INVESTIGATING ORIGIN AND FUNCTIONAL IMPACT OF GENOMIC STRUCTURAL VARIANTS WITH NEXT-GENERATION SEQUENCING

Genomic variants play an important role in phenotypic variation and have significant impact on a disease development. Due to the technology limitations, inference of genomic variants and their potential consequence on phenotype was until recently restricted. Only with the advent of next-generation sequencing (NGS) approaches, could a vast majority of genomic variants be successfully identified for the first time. In my PhD Thesis I will present my work on structural variants (SVs), their formation mechanism and their functional impact. The first part of my Thesis focuses on structural variants in non-human primates, studies of which using NGS have not been pursued prior to the research studies we carried out. In order to inspect the origin and functional impact of SV formation mechanisms, we constructed a comprehensive SV map based on the fibroblast-derived DNA from three different species: chimpanzee, orangutan and rhesus macaque. We noted striking differences in the activity of homology-related SV formation mechanisms between the great apes and rhesus macaques, with a third of the chimpanzee and orangutan SVs inferred to be formed by non-allelic homologous recombination compared with only 2% of the macaque SVs. One additional key finding was the presence of a markedly higher mobile element activity in macaques compared to the other non-human primates studies. Additionally, we could show that long L1 elements surpassed Alu activity in chimpanzee and orangutan as opposed to macaque where AluMacYa3 dominates the genomic landscape causing a burst of relatively short SVs. In addition to inserting into genome, active L1 elements possess the ability to mobilize 3’ flanking DNA to different genomic loci as transductions. By combining translocation and L1 discovery pipelines we further developed a novel computational methodology, termed TIGER, for the discovery of polymorphic L1-mediated 3’ transductions. We employed TIGER to a deeply sequenced human genome and to aforementioned non-human primates species to characterize transductions. TIGER enables studying germline L1-mediated 3’ transductions, making a relevant structural variation class amenable for population and disease studies for the first time. In the second part of my Thesis, I discuss the differences in the formation mechanisms of both germline and somatic SVs in the human genome. Our de novo mechanism classification analyses performed on four previously published SV datasets revealed that almost half of germline human SVs are due to mechanisms independent of homology, followed by homology-related DNA repair, mobile elements and variable number of tandem repeats. We also investigated the formation of somatic SVs in four medulloblastoma brain tumor patients with a germline TP53 mutation (Li- Fraumeni syndrome). In contrast to the germline SVs, our analyses of rearrangement breakpoints in medulloblastoma in the context of mutated TP53, rather support a model of massive DNA double strand breaks known as chromothripsis, followed by exclusive homology-independent repair

Malignancy-related hyponatremia: Case report

Hiponatrijemija je čest elektrolitski poremećaj kod hospitalizovanih bolesnika. Nakon potvrđivanja laboratorijskog nalaza, potrebno je pojavu hiponatrijemiju etiološki razjasniti. Prikaz bolesnika: Bolesnica stara 56 godina, hospitalizovana je zbog evaluacije grčeva nogu i malaksalosti. U biohemijskim nalazima se registruje hipotona hiponatrijemija, te je posumnjano na SIADH (engl. Syndrome of Inappropriate Antidiuretic Hormone Secretion). Evaluacijom SIADH-a, nije nađen uzrok hiponatrijemije. Primenom simptomatske terapije i korekcijom hiponatrijemije, pacijentkinja je otpuštena lako poboljšana. Četiri meseca nakon hospitalizacije, javlja se konfuzna, sa gušenjem, sindromom hiperglikemije i teškim opštem stanjem. Pored toga, kod pacijentkinjw su laboratorijski potvrđene hipotone hiponatrijemije, ultrasonografijom abdomena su viđene metastatske promene na jetri. Ubrzo nakon prijema, dolazi do smrtnog ishoda. Rodbina je odbila obdukciju, te primarno ishodište malignoma nije utrvđeno. Hipotona hiponatrijemija zahteva ozbiljnu kliničku evaluaciju. SIADH je najčešći uzrok hipotone hiponatrijemije, a najvažniji uzrok SIADH-a je maligna bolest. Pažljiva korekcija hiponatrijemije i lečenje osnovne bolesti je osnova lečenja SIADH-a.Hyponatremia is a common electrolyte disorder in hospitalized patients. In the case of repetitive biochemically confirmed hyponatremia, it is necessary to find its cause. Case report: a 56-year-old woman was admitted to hospital due to leg cramps and malaise. Routine biochemical analysis revealed hypotonic hyponatremia and Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH) was suspected. The cause of hyponatremia was not discovered by means of evaluating SIADH. The patient was simptomatically treated, and discharged in better general condition, with partially corrected hyponatremia. She was advised to restrict water intake and to repeat serum electrolytes and BUN regularly. Four months later, she presented with dyspnea and hyperglycemic syndrome, she was confused and her life was threatened. Beside severe hyponatremia and hyperglycemia, an urgent abdominal ultrasonography showed liver secondaries. Shortly after admission, the patient passed away. Unfortunately, the patient's family refused an autopsy, so the origin of neoplasia has remained unknown. It is necessary to do a thorough clinical evaluation of hypotonic hyponatremia. SIADH, as the most frequent cause of hypotonic hyponatremia, is often a paraneoplastic syndrome. Careful correction of hypotonic hyponatremia and management of the underlying disease is the mainstay of SIADH treatment

Correlation of metabolic profile and Bone mineral density in treatment: Naive females with subclinical thyroid dysfunction: A pilot study

Uvod: Poznat je uticaj manifestnih tireoidnih disfunkcija na promene u koštanoj masi koji se ispoljava kroz izmene u koštanom prometu, koje dovode do promena koštane gustine i mogućeg povećanog rizika od pada i preloma. Takođe, uticajem na metaboličke parametre, prevashodno na lipide, tireoidne disfunkcije predstavljaju važan uzrok akceleracije ateroskleroze i povećanja cerebrovaskularnog i kardiovaskularnog rizika. Ovom studijom je ispitivan metabolički profil i koštana gustina kod pacijenata sa dijagnostikovanom supkliničkom tireoidnom disfunkcijom. Materijal i metode: Pilot studija preseka obuhvatila je 45 selektovanih ispitanica, koje su ispunile inkluzione kriterijume. Na osnovu nivoa tireo-stimulišućeg hormona (TSH) i slobodnog tiroksina (fT4) ispitanice su svrstane u 3 grupe, po 15 ispitanica u svakoj grupi: -grupa: supklinička hipotireoza (SHipo), grupa: supklinička hipertireoza (SHiper), i grupa: eutireodna-tj. kontrolna grupa. Od faktora rizika, praćeni su godine života, podaci o menopauzi i prethodnim prelomima, a od metaboličkih parametara indeks telesne mase (engl. Body Mass Index, BMI), obim struka (OS), obim kuka (OK), nivo tri-glicerida i nivo ukupnog holesterola. Kod svih ispitanica je merena koštana gustina na lumbalnoj kičmi i levom kuku (LK) i izražena u T skorovima. Dobijeni podaci su analizirani koriščćenjem statističkog paketa SPSS za Windows. Statistički značajnom je smatrana verovatnoća manja od 0,05 (p<0,05). Rezultati: Grupe ispitanica su se značajno razlikovale po nivoima ukupnog holesterola, kao i T skorovima lumbalne kičme i LK. Smanjena koštana gustina je registrovana kod 35 (77,8%) ispitanica. Vrednosti T skorova lumbalne kičme i LK su unutargrupno međusobno korelisali. Od faktora rizika, postojanje prethodnih preloma i menopauze, značajno utiču na T skorove, što nije slučaj ni sa jednim metaboličkim parametrom. Najniži T skorovi, kao i sa kliničkog aspekta najznačajnija dislipidemija, su registrovani u grupi SHipo. Diskusija: U okviru ove pilot studije pokazano je smanjenje koštane gustine u populaciji ispitanica sa supkliničkim tireoidopatijama, najviše izražena u SHipo grupi. U istoj grupi je zabeležen i klinički najaterogeniji lipidni profil (hiperholesterolemija). Metabolički parametri kao i hormoni nisu uticali na T skorove lumbalne kičme i LK. Zaključak: Supkliničke tireoidne disfunkcije su povezane sa smanjenjem koštane gustine, izražene kroz T skorove na lumbalnoj kičmi i LK, i na njih nisu uticali metabolički parametri. Pravovremenom dijagnostikom i lečenjem tireoidnih disfunkcija, moguće je usporiti gubitak koštane mase, smanjiti rizik kako od pada tako i od preloma, pri čemu korekcijom lipidnog disbalansa se može smanjiti potencijalni cerebrovaskularni i kardiovaskularni rizik.Introduction. It is well-known that overt thyroid dysfunctions have an influence on bone mineral density (BMD). It is noticed as a change in bone turnover and BMD, with subsequent increased risk of fall-induced fractures. Additionally, promoting the lipid profile derangement, subclinical thyroid dysfunction seems to be an important factor of atherosclerosis acceleration and the risk of cerebrovascular and cardiovascular incidents. In this study we examined the relationship between some of osteoporosis risk factors, metabolic parameters and BMD, in subclinical thyroidopathies. Material and Methods. Pilot cross-sectional study involved 45 females selected on fulfilled inclusion criteria. Three groups of 15 examinees were formed, based on TSH and fT4 serum levels (SHypo, SHyper, and euthyroid-control). As for risk factors, age, records on menopause and history of past fractures as well as metabolic parameters Body Mass Index (BMI), waist and hip circumference, cholesterol and triglyceride levels were observed. The entire study population was referred to DEXA (Dual Energy X Ray Absorptiometry) scan for BMD measurement on lumbar spine (LS) and left hip (LH), expressed through T scores. The obtained data were analyzed by SPSS for Windows 18.0 statistical package. The level of statistical significance was 0.05. Results. The groups of patients differed on cholesterol levels and LS/LH T scores. Decreased BMD was detected in 35 (77.4%) patients. Intergroup correlations of LS/LH T scores were registered. A history of past fractures and records on menopause significantly influenced LS/LH T scores, which was not the case with metabolic parameters. Both the lowest T scores and clinically important dyslipidemia were presented in SHypo group. Discussion. The pilot study pointed out a decrease in BMD (mostly on osteopenia level) in subclinical, presumably hypothyroid disorder. The presence of hypercholesterolemia was detected in the same group. Metabolic parameters and thyroid hormones (TSH and fT4) did not have an effect on BMD. Conclusion. Subclinical thyroid dysfunctions are associated with a decrease in BMD, mostly presented as osteopenia, with a higher degree of severity in SHypo group. There is no registered influence of metabolic parameters on LS/LH T scores. It can be possible to slow down progression of decrease in BMD and subsequent fracture risk, with proper diagnostics and management of subclinical thyroid dysfunctions. Additionally, with correction of lipid disorders, cardiovascular and cerebrovascular risk could be minimized

Computational analysis of plasma glycome and genotypes in human populations

Genski uvjetovane bolesti se u kliničkoj praksi izražavaju kroz različite fenotipske promjene. Kako bi bilo moguće što jednostavnije identificirati gen odgovoran za bolest ili ustanoviti najbolju dijagnostičku metodu, potrebno je rasvijetliti odnos genotipskih i fenotipskih značajki. Za uspješno povezivanje genotipa i odgovarajuće fenotipske značajke primjenjuje se studija povezanosti na razini genoma (eng. GWAS, genome-wide association studies). Za analizu povoljan genetski biljeg mora odražavati različitosti između pojedinaca u populaciji. Stoga se za tu svrhu koriste nukleotidni polimorfizmi (eng. SNP, single nucleotide polimorphism), koji nastaju uslijed točkaste mutacije. Jedna od fenotipskih značajki prikladna za analizu u sklopu genetskih promjena je glikozilacija, proces koji obuhvaća dodavanje razgranatih lanaca ugljikohidrata – glikana na proteine i lipide. Glikozilacija je najsloženiji i najzastupljeniji oblik post-translacijske modifikacije. Ciljevi ovog istraživanja su pronalazak moguće veze između glikozilacije i SNP-ova u izoliranim populacijama uporabom bioinformatičkih alata i algoritama strojnog učenja, te razvoj reproducibilne i statistički utemeljene metode. Razvila sam skup računalnih metoda za analizu i prikaz korelacije glikana i genotipskih parametara koje se zasnivanju na hijerarhiskom grupiranju uzoraka iz izračuna međusobne udaljenosti u vrijednostima glikana i IBD (identity by descent) parametara genotipa. Rezultati pokazuju da je takvim grupiranjem moguće izlučiti profile glikozilacije svojstvene podgrupama u populaciji i pronaći njihovu povezanost s rezultatima genotipizacije.Clinical diseases are characterized by distinct phenotypes. To identify disease–related genes or to develop appropriate diagnostic tests, it is necessary to elucidate the gene–phenotype relationships. Genome–wide association studies (GWAS) are used for identifying genetic associations with phenotypic traits by analyzing a set of single nucleotide polymorphisms (SNPs) as the genetic markers. SNPs arise from point mutations in DNA and are the major source of diversity among individuals. Phenotypic trait that can be analyzed in a context of genetic changes is glycosylation. This process involves the addition of glycans (sugar chains) to both proteins and lipids, and is the most complex and abundant post-translational modification. The goal of this research is to find the possible relationship between glycosylation profiles and SNPs in isolated human populations by using bioinformatics tools and machine learning algorithms, and to develop an analysis pipeline that would be reproducible and statistically relevant. I have developed a method and a set of computational tools to analyze and visualize correlations between glycans and genotypes based on hierarchical clustering of glycan profile distance data and identity by descent (IBD) values in genotypes. Analysis performed on two distinct datasets from two isolated populations in Croatia and Scotland show that the method is able to identify distinct glycan profiles in subpopulations and find their correlation to genotype results

Analysis of energetic exploitation of straw in Vojvodina

The Autonomous Province of Vojvodina is an autonomous province in the Republic of Serbia. It is located in the northern part of the country, in the Pannonia plain. Vojvodina is an energy-deficient province. The average yearly quantity of the cellulose wastes in Vojvodina amounts to about 9 millions tons barely in the agriculture, and the same potential on the level of Serbia estimates to almost 13 million tons. This study gives the analysis of energetic exploitation of straws from stubble cereals processed in different forms. Costs for the equipment that uses biomass in the EU are approximately two times higher with respect to those for the equipment for combustion of natural gas or of fuel oil. Costs of investments for combustion of biomass in Vojvodina if compared with the cited data are approximately for 40-50% lower. The difference of the investment costs for the construction of such units is because units for straw combustion designed and constructed in our country, have neither the complicated devices for manipulation of fuels, nor the devices for the waste gasses processing. The definite conclusions about the economic justification of the energetic exploitation of stubble straws can be obtained only by comparison of costs of the so obtained energy, with the costs of energy obtained through the combustion of classical fuels. Previous comparisons were the most often based on the comparisons of value of prices of the equivalent straw quantity with the process of fuel oil of other classical fuels. Such the comparisons leaded to the very positive evaluations of the economical effects of straws, without taking into account the realizability of the named method. Namely, comparisons of straw and fuel oil hardly could lead to the conclusion that these two fuels are mutually substitutable. According to its physical properties, straw is most similar to firewood, but the preciousness and lacking of this the very resource excludes it from the comparative analysis, so that comparisons were performed with respect to coal. The basic reason for the application of coal as the alternative energent is the circumstance that coal represents very often-used energent whose technology of combustion is the most similar to those used for straw, and at the same time, it has not any other application.Biomass Energy Combustion Straw Vojvodina

Subclinical hypothyroidism

Subklinički hipotiroidizam je asimptomatska ili simptomatska tiroidna disfunkcija, koja se karakteriše umerenim porastom nivoa TSH i normalnim nivoima perifernih tiroidnih hormona. Često se otkriva uzgredno. Etiologija subkliničkog hipotiroidizma je identična etiologiji kliničkog hipotiroidizma. Dijagnostikuje se laboratorijski, određivanjem nivoa TSH i perifernih tirodnih frakcija. S'obzirom da je od kliničkog značaja samo perzistentni subklinički hipotiroidizam, za definitivnu potvrdu je neophodno učiniti hormonsku reevaluaciju 6-12 meseci nakon incijalne. Značaj subkliničkog hipotiroidizma je prevashodno u postojanju određenih propratnih pojava, koje mogu značajno uticati na kvalitet života bolesnika (dislipidemija, kardiovaskularni i neurokognitivni poremećaji, infertilitet, poremećaj intrauterinog razvoja ploda). Upravo stoga, lečenje subkliničkog hipotiroidizma malim dozama levotiroksina treba sprovesti u slučajevima gde je korist od supstitucije daleko veća od njenih potencijalnih rizika. Ovaj revijski rad je pokušaj argumentovanog razmatranja kliničkih i terapijskih aspekata subkliničkog hipotiroidizma iz literature i našeg kliničkog iskustva.Subclinical hypothyroidism is asymptomatic or symptomatic thyroid dysfunction, presented as slightly elevated TSH level and reference range values of peripheral thyroid hormons. Its etiology is the same as the etiology of clinical hypothyroidism. By measurement of TSH serum level as well the levels of peripheral free thyroid hormone fractions, it is easy, but more frequently incidental, diagnosed. Because of clinical significance is just persistent subclinical hypothyroidism, obliged hormonal confirmation would be done 6-12 months after initial measurement. The importance of subclinical hypothyroidism lay in the co-existence of some accompanying phenomenons, which can significantly influence on the patients life quality (lipid disorders, cardiovascular and neurocognitive dysfunctions, infertility, inadequate fetal growth and deveolpment). For such reasons, it is necessary to substitute subclinical hypothyroidism with small doses of levothyroxine, where we can expect more benefit than the risk of therapy. This review article is an attempt to unite present important clinical and therapeutical aspects of subclinical hypothyroidism from reviews and our experience

Next-generation sequencing-based detection of germline L1-mediated transductions

Background: While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking. Results: Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species. Conclusions: By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2670-x) contains supplementary material, which is available to authorized users

Dog Therapy Utilization with Children Affected by Hyperactivity Syndrome

Title of Diploma Thesis: Dog therapy utilization with children affected by hyperactivity syndrome Aim of Diploma Thesis: Identify whether and how long the children with hyperactivity syndrome are able to calm down, work and relax during the dog therapy lesson. Methods: Research has been based on the structured monitoring of three children with hyperactivity syndrome. Reduction of motion fidget during the dog therapy lesson should proofpositive influence ofthe therapy. Monitoring was repeated to confirm long time effect ofthe therapy. Evaluation by observers has been based on five grades scale. For research has been used in children care reports persona! documents from pedagogic- psychology examination. Results: The assumption, that during dog therapy lesson are children with hyperactivity syndrome able to concentrate on their work longer time, is confirmed. It is possible to use dog therapy as a supporting curative method for children with hyperactivity syndrome. Key words: dog therapy, animal therapy, zoonoss, hyperactivity, Attention Deficit Hyperactivity Disorder, positionin