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Proteini BPM iz vrste Arabidopsis thaliana primarnu ulogu ostvaruju u putu proteasomalne razgradnje ovisne o ubikvitinu, u sklopu kojeg prepoznaju specifične supstrate za razgradnju. Prethodno je utvrđena interakcija proteina BPM1 s proteinima RDM1 i DMS3, komponentama metilacijskog mehanizma RdDM. Uloga proteina BPM1 u mehanizmu RdDM u ovom je radu ispitana primjenom kromatinske imunoprecipitacije u svrhu identifikacije novih ciljnih gena ovog mehanizma u čijoj regulaciji metilacije posreduje protein BPM1. Identificirani su geni FBW2, RKP i AGL14, a kao referentni odabran je gen CML41. Analizom metilacije ovih gena metodom bisulfitnog sekvenciranja i pirosekvenciranja u linijama s prekomjernom ekspresijom proteina od interesa (oeBPM1, oeDMS3), te linijama s narušenom funkcijom mehanizma RdDM (dms3-1, nrpe1-10), potvrđen je stimulativni učinak proteina BPM1 na aktivnost mehanizma, koji je najizraženiji tijekom embriogeneze, a sam mehanizam RdDM značajniji je za zigotnu nego za somatsku embrigenezu. Ovisno o poziciji, učinci metilacijskih događaja reflektiraju se na ekspresiju svih analiziranih gena. Proteins BPM of Arabidopsis thaliana have a primary role in the ubiquitin-dependent proteasomal degradation pathway, where they recognize specific substrates for degradation. The interaction of BPM1 protein with RDM1 and DMS3, components of RdDM methylation mechanism, has previously been established. Here, the role of BPM1 protein in RdDM was analyzed using chromatin immunoprecipitation for identification of new RdDM targets whose methylation is mediated by BPM1 protein. Genes FBW2, RKP and AGL14 were identified as targets, and CML41 was chosen as a reference gene. Methylation analysis of these genes was conducted using bisulfite sequencing and pyrosequencing in lines with overexpression of BPM1 (oeBPM1) or DMS3 (oeDMS3), and lines with impaired RdDM function (dms3-1, nrpe1-10). The results confirmed a stimulative role of BPM1 protein in RdDM. This mechanism proved to be more significant in zygotic than somatic embyrogenesis. Depending on the position, methylation had a diverse effect on expression of analyzed genes.

Glavna uloga proteina BPM1 u uročnjaku (Arabidopsis thaliana L.) je u kompleksu kulin3- ovisnih E3 ligaza gdje domenom MATH prepoznaje specifične proteinske supstrate usmjeravajući ih u ubikvitinaciju i degradaciju na proteasomu 26S. Metodama ko-imunoprecipitacije i spektrometrije masa utvrđena je interakcija proteina BPM1 s DMS3 i RDM1 komponentama kompleksa DDR uključenog u RNA-posredovanu metilaciju DNA (RdDM). Kompleks DDR regrutira Polimerazu V te sudjeluje u pozicioniranju metilacijskog kompleksa na genom. Navedene interakcije nisu posredovane domenom MATH, ukazujući kako se kroz ovu interakciju proteini DMS3 i RDM1 ne degradiraju. U svrhu razjašnjena uloge proteina BPM1 u RdDM-u, odabrano je pet gena za analizu metilacije bisulfitnim mapiranjem i pirosekvenciranjem. Geni FWA i CML41 korišteni su kao kontrole budući da je njihova regulacija RdDM-om poznata od ranije. Kromatinskom imunoprecipitacijom identificirali smo zajedničke vezne regije proteina BPM1 i DMS3 na genomu linije s nefunkcionalnom proteasomalnom degradacijom, te izdvojili gene AGL14, RKP i FBW2 kao nove mete proteina BPM1 i RdDM-a. Metilacijski obrasci za svako metilacijsko mjesto i metilacijski kontekst, uspoređeni su između divljeg tipa te linije s overekspresijom proteina BPM1 i linije s nefunkcionalnim proteinom DMS3. Rezultati su pokazali značajno više razine metilacije povezane s RdDM-om u biljkama s povišenom ekspresijom proteina BPM1 te gubitak metilacije u RdDM nefukcionalnoj mutanti dms3.

U biljaka proces embriogeneze, može započeti iz oplođene jajne stanice (zigotna embriogeneza), ali i indukcijom somatskih stanica (somatska embriogeneza). U oba slučaja dolazi do značajnog epigenetičkog reprogramiranja, a jedan od ključnih mehanizama je metilacija DNA posredovana malim molekulama RNA (engl. RNA-directed DNA methylation, RdDM) koja osigurava metilaciju de novo. Uročnjakov protein BPM1 dio je velike porodice proteina MATH-BTB koji sudjeluju u brojnim razvojnim procesima biljaka i životinja kao komponente proteina ubikvitinskih ligaza ovisnih o kulinu. Dodatno, protein BPM1 stupa u interakciju s proteinima DMS3 i RDM1, važnim komponentama u procesu RdDM-a. Interakcije se ostvaruju neovisno o kulinu, upućujući na ulogu proteina BPM1 u regulaciji metilacije DNA de novo. Kromatinskom imunoprecipitacijom identificirane su zajedničke vezne regije proteina BPM1 i DMS3 na genomu uročnjaka te su odabrani geni na kojima su provedene analize ekspresije i metilacije u somatskim i zigotnim embrijima uročnjaka. Također, ispitana je učinkovitost indukcije somatskih embrija u transgeničnim linijama s prekomjernom ekspresijom proteina BPM1 ili DMS3, u liniji s utišanim genima BPM te liniji s nefunkcionalnim proteinom DMS3. Embriogeni potencijal linije s utišanim genima BPM bio je smanjen, dok u linijama s prekomjernom ekspresijom nije došlo do značajnih promjena u odnosu na divlji tip. Dobiveni rezultati upućuju na novu ulogu proteina MATH-BTB u regulaciji metilacije DNA tijekom embriogeneze.

The protein DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) is one of the key components of RNA-directed DNA methylation (RdDM). By mediating DNA methylation, RdDM has a role in controlling genome integrity and gene expression, particularly during plant acclimation to fluctuating environmental conditions. The aim of this study was to question the impact of elevated temperature on selected physiological and biochemical parameters in Arabidopsis thaliana with modified DMS3 expression. Seedlings with overexpressed DMS3 gene (oeDMS3 line) or non-functional DMS3 protein (dms3-1 line) together with the wild type (wt), were cultured at temperature of 24 °C and subsequently exposed to 37 °C for six hours. Plant material was collected at two time points - immediately after the treatment and after a 24-hour recovery period at 24 °C. In dms3-1 line, exposure to 37 °C resulted in a decrease of photosynthetic performance at both time points. Increased proline and MDA content were observed only in dms3-1 after recovery. In oeDMS3 line, guaiacol peroxidase (G-POD), ascorbate peroxidase (APX), and catalase (CAT) activity increased immediately after the treatment, whereas superoxide dismutase (SOD) increased after recovery. In dms3-1 line, only CAT activity increased immediately after the treatment, whereas G POD decreased at both time points. It can be concluded that dms3-1 was more heat-sensitive than oeDMS3 and wt, suggesting that the DMS3 protein is required for heat tolerance in Arabidopsis.

A member of a small family of Arabidopsis thaliana MATH-BTB proteins, BPM1 functions as a substrate-specific adaptor of a cullin3-based E3 ubiquitin ligase complex. Its N-terminal MATH domain binds target proteins and designates them for ubiquitination and subsequent proteasomal degradation. Recent studies reported that BPM1 mediates proteasomal degradation of transcription factors (ERF/AP2, R2R3 MYB and Homeobox) and phosphatases (PP2Cs). By using immunoprecipitation, TAP-tag purification, fluorescence-lifetime imaging microscopy, yeast-2-hybrid, pull down and microscale thermophoresis we confirmed BPM1 interaction with DMS3 and RDM1, key regulators of de novo DNA methylation in A. thaliana. Chromatin immunoprecipitation was used to determine regions of overlap between DMS3 and BPM1 chromatin binding sites. The DNA methylation status of identified regions was analysed by pyrosequencing after bisulphite conversion in A. thaliana plants overexpressing BPM1, in plants with downregulated BPMs as well as in DNA methylation mutants. Methylation patterns were assessed during plant development and after exposure to different abiotic stresses. Our results indicate that BPM1 does not participate in degradation of DMS3 and RDM1. Rather, it seems BPM1 recruits DNA methylation machinery to specific chromatin positions for de novo DNA methylation establishment.

Dizalica je jedan od ključnih transportnih sredstva unutrašnjeg gradilišnog transporta u visokogradnji, te kao takva ima vrlo bitan utjecaj na gradilištu. U ovom završnom radu su opisane vrste toranjskih i mobilnih dizalica, njihove karakteristike i prednosti i mane. Prikazan je postupak proračuna učinka dizalica, montaža i demontaža, zahtjevi za odabir stroja kao i njihov način rada. Također su navedena propisana pravila zaštite na radu kao i ilustracije dizalica s primjerima u praksi.

The GH3 family of acyl-acid-amidosynthetases catalyze the formation of amino acid conjugates to modulate levels of active auxins in plants. The aim of this study was to regenerate plants with overexpression of different GH3 genes. Wild type Arabidopsis thaliana plants, ecotypes WS and Col-0, were previously transformed via floral dip with Agrobacterium tumefaciens (strain GV3101, pMP90) to obtain AtGH3.3, PbGH3 and PpGH3.2 overexpressing plants. Primary transformants (T1) were analyzed for the presence of transgenes by DNA isolation, PCR amplification and gel electrophoresis. Because PpGH3.2 transgenic plants could not be obtained, floral dip transformation was conducted with new plant material. Secondary transformants (T2) were grown on hygromicin selective plates and lines with one T-DNA insertion were selected. AtGH3.3-His6 and PbGH3-His6 transgenic proteins were extracted from T2 plants and purified on Ni-NTA agarose. Western-blotting and immunodetection showed successful purification of transgenic PbGH3-His6, while AtGH3.3-His6 was degraded. T3 seedlings were grown on selective plates and homozygous lines were determined. These lines were used for RNA isolation and qPCR for measuring transgene overexpression. Seeds with overexpression of GH3 genes, regenerated in this research, will be further used to study the auxin metabolism in plants.

Tkivno inženjerstvo još je uvijek daleko od zadanog cilja, izrade funkcionalnih zamjenskih humanih organa te je razumno pretpostaviti da tehnologija biofabrikacije vjerojatno neće omogućiti stvaranje 100% autentičnih funkcionalnih kopija organa. Realniji tehnološki cilj je projektiranje tkivnih i organskih konstrukata sa sposobnošću obnavljanja funkcije oštećenih organa. Zahvaljujući brzo-razvijajućem biomimetičkom pristupu koji predstavlja integraciju inženjerstva i razvojne biologije, taj je cilj postao ostvariv. Brzom napretku ovog područja osobito je pridonio početak upotrebe 3D printera u izradi bioloških struktura, odnosno bioprintanje. Ovaj rad opisuje tradicionalne pristupe tkivnog inženjerstva, ukazuje na njihove nedostatke, uspoređuje ih s novijim metodama biomimetičkog pristupa te predstavlja nedavni napredak u izradi živih struktura koji je omogućila revolucionarna tehnologija bioprintanja. Tissue engineering is still far from the set goal, production of functional replacement human organs and it is reasonable to assume that biofabrication technology will probably not allow the creation of 100% authentic copies of functional organs. A more realistic technological goal is to design tissue and organ constructs with the ability to restore function of damaged organs. Thanks to the fast-evolving biomimetic approach which represents the integration of engineering and developmental biology, that goal has become feasible. The rapid progress in this area was especially stimulated by the beginning of use of 3D printers in the development of biological structures or bioprinting. This paper describes the traditional approaches to tissue engineering, highlights their shortcomings, compares them with newer methods of biomimetic approach and presents recent advances in the development of the living structures which were enabled by the revolutionary technology of bioprinting.