastnost TFA AlCl3

1

Vloga Katalizator in topilo Katalizator Na aromatskih spojinah z acetanhidridom v

prisotnosti TFA



Nevarnost Korozivna, vendar manj nevarni Zelo koroziven, hitro reagira z stranski produkti DRUGI TIP REAKCIJE vlago do HCl



Okoljski vpliv in Možnost recikliranja z zaradi hidrolize; Težko recikliramo

recikliranje 2 destilacijo; manj odpadkov

več odpadkov



Uporaba v sintezi Pogosta v 1,5 Primerna za zeleno kemijo 1 klasičnih sintezah

Na benzojski kislini z 1,3-dimetoksibenzenom v prisotnosti

trifluoroacetanhidrida (TFAA) in TFA





The effect of Staphylococcus capitis growth rate




on the effectiveness of bacteriophage K

Špela Blaznik, Ana Lisac, Aleš Podgornik

Faculty of chemistry and chemical technology, Ljubljana, Slovenia



INTRODUCTION METHOD

Phage parameters are key for optimizing bacteriophage

production and evaluating phage therapy. The adsorption

constant (k a) mL/(CFU·h)] quantifies phage attachment to

bacteria, based on the decline in phage concentration over

time. The latent period (LP) [h] is the time from infection to

lysis, while the burst size (BS) measures viruses released per

cell. These values depend on the host's physiological state,

regulated by adjusting the dilution rate in a chemostat.

Bacteriophage replication, relies on this state, utilizing the

host's metabolism and components [1, 2]. To prevent

bacterial washout, the dilution rate D [h-1] must not exceed

the maximum growth rate µmax [h-1] [3]. Figure 1: A schematic representation of a chemostat,which enables stationary growth

of microorganisms by regulating the inflow of medium.



RESULTS AND DISCUSION





During the exponential growth

phase, S. capitis exhibits a

maximum specific growth rate of

0,6602 h⁻¹ (Figure 2). At lower

dilution rates, bacterial

concentration remains stable

(Figure 3), but as the dilution rate

nears the maximum growth rate, it

decreases, approaching washout

Figure 2: S. capitis growth curve with determination of conditions. Even in suboptimal µ max Figure 3: Physiological state of the bacterial culture at different

dilution rates states, S. capitis remains

susceptible to phage infection,



with phage concentration

increasing up to a dilution rate of

0,288 h⁻¹ (Figure 4). In the absence

of infection, the initial phage

concentration (P₀) would be equal

to the generated phage

concentration (GB). Both bacterial

and phage dynamics remain

relatively stable (Figure 5), despite

Figure 4: Equilibrium concentration of initial phage Figure 5: Eclipse and latent period during phage production changes in the physiological state.

concentration, generated bacteriophages and infected

bacteria Improved bacterial conditions

enhance phage production (BS)



(Figure 6) and increase the



adsorption constant at higher



dilution rates (Figure 7).





CONCLUSION



Improved bacterial physiological



states enhance phage production

and adsorption rates, highlighting

Figure 6: Burst size (calculated in two ways) at different dilution Figure 7: Adsorption constant of bacteriophage K at different the critical role of growth

rates growth rates of S. capitis conditions in optimizing phage

therapy. Future experiments with

REFERENCES intermediate dilution rates

[1] K. Šivec, A. Podgornik: Determination of Bacteriophage Growth Parameters under Cultivating Conditions. Appl Microbiol Biotechnol ⁻ between 0,09 and 0,288 h¹ will be

2020, 104, 8949–8960. necessary to gather a more

[2] D. Nabergoj, N. Kuzmić, B. Drakslar, A. Podgornik: Effect of Dilution Rate on Productivity of Continuous Bacteriophage Production in comprehensive dataset for better

Cellstat. Appl Microbiol Biotechnol 2018, 102, 3649–3661.

[3] P. Žnidarščič Plazl, A. Pavko: Praktikum Iz Biokemijskega Inženirstva. Ljubljana: UL, Fakulteta za kemijo in kemijsko tehnologijo 2005. analysis.





Raziskovanje strukture signalnega kompleksa




FHL2:β-katenin v povezavi z EpCAM



Tina Logonder, Aljaž Gaber

Oddelek za kemijo in biokemijo, Fakulteta za kemijo in kemijsko tehnologijo, Univerza v Ljubljani

Večna pot 113, 1000 Ljubljana, Slovenija



UVOD REZULTATI



Epitelijska celična adhezijska molekula (EpCAM) je transmembranski glikoprotein

pomemben pri morfogenezi epitelija in karcinogenezi epitelijskih celic. Je eden





najpogosteje uporabljenih diagnostičnih označevalcev za karcinome. Po cepitvi in in FHL2:β-katenin nn + tete β-katenin a 2 La EpCAM-a se sprosti topna znotrajcelična domena ( EpIC : aa 289–314), ki tvori -k H-k β FHL2 Fβ signalni kompleks s proteinoma FHL2 in β-katenin v kanonični signalni poti Wnt. To )

U

vodi do izražanja onkogenov in posledično proliferacije rakavih celic (slika 1). Kljub A

m β-katenin

(

terapevtskemu interesu je strukturnih informacij o tem kompleksu malo (slika 2). can

a

rb

Glavna omejitev dosedanjih študij je bila, da je bil FHL2 v prejšnjih raziskavah sob

A

izražen kot fuzijski protein z oznako. Namen dela je strukturna karakterizacija FHL2

signalnega kompleksa FHL2:β-katenin z in brez vezanega proteina EpIC, kar

bi prineslo vpogled v mehanizem karcinogeneze proteina EpCAM in razkrilo nove

tarče za zdravljenje raka.

Retencijski volumen (ml)

Slika 3: Analiza interakcije med proteinoma FHL2 in β‑katenin z velikostno izključitveno

kromatografijo. Levo: kromatogram z vzorci β‑katenin + FHL2, β‑katenin and FHL2. Desno:

analiza frakcij, ki so na kromatogramu označene s sivo barvo, z SDS-PAGE.



[FHL2] = 235 μM, [β‑katenin] = 13 μM





Kd= 930 ± 140 nM



n = 1,82 ± 0,03





Čas (min) Molarno razmerje



Slika 4: ITC meritev titracije FHL2 v β-katenin. Levo: graf odvisnosti diferencialne moči (DP) v



odvisnosti od časa. Desno: graf po integraciji površin vrhov in normalizaciji prikazuje spremembo



Slika 1: Shematski prikaz signalne poti proteina EpCAM, ki se začne z regulirano

entalpije v odvisnosti od molarnega razmerja FHL2/β-katenin.

znotrajmembransko proteolizo (RIP).



PREDHODNE RAZISKAVE

1. Prvič uspešno izražen in očiščen protein FHL2 brez oznake

Interakcija EpIC:FHL2 2. Analiza s SEC (velikostna izključitvena kromatografija): Y2H, Co-IP, SEC: potrjena tvorba stabilnega kompleksa FHL2:β‑katenin (slika 3) • vezava EpIC-a na LIM4 FHL2

(Maetzel, 2009) 3. ITC (izotermna titracijska kalorimetrija) in SLS (statično sipanje svetlobe) :

stehiometrija FHL2:β‑katenin = 2:1

Interakcija FHL2:β-katenin

Y2H, Co-IP: 4. ITC: K v mikromolarnem območju (slika 4) d

• vse štiri domene LIM (razen LIM0) nujne za vezavo na β-katenin

• vezava FHL2 na N-konec β-katenina

(Martin, 2002; Wei, 2003)



NADALJEVANJE RAZISKAVE

EpIC

3,1 kDa

Določitev strukture kompleksa EpIC:FHL2:β‑katenin s

• pristopom integrativne strukturne biologije





FHL2

Strukture PDB

32 kDa

LIM0 LIM4

LIM1 LIM3 LIM2



β-katenin Modeli AlphaFold Cross-Linking Mass Spectrometry Integrative

ang. CLMS IMP

85 kDa Modeling



Platform

N C





Model strukture



signalnega kompleksa



ARM1-12 EpIC:FHL2:β‑katenin



SAXS cryo-EM



Slika 2: Shematski prikaz interakcij znotraj signalnega kompleksa EpIC:FHL2:β-katenin. ang. Small-Angle X-ray Scattering ang. Cryo‐Electron Microscopy





REFERENCE ZAHVALA



1. Gires, O., Pan, M., Schinke, H., Canis, M. & Baeuerle, P. A. Expression and function of epithelial cell adhesion molecule EpCAM: where are we after 40 years? Cancer Metastasis Rev.39, 969–987 (2020). Delo je bilo podprto s strani Javne



2. Habibe, J. J., Clemente-Olivo, M. P. & de Vries, C. J. How (epi)genetic regulation of the LIM-domain protein FHL2 impacts multifactorial disease. Cells 10, 2611 (2021).



3. Shang, S., Hua, F. & Hu, Z.-W. The regulation of β-catenin activity and function in cancer: therapeutic opportunities. Oncotarget8, 33972–33989 (2017). agencije za znanstvenoraziskovalno in



4. Valenta, T., Hausmann, G. & Basler, K. The many faces and functions of β-catenin. EMBO J. 31, 2714–2736 (2012). inovacijsko dejavnost Republike



5. Cao, C. Y., Mok, S. W.-F., Cheng, V. W.-S. & Tsui, S. K.-W. The FHL2 regulation in the transcriptional circuitry of human cancers. Gene. 572, 1–7 (2015).



6. Cai, T. et al. FHL2 promotes tubular epithelial‐to‐mesenchymal transition through modulating β‐catenin signalling. J. Cell. Mol. Med.22, 1684–1695 (2018). Slovenije, raziskovalni program



7. Tretter, J. Innovative therapy modalities for solid EpCAM-positive tumors. (Ludwig-Maximilians-Universität München, 2017). P1‑0140 in raziskovalni projekt



8. Trzpis, M., Bremer, E., McLaughlin, P. M. J., De Leij, L. F. M. H. & Harmsen, M. C. EpCAM in morphogenesis. Frontiers in Bioscience.13, (2008).



9. Maetzel, D. et al. Nuclear signalling by tumour-associated antigen EpCAM. Nat. Cell Biol. 11, 162–171 (2009). Z1‑2637.



10. Martin, B. et al. The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts. J. Cell Biol.159, 113–122 (2002).



11. Wei, Y. et al. Identification of the LIM protein FHL2 as a coactivator of beta-catenin. J. Biol. Chem. 278, 5188–5194 (2003).





In vitro antioxidant activity and total phenolic content of different extracts of Himalayan Balsam



(Impatiens glandulifera Royle)



Marcel Žafrana,b, Ana Gajićc, Lovro Žibernac a , Alen Albreht

aNational Institute of Chemistry, Department of Analytical Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia

bFaculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia

cFaculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, SI-1000 Ljubljana, Slovenia



PURPOSE OF THE RESEARCH

INTRODUCTION

This paper reports preliminary results on the in vitro antioxidant activity of HB

extracts. Given the considerable number of possible different HB extracts, chemical

Himalayan Balsam (HB) is a large annual plant (therophyte) that is classified

characterization was performed to determine the total phenolic content (TPC).

as an invasive alien plant species. It is considered one of the most virulent

plants in Europe and elsewhere in the world, as it is one of the main causes of

biodiversity loss. As a result, alternative uses are being sought [1]. EXPERIMENTAL METHODS

At the molecular level, HB is known to contain several compounds of academic

and industrial interest [2]. It has been suggested that extracts of Impatiens

species could be used as natural sources of antioxidants. Several studies have HB extracts were obtained by maceration with three different solvents (ethanol

indicated that certain HB extracts could have some degree of antioxidant (EtOH), water (H O) and acetone) at three extraction temperatures (room 2

activity, but the main contributing compounds have not been identified in these temperature (RT), 50 °C and 70 °C) where possible. Otherwise, the extracts were

mixtures. It is assumed that phenolic compounds (polyphenols) are at least obtained at RT and under reflux. The extracts were prepared at a ratio of 1:100 to

partially, if not largely, responsible for the observed biological activity [3]. 1:100 000. In vitro antioxidant properties were determined by a combination of

complementary colorimetric assays (DPPH, ABTS and FRAP). The TPC

determination based on the Folin-Ciocalteu reagent was also carried out

spectrophotometrically.



RESULTS



] RT acetone 20,0

[% reflux EtOH H 2 O

16,0

fect acetone (reflux)



ef 12,0 RT 50 °C 70 °C ing acetone (RT) RT 50 °C 70 °C 8,0 eng v

sca 4,0 H2O (70 °C)

H



DPP 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ex 0 0,0 tracts H2O (50 °C)

1 00 000 :1 00 00 :1 00 000 :1 00 000 :1 00 000 :1 00 000 :1 00 000 :1 00 000 ent :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 H2O (RT) 1 :1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0



:1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 :1 :1 :1 :1 :1 :1 :1 er 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 1 1 1 1 1 1 1diff EtOH (70 °C) different extracts



RT acetone EtOH (50 °C)

] reflux EtOH H 2 O

[% 24,0 EtOH (RT)



ect RT 50 °C 70 °C RT 50 °C 70 °C 0 100 200 300 400 500 600 18,0 eff TPC [μg GAE/g extract]

ing

12,0



eng Fig. 2. Determination of the TPC of EtOH, H v2O and acetone extracts, expressed in μg of gallic acid equivalent per g of extract. 6,0 sca

S

0,0



ABT 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 :1 00 000 :1 00 000 :1 00 000 :1 00 000 :1 00 00 :1 00 000 :1 00 000 :1 00 000 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 00 CONCLUSIONS 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 :1 :1 :1 :1 :1 :1 :1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 1 1 1 1 1 1 1

different extracts The results of all three in vitro antioxidant assays

2,0 (Fig. 1) showed weak antioxidant activity of the

EtOH H extracts tested. The presence of TPC (Fig. 2) was 2 O acetone



L] detected in all extracts tested. Their concentrations RT RT 50 °C 70 °C 50 °C 70 °C RT reflux ranged from 231.74 to 504.37 μg GAE/g extract. /m 1,5 g μ Since phenolic compounds are considered potent [ antioxidants, we will perform further in vitro

1,0 measurements of antioxidant properties in the

RAP) future by increasing the amount of plant material or

(F decreasing the amount of extraction solvents used, 0,5

γ AAE as we conclude that the extracts obtained in this case were not sufficiently concentrated. We will also

0,0 perform a more detailed chemical characterization



0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 of the extracts to determine which group of 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 :1 00 00 :1 00 000 :1 00 000 :1 00 000 :1 00 000 :1 00 000 :1 00 000 :1 00 000 compounds contributes the most to their 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 :1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 :1 :1 :1 :1 :1 :1 :1 :1 antioxidant activity. 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 :1 1 1 1 1 1 1 1 1

different extracts

Fig. 1. In vitro antioxidant properties of HB extracts of the whole plant. The abbreviation AAE stands for ascorbic acid equivalent.



REFERENCES



[1] M. Langmaier et al., (2020), A Systematic Review of the Impact of Invasive Alien Plants on Forest Regeneration in European Temperate Forests, Front. Plant Sci., 11, 524969.

[2] K. Helsen et al., (2021), Biological flora of Central Europe: Impatiens glandulifera Royle, Perspect. Plant Ecol. Evol. Syst., 50, 125609.

[3] V. B. Schini-Kerth et al., (2011), Vascular Protection by Natural Product-Derived Polyphenols: In Vitro and In Vivo Evidence, Planta Med., 77, 1161–1167.





Analysis of Adhesive Joint Using Beech Wood





Tjaša Likeb 1 2 1 2 , Martin Capuder*, Tina Skalar, Andreja Pondelak

1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia, tl8013@student.uni-lj.si

2 Slovenian national building and civil engineering institute, Dimičeva ulica 12, 1000 Ljubljana, Slovenia



INTRODUCTION RESULTS



Wood is an anisotropic, porous material with key 20 PUR

features like longitudinal tracheids in softwoods and a] 18 P 16

vessel elements in hardwoods, facilitating resin flow. M 14 [

th 12



Glue is the primary bonding material in wood products, 10 eng 8 with adhesives classified as organic, semi-synthetic, or str 6



synthetic. 4 2 Shear

0

RR RT TT



MATERIAL & METHODS Figure 1: Shear strength results

4000 3000 7_1

7_4

Samples were prepared with two compression times 3500 2500

] 3000 ] 7_6



radial-radial f 2000 f 1500 d d (RR), radial-tangential (RT), and 8 7_10 ar 1500 ar d d n n 1000 7_11 1000 tangential-tangential (TT). Polyurethane glue (PUR) (60 min, 120 min) and three wood ring orientations: rce rce 2o 7_9 o N N [ 2000 7_8 [ 2500

Sta Sta 7_12 500 500

was used. The samples were exposed to UV radiation 7_13 0

-500 0 2000 4000 6000 0 7_14

and moisture for 3 months. Standard travel [mm] 0 500 1000 1500 2000 2500 3000 3500 Standard travel [mm]

The shear strength (EN302) and microstructure (by Figure 2: Tensile-shear test results for the samples before and after aging (1 month)

optical microscope, SEM, EDS, FTIR, XRD, and epi- 7000

fluorescence microscopy) were analyzed. 6000

5000

y

4000 Beech wood t= 0 months

tensit 3000 Beech wood t= 3 months

In

PENETRATION DEPTH (t = 0) 2000 Polyurethane t = 0 months

1000 Polyurethane t = 3 months



Table 1: Adhesive joint thickness and depth of penetration of the adhesive into 0

5 10 15 20 25 30 35 40

the wood microstructure (annual rings orientation: RR, TT) 2θ (°)

RR TT Figure 3: XRD results for beech wood and polyurethane glue analysis before and after aging

60 min 120 min 60 min 120 min



Adhesive joint thickness [µm] 128.2 75.8 97.2 45.6

Penetration into the upper plate s [µm] Penetration into the bottom plate [µm] 339.8 37.9 324.5 85.5

25.8 478.6 77.9 110.2 th

Table 2: Adhesive joint depth of penetration of the adhesive into the wood mon

microstructure by area by plates (annual rings orientation: RR, TT) 0 = t

Orientation A(P1) [%] A(P2) [%]

RR 70,17 29,83



TT 38,72 61,28 s



th



RT on

Adhesive joint thickness 3 [µm] 60 min 120 min A(P1) [%] m

253.9 8.3 / =

Penetration into R orientated plate [µm] 22.9 299.2 84.38 t

Penetration into T orientated plate [µm] 89.6 140.4 15.62

Table 3: Adhesive joint depth of penetration of the adhesive into the wood

Figure 4: SEM image of a sample PUR 120 TT (time of aging: 0 and 3 months,

microstructure by distance and by area by plates (annual rings orientation: RT)

A – polyurethane glue, B – beech wood)



CONCLUSION



This study focuses on the aging process of wood joints, essential for understanding the durability of wooden structures. We investigated how environmental factors like moisture, temperature changes, and mechanical stress affect wood over time. Our results showed that samples compressed for 120 minutes with radial-tangential (RT) orientation had the best penetration. To fully assess aging effects, prolonged exposure is necessary, as both wood and polyurethane glue degrade over time. Identifying weaknesses through this research supports the development of stronger joint designs and the selection of more resilient materials, enhancing the longevity of wooden constructions.





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