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1.
The detection and study of biologically active compounds in environmental processes and samples
Mojca Žorž Furlan, doctoral dissertation

Abstract: Environmental pollution in the 21th century still represents a global problem for human and animal health. Despite general awareness about released substances and their degradation products their fate and possibilities of removal are not well investigated. Even though the chemicals released are dispersed and diluted in water cycles, their poor biodegradability and/or strong accumulation can result in the intoxication of exposed organisms. Similarly, as a part of the environment, food can get contaminated by bioactive substances during different steps of preparation. Not only artificial compounds such as pesticides or pharmaceuticals, but also natural toxins enter the food chain and impact negatively on humans' and animals' health. In addition, the activity of some bacteria can influence the production of amines from amino acids after fermentation, to which the human body responds with several symptoms of intoxication. Several analytical methods for the determination of trace levels of broad range contaminants have been developed. Due to the largely robust, selective and sensitive features of the conventional (rearguard) techniques, they represent the first choice for analysing multiple organic compounds in frequently very complex matrices. However, screening (vanguard) methods are paving the way in the chemical analytics as a solution that provides simplicity and rapid analytical responses with binary (yes/no) answers. They require little or no sample treatment as well as more economically-efficient instrumentation. The combination of vanguard-rearguard analytical strategies hence offers a compromise between classical analytical figures of merit and productivity-related characteristics. In the first part of our research feasibility studies for the application of TLS and/or TLM in novel analytical methods for the determination of lipid-lowering drug atorvastatin and a mycotoxin ochratoxin A . The survey on atorvastatin performed spectrophotometrically has shown a decrease of ATV-sulpho-vanillin product at the wavelength of its maximum absorbance after dilution by organic solvent, which was investigated due to the possible increasing of the method sensitivity. As the predicted LODs that could be obtained by TLM (0.3 mg/L) could not reach the concentration of ATV usually present in the environment (ng/L-g/L) further experiments on this subject were therefore not justified. On the other hand, the ELISA assay for the determination of ochratoxin A was performed. In case of μFIA-TLM, the measurements were influenced by high background signal resulting in high LODs of TLM (470 pg/mL), which is known as a background limited technique. It was estimated that the LODs of standard ELISA assay could not be significantly improved, therefore no further research was conducted in this direction. In the second part of the dissertation, a sensitive rearguard system by coupling HPLC and TLS for the determination of biogenic amines in wine samples was developed. Putrescine, cadaverine, histamine and tyramine were separated and detected on a HPLC-TLS system after derivatization by dabsyl chloride. The method was optimized in terms of chromatographic conditions and in terms of TLS parameters. Also, the sensitivity of the newly developed method was evaluated by comparing the TLS detection with DAD detection in terms of LOD values, where TLS showed 3.6-fold improvement compared to DAD. Afterwards, the standard addition calibration was performed and evaluated for its recoveries (86−117%) in the determination of the four BAs. The applicability of the novel method was tested by the analysis of real white and red wine samples and by comparing the results to the standard HPLC-FL method and concentrations of BAs in wine samples were in good accordance. In addition, the dabsylated BAs showed better stability compared to the OPA derivatives as they have not lost the peak intensity after 17h of storage. In the third part, a vanguard system for detection of the overall biogenic amines concentration was developed by employing μFIA-TLM. Initially, NH4Cl standard solutions were applied in the indophenol reaction for batch mode, off-line μFIA-TLM and in an on-line indophenol formation for μFIA-TLM detection. By adding 50 % of EtOH to indophenol we obtained 9-fold improvement. In addition, indophenol showed good stability under TLM conditions. We optimized the microfluidic and TLM parameters in the off-line and on-line indophenol reaction. The addition of 5% ethanol to the reagent in the on-line reaction resulted in the 3-fold improvement of the signal-to-noise ratio. Further on, the overall reaction, including the enzymatic and the following indophenol reaction, was optimized by choosing the optimal buffer (pH=7, 0.5 M) and alkaline conditions (2M NaOH). The influence of interferences from amino compounds was also evaluated and discussed. The off-line and on-line μFIA-TLM were evaluated by their performance characteristics. The LOD for ammonia detection reached 2.3 μM and the applicability in ammonia detection in water samples was discussed. Similar LOD of 3.2 μM was obtained for the overall concentration of BAs and LOD of 3.8 μM for histamine, which is more than 4-folds lower value as the lowest suggested limits of intake for histamine in wine samples (2 mg/L; 18 μM). Finally, an immobilization procedure on magnetic nanoparticles was developed for the possible implementation of the selected enzyme in a miniaturized biosensor.
Keywords: thermal lens spectrometry, thermal lens microscopy, high performance liquid chromatography, microfluidics, biogenic amines, microbial transglutaminase, indophenol (Berthelot) reaction
Published: 04.06.2018; Views: 119; Downloads: 38
.pdf Fulltext (2,90 MB)

2.
MODIFIED U1 RNAs AS SPLICING CORRECTORS IN HUMAN GENETIC DISORDERS
Katarzyna Rajkowska, 2018, doctoral dissertation

Abstract: The experimental work of this thesis was performed at the International Centre for Genetic Engineering and Biotechnology (ICGEB) in the Human Molecular Genetics Group, under the scientific direction of Prof. Franco Pagani. The project was developed during the academic years 2014-2017. Modified U1 RNAs, also named Exon Specific U1s (ExSpeU1s) represent a novel class of small RNA-based molecules that correct exons splicing defects. To evaluate their therapeutic potential focused on Familial Dysautonomia (FD), a rare autosomal recessive disorder characterized by progressive degeneration of the sensory and autonomic nervous system. More than 99% of patients are homozygous for the T to C transition in position 6 of the IKBKAP intron 20 (c.2204+6T>C). This substitution modifies the exon 20 5’ splice site (5’ss) inducing exon skipping in a tissue-specific manner and reducing the total amount of IKAP protein. The molecular mechanisms underlying the IKBKAP mis-splicing are not completely clear and there are no effective treatments. In this thesis, I investigated the therapeutic potential of ExSpeU1s and the role of cis- and trans-acting factors that regulates IKBKAP splicing. Using a splicing functional assay, I identified ExSpeU1s that bind to intron 20 sequences and rescue the exon 20 skipping defect. Interestingly, their rescue activity was modulated by several splicing factors and requires a critical exonic splicing enhancer element. Transfection experiment showed the involvement of both enhancing (TIA1, PTBP1 and PTB4) and inhibitory (SRSF3, hnRNPA1, FOX and FUS) splicing factors in IKBKAP splicing. To better evaluate the ExSpeU1s therapeutic efficacy, I transduced FD patient’s fibroblasts with a lentiviral vector expressing the most active ExSpeU1. This resulted in a complete rescue of the exon skipping defect and improvement in IKAP protein expression. Most importantly, intraperitoneal delivery of ExSpeU1s by AAV9 into a transgenic mouse model, that recapitulates the tissue-specific mis-splicing seen in FD patients, corrected the aberrant splicing patterns in several tissues increasing the amount of the corresponding IKAP protein. All together, these results identify novel regulatory splicing factors involved in the IKBKAP exon 20 regulation and provide the proof of principle that ExSpeU1s delivered in vivo by AAV vectors represent a novel therapeutic strategy for FD.
Keywords: Familial Dysautonomia, IKBKAP, IKAP, splicing, splicing defects, ExSpeU1, U1 snRNA, mouse model, AAV
Published: 26.03.2018; Views: 420; Downloads: 8
.pdf Fulltext (13,96 MB)

3.
Speleogenetic factors and processes in the karst conduits of Zagorska Mrežnica Spring Cave (Croatia)
Petra Kovač Konrad, 2018, doctoral dissertation

Abstract: Zagorska Mrežnica spring cave is in the Desmerice village, 7.59 km SW from the town of Ogulin. The coordinates of the entrance are X: 399563 m, Y: 5006974 m, and Z: 314 m (HTRS96). The cave system is positioned in the contact zone of Jurassic limestone and the thrust front composed of Triassic Dolomite. The karst drainage system has elements of point recharge through a set of ponors in the hinterland karst poljes and diffuse infiltration through numerous dolines on Velika Kapela Mountain. The karst of Ogulinsko Zagorje area and its epiphreatic and phreatic cave systems have been intensively explored for the last eight years, resulting in the mapping of 1134 m of submerged passages of the Zagorska Mrežnica spring cave. Until 2014 it was the longest mapped submerged system in Croatia explored by cave diving techniques only. Altogether, over 3.5 km of submerged passages in seven caves of Ogulinsko Zagorje (Zagorska Mrežnica spring cave, Spring of Rupečica, Ponor of Rupečica, Cave system Pećine-Veliko vrelo, Spring of Bistrac, Cave Zagorska Peć and Pit Klisura) were explored during 500 hours of diving over a ten-year period. The goal of the research was to determine speleogenetic factors and processes in phreatic conditions. A new methodology for mapping of cave cross-sections, microrelief forms, and structural elements was developed, sediment and petrographic analysis were done, hydrological analysis before and after the building of the accumulation lake Sabljaci, 48 cross-sections of the cave passaged and a 3D model of the cave system was created, water chemistry as well as geomorphological analysis was carried out. In the end, a simplified (modified) vulnerability assessment was also done. The results of my research showed that the distribution of the cave passages is a result of complex tectonic activities that are reflected in the orientation of cave passages. The general orientation of the measured fissures, measured during cave diving, show a dominance of a NWW-SEE direction but also the pattern of cave passages shows a significant dominance of the NE-SW orientation indicating the existence of multiple secondary traverse faults originating from main NW-SE faults. The morphology of the cave passages shows a transition between epiphreatic (possibly vadose) and phreatic phases as well as the presence of paragenetic developments in the ceiling. This indicates the existence of several epiphreatic (possibly vadose) and phreatic speleogenetic phases. The initial shape of the cross-sections was identified by sub-horizontal beds and bedding planes. The mapping of microrelief forms showed that the mechanical erosion was a very intensive process shaping the morphology of the cave passages. The sediment mineralogy coincides with the lithology of the catchment area, and the sub- angular grains reveal relatively short transport distances. Further analysis of the catchment area’s geomorphology and its relevance for the speleogenesis of Zagorska Mrežnica cave show influence of relief structures that have a Dinaric orientation (NW-SE) with cave structures and passage orientations, great relative relief, steep slopes on Velika Kapela, and flat areas of karst poljes dictate the allogenic and autogenic hydrological regime of the cave. A 3D model of the cave system reveals a relationship between the cave system and the landscape, as well as a possible connection with the nearby Zagorska Peć cave. The modified karst vulnerability assessment shows that the catchment area of Zagorska Mrežnica spring cave has a high vulnerability ranking, mainly due to high doline density and the potential large diffuse capacity of infiltration of pollutants.
Keywords: Ogulin Zagorje, karst, spring, speleogenesis, Zagorska Mrežnica spring cave
Published: 02.03.2018; Views: 323; Downloads: 11
.pdf Fulltext (5,26 MB)

4.
Ecological evaluation of aquatic and terrestrial subterranean fauna in a karst cave
Peter Kozel, 2018, doctoral dissertation

Abstract: Studies on environmental parameters−subterranean fauna relationship and spatial and temporal patterns of subterranean fauna have dealt with either aquatic or terrestrial faunas. So far, no simultaneous ecological evaluation of aquatic and terrestrial faunas has been performed. To address this issue, we conducted a pilot study dealing in parallel with these two faunas in the cave Zguba jama near Postojna, Slovenia, applying monthly sampling over one year. Aquatic fauna was sampled from permanent water drips, and terrestrial fauna by visual inspection and advanced baited pitfall trapping, along with recording the main abiotic parameters. In percolating water, we recorded aquatic fauna, Copepoda being the dominant taxon, and species-rich troglobiotic fauna, which most probably coexist with aquatic species in a semi-aquatic epikarst environment. Analyses revealed that temperature, distance from the entrance and ceiling thickness are the most important parameters that influence the presence of stygobionts; higher values of these parameters result in a higher probability of the presence of stygobionts in percolation water. The recorded spatial distribution of stygobionts varied distinctly among the sampling sites. This indicates that fauna in sampled drips originated from aquatic or semiaquatic epikarst microhabitats, being completely or nearly completely separated from each other, and probably varied according to environmental characteristics. The temporal pattern of the stygobiotic fauna dynamics presumably occurred because of variable water flow rates and the specific physical and chemical characteristics of the water. The abundance of troglobionts showed distinctive spatial distribution pattern over the seasons. In spring and summer, the highest abundances and the highest probability of presence were found near the entrance. In autumn and winter, unfavorable conditions in the entrance zone most likely triggered the migration of troglobionts towards the climatically more stable deep cave zone, and from the cave into the adjacent fissure network. The highest abundance of troglobionts was found at 7–9°C and relatively high ground substrate moisture. Additionally, we found a higher probability of the presence of troglobionts in cave sections with a larger passage cross-section size, which is probably due to the greater variability of microhabitat types in spacious cave sections. In addition, the higher probability of presence relates to higher substrate pH. One potential explanation for this could be that the specific microorganism communities present in such conditions support the nutritional needs of troglobionts. Most troglobiotic species preferred the deep cave zone where the highest species richness and diversity were also observed. Species richness, abundance and diversity of troglophiles and trogloxenes were highest in the entrance zone and showed relatively similar patterns within the cave. However, troglophiles were more abundant in the transition zone and the initial part of the inner zone. The temporal pattern of trogloxenes and presumably of most troglophiles occurred because of migration between the surface and the cave. The probability of the presence of trogloxenes was highest in the entrance zone and in winter, owing to massive overwintering in the cave. A modified technique of pitfall trapping by placing traps in two parallel sets, a ground and an upper one along the cave, resulted in recording greater species diversity in comparison with the traditional method, i.e., ground pitfall trapping alone. It turned out that such sampling contributes at the same time to both more effective and less invasive inventory of subterranean fauna. The dynamics of relative abundance and species richness of stygobionts and troglobionts within the cave showed partly comparable annual patterns. Nevertheless, our findings point toward the need for a long-term and more detailed study in future to address this question properly.
Keywords: biological inventories, epikarst fauna, faunal dynamics, microhabitats, stygobionts, troglobionts, troglophiles, trogloxenes, Zguba jama
Published: 16.02.2018; Views: 236; Downloads: 105
.pdf Fulltext (5,25 MB)

5.
SIGNALING STUDIES IN THE EMERGING KIWIFRUIT PATHOGEN Pseudomonas syringae pv. actinidiae
Sree Gowrinadh Javvadi, 2017, doctoral dissertation

Abstract: In the past two decades emerging and re-emerging plant pathogens have caused new threats to the production of several economically important crops, one among them is P. syringae pv. actinidiae (PSA) which causes canker or leaf spot on kiwifruit plants. PSA enters plant through wounds and remains dormant in cortex tissue of the branches, and spreads in the tissue to cause severe symptoms from winter to early spring. The disease can be visualized by brown discoloration of buds, dark brown angular spots surrounded by yellow haloes on leaves, cankers with white to reddish (oxydation) exudate on twigs and trunks, fruit collapse, wilting and eventually plant mortality. Current control methods have their own significance in disease control, however there is considerable lack of clear understanding of PSA pathogenicity. Virulence of plant pathogens often relies on the synchronized/coordinated expression of pathogenicity factors via quorum sensing (QS). Therefore, investigations on QS in PSA may lead to develop novel disease control strategies and reliable methods to curb the disease. It is currently unknown whether PSA produces a QS signal molecule thus the aim of this thesis is to investigate whether PSA possesses a QS system. As genome mining did not reveal the presence of any currently known QS system, this study initially by metabolomics was aimed at identifying potentially low molecular weight secondary metabolite QS molecules produced by PSA. Azelaic acid was discovered to be produced by PSA, this is the first report of azelaic acid production by bacteria. The characterization and possible role of azelaic acid in QS is presented. Since azelaic acid is ubiquitous in nature, in addition to determining its biological role, the catabolism of azelaic acid in bacteria using the efficient degrader Pseudomonas nitroreducens DSM 9128 was also studied.
Keywords: Quorum sensing, Azelaic acid, Metabolomics, Catabolism
Published: 19.12.2017; Views: 411; Downloads: 27
.pdf Fulltext (4,41 MB)

6.
Development of Slovenian linguistic terminology in Slovenian grammars in the 18th and 19th centuries
Mitja Trojar, 2017, doctoral dissertation

Abstract: The dissertation describes the problems of metalanguage and peculiarities of linguistic terminology. Special attention is paid to autonymy, which plays a central role in metalanguage. The dissertation presents the life and work of Eugen Wüster, the founder of traditional terminology, and the principal tenets of his approach to terminology. Traditional terminology is critically evaluated; we show its inability to describe linguistic terminology: it lacks a coherent theoretical apparatus enabling description, its semantic theory is based on classical definition, which in many cases cannot be formulated, which means that traditional terminology is also unable to account for translation of terms and comparison of concepts across different concept systems. A sample of 50 basic linguistic terms from 19 grammars from the 18th, 19th and 20th centuries was used to examine the development of Slovenian linguistic terminology and to ascertain who played the most important role in it. The research shows that Anton Janežič made by far the most important contribution to Slovenian linguistic terminology: he introduced 27 terms (in both his 1854 and 1863 grammars), which represents 54% of the 50 terms retained by Breznik. The dissertation also contains some remarks on the semantics of linguistic terms. The scientific relevance of the thesis lies in its critical evaluation of traditional terminology; the results show that traditional terminology faces a number of theoretical problems; a new theory of terminology is needed, one that will be able to account for translation of terms as well as for diachronic conceptual change. The scientific relevance of results related to the second research goal (determining the contributions of individual grammarians to the development of Slovenian linguistic terminology) consists in proposing a model of research that could be used in order to measure the influence of a specific author on the introduction of new terms.
Keywords: metalanguage, linguistic terminology, history of terminology, development of linguistic terminology, diachrony
Published: 27.11.2017; Views: 539; Downloads: 113
.pdf Fulltext (4,06 MB)

7.
THE IMPACT OF EUROPEANISATION ON THE NATURE PROTECTION SYSTEM OF SELECTED COUNTRIES OF SOUTHEAST EUROPE ON THE EXAMPLE OF THE ESTABLISHMENT OF MULTI-LEVEL GOVERNANCE SYSTEM OF NATURA 2000
Aleksandar Šobot, 2017, doctoral dissertation

Abstract: The Europeanisation of Southeastern Europe led to the pre-accession and accession process of joining countries of the former Yugoslavia to the European Union. Slovenia's accession to the formal process of accession to the European Union started in 1999. This process lasted until 2003. Then, in 2004, Slovenia joined the European Union. Croatia's access to the formal process of accession to the European Union started in 2003. This process lasted until 2012. Then, in 2013, Croatia joined the European Union. Bosnia and Herzegovina entered the pre-accession process with the process of stabilization and association with the European Union, which lasted from 1999 to 2015. Then, in 2016, Bosnia and Herzegovina officially submitted its application for European Union membership. This paper examines the impact of the Europeanisation during this period on the nature protection system in the case of Slovenia, Croatia and Bosnia and Herzegovina while establishing multi-level governance system of protected areas within NATURA 2000 network. The analysis of the establishment of multi-level governance system is performed according to the morphogenetic model in three cycles as a case study. The case study is organized with the help of interviews and desk analysis of the relevant documents. In the first cycle were explained the projects/process of implementation of NATURA 2000 in Slovenia, Croatia and Bosnia and Herzegovina and its changing impacts on national legislation in the area of nature protection (environmental policy). The second cycle explains the role of the main actors in the establishment of multi-level governance system of NATURA 2000 such as the European Union institutions, competent ministries, national parks and non-governmental organisations (environmental polity). The third cycle outlines the contribution to the establishment of multi-level governance system of NATURA 2000 on the nature protection systems of Slovenia, Croatia and Bosnia and Herzegovina and their future challenges (environmental politics). The main conclusions point to the major changes in the nature protection systems of Slovenia, Croatia and Bosnia and Herzegovina during the process of implementation/establishing multi-level governance system of NATURA 2000. Main legal and institutional points of implementation of NATURA 2000 with all positive and negative sides are presented here. In the end, implementation of the European ecological network NATURA 2000 in Slovenia is marked as an excellent example with significant changes in legal (laws on nature protection) and institutional (transformation of governmental institutions, establishment of new institutions and inclusion of NGOs in decision making) framework. This process in Croatia was marked as good with significant changes in legal (laws on nature protection, adopting new strategy of sustainable development) and institutional (transformation of governmental organisation, establishment of new institutions and inclusion of NGOs in decision making) framework. This process in Bosnia and Herzegovina was marked as satisfactory with significant changes in legal (adopting new entity laws on nature protection) and institutional (adopting coordination mechanism for inter-entity and inter-institutional cooperation as well as inclusion of NGOs in decision making) framework. There are also significant weak points during the process of N2000 in all three countries (such as the late change of national legislative, not following the national legislative from governmental institution and not including NGOs into decision making), which during the proces of implementation of N2000 (for Croatia and Slovenia) or the process of preparation of implementation (for Bosnia and Herzegovina) cause significant problems, that call for action in the future in order to improve governance of natural resources /NATURA 2000. On the other hand, these points could serve as an example for all countries that underwent/or should undergo ...
Keywords: The Europeanisation of Southeastern Europe, the pre-accession process, the accession process, Slovenia, Croatia, Bosnia and Herzegovina, the nature protection system, NATURA 2000 protected areas
Published: 06.11.2017; Views: 329; Downloads: 56
.pdf Fulltext (2,77 MB)

8.
Study of the properties of air flow over orographic barrier
Maruška Mole, 2017, doctoral dissertation

Abstract: Earth’s atmosphere is a complex system. All weather phenomena take place in its lowest layer, the troposphere, which is strongly influenced by human activities and the underlying surface orography. A good example of the influence the orography has on the behavior of air flows is the appearance of strong north-east downslope wind in Vipava valley, called Bora. Numerical models used to analyze flows in complex terrain need meteorological data both for setting the initial conditions and the verification of modeling results. Obtaining spatial distributions of meteorological observables can be challenging, especially in the case of strong winds, such as Bora, where traditional methods may be inadequate due to prohibitive wind speeds. In most cases, vertical properties of the atmosphere can be obtained using remote sensing techniques. Contrary to vertical profile measurements with traditional methods, remote sensing techniques do not require the measuring device to be placed within the flow and are therefore more appropriate for measurements in severe weather conditions such as strong winds. The aim of this thesis is a detailed analysis of wind and tropospheric structure properties in and above the Vipava valley in a variety of typical atmospheric conditions, including strong wind events. It employs a combination of high resolution wind and lidar data in addition to standard meteorological measurements. In Ajdovščina, there are four predominant wind directions, two of them directly connected to Bora. In the case of Bora, periodicity analysis of wind data from Ajdovščina yielded a range of possible wind gust periods between 1 and 7 minutes. The periods were not stable, with the periodogram less noisy for stable wind directions. Wavelike structures were found to be present in the troposphere in half of the investigated cases, regardless of the presence of Bora. In statically stable conditions, gravity waves propagated throughout the planetary boundary layer (PBL). In the case of Bora, the PBL experienced oscillations with periods between 1 and 2 minutes. A shear layer was present above the PBL, causing Kelvin-Helmholtz waves at its boundaries with periods ranging from 3 to 6 minutes. In some cases, periodic structures were observed above the shear layer as well, which were found to have longer periods than those within the PBL.
Keywords: remote sensing, Vipava valley, wind properties, Bora, wind gusts, wind periodicity, tropospheric structures, Kelvin-Helmholtz waves
Published: 18.09.2017; Views: 857; Downloads: 40
.pdf Fulltext (45,11 MB)

9.
Graphene/organic semiconductor systems
JINTA MATHEW, 2017, doctoral dissertation

Keywords: GRAPHENE, PDIF-CN2, ATOMIC FORCE MICROSCOPY, ORGANIC THIN FILM TRANSISTOR, OSTWALD RIPENING.
Published: 15.09.2017; Views: 692; Downloads: 17
.pdf Fulltext (41,84 MB)

10.
Study of expression systems for large-scale protein production in CHO cells
Juan Pedro Menvielle, 2013, doctoral dissertation

Published: 28.07.2017; Views: 1132; Downloads: 51
.pdf Fulltext (3,44 MB)

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