Alternative splicing in higher plants

Alternativno izrezivanje introna proces je kojim se iz jednog genskog transkripta dobivaju različite izoforme zrele mRNA. Prisutno je u svim višim eukariotima i ima ulogu u funkcionalnom proširenju proteoma, kao i u posttranskripcijskoj regulaciji ekspresije gena. Procesi alternativnog izrezivanja introna u biljaka nisu dovoljno istraženi i tek se očekuju brojna istraživanja na tom području sukladno s razvojem novih metoda detekcije tog procesa. Sekvencioniranje genoma A. thaliana (a i drugih biljnih vrsta u novije vrijeme) omogućilo je nova saznanja o alternativnom izrezivanju introna u biljaka, a uročnjak je biljka koja se najčešće koristi u ovim istraživanjima. U praktičnome dijelu rada navedene su neke hipotetske izoforme proteinskih produkata BPM gena uročnjaka dobivene alternativnim izrezivanjem introna zbog alternativnih donorskih i akceptorskih mjesta.Alternative splicing is a process of producing different isoforms of mature mRNA which originate from a common locus. Alternative splicing is present in all higher eukaryotes and has roles in expanding proteome diversity and post-transcriptional regulation. Alternative splicing in higher plants has not been studied extensively and new studies are anticipated as the new tools to accurately predict and visualize alternative splicing are being developed. Sequencing of the A. thaliana genome (and of other plant species, recently) has provided new information about alternative splicing in higher plants, Arabidopsis being the most commonly used species in these researches. Here, in the practical part of this work, the BPM genes of Arabidopsis are studied and some of the hypothetical isoforms of protein products derived from alternative splicing using the alternative donor and acceptor splice sites are created

Alternative splicing in higher plants

Alternativno izrezivanje introna proces je kojim se iz jednog genskog transkripta dobivaju različite izoforme zrele mRNA. Prisutno je u svim višim eukariotima i ima ulogu u funkcionalnom proširenju proteoma, kao i u posttranskripcijskoj regulaciji ekspresije gena. Procesi alternativnog izrezivanja introna u biljaka nisu dovoljno istraženi i tek se očekuju brojna istraživanja na tom području sukladno s razvojem novih metoda detekcije tog procesa. Sekvencioniranje genoma A. thaliana (a i drugih biljnih vrsta u novije vrijeme) omogućilo je nova saznanja o alternativnom izrezivanju introna u biljaka, a uročnjak je biljka koja se najčešće koristi u ovim istraživanjima. U praktičnome dijelu rada navedene su neke hipotetske izoforme proteinskih produkata BPM gena uročnjaka dobivene alternativnim izrezivanjem introna zbog alternativnih donorskih i akceptorskih mjesta.Alternative splicing is a process of producing different isoforms of mature mRNA which originate from a common locus. Alternative splicing is present in all higher eukaryotes and has roles in expanding proteome diversity and post-transcriptional regulation. Alternative splicing in higher plants has not been studied extensively and new studies are anticipated as the new tools to accurately predict and visualize alternative splicing are being developed. Sequencing of the A. thaliana genome (and of other plant species, recently) has provided new information about alternative splicing in higher plants, Arabidopsis being the most commonly used species in these researches. Here, in the practical part of this work, the BPM genes of Arabidopsis are studied and some of the hypothetical isoforms of protein products derived from alternative splicing using the alternative donor and acceptor splice sites are created

Alternative splicing in higher plants

Alternativno izrezivanje introna proces je kojim se iz jednog genskog transkripta dobivaju različite izoforme zrele mRNA. Prisutno je u svim višim eukariotima i ima ulogu u funkcionalnom proširenju proteoma, kao i u posttranskripcijskoj regulaciji ekspresije gena. Procesi alternativnog izrezivanja introna u biljaka nisu dovoljno istraženi i tek se očekuju brojna istraživanja na tom području sukladno s razvojem novih metoda detekcije tog procesa. Sekvencioniranje genoma A. thaliana (a i drugih biljnih vrsta u novije vrijeme) omogućilo je nova saznanja o alternativnom izrezivanju introna u biljaka, a uročnjak je biljka koja se najčešće koristi u ovim istraživanjima. U praktičnome dijelu rada navedene su neke hipotetske izoforme proteinskih produkata BPM gena uročnjaka dobivene alternativnim izrezivanjem introna zbog alternativnih donorskih i akceptorskih mjesta.Alternative splicing is a process of producing different isoforms of mature mRNA which originate from a common locus. Alternative splicing is present in all higher eukaryotes and has roles in expanding proteome diversity and post-transcriptional regulation. Alternative splicing in higher plants has not been studied extensively and new studies are anticipated as the new tools to accurately predict and visualize alternative splicing are being developed. Sequencing of the A. thaliana genome (and of other plant species, recently) has provided new information about alternative splicing in higher plants, Arabidopsis being the most commonly used species in these researches. Here, in the practical part of this work, the BPM genes of Arabidopsis are studied and some of the hypothetical isoforms of protein products derived from alternative splicing using the alternative donor and acceptor splice sites are created

Effect of lucerne - grass mixtures on weed infestation and forage yield

Cilj ovoga istraživanja bio je utvrditi kako će dodavanje raznih vrsta trava lucerni utjecati na pojavu korova i prinos krme. Postavljen je poljski pokus po shemi slučajnog blok sustava u listopadu 2014. godine u Tenji (Repulika Hrvatska). Pokus je proveden u tri ponavljanja na površini osnovne parcele od 6 m2. Pokusne parcele su prekrivene agrilnom folijom „Lutrasil“ zbog kasnog roka sjetve, a krajem veljače folija je skinuta uz odlično prezimljenje zasijanih biljaka u pokusu. Floristička opažanja su obavljena 05. travnja 2015. godine. Košnja pokusa obavljena je u 2 roka: 08. svibnja 2015. i 16. lipnja 2015. U oba roka košnje lucerna je bila u fazi početka cvatnje, a trave su u prvom roku košnje bile u fazi početka metličanja, odnosno klasanja, dok su u drugom roku bile u vegetativnoj fazi. U pokusu je florističkom analizom utvrđena prisutnost 13 korovnih vrsta, a dominatni su bile jednogodišnji širokolisni korovi. Najmanji broj korovnih vrsta po m2 su imale smjese lucerne s velikim brojem trava (5. i 6. pokusna varijanta) i to 43, odnosno 42 korova/m2, monokultura lucerne je imala 98 korova/m2, a najveći broj korova/m2 je imala esparzeta, njih 166. Najprinosnija pokusna varijanta što se tiče zelene mase je bila 5. pokusna varijanta (lucerna, vlasulja nacrvena, vlasulja trstikasta, klupčasta oštrica i engleski ljulj) s prinosom od 50,47 t/ha u 2 otkosa, lucerna u monokulturi je imala 41,5 t/ha, a najmanji prinos ZM je imala esparzeta (23,19 t/ha ZM). U pogledu ST najprinosnija se pokazala lucerna u monokulturi koja je u 2 otkosa imala prinos 10,13 t/ha ST, s očekivanim godišnjim prinosom od 18,41 t/ha ST. Preostale pokusne varijante su imale prinos ST između 14,04 i 16,85 t/ha koliko je imala smjesa lucerne i vlasulje trstikaste, a najmanji prinos ST, kao i kod ZM je imala esparzeta koja je ostvarila 4,66 t/ha ST u 2 otkosa.The aim of this study was to determine how the addition of different grasses to lucerne will affect on weed appearances and forage yield. Field experiment was set by randomized complete block design in October 2014 in Tenja (Republic of Croatia). The experiment was conduct in three repetitions on plot surface of 6 m2. Because of the late sowing date, experimental plots were covered with agrilic foil „Lutrasil“ and by the end of February the foil was removed with great overwintering of the planted plants in experiment. Floristic observations were made on April 05, 2015. The mowing was done in 2 terms: May 08, 2015 and June 16, 2015. In both mowing terms, lucerne was in the stage of flowering, while grasses were in the budding stage on first mowing term and in vegetative stage on second mowing term. By floristic analysis presence of 13 weed species were determined in experiment and dominant ones were annual broad leaf weeds. The smallest number of weeds per m2 had lucerne mixtures with large number of grasses (5th and 6th experimental treatment) – 43 and 42 weeds/m2, lucerne monoculture had 98 weeds/m2 and the largest number of weeds/m2 had esparcet, 166 of them. Highest yielding experimental treatment regarding to green mass (GM) was treatment number 5 (which included: Lucerne, Red fescue, Tall fescue, Orchard grass and Perennial Ryegrass) with yield of 50,47 t/ha of GM in 2 mowing terms, lucerne monoculture had 41,5 t/ha of green mass and smallest yield of green mass had the esparcet monoculture (23,19 t/ha GM). In terms of dry matter (DM), highest yielding was lucerne monoculture which had yield of 10,13 t/ha DM with expected annual yield of 18,41 t DM/ha. The remaining experimental treatments had dry matter yield between 14,04 and 16,85 t/ha how much had the mixture of lucerne and tall fescue, while the smallest yield of dry matter, same as the green mass, had the esparcet monoculture with 4,66 t DM/ha in 2 mowing

Konstrukcija molekularnih alata temeljenih na sustavu CRISPR/Cas9 za epigenetičku modulaciju ekspresije gena

Repurposing of platforms for targeted genome engineering, zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs) and especially CRISPR/Cas9 system, has enabled targeted manipulation of epigenetic marks and new insights into epigenetic regulatory mechanisms. In this work, I have fused the catalytic domain of human TET1 (Ten-Eleven Translocation 1) protein Cterminally to the catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes to create the dCas9- TET1 molecular tool. I have also tested the activity of N-terminal fusion of TET1-dCas9. The TET1 protein is a methylcytosine dioxygenase involved in active demethylation of 5-methylctosines to 5- hydroxymethylcytosines. In context of gene expression, methylated CpG dinucleotides within gene promoters are usually associated with gene silencing, and unmethylated CpGs with active gene transcription. Constructed molecular tools were successfully used for targeted CpG demethylation in promoter regions of two candidate genes, MGAT3 and LAMB1. I also showed that following demethylation the MGAT3 gene changed expression level. I have compared MGAT3 gene activation rates achieved following demetlylation using dCas9-TET1 with a more conservative approach of VPR-dCas9 mediated direct activation. For this approach, I used an N-terminal fusion of VPR activation domain and dCas9 from Staphylococcus aureus (dSaCas9).Prenamjene alata za genetičko inženjerstvo, baziranih na zinc finger nukleazama (ZFN), transcription activator-like effector nukleazama (TALEN) te posebno sustavu CRISPR/Cas9, pružile su mogućnost ciljanog modificiranja epigenoma i time razjašnjavanje uloge epigenetičkih modifikacija u regulaciji ekspresije gena. U ovom radu napravila sam C-terminalnu fuziju katalitičke domene proteina TET1 (Ten-Eleven Translocation 1) i katalitički inaktivne nukleaze Cas9 (dCas9) izolirane iz vrste Streptococcus pyogenes i konstruirala dCas9-TET1 molekularni alat za ciljanu demetilaciju DNA. Testirala sam i aktivnost N-terminalne fuzije TET1-dCas9. Protein TET1 katalizira pretvorbu 5- metilcitozina u 5-hidroksimetilcitozin, što predstavlja prvi korak u aktivnoj demetilaciji DNA. Korelacijske analize povezuju hipermetilirane CpG dinukleotide u promotorima gena s represijom transkripcije, a hipometilirane CpG dinukleotide s aktivnom transkripcijom. Konstruirani alati uspješno su upotrijebljeni za ciljanu demetilaciju CpG dinukleotida unutar promotora kandidat gena MGAT3 i LAMB1. Pokazala sam da je nakon demetilacije ciljanih CpG mjesta u promotoru gena MGAT3 porasla razina transkripata ovoga gena. Usporedila sam efekt aktivacije gena MGAT3 postignut ciljanom demetilacijom dCas9-TET1 alatom s konzervativnijim pristupom direktne aktivacije pomoću VPR-dCas9 alata. U ovu svrhu upotrijebila sam N-terminalnu fuziju aktivacijske domene VPR i dCas9 iz vrste Staphylococcus aureus (dSaCas9)

Determination of biogas potential of Jerusalem artichoke (Helianthus tuberosus L.)

Razvojem tehnologije i društva razvila se svijest o sve većem zagađenju okoliša te se uvidjela neodrživost postojećeg načina iskorištavanja energije. U budućnosti će biti potrebno što je više moguće energiju dobivati iz obnovljivih izvora, a jedan od tih izvora je proizvodnja električne i toplinske energije iz bioplinskih postrojenja. Čičoka (Helianthus tuberosus L.) je biljka široke primjene, prvenstveno u ljudskoj prehrani, hranidbi stoke, u medicinske svrhe, industrijskoj preradi i sl., ali predstavlja potencijalni izvor biomase za proizvodnju bioplina. Prinos gomolja čičoke dostiže u optimalnim uvjetima uzgoja do 40 t/ha, a nadzemni dio, odnosno stabljika i ostala zelena masa doprinosi s do 55 t/ha. Proizvodnja bioplina iz jedne tone gomolja čičoke iznosi 50,46 m3, a iz jedne tone nadzemne mase 82,89 m3 i najbolje bi bilo gomolj koristiti u ranije navedene svrhe, dok bi nadzemna masa bila pogodnija za proizvodnju bioplina i zatvaranje cjelokupnog procesa proizvodnjeThe development of technology and society developed awareness of the increasing enviroment pollution and unsustainability of the existing ways of using energy. In the future, it will be necessary to use energy from renewable sources as much as possible and one of these sources is production of electrical and thermal energy from biogas plants. Jerusalem artichoke (Helianthus tuberosus L.) is a plant of wide range of usage, primarily in human nutrition, animal husbandry, for medical purposes, industrial processing, etc., but it is also a potential source of biomas for biogas production. The yield of tubers of Jerusalem artichoke in optimal growing conditions reaches up to 40 t/ha, and above-ground parts, stem and other green mass contributes up to 55 t/ha. Biogas production from one ton of Jerusalem artichoke tuber is 50,46 m3 and from one ton of aboveground mass is 82,89 m3 and it would be the best to use tubers in before mentioned purposes, while the above-ground mass would be more suitable for biogas production and closure of the entire production proces

Konstrukcija molekularnih alata temeljenih na sustavu CRISPR/Cas9 za epigenetičku modulaciju ekspresije gena

Repurposing of platforms for targeted genome engineering, zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs) and especially CRISPR/Cas9 system, has enabled targeted manipulation of epigenetic marks and new insights into epigenetic regulatory mechanisms. In this work, I have fused the catalytic domain of human TET1 (Ten-Eleven Translocation 1) protein Cterminally to the catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes to create the dCas9- TET1 molecular tool. I have also tested the activity of N-terminal fusion of TET1-dCas9. The TET1 protein is a methylcytosine dioxygenase involved in active demethylation of 5-methylctosines to 5- hydroxymethylcytosines. In context of gene expression, methylated CpG dinucleotides within gene promoters are usually associated with gene silencing, and unmethylated CpGs with active gene transcription. Constructed molecular tools were successfully used for targeted CpG demethylation in promoter regions of two candidate genes, MGAT3 and LAMB1. I also showed that following demethylation the MGAT3 gene changed expression level. I have compared MGAT3 gene activation rates achieved following demetlylation using dCas9-TET1 with a more conservative approach of VPR-dCas9 mediated direct activation. For this approach, I used an N-terminal fusion of VPR activation domain and dCas9 from Staphylococcus aureus (dSaCas9).Prenamjene alata za genetičko inženjerstvo, baziranih na zinc finger nukleazama (ZFN), transcription activator-like effector nukleazama (TALEN) te posebno sustavu CRISPR/Cas9, pružile su mogućnost ciljanog modificiranja epigenoma i time razjašnjavanje uloge epigenetičkih modifikacija u regulaciji ekspresije gena. U ovom radu napravila sam C-terminalnu fuziju katalitičke domene proteina TET1 (Ten-Eleven Translocation 1) i katalitički inaktivne nukleaze Cas9 (dCas9) izolirane iz vrste Streptococcus pyogenes i konstruirala dCas9-TET1 molekularni alat za ciljanu demetilaciju DNA. Testirala sam i aktivnost N-terminalne fuzije TET1-dCas9. Protein TET1 katalizira pretvorbu 5- metilcitozina u 5-hidroksimetilcitozin, što predstavlja prvi korak u aktivnoj demetilaciji DNA. Korelacijske analize povezuju hipermetilirane CpG dinukleotide u promotorima gena s represijom transkripcije, a hipometilirane CpG dinukleotide s aktivnom transkripcijom. Konstruirani alati uspješno su upotrijebljeni za ciljanu demetilaciju CpG dinukleotida unutar promotora kandidat gena MGAT3 i LAMB1. Pokazala sam da je nakon demetilacije ciljanih CpG mjesta u promotoru gena MGAT3 porasla razina transkripata ovoga gena. Usporedila sam efekt aktivacije gena MGAT3 postignut ciljanom demetilacijom dCas9-TET1 alatom s konzervativnijim pristupom direktne aktivacije pomoću VPR-dCas9 alata. U ovu svrhu upotrijebila sam N-terminalnu fuziju aktivacijske domene VPR i dCas9 iz vrste Staphylococcus aureus (dSaCas9)

Nano-and microcarriers as drug delivery systems for usnic acid: Review of literature

Usnic acid is one of the most investigated lichen secondary metabolites, with several proven biological properties with potential medical relevance. However, its unfavorable physico-chemical properties, as well as observed hepatotoxicity, have discouraged wide-range utilization of usnic acid as a promising therapeutic agent. In accordance with the growing research interest in the development of nanotechnology, especially in the arena of preparations based on natural sources of medicinal compounds, usnic acid incorporated into nano-and microsized colloidal carriers has been a subject of a large number of publications. Therefore, this review discusses the overall results of the studies dealing with usnic acid encapsulated into lipid-based, polymeric and nonorganic micro-and/or nanocarriers, as potential drug delivery systems for this natural compound, in an attempt to introduce its usage as a potential antitumor, antimicrobial, wound-healing, antioxidative and anti-inflammatory drug

Chemical and antimicrobial analyses of Sideritis romana L. subsp. purpurea (Tal. ex Benth.) Heywood, an endemic of the Western Balkan

A comprehensive study on essential oil and different solvent extracts of Sideritis romana L. subsp. purpurea (Tal. ex Benth.) Heywood (Lamiaceae) from Montenegro is reported. The gas chromatography-mass spectrometry analysis of the essential oil revealed a total of 43 components with bicyclogermacrene (23.8%), germacrene D (8%), (E)-caryophyllene (7.9%) and spathulenol (5.5%) as the major ones. Sesquiterpenoid group was found to be the most dominant one (64.8%), with 19.9% of the oxygenated forms. In the crude methanol extract of the investigated plant, obtained by Sohhlet exraction, the total phenol content was 14.7 ± 0.4 mg of GA/g, the total flavonoids were 0.29 ± 0.03% expressed as hyperoside percentage, whereas the total tannins content was 0.22 ± 0.04% expressed as pyrogallol percentage. For the antimicrobial activity determination, the following microorganisms have been used: methicillin-susceptible Staphylococcus aureus (MSSA (American Type Culture Collection (ATCC) 29213)) and methicillin-resistant S. aureus (MRSA (clinical strain)), Escherichia coli (ATCC 25922), carbapenem-susceptible Klebsiella pneumoniae (clinical strain), carbapenem-resistant K. pneumoniae (clinical strain) and Candida albicans (ATCC 14053). The essential oil showed high potency against MSSA and MRSA, both at high (~5 × 10 5 CFU/mL) and low (~5 × 10 3 CFU/mL) inoculum. With respect to MSSA, the minimal inhibitory concentration (MIC) value was 0.307 mg/mL, with bactericidal activity obtained at 0.615 mg/mL, while, in the case of MRSA, the MIC and minimal bactericidal concentration (MBC) values were 0.076 and 0.153 mg/mL, respectively. Regarding anti-Candida albicans activity, the MIC value was 2.46 mg/mL without reaching fungicidal activity. In addition to the observed essential oil efficacy, different solvent extracts were analyzed for their antimicrobial activity. Similarly to the essential oil, thehighest efficacy was observed against both MSSA and MRSA strains, at high and low inoculums, in the case of the 1,2-dichloroethane and methanol extracts. A potent fungicidal activity has been also found for the n-hexane and 1,2-dichloroethane extracts. It can be concluded that Sideritis romana L. subsp. purpurea (Tal. ex Benth.) Heywood provides a wide range of application in different fields such as phytochemistry, pharmacology, toxicology or pharmacognosy