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Raziskave že desetletja uvrščajo navadno konopljo (Cannabis sativa L.) med hitro rastoče, nezahtevne, hiperakumulativne rastline. Pripisujejo ji visoko stopnjo privzema težkih kovin, medtem ko geni, odzivni na prisotnost potencialno toksičnih kovin, pri navadni konoplji še niso sekvencirani in anotirani, s tem pa tudi ni dodobra raziskana njihova potencialna vključenost v privzem kovin. Ta raziskava skuša zapolniti vrzel nasprotujočih si ali nepopolnih podatkov v literaturi in preučuje vključenost genov GSR 1, PLD α, PDR 12 in HMA 4 v metabolizmu potencialno toksičnih kovin v navadni konoplji. Raziskava temelji na lastnih eksperimentalnih podatkih ter analizah v kontroliranem lončnem poskusu, pri čemer smo preučevali vključenost genov na dve potencialno toksični kovini, svinec in kadmij, pri dveh sortah navadne konoplje, Futura 75 in Tisza. Z načrtovanjem začetnih oligonukleotidov na podlagi medvrstno ohranjenih genskih zaporedij - ortologov v že anotiranih vrstah, kot so Arabidopsis thalinana, Brassica juncea, Lactuca sativa, Triticum urartu ter Vitis vinifera, pa raziskava nudi tudi posredni vpogled v podobnost nukleotidnih zaporedij tovrstnih genov v navadni konoplji. Research has classified industrial hemp (Cannabis sativa L.) as a fast-growing, undemanding, hyperaccumulative plant for decades. It is also attributed a high level of heavy metal uptake, while genes responsive to the presence of potentially toxic metals in industrial hemp have not been sequenced and annotated, and their involvement in their uptake is not well understood. The study tries to fill the gap of conflicting or incomplete data in literature, and investigates the involvemenet of GSR 1, PLD α, HSP70, PDR 12, and HMA 4 genes in the response of industrial hemp to potentially toxic metals. The study is based on our own experimental data and analyses in a controlled pot experiment, whereby we evaluated the involvement of genes in the response to two potentially toxic metals, lead and cadmium, in two separate varieties of hemp, Futura 75 and Tisza. By designing the initial oligonucleotides on the basis of interspecific preserved gene sequences - orthologs in already annotated species such as Arabidopsis thalinana, Brassica juncea, Lactuca sativa, Triticum urartu and Vitis vinifera, the study also offers an indirect insight into the similarity of nucleotide sequences of these genes in industrial hemp.

Glive rodu Verticillium predstavljajo ene izmed najbolj škodljivih rastlinskih patogenov. Predstavniki rodu lahko parazitirajo širok spekter gostiteljskih rastlin, med katerimi je tudi veliko gospodarsko in kmetijsko pomembnih kultur. V Sloveniji največ škode povzročata vrsti V. nonalfalfae, ki povzroča blago in letalno obliko verticilijske uvelosti hmelja in V. dahliae. Za lažje nadzorovanje in preprečevanje širjenja bolezni, je ključnega pomena razvoj ustreznih diagnostičnih metod, ki bi omogočile hitro in zanesljivo identifikacijo ter razločevanje med posameznimi vrstami in patotipi. Cilj naše raziskave je vključeval razvoj metode LAMP za hitro in učinkovito določanje vrst rodu Verticillium ter letalnega patotipa vrste V. nonalfalfae (PG2). Ker je za obravnavan rod fitopatogenih gliv značilna velika heterogenost, smo izvedli tudi molekularno analizo variabilnosti. Preko pomnoževanja DNA z začetnimi oligonukleotidi, specifičnimi za letalno regijo patotip PG2, smo želeli pokazati veliko heterogenost znotraj posameznih vrst gliv rodu Verticillium in težavnost uporabe ustrezne diagnostične metode ter načrtovanja začetnih oligonukleotidov, ki bi omogočili razločevanje med vrstami in patotipi tega rodu. Fungi from the genus Verticillium are known to be one of the most harmful plant pathogenic organisms. The genus consists of species that can infect a broad spectrum of plants, many of which are agriculturally relevant crops. In Slovenia a great amout of harm is caused by two species, V. nonalfalfae and V. dahliae. By infecting the hop plant, they can cause a mild or lethal form of a disease, called verticillium wilt. In order to control and potentially even prevent the disease from spreading, the development of suitable diagnostic methods for rapid detection and identification is crucial. The aim of our study was to develop the LAMP method, which can enable a rapid and reliable detection of species of the genus Verticillium and the pathotype PG2. Due to the genus' heterogeneity, we analised a series of isolates via PCR primers specific for the amplicifation of different regions from lethal region of PG2 isolate, by which we wanted to emphasize the difficulties of development of the suitable method and designe of specific primers for molecular diagnostics of the genus Verticillium.

Izhodišče: Rak ustne votline in ustnega dela žrela je eden izmed najpogostejših rakov na svetu. Tradicionalno je povezan s kajenjem tobaka in pitjem alkohola, vendar se s to vrsto raka vedno bolj povezuje okužba HPV. HPV so zelo razširjeni majhni DNA virusi, ki povzročajo različne novotvorbe. V HPV pozitivnih rakih je najpogosteje mutiran PIK3CA gen, ki je zelo povezan z invazivnostjo raka. Prisotnost aktivacijskih mutacij v genu PIK3CA povzroči okrepitev PIK3K signalne poti v tumorskih tkivih, kar nakazuje, da je PIK3CA vpleten tudi v oblikovanje tumorjev. Metodologija: Opravili smo analizo DNA 108 tumorjev in 88 vzorcev zdravih tkiv ustne votline in ustnega dela žrela. Za določanje prisotnosti HPV, določitev tipa HPV in določanje najpogostejših aktivacijskih mutacij v genu PIK3CA smo uporabili metodo verižne reakcije s polimerazo (PCR). Dobljene produkte PCR smo analizirali s pomočjo elektroforeze v agaroznem gelu. Rezultati: Ugotovili smo, da lahko z metodo verižne reakcije s polimerazo določimo prisotnost HPV DNA v vzorcu kakor tudi mutacije v genu PIK3CA. Sklep: PCR reakcija se je izkazala za uspešno metodo za dokazovanje prisotnosti mutacij v genu PIK3CA kakor tudi za določanje prisotnosti HPV DNA v vzorcu. Za potrditev, da je ta metoda primerna tudi za določitev tipa HPV, pa bi morali celotno analizo opraviti na večjem številu vzorcev. Starting point Head and neck cancer is one of the most common cancers worldwide. It is traditionally associated with smoking and drinking but this type of cancer is increasingly associated with HPV infection. HPVs are very extended, small DNA viruses that cause a variety of neoplasms. In HPV positive cancers the PIK3CA gene is the most mutated gene and it is connected with cancer invasiveness. The presence of the activating mutations in PIK3CA gene strengthens the PIK3K signalling pathway in tumour tissues, which suggests that the PIK3CA gene is involved in tumour formation.. Methodology: A DNA analysis of 108 examples of tumour tissues and 88 examples of healthy tissues of the oral cavity and oral part of the larynx was done. To determine the presence of HPV, the type of HPV and the most common activating mutations in the PIK3CA gene, a polymerase chain reaction was used. The obtained PCR products were analysed with electrophoresis in the agarose gel. Results: It was determined that the polymerase chain reaction is an adequate method for determining the presence of the HPV DNA in the sample as well for determining mutations of the PIK3CA gene. Conclusion: PCR reaction has proven to be a successful method for determining the presence of PIK3CA mutations as well as for determining the presence of HPV DNA in the sample. In order to confirm that this method is also appropriate for determining a type of HPV, an analysis should be carried out on a larger number of samples.

Humani papiloma virusi predstavljajo družino izredno heterogenih in razširjenih majhnih DNA virusov, ki povzročajo različne novotvorbe pri ljudeh in živalih. So etiološko povezani z različnimi benignimi in malignimi novotvorbami epitelija in kože in so bili zadnjih 30 let predmet obsežnih raziskav po vsem svetu. V tem času so razvili številne metode za dokazovanje HPV. Namen naloge je bil, na vzorcu 51 tumorjev ORL področja določiti prisotnost HPV DNA. Uporabili smo metodo verižne reakcije s polimerazo ali PCR metodo, ki je trenutno najobčutljivejša metoda za dokazovanje okužb s HPV. Dokazovanje virusov s PCR metodo temelji na in vitro pomnoževanju za virus značilnega majhnega odseka genoma. PCR reakcija je sestavljena iz 25-40 ponovitev temperaturnih ciklov zaporedne denaturacije osamljene DNA, spajanja izbranih začetnih oligonukleotidov s komplementarnimi zaporedji, ter sinteze nove komplementarne DNA v območju med začetnima oligonukleotidoma. Vsaka novo sintetizirana kopija odseka DNA v naslednjem ciklu reakcije služi kot matrica. Ugotovili smo, da je metoda verižne reakcije s polimerazo ali PCR metoda primerna, hitra in enostavna za določanje prisotnosti HPV DNA v ORL tumorjih. The human papilloma viruses represent a family of highly heterogeneous and spread small DNA viruses that cause a variety of neoplasms in humans and animals. HPV are etiologically associated with various benign and malignant neoplasms of epithelial and skin and have been the subject of extensive worldwide research over the past 30 years. In the meantime, a number of methods have been developed to determinate HPV. The purpose of the research was to determine the presence of HPV DNA on the sample of 51 tumours of ORL area. For this purpose, we used the method of polymerase chain reaction or the PCR method. This reaction is currently the most sensitive method for the demonstration of the HPV presence. The determination of viruses by PCR method is based on the in vitro amplification for the virus of the small specific segments. The reaction consists of 25-40 repeated temperature denaturation cycles of isolated DNA sequence, annealing of primers to complementary sequences, and the synthesis of the new complementary DNA in the region between the primers. Each newly synthesized copy of the DNA segment in the next cycle of the reaction serves as a matrix. We determined that the PCR method is adequate, quick and easy for determining the presence of the HPV DNA in the ORL tumours.