Search

Odvisnost od alkohola je posledica okoljskih in genetskih dejavnikov. Na možganski center za nagrajevanje alkohol deluje posredno predvsem preko μ-opioidnega receptorja, ki ga v genomu kodira gen OPRM1. Tema naše raziskave je bilo iskanje vzročno-posledične povezave med odvisnostjo od alkohola in izraženostjo sopojavnih duševnih motenj (SDM) ter razporeditvijo genotipov polimorfizmov v genu OPRM1 v skupinah akutno odvisnih preiskovancev, abstinentnih preiskovancev in zdravih kontrol. Zanimalo nas je, ali se značilnosti preiskovancev in izraženost SDM med posameznimi skupinami statistično razlikujejo glede na razporeditev genotipov polimorfizmov OPRM1 rs1799971 in rs677830. Ker je genska ekspresija natančno uravnavana preko sistema molekul miRNA, smo v raziskavo vključili tudi dva polimorfizma genov za molekule miRNA, ki uravnavata izražanje gena OPRM1. To sta polimorfizem rs1011784 v genu MIR23B in polimorfizem rs2296616 v genu MIR107. Za oceno izraženosti sopojavne simptomatike smo uporabili ustrezne vprašalnike. Vzorce DNA smo analizirali z metodo KASP, na koncu pa je sledila še statistična analiza. Ugotovili smo, da alternativen alel T polimorfizma rs677830 poveča tveganje za pojav odvisnosti od alkohola ob upoštevanju stanu, starosti, izobrazbe in kajenja. Pri ostalih polimorfizmih nismo opazili vpliva na pojav odvisnosti od alkohola, smo pa opazili vpliv na izraženost SDM. Alel G polimorfizma rs1799971 je bil povezan z nižjo izraženostjo sovražnosti v skupini zdravih kontrol. Heterozigotiza ta polimorfizem v skupini abstinentnih preiskovancev so imeli večjo izraženost socialne anksioznosti. V skupini abstinentnih pacientov so imeli nosilci vsaj enega polimorfnega alela T polimorfizma rs677830 nižjo izraženost socialne anksioznosti. Referenčni alel G polimorfizma rs1011784 je bil pri akutno odvisnih pacientih povezan z višjo izraženostjo kompulzivnih in anksioznih simptomov. Največji vpliv na izraženost sopojavne simptomatike je imel polimorfizem rs2296616, pri katerem so nosilci vsaj enega polimorfnega alela A v skupini zdravih kontrol imeli višjo izraženost depresivne in anksiozne simptomatike, v skupini abstinentnih pacientov pa je bila bolj izražena anksiozna simptomatika pri homozigotih za referenčni alel G. V skupini zdravih kontrol smo opazili, da so nosilci vsaj enega polimorfnega alela A pogosteje uživali alkoholne pijače, vendar so bile razlike statistično značilne samo pri heterozigotih. Dobljeni rezultati bi nam lahko pomagali oceniti tveganje za izraženost SDM, kar bi povečalo uspešnost uspešnost z dravljenja odvisnosti. Alcohol dependence is caused by environmental and genetic factors. Alcohol acts indirectly on the brain's reward center, mainly via the μ-opioid receptor, which is encoded by the OPRM1 gene. Our study aimed to investigate the cause-effect relationship between alcohol dependence and the expression of co-occurring psychiatric disorders, and the genotype distribution of polymorphisms in the OPRM1 gene in groups of acutely dependent subjects, abstinent subjects, and healthy controls. We were interested in whether subjects differed statistically between groups and in the expression of co-occurring disorders with regard to the distribution of genotypes in the rs1799971 and rs677830 polymorphisms. Since gene expression is precisely regulated through the miRNA molecule system, two polymorphisms of the miRNA molecule genes regulating OPRM1 gene expression were also included in the study. These are the rs1011784 polymorphism in the MIR23B gene and the rs2296616 polymorphism in the MIR107 gene. The corresponding questionnaires were used to assess the expression of co-occurring disorders. DNA samples were analyzed by KASP, followed by statistical analysis. We found that the T allele of rs677830 polymorphism increased the risk of alcohol dependence when partnership, age, education, and smoking were also taken into account. For the other polymorphisms, we did not observe any effect on the occurrence of alcohol dependence, but we did observe an effect on the expression of comorbid symptomatology. The G allele of the rs1799971 polymorphism was associated with a lower expression of hostility in the group of healthy controls. Heterozygotes for this polymorphism in the abstinent group had a higher expression of social anxiety. In the abstinent group, carriers of at least one T allele of the rs677830 polymorphism had a lower expression of social anxiety. The reference G allele of the rs1011784 polymorphism was associated with a higher expression of compulsive and anxious symptoms in acutely dependent patients. The polymorphism rs2296616 had the greatest impact on the expression of comorbid symptomatology, with carriers of at least one polymorphic A allele in the healthy control group having a higher expression of depressive and anxiety symptoms. In the abstinent group, anxiety was expressed more in those homozygous for the reference G allele. In the healthy controls group, carriers of at least one polymorphic A allele were more likely to consume alcoholic beverages, but the differences were statistically significant only in heterozygotes. These results could help us assess the risk of co-occurring disorders, which would increase the success rate of addiction treatment.

Eritrocitoza je redko bolezensko stanje s povišano maso rdečih krvnih celic, povišanim hematokritom in/ali s povišano koncentracijo hemoglobina. Bolezen lahko nastane iz več razlogov, največkrat pa je diagnosticirana kot policitemija vera, kot posledica pridobljenih nukleotidnih različic gena JAK2, najpogostejše različice V617F v eksonu 14 ali večih nukleotidnih različic v eksonu 12. Po drugi strani so prirojena nagnjenja za eritrocitozo redka, predvsem pa je genetsko ozadje le-teh precej raznoliko. Vloga gena JAK2 je do sedaj pri razvoju družinske eritrocitoze še precej slabo poznana. Veliko bolnikov s sumom na družinsko eritrocitozo (okoli 70 %) še vedno ostane brez ugotovljenega vzroka bolezni in jih tako diagnosticirajo kot bolnike z eritrocitozo neznanega vzroka. Namen raziskave je bil preveriti, ali so zarodne različice gena JAK2 prisotne pri izbranih bolnikih s potrjeno eritrocitozo neznanega vzroka in optimizirati metodo PCR za pomnoževanje izbranih eksonskih regij z vključenimi obmejnimi regijami intronov gena JAK2. Analizirali smo dva vzorca bolnikov iste družine in sedem kontrolnih vzorcev zdravih oseb. S sekvenciranjem po Sangerju smo potrdili dve, z analizo NGS v genu JAK2 predhodno odkriti različici, in sicer v eksonu 13 (rs1362123436) in v intronu 19 (rs780797578). Pri obeh bolnikih sta omenjeni različici heterozigotni, vendar za zdaj še brez pojasnjenega kliničnega pomena. Hkrati smo odkrili tudi nove različice v intronu 12 pri obeh bolnikih ter pri enem bolniku v intronu 18 ter 24. V namen ovrednotenja pomena vseh na novo odkritih nukleotidnih različic so potrebne nadaljne funkcijske preiskave. Erythrocytosis is a rare condition caused by an increase in the red blood cell mass, hematocrit and/or heamoglobin. While the disease can occur due to several diverse causes, it is mostly diagnosed as polycythemia vera, an acquired type of erythrocytosis. Polycythemia vera develops upon acquired nucleotide variants in the JAK2 gene, most commonly the V617F variant in exon 14 or multiple other nucleotide variants in exon 12. On the other hand, congenital predispositions for familial erythrocytosis are rare and overall, their genetic background is relatively heterogeneous. The role of the JAK2 gene in the development of familial erythrocytosis is for now still poorly understood. Many patients with suspected familial erythrocytosis (about 70 %) remain without an identified cause of the disease and are thus diagnosed as having erythrocytosis of unknown cause. The aim of the study was to verify whether germline variants of the JAK2 gene are present in selected cohort of patients with confirmed erythrocytosis of unknown cause and to optimise the PCR method for amplification of selected exon and intron regions of gene JAK2. We included two samples of patients from the same family and seven control samples of healthy individuals. Two previously detected variants by NGS analysis in JAK2 gene were confirmed, one in exon 13 (rs1362123436) and another in intron 19 (rs780797578). Both variants are heterozygous, but yet without any significant clinical relevance. In addition, new nucleotide variants were detected in intron 12 in both patients and in introns 18 and 24 in one patient. To evaluate the significance of the newly discovered nucleotide variants, further functional tests will need to be performed in the future.

In Bosnia and Herzegovina, pedunculate oak (Quercus robur L.) occurs at the southern margin of its distribution range, close to the glacial refugia of this species. To assess the patterns of genetic diversity distribution at the rear edge of the Holocene colonization, we studied genetic variation in 20 pedunculate oak populations using 14 allozyme loci. Despite considerable differences among populations, neither the numbers of alleles nor genetic diversity showed any geographical trend within the studied area, although small isolated populations showed generally lower allelic richness. The Bayesian analysis of population structure indicated a kind of geographical pattern. We identified no signs of a recent bottleneck. The proximity to multiple glacial refugia explains the outcomes. Nasl. z nasl. zaslona. Opis vira z dne 23. 12. 2021. Bibliografija: str. 38-40. Abstract.

Metalloproteinases (MMPs) have an important role in tissue remodeling and have been shown to have an effect on tumor progression, invasion, metastasis formation, and apoptosis in several tumors, including mesothelioma. Mesothelioma is a rare tumor arising from pleura and peritoneum and is frequently associated with asbestos exposure. We have performed a systematic search of PubMed.gov and ClinicalTrials.gov databases to retrieve and review three groups of studies: studies of MMPs expression in tumor tissue or body fluids in patients with mesothelioma, studies of MMPs genetic variability, and studies of MMPs as potential novel drug targets in mesothelioma. Several studies of MMPs in mesothelioma tissues reported a link between higher expression levels of commonly studied MMPs and clinical parameters, such as overall survival. Fewer studies have investigated genetic variability of MMP genes. Nevertheless, these studies suggested that certain genetic variants in MMP genes can have either protective or tumor-promoting effects on mesothelioma patients. MMPs have been also reported as novel drug targets, but so far no clinical trials of MMP inhibitors are registered in mesothelioma. In conclusion, MMPs play an important role in mesothelioma, but further studies are needed to elucidate the potentials of MMPs as biomarkers and drug targets in mesothelioma.

Interlevkin 21 (IL21) je imunomodulatorni citokin, ki deluje na prirojeni in pridobljeni imunski odziv. Povezan je z nastankom različnih bolezenskih stanj. Študije so pokazale, da ima povečanje ali zaviranje aktivnosti IL21 pozitivni učinek pri zdravljenju širokega spektra bolezni. Bolezenska stanja so posledica odstopanja v sintezi/aktivnosti IL21, do katerih lahko pride med drugim tudi zaradi sprememb v zaporedju DNA. Zanimiv model za raziskave IL21 v povezavi z bolezenskimi stanji je kokoš, saj lahko pri tej vrsti v eksperimentalnih, kontroliranih pogojih induciramo avtoimunski artritis z bakterijo Mycoplasma synoviae. Cilj te raziskave je bil preučiti genetsko variabilnost gena IL21 pri kokoši in preveriti, če se te variante nahajajo znotraj regulatornih regij. Določili smo zaporedje dela gena IL21 pri trajni celični liniji kokošjih makrofagov HD-11. Eksperimentalno smo potrdili prisotnost osmih že znanih različic nukleotidnega zaporedja (SNP-jev). Z uporabo bioinformacijskih metod smo napovedali 16 motivov in tri domene v zaporedju proteina, štiri mesta za vezavo mikro RNA ter lokacije in dolžine α-vijačnic. Naredili smo tudi pregled genetskih variant pri človeku, ki so bile povezane z avtoimunskimi boleznimi. Ena se nahaja v regiji, ki ima ortologno zaporedje v genu IL21 pri kokoši, vendar na tej regiji pri kokoši še ni opisanih genetskih variant. Rezultati te študije bodo omogočili nadaljevanje preučevanja gena IL21 pri kokoši in njegove morebitne povezave z nastankom avtoimunskih bolezni. Interleukin 21 (IL21) is an immunomodulatory cytokine that is part of innate and adaptive immune response. It has been associated with the onset of various inflammatory and autoimmune diseases. Studies have demonstrated that increasing or inhibiting IL21 activity has a positive effect in the treatment of a wide range of diseases. Changes in DNA sequence are one of the causes for differences in IL21 synthesis and/or activity. Chicken is an interesting model for studies of IL21 and its association with diseases, as in this species autoimmune arthritis can be induced by the bacterium Mycoplasma synoviae under experimental, controlled conditions. The aim of the present study was to examine genetic variability of the IL21 gene in chicken and to analyze if these variants are located within regulatory regions. We determined the sequence of part of the IL21 gene in macrophage-like immortalized cell line derived from chicken bone HD-11. Eight previously known single nucleotide polymorphisms (SNPs) were experimentally confirmed. With the use of bioinformatic tools we identified 16 predicted motifs and three domains in IL21 protein sequence in chicken. We predicted four miRNA binding sites and locations and lengths of α-helixes. We also performed the review of genetic variants of IL21 gene in human that were associated with autoimmune diseases. One human variant is located in a region that has an orthologous sequence in the IL21 gene in chicken. The results of this study present a baseline for continuation of the study of the IL21 gene in chicken and its possible association with the development of autoimmune diseases.

Olive is considered one of the oldest and the most important cultivated fruit trees in Albania. In the present study, the genetic diversity and structure of Albanian olive germplasm is represented by a set of 194 olive genotypes collected in-situ in their natural ecosystems and in the ex-situ collection. The study was conducted using 26 microsatellite markers (14 genomic SSR and 12 Expressed Sequence Tag microsatellites). The identity analysis revealed 183 unique genotypes. Genetic distance-based and model-based Bayesian analyses were used to investigate the genetic diversity, relatedness, and the partitioning of the genetic variability among the Albanian olive germplasm. The genetic distance-based analysis grouped olives into 12 clusters, with an average similarity of 50.9%. Albanian native olives clustered in one main group separated from introduced foreign cultivars, which was also supported by Principal Coordinate Analysis (PCoA) and model-based methods. A core collection of 57 genotypes representing all allelic richness found in Albanian germplasm was developed for the first time. Herein, we report the first extended genetic characterization and structure of olive germplasm in Albania. The findings suggest that Albanian olive germplasm is a unique gene pool and provides an interesting genetic basis for breeding programs.

V raziskavi smo proučevali genetske in morfološke značilnosti lesnike (Malus sylvestris (L.) Mill.) na območju Slovenije in Hrvaške. Analizirali smo 14 morfoloških znakov na listih, genetska analiza pa je bila opravljena na 10 lokusih z mikrosatelitskimi markerji. Analiza populacij lesnike je pokazala zelo visoko stopnjo genetske variabilnosti lesnike (HT = 0,77) in nizko vrednost fiksacijskega indeksa (FIS = 0,08). Ti rezultati skupaj z visoko stopnjo genetske diferenciacije (FST = 0,18) nakazujejo na veliko genetsko distanco med populacijami in nizko stopnjo razmnoževanja v sorodstvu, kar so značilnosti populacij v ledenodobnih zatočiščih. Analizirana drevesa so bila na podlagi morfometrijske analize razvrščena v skupine lesnik (56,52 %), križancev (39,12 %) in žlahtnih jablan (4,35 %), na podlagi genetske analize pa se deleži dreves po posameznih genetsko določenih skupinah razlikujejo: lesnika (73,91%), križanci (19,57 %) in žlahtne jablane (6,52 %). Le 52 % botanično določenih lesnik se je izkazalo za lesnike tudi na podlagi genetskih raziskav, kar potrjuje dejstvo, da je lesniko na podlagi posameznih morfološih znakov botanično težko povsem zanesljivo določiti. Ugotavljamo, da obstajata dva ločena genska sklada lesnike in žlahtne jablane s številnimi drevesi, ki nosijo mešanico genov obeh vrst. Edini semenski objekt lesnike v Sloveniji ni primeren za uporabo v gozdarstvu, saj niti semenska drevesa niti sadike niso genetsko ustrezne. Podajamo smernice za ohranjanje genskih virov lesnike v prihodnje in priporočamo načine gospodarjenja z gozdom, ki bi lesniki omogočili dolgoročni obstoj in čim večjo gensko pestrost. Genetic and morphological characteristics of European crab apple (Malus sylvestris (L.) Mill.) in the territory of Slovenia and Croatia were studied. We analysed 14 morphological characteristics on the leaves. Genetic analysis was performed using 10 microsatellite markers. The analysis of the population of European crab apple tree population has revealed a very high degree of genetic variability among European crab apple trees (HT = 0.77). The low fixation index (FIS = 0.08) combined with a high degree of differentiation (FST = 0.18) indicates a significant genetic distance between populations and a low rate of kin reproduction, typical of populations in glacial refugia. Based on morphological analysis, the studied trees were classified as European crab apple (56.52%), hybrids (39.12%) and domesticated apple (4.35%), based on genetic analysis, the classification differs: European crab apple (73.91%), hybrids (19.57%), domestocated apple (6.52%). Only 52 % of botanically identified European crab apples were identified as European crab apple based on genetic data, and we confirm that individual morphological characters are not completely reliable for species identification. There are two separate gene pools of European crab apple trees and domesticated apple with a number of trees with a genetic mixture of the two species. The only seed object for European crab apple in Slovenia is not suitable for forestry use. Niether the seed trees nor the seedlings are genetically unsuitable. We have prepared guidelines for the conservation of the European crab apple genetic stock and proposed appropriate silvicultural measures that could enable long-term existence and high genetic diversity.

Opioidni receptor mu, ki ga kodira OPRM1, je ključen za protibolečinsko delovanje opioidnih zdravil. Je tarča tramadola, ki spada med centralno delujoče opioidne analgetike za lajšanje zmernih do močnih akutnih in kroničnih bolečin. Genetski polimorfizmi OPRM1 in genov za miRNA lahko zaradi spremenjenega izražanja ali občutljivosti receptorja vplivajo na izid zdravljenja s tramadolom. V raziskavi smo želeli z genotipizacijo s kvantitativno alelno specifično verižno reakcijo s polimerazo določiti frekvence polimorfizmov OPRM1 rs1799971, OPRM1 rs677830, MIR23B 1011784 in MIR107 rs2296616 v skupini preiskovank po operaciji raka dojke in ovrednotiti njihov vpliv na jakost akutne bolečine in prisotnost neželenih učinkov ter kronične in nevropatske bolečine. Nosilke vsaj enega polimorfnega alela MIR107 rs2296616 A so imele manjše tveganje za pojav slabosti v tretjem tednu zdravljenja, preiskovanke z genotipom MIR23B 1011784 GG pa večje tveganje za pojav slabosti v četrtem tednu. Večje tveganje za pojav zaprtosti kadarkoli v prvem mesecu smo potrdili pri nosilkah vsaj enega polimorfnega alela OPRM1 rs1799971 G in v tretjem tednu pri nosilkah vsaj enega polimorfnega alela OPRM1 rs677830 T. Prisotnost polimorfizma MIR107 rs2296616 je povečala tveganje za kronično bolečino, prisotnost polimorfizma MIR23B 1011784 je povečala tveganje za nevropatsko bolečino, prisotnost polimorfizma OPRM1 rs677830 pa je zmanjšala tveganje za nevropatsko bolečino. Vpliva polimorfizmov na jakost akutne bolečine nismo potrdili. Mu opioid receptor encoded by OPRM1 is crucial for the analgesic effect of opioid drugs. It is the target of tramadol, a centrally acting opioid analgesic for the treatment of moderate to severe acute and chronic pain. Genetic polymorphisms of the OPRM1 and miRNA genes may affect the outcome of tramadol treatment due to altered expression or receptor sensitivity. The aim of this study was to determine the frequencies of OPRM1 rs1799971, OPRM1 rs677830, MIR23B 1011784 and MIR107 rs2296616 polymorphisms in the group of patients after breast cancer surgery. Genotyping with quantitative allele-specific polymerase chain reaction was performed to evaluate the impact of these polymorphisms on the severity of acute pain and the presence of adverse effects, chronic and neuropathic pain. Carriers of at least one polymorphic MIR107 rs2296616 A allele had a lower risk of nausea in the third week of treatment, and patients with MIR23B 1011784 GG genotype had a higher risk of nausea in the fourth week. A higher risk of constipation during the first month was confirmed in carriers of at least one polymorphic OPRM1 rs1799971 G allele and in the third week in carriers of at least one polymorphic OPRM1 rs677830 T allele. The presence of MIR107 rs2296616 polymorphism increased the risk of chronic pain, the presence of MIR23B 1011784 polymorphism increased the risk of neuropathic pain, and the presence of the OPRM1 rs677830 polymorphism reduced the risk of neuropathic pain. The effect of the polymorphisms on the intensity of acute pain has not been confirmed.

Rak želodca je maligni tumor, ki vznikne v želodčni sluznici, lahko pa tudi iz žleznih ali limfatičnih celic v steni želodca. Je peto najpogostejše rakavo obolenje na svetu in tretji najpogostejši vzrok smrti, povezane z rakom. Kljub napredkom v diagnostiki in zdravljenju ostaja petletno preživetje bolezni slabo. Razvoj bolezni je kompleksen proces, na katerega lahko vpliva veliko okoljskih dejavnikov tveganja, povežemo pa ga lahko tudi z različnimi genetskimi dejavniki, med katerimi so tudi polimorfizmi posameznega nukleotida (SNP), ki lahko vplivajo na pojav in potek bolezni. Glavni namen magistrske naloge je bil določanje kandidatnih genetskih variacij ali različic z uporabo bioinformacijskih pristopov na podlagi vnaprej izbranih kriterijev, kot so pogostnost alelov, lokacija v bližini funkcionalnih elementov in/ali lokacija v promotorskem področju ali 3' koncu gena (v nadaljevanju 3'UTR regija). Analizo kandidatnih genetskih različic smo izvedli na vzorcih DNA iz tkiv bolnikov z rakom želodca in na vzorcih DNA iz krvi zdravih posameznikov. Raziskovalna naloga je bila zastavljena kot študija primerov s kontrolami. S statistično analizo genetskih različic v populacijah bolnikov z rakom želodca in zdravih posameznikov sem opredelila povezavo med izbranimi različicami in dovzetnostjo za razvoj bolezni. Gastric cancer is a malignant carcinoma of the digestive tract and as such the fifth most common cancer malignancy worldwide and the third most common cause of cancer related deaths in the world. Although many advances in diagnosing and treatment in the last few decades, the 5-year survival rate remains poor. The developement of gastric cancer is a complex, multifactorial process with various potential environmental risk factors, but however it can also be related to different genetic factors, including single nucleotide polymorphisms (SNPs), which may contribute to the developement of the disease. The aim of my master's thesis was to determine candidate genetic variations with selected bioinformatic tools. Selected variations had to meet our criteria, such as allele frequency over 0,1, location of polymorphisms in the promoter region or in the 3'-untranslated region of the gene. The study was performed on DNA derived from tissue of patients with gastric cancer and blood samples from healthy subjects of control group. The correlation between selected polymorphisms and susceptibility to the disease was tested by statistically analysing selected genetic variations of gastric cancer patients and healthy subjects.

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder of multifactorial etiopathology likely to involve the interactions between genetics and lifestyle. Chronic inflammation and oxidative stress (OS) may participate in the pathophysiology of the syndrome. The question of the extent to which OS and inflammation are causally related to the development of the syndrome and metabolic complications remains unanswered. By our knowledge, the role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome as an important trigger of inflammatory pathways and NLRP3 and CARD8 polymorphisms has never been addressed in PCOS yet. We conducted a case-control study conducting of total 169 Slovenian PCOS patients and 83 healthy blood donors. They were genotyped for polymorphisms in antioxidative (SOD2 rs4880, CAT rs1001179, PON1 rs854560, and rs662) and inflammatory pathways genes (NLRP3 rs35829419, CARD8 rs2043211, TNF rs1800629, IL1B rs1143623, and rs16944, IL6 rs1800795) using competitive allele-specific polymerase chain reaction (PCR). Logistic regression and the Mann&ndash, Whitney test were used in the statistical analysis. SOD2 rs4880, CARD8 rs2043211, and IL1B rs16944 were associated with the risk of developing PCOS. Furthermore, the interactions between CARD8 rs2043211 and IL6 rs1800795 and between IL1B rs1143623 and IL6 rs1800795 also significantly affected the risk for PCOS. With regard to glucose homeostasis, CAT rs1001179, SOD2 rs4880, PON1 rs854560, NLRP3 rs35829419, and TNF rs1800629 were significantly associated with response to the glycemic load. Our data indicate that the genetic variability in the antioxidative and inflammatory pathways influences the development of PCOS and glucose homeostasis in PCOS patients.

Mikro RNA so kratke nekodirajoče RNA, ki uravnavajo izražanje genov. Preko vplivov na razne biološke poti v organizmu lahko prispevajo tudi k spremembi fenotipa in posledično k uspešnosti prireje rejnih živali. S tem postanejo miRNA uporabne tudi kot biooznačevalci v selekciji živali. Za analizo smo izbrali miRNA gen gga-mir-1658 pri kokoši. Namen te naloge je bil: 1. pridobiti znane genomske podatke o gga-mir-1658 pri kokoši in 2. z molekularno-genetskimi metodami analizirati povezavo med SNP-ji (rs316300182 in rs1059378319) v gga-mir-1658 ter desetimi pitovnimi in klavnimi lastnostmi kokoši. V prvem delu smo informacije o gga-mir-1658 iz osmih podatkovnih zbirk in bioinformacijskih orodij združili v atlas regulatornih elementov. Z asociacijsko analizo smo nato potrdili povezavo med SNP-jem rs1059378319 in maso jeter (p = 0,05). Živali z genotipom CC so imele za 5,91 g težja jetra (p = 0,02) kot živali z genotipom CT. Povezava med rs316300182 in maso jeter je bila le nakazana (p = 0,07). Živali z genotipom CC so imele za 5,66 g težja jetra (p = 0,06) kot živali z genotipom CG. Povezave ostalih izmerjenih lastnosti s prej omenjenima SNP-jema niso bile značilne. Rezultati nakazujejo na to, da je gga-mir-1658 zanimiva za nadaljnje raziskave. S testiranjem na večji in pasemsko bolj raznoliki populaciji bo možno bolje raziskati njen funkcionalni vpliv in njen potencial biooznačevalca v selekciji kokoši. MicroRNAs are small noncoding RNAs that regulate gene expression. Through effects on various biological pathways in organism, miRNAs contribute to phenotype changes and consequently on efficiency of animal production. That is why miRNAs became useful as biomarkers in animal selection. For the analysis we selected miRNA gene gga-miR-1658. The aim of this study was: 1.) to retrieve known genomic information about gga-mir-1658 in chicken and 2.) to analyze the association between two SNPs (rs316300182 and rs1059378319) in gga-mir-1658 gene and ten growth and carcass traits using molecular-genetic methods. Firstly, we designed an atlas of regulatory elements for gga-mir-1658 from eight databases and bioinformatics tools. In the second part, we confirmed an association between rs1059378319 and liver mass (p = 0.05). Animals with CC genotype had 5.91g heavier liver (p = 0.02) than animals with CT genotype. Association between SNP rs316300182 and liver mass (p = 0.07) did not meet the level of significance, but was close. Animals with CC genotype had for 5.66g heavier liver (p = 0.06) as animals with a genotype CG. Other growth and carcass traits did not reach significant associations with these two SNPs. Results suggest that gga-mir-1658 is interesting for further studies. Testing on a larger population and including additional breeds is required to functionally characterize it and potentially apply it as a biomarker in chicken selection programs.

Družinska eritrocitoza je redka, prirojena bolezen s heterogenim genetskim ozadjem, opredeljena s povišanim številom eritrocitov ter povečanim hematokritom in hemoglobinom v krvi. S hipoksijo inducirani transkripcijski dejavniki alfa (HIFA) vplivajo na povišanje števila eritrocitov z aktivacijo prepisovanja tarčnih genov pri nizkih koncentracijah kisika. Različice nukleotidnega zaporedja v genu za eno izmed treh izoform HIFA, za endotelijsko PAS domeno 1 (EPAS1), vodijo do razvoja družinske eritrocitoze tipa 4. Z raziskavo smo ob sodelovanju s Specializiranim hematološkim laboratorijem Kliničnega oddelka za hematologijo Univerzitetnega kliničnega centra (UKC) Ljubljana postavili diagnostični test za identifikacijo različic, povezanih z družinsko eritrocitozo, v nukleotidnem zaporedju gena EPAS1. Pri tem smo optimizirali reakcijo PCR za pomnoževanje treh izbranih regij gena EPAS1 ter z metodo po Sangerju določili zaporedje izbranih regij v skupni velikosti 1374 baznih parov. Z novo razvito diagnostično metodo smo analizirali vzorce štirih bolnikov iz treh družin in dveh zdravih preiskovancev kontrolne skupine. Rezultati so pokazali, da so vsi bolniki homozigoti (GG) na mestu različice nukleotidnega zaporedja c.C1035-7G (rs7557402) in trije bolniki homozigoti (TT) na mestu različice nukleotidnega zaporedja c.C1249+151T (rs11678817). Na obeh lokusih je bil sorodni preiskovanec kontrolne skupine heterozigot, medtem ko je bil nesorodni preiskovanec kontrolne skupine homozigot (CC). Za ovrednotenje pomena identificiranih različic nukleotidnega zaporedja pri nastanku eritrocitoze so potrebne nadaljnje raziskave. Familial erythrocytosis is a rare congenital disorder with heterogenous genetic background, defined by increased red blood cell number, elevated haematocrit and haemoglobin in blood. Hypoxia-inducible factors alpha (HIFA) activate transcription of target genes in low-oxygen conditions and consequently upregulate red blood cell production. Sequence variants in gene for one of three HIFA isoforms, endothelial PAS domain protein 1 (EPAS1), lead to the development of familial erythrocytosis type 4. In collaboration with the Specialized haematological laboratory of the Clinical department of haematology, University Medical Centre Ljubljana, we established diagnostic test for identification of EPAS1 gene variants associated with erythrocytosis. We optimized PCR reaction for amplifiqation and sequenced three selected regions, with total length of 1374 base pairs, using Sanger method. Newly developed diagnostic method was used to analyse samples of four patients with unknown eythrocytosis from three different families and two healthy individuals from control group. All four patients were homozygous (GG) for sequence variant c.C1035-7G (rs7557402) and three patients were homozygous (TT) for sequence variant c.C1249+151 (rs11678817). Related individual from control group was heterozygous, whereas unrelated individual from control group was homozygous (CC) at both loci. To elucidate the effect of identified variants on development of erythrocytosis, additional research is needed in the future.

Družinska eritrocitoza je redka dedna bolezen, za katero je značilno povišano število rdečih krvničk, lahko tudi povišan hematokrit in hemoglobin. Bolniki s to boleznijo se srečujejo s simptomi, kot so glavoboli, slabosti, krvavitve iz nosu, tromboze in krvavitve, ki lahko vodijo tudi do smrti. Tumor-supresorski protein von Hippel-Lindau (VHL) je del kompleksa, ki sodeluje pri razgradnji proteinov, tudi transkripcijskega dejavnika endotelijske PAS domene, ki se aktivira ob pomanjkanju kisika. Različice nukleotidnega zaporedja v genu VHL lahko vodijo do razvoja družinske eritrocitoze tipa 2. V raziskavi smo v sodelovanju s Specializiranim hematološkim laboratorijem kliničnega oddelka za hematologijo, interna klinika Univerzitetnega kliničnega centra Ljubljana, postavili diagnostično metodo za analizo različic nukleotidnega zaporedja gena VHL povezanih z družinsko eritrocitozo. Optimizirali smo reakcijo PCR, pomnožili odseke gena VHL ter jih sekvencirali z metodo po Sangerju. Z novo razvitim diagnostičnim testom smo analizirali DNA štirih bolnikov z eritrocitozo neznanega vzroka. Na podlagi sekvenčnih reakcij smo ugotovili, da nihče od preiskovanih bolnikov nima različice nukleotidnega zaporedja v genu VHL. S tem smo pri izbranih bolnikih izključili bolezen družinsko eritrocitozo tipa 2. V prihodnje se bo uveden molekularno-genetski test v kombinaciji z drugimi testi ugotavljanja različic v genih, povezanih z družinsko eritrocitozo, uporabljal za rutinsko diagnostiko pri bolnikih z eritrocitozo neznanega vzroka. Familial erythrocytosis is a rare inherited disease characterized by an increased number of red blood cells, often also elevated hematocrite and hemoglobin. Patients have symptoms such as headaches, nausea, thrombosis and bleeding, which can lead to death. Von Hippel-Lindau tumor suppressor protein (VHL) is part of a complex involved in the degradation of proteins, including the Endothelial PAS domain-containing protein 1 transcription factor, which is activated in the presence of oxygen deficiency. Sequence variants in VHL gene can lead to the development of familial erythrocytosis type 2. In collaboration with the Specialized haematological laboratory of the Clinical department of hematology, internal clinic University Medical Centre Ljubljana, we established a diagnostic method for the VHL variants in patients with familial erythrocytosis. We optimized PCR, amplified fragments of VHL gene and sequenced then with Sanger method. The new diagnostic test was used to analyse four patients with unknown causes of erythrocytosis. Based on sequential reactions, we confirmed that none of the examined patients had a variation in the VHL gene and therefore we excluded familial erythrocytosis type 2. In the future, this molecular-genetic test, with other tests on different genes, could be used for routine screening of variants in patients with idiopathic erythrocytosis.

Jablana (Malus domestica Borkh.) je sadno drevo iz družine rosaceae. Je tretja najbolj gojena sadna vrsta, njena pridelava pa je razširjena po celem svetu. Na Finskem jablana sodi med ekonomsko manj pomembne kulturne rastline, vendar je priljubljeno vrtno in kmetijsko drevo ter ima zgodovinsko in kulturno pomembno vlogo. Zaradi lege Finske, ki predstavlja najsevernejšo regijo, kjer jablana še uspeva, so sorte, ki rastejo tu, posebej zanimive z vidika genetske raznovrstnosti. V tej študiji smo analizirali finske sorte jablan z namenom določitve jedrne zbirke. Za namene te študije smo zbrali 704 vzorcev jablan, ki rastejo širom Finske in izolirali njihovo DNK. Genetska identiteta vzorcev je bila analizirana s 7 mikrosatelitskimi markerji SSR (simple sequence repeat). Študije genetske strukture so pokazale, da je raven genetske variabilnosti finskih jablan (He = 0,8, Ae = 6,2) primerljiv z drugimi evropskimi populacijami jablan. Na podlagi dobljenih genetskih podatkov smo določili genotipe, ki predstavljajo jedrno zbirko, ki jo sestavlja 37 enot. Uporabili smo šest različnih metod izgradnje jedrne zbirke in jih primerjali med sabo. Na osnovi primerjav različnih parametrov smo izbrali najboljšo med njimi, in sicer jedrno zbirko, osnovano s programom CoreFinder. Podane so tudi smernice za implementacijo, vzdrževanje in uporabo pripravljene jedrne zbirke. The apple (Malus domestica Borkh.) is a fruit tree of the Rosaceae family. It is grown all across the world, and is the third most widely produced fruit crop globally. Although it is a less important crop in Finland economically, its historical, cultural and societal significance is considerable, and it is widely popular in home gardens. At the same time, Finland is the northernmost region where apples can be cultivated, and because of the growing conditions, Finnish apple trees contain valuable genetic variations. In this thesis, we studied Finnish apple germplasm with the aim of compiling a core collection. To perform this task, we studied 704 apple accessions inventoried from southern and central Finland, and Southern Ostrobothnia. The genetic identity of the samples was observed from isolated DNA using 7 microsatellite simple sequence repeat (SSR) markers. The study of the genetic structure showed a rate of variability and diversity comparable to other European apple populations. Based on microsatellite data, we constructed a core collection consisting of 37 entries of Finnish apple trees. Six different methods of core collection construction were tested and compared. Based on a comparison of heterozygosity, allelic diversity and population structure, the core collection compiled with CoreFinder was selected as the best approach. Moreover, guidelines are presented for the implementation of the proposed core collection, with its maintenance and further development in mind.

Raziskava obravnava genetsko variabilnost, genetsko raznolikost in genetsko diferenciacijo bukve (Fagus sylvatica L.) v Sloveniji. S pomočjo izoencimske analize smo pregledali 13 avtohtonih bukovih populacij. Polimorfizem smo odkrili na 11 od 12 analiziranih izoencimskih lokusih, 9 lokusov je polimorfnih v vseh populacijah. Opazen je rahel trend naraščanja genetske variabilnosti v smeri od severa proti jugu, medtem ko povprečna ugotovljena heterozigotnost Ho od severa proti jugu upada. Genetska diferenciranost je razmeroma majhna, med populacijami pa nismo odkrili diferenciranosti, ki bi ji lahko pripisali jasno geografsko pogojenost. The study investigates the genetic variability, genetic diversity and genetic differentiation of the beech (Fagus sylvatica L.) in Slovenia. On the basis ofisozyme analysis, 13 indigenous beech populations were examined. Polymorphism was revealed in 11 isozyme loci, 9 loci show polymorphism in all treated populations. The trend of slight north-south increase of genetic variability is present, while mean observed heterozygosity Ho decreases in thenorth-south direction. Genetic differentiation is relatively weak and we revealed no geographically conditioned differentiation among the populations. The study investigates the genetic variability, genetic diversity and genetic differentiation of the beech (Fagus sylvatica L.) in Slovenia. On the basis ofisozyme analysis, 13 indigenous beech populations were examined. Polymorphism was revealed in 11 isozyme loci, 9 loci show polymorphism in all treated populations. The trend of slight north-south increase of genetic variability is present, while mean observed heterozygosity Ho decreases in thenorth-south direction. Genetic differentiation is relatively weak and we revealed no geographically conditioned differentiation among the populations. The study investigates the genetic variability, genetic diversity and genetic differentiation of the beech (Fagus sylvatica L.) in Slovenia. On the basis ofisozyme analysis, 13 indigenous beech populations were examined. Polymorphism was revealed in 11 isozyme loci, 9 loci show polymorphism in all treated populations. The trend of slight north-south increase of genetic variability is present, while mean observed heterozygosity Ho decreases in thenorth-south direction. Genetic differentiation is relatively weak and we revealed no geographically conditioned differentiation among the populations.

V prispevku so predstavljene raziskave, ki obravnavajo razširjenost navadne jelke (Abies alba Mill.) na ozemlju današnje Slovenije v pleistocenu in holocenu. Zbrani so podatki o paleobotaničnih ostankih (fosilni ostanki iz terciarja, pelod, makroskopski rastlinski ostanki) v paleolitskih postajah in o genetskih značilnostih današnjih jelovih populacij. V prispevku razpravljamo o možnem obstoju ledenodobnih zatočišč in lokalnem postglacialnem razvoju populacij jelke v Sloveniji. The paper reviews the research dealing with the presence of silver fir (Abies alba Mill.) within modern-day Slovenian territory in the Pleistocene and Holocene. The data on paleobotanical remains (Tertiary fossil remains, pollen,macroscopic plant remains) on Paleolithic sites and on genetic properties of the present silver fir populations are presented. The possible existence of glacial refugia and local postglacial development of silver fir populations in Slovenia are discussed.

Izhodišča: Clostridium difficile, črevesna patogena bakterija, je ena izmed najpomembnejših povzročiteljev bolnišničnih okužb na svetu. Bolezen povzročajo toksigeni sevi, ki izdelujejo toksin A in/ali toksin B. Oba sta zapisana na toksinskem lokusu PaLoc. PaLoc se nahaja v bakterijskem kromosomu in je zelo dobro raziskan genetski element. Glede na razlike v PaLoc-u lahko seve razdelimo v različne toksinotipe. Nekateri sevi C. difficile izdelujejo še tretji toksin, binarni toksin CDT, katerega vloga pri razvoju bolezni pa še ni povsem pojasnjena, vendar zadnje raziskave potrjujejo, da postaja pomemben člen v patogenezi okužbe s C. difficile. Zapis za binarni toksin se prav tako nahaja v kromosomu, na lokusu imenovanem lokus Cdt (CdtLoc), ki je mnogo slabše raziskan kot PaLoc. Metodologija: V raziskavi smo z bioinformatskimi orodji (Artemis, BioEdit Sequence Alignment Editor in MEGA) analizirali lokus Cdt in gene, ki se nahajajo navzgor in navzdol od CdtLoc-a, ter tako preverili ali se nahaja vedno na istem mestu v genomu. Genetsko variabilnost genov, ki sestavljata CdtLoc (cdtA in cdtB) smo določili na nukleotidnem in aminokislinskem zaporedju. Sorodnosti med sevi smo predstavili s konstrukcijo filogenetskih dreves za kar smo uporabili algoritem združevanja sosedov. Rezultati: Ugotovili smo, da lokus Cdt pri vseh predstavnikih toksinotipov vključenih v analizo leži na istem mestu v kromosomu. Analiza nukleotidnega in aminokislinskega zaporedja nam je pokazala, da se v genih, ki sestavljata CdtLoc, pojavljajo le točkovne mutacije, ki so večinoma enakomerno razporejene po celotni dolžini gena, rahlo je povišano le njihovo število na 5'-koncu cdtB gena. Filogenetska analiza na podlagi vseh treh genov, ki sestavljajo CdtLoc in združenih MLST lokusov, pa je pokazala enake sorodstvene odnose med sevi. Sklep: Naši rezultati kažejo, da se CdtLoc nahaja vedno na istem mestu v genomu in da k variabilnosti obeh toksinskih genov največ prispevajo točkovne mutacije. Glede na ujemanje filogenetskih dreves na podlagi genov CdtLoc-a in MLST lahko sklepamo, da se lokus Cdt verjetno prenaša primarno vertikalno. Introduction: Clostridium difficile is a nosocomial pathogen and it is one of the most important causes of health care-associated infections. Disease is caused only by toxigenic strains that produce toxin A and/or toxin B, which are the main virulence factors encoded in the pathogenicity locus PaLoc. PaLoc, a well defined genetic element, is located in the chromosome. Based on the changes in the PaLoc strains can be distributed into different toxinotypes. Some C. difficile strains can produce also a third toxin, binary toxin CDT. Even though its role in disease is still unclear, latest studies suggests that CDT could play an important part in the pathogenesis of C. difficile infection. Binary toxin is encoded in the binary toxin locus, named CdtLoc. Like PaLoc, CdtLoc is also found in the chromosome, but its genetic variability is less studied compared to the PaLoc. Methodology: The aim of this study was to analyze the binary locus Cdt and to check whether CdtLoc is integrated always at the same location in the genome by analysis of genes located upstream and downstream of the CdtLoc, using different bioinformatics tools (Artemis, BioEdit Sequence Alignment Editor and MEGA). Genetic variability of both toxin genes (cdtA and cdtB) was defined on the nucleotide and amino acid sequences. Furthermore, phylogenetic relationships between different strains based on binary toxin genes were explored with the construction of phylogenetic trees using neighbor-joining algorithm. Results: In all 20 analyzed strains was CdtLoc inserted in the same chromosomal site. Analysis of nucleotide and amino acid sequences demonstrated that genetic variability of CdtLoc is due to accumulation of point mutations. Point mutations were more or less equally distributed through the toxin genes, their number is slightly increased only on the 5'-end of the cdtB gene. Also, phylogenies based on all three genes, encoded in the CdtLoc (cdtA, cdtB and cdtR) were congruent with phylogeny demonstrated with concatenated MLST loci. Conclusion: Our results indicate the same chromosomal integration site of CdtLoc and that point mutation are responsible for the genetic variability of both toxin genes. Congruent phylogenetic relationships based on CdtLoc genes and MLST indicate that the transfer of the binary locus Cdt is primarily vertical.

Vpliv onesnaževanja ozračja na genetsko strukturo bukovih populacij v Sloveniji

V prispevku so predstavljene raziskave, ki obravnavajo razširjenost navadne jelke (Abies alba Mill.) na ozemlju današnje Slovenije v pleistocenu in holocenu. Zbrani so podatki o paleobotaničnih ostankih (fosilni ostanki iz terciarja, pelod, makroskopski rastlinski ostanki) v paleolitskih postajah in o genetskih značilnostih današnjih jelovih populacij. V prispevku razpravljamo o možnem obstoju ledenodobnih zatočišč in lokalnem postglacialnem razvoju populacij jelke v Sloveniji. The paper reviews the research dealing with the presence of silver fir (Abies alba Mill.) within modern-day Slovenian territory in the Pleistocene and Holocene. The data on paleobotanical remains (Tertiary fossil remains, pollen,macroscopic plant remains) on Paleolithic sites and on genetic properties of the present silver fir populations are presented. The possible existence of glacial refugia and local postglacial development of silver fir populations in Slovenia are discussed.

Namen naše raziskave je bil s pomočjo vzorčenja in proučevanja nekaterih enostavnih morfološko-funkcionalnih parametrov (višina rastlin, masa semen, število semen na plod, število plodov na rastlino) na primerkih vrste C. cava na treh geografsko različnih območjih (UZ Maribor, Spodnji Duplek, Pavlovci) pokazati negativni vpliv fragmentacije habitatov in populacij na te morfometrične parametre. S tem smo nakazali na zmanjšano viabilnost primerkov kot posledico fragmentacije. V raziskavo smo vključili popise, ki smo jih naredili leta 2006. Takrat je bilo na območju UZ Maribor najdenih 60 poligonov z vrsto C. cava. Na 3 poligonih so bila najdena rastišča tako belih kot tudi vijoličnocvetnih predstavnikov, na preostalih 57 poligonih pa le vijoličnocvetni predstavniki vrste. Na podlagi opravljenih popisov območja UZ Maribor smo ugotovili 6,7 % izgubo habitata vrste C. cava v le štirih letih. Ugotovili smo, da je število cvetočih osebkov C. cava v habitatnih fragmentih znotraj UZ Maribor odvisno od površine fragmenta. Število plodov na rastlino in masa semen kot najenostavnejša reproduktivna znaka izražata manjše vrednosti v maloštevilnih populacijah. Ugotovili smo, da se belocvetni primerki pojavljajo le znotraj velikih površin in da jih na manjših fragmentiranih površinah ni zaslediti. S tem sklepamo na negativni vpliv genetskega zdrsa ter na dolgoročno zmanjšanje variabilnosti v populaciji. Višina rastlin je v upadu pri obeh barvah. V upadu je tako število plodov na rastlino kot tudi število semen na plod, kar izraziteje velja za vijoličnocvetne primerke vrste. Tehtanje semen je pokazalo porast mase semen, kar je edini znak, v katerem je zazanati povišanje vrednosti na fragmentirani lokaciji v Spodnjem Dupleku. The purpose of our research was to indicate the negative influence of the fragmentation of habitats and populations on these morphometrical parameters with the help of sampling and studying of simple morphological-functional parameters (height of plants, mass of seeds, number of seeds per fruit, number of fruits per plant) on the samples of the C. cava species on three geographically diverse regions (UP of Maribor, Spodnji Duplek, Pavlovci). With that we indicated on the reduced viability of samples as the consequence of the fragmentation. All surveys done in 2006 were included in this research. Sixty ranges with the C. cava species were found then in the region of UP of Maribor. There were found the representatives of white and purple holewrts flowers at three ranges. At the remaining 57 ranges only the representatives of the species of purple flowers were found. On the basis of the surveys in the region of UP of Maribor, the loss of 6,7% of natural habitats of holewort was established in only four years. We estimated, that the number of flowering specimens of holewort in the habitat fragments within UP of Maribor depends on the surface area of the fragment. The number of fruits per plant, the number of seeds per fruit and the mass of seeds, as the simpliest reproductive signs, express lower values in populations that are small in number. We estimated, that white holewort flowers appear only within big surface areas and that they aren't traced on smaller fragmentary surface areas. According to this, we can conclude to the negative impact of genetic drift and to the long-term diminishing of variability in population.The height of plants is in decrease by both colours. There is also the decrease in the number of fruits per plant as well as the number of seeds per fruit, which outstandingly goes for purple flowers samples of the species.The weighing of the seeds showed the increase of the mass of the seeds, that is the only sign in which the increase of the value on the fragmentary location in Spodnji Duplek is perceived.