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11.
Correlations between photocatalytic activity and chemical structure of Cu-modified TiO2-SiO2 nanoparticle composites
T. Čižmar, doktorska disertacija

Opis: The objective of this dissertation was to examine how copper modification can improve the photocatalytic activity of TiO2-SiO2 and to explain the correlation between Cu concentration and chemical state of Cu cations in the TiO2-SiO2 matrix, as well as the photocatalytic activity under the UV/solar irradiation. The Cu-modified TiO2-SiO2 photocatalysts were prepared by a low temperature sol-gel method based on organic copper, silicon and titanium precursors with varied Cu concentrations (from 0.05 to 3 mol%). The sol-gels were dried at 150 °C to obtain the photocatalysts in the powder form. To test thermal stability, additional set of photocatalysts was obtained by calcinating dried samples in air at 500 °C for 1 h. The photocatalytic activity was determined by a fluorescence-based method of terephthalic acid decomposition. Up to three times increase in photocatalytic activity of air-dried samples is obtained when TiO2-SiO2 matrix is modified with Cu in a narrow concentration range from 0.05 to 0.1 mol%. At higher Cu loadings the photocatalytic activity of Cu-modified photocatalyst is smaller than in the unmodified reference TiO2-SiO2 photocatalyst. Calcined samples showed significantly reduced photocatalytic activity compared to air-dried samples. XRD analysis showed that all Cu-modified TiO2-SiO2 nanocomposites with different Cu concentrations have the same crystalline structure as unmodified TiO2-SiO2 nanocomposites (air-dried or calcined). The addition of Cu does not change the relative ratio between the anatase and brookite phase or unit cell parameters of the two TiO2 crystalline structures. TEM analysis showed that the addition of Cu does not change the morphology of TiO2-SiO2 catalyst dried at 150 °C. The Cu K-edge XANES and EXAFS analysis were used to determine valence state and local structure of Cu cations in Cu-modified TiO2-SiO2 photocatalyst. The results elucidate the mechanism responsible for the improved or hindered photocatalytic activity. In the air-dried samples with low Cu content, which exhibit largest activity, Cu-O-Ti connections are formed, suggesting that the activity enhancement is due to Cu(II) cations attachment on the surface of the photocatalytically active TiO2 nanoparticles, so Cu(II) cations may act as free electron traps, reducing the intensity of recombination between electrons and holes at the TiO2 photocatalyst’s surface. At higher Cu loadings no additional Cu-O-Ti connections are formed, instead only Cu-O-Cu connections are established, indicating the formation of amorphous or nanocrystalline Cu(II) oxide, which hinders the photocatalytic activity of TiO2. Calcination of Cu-modified TiO2-SiO2 photocatalysts at 500 °C induces significant structural changes: Cu-O-Ti connections are lost, Cu partially incorporates into the SiO2 matrix and amorphous copper oxides, which again reduce the photocatalytic activity of the material, are formed.
Ključne besede: titanium dioxide, Cu-modified TiO2-SiO2 photocatalyst, photocatalytic activity, Cu K-edge XANES, EXAFS.
Objavljeno: 17.12.2018; Ogledov: 294; Prenosov: 22
.pdf Polno besedilo (3,05 MB)

12.
ACCESSIBILITY AS A NON-PREJUDICIAL APPROACH: IS CULTURE BACK TO NORMALITY?
Ilaria Bollati, 2018, doktorska disertacija

Opis: This research explores the general and actual accessibility to cultural spaces and organizations, focusing on the network of relationships between contents, innovation, and participation. It investigates how Culture can be perceived as a normal experience, actually able to set a rich dialogue with each of us, normal ordinary people/consumers. Normality means inclusion and sharing. The proposed investigation is based on a triad of interactions among culture, economics, and design. Assuming that Culture generates its value from a cognitive approach, or from a dynamic and context-dependent value chain that is subject to a cognitive evolution, this research acknowledges that the cultural experience is subject to a double issue of access: - The horizontal question is related to complications associated with the structure. Believing that ‘culture is special’ implies the risk of progressively widening the gap between cultural supply and society. Culture is ‘locked’ in specific sites and a big slice of the world’s adult population has yet to be involved in any cultural experience. - Once physically inside the cultural structure, the vertical issue is generated by the difficulty in entering in contact with the offered contents. This research focuses only on museums and multimedia exhibitions in which the learning process has changed: the research presents itself as a conversation where both ‘those who know’ and ‘those who learn’ play equally active parts in a relative process of understanding. Starting from the existing processes, forms, previous studies and case studies, the survey yearns for their systematization in innovative models. The process, from theory to practice and vice versa, goes beyond a traditional mechanism of deduction: it moves from specific contexts to the abstraction of replicable approaches. The question of how the narration emerges guides you toward a new method of analysis, study, and cataloguing; a schematization capable of investigating not only knowledge, but also the visitor's ‘cognitive metabolism’ (how knowledge is acquired) during the multimedia experience; an innovative multipurpose tool, useful for both the museum institution and the designer. The research finally assesses and takes into account an actual experiment, the outcomes of which may prove to be useful in feeding theoretical implications with empirical experiences: RovelloDue - Piccolo Spazio Politecnico, a temporary multimedia space.
Ključne besede: audience development, cognitive accessibility, cultural addiction, culture, human, emotion, immersion, innovation, interactive exhibitions, participation, natural interfaces, normality, technologies
Objavljeno: 13.12.2018; Ogledov: 193; Prenosov: 7
.pdf Polno besedilo (45,89 MB)

13.
STUDY OF ATMOSPHERIC AEROSOL PROPERTIES IN THE VIPAVA VALLEY
Longlong Wang, doktorska disertacija

Opis: The aim of the dissertation was to study aerosol loading distributions and properties over the Vipava valley, a representative hot-spot for complex mixtures of different aerosol types of both anthropogenic and natural origin. An infrared Mie and a two-wavelength polarization Raman lidar systems were used as main detection tools. The polarization Raman lidar, which provides the capability to extract the extinction coefficient, backscatter coefficients, depolarization ratio, backscatter Ångström exponent, lidar ratio and water vapor mixing ratio profiles, was itself designed, built and calibrated as a part of this thesis. Lidar data, combined with in-situ measurements, was used to determine detailed information on different aerosol types. Vertical profiles of aerosol mass concentration were extracted from the Mie lidar data taken in April 2016, where the in-situ measurements of aerosol size distribution and number concentration as well as aerosol absorption coefficient and black carbon mass concentration were used to estimate the mass extinction efficiency (MEE). Aerosol morphology and chemical composition determined by SEM-EDX on sampled particles were used for the identification of primary aerosol types. Two cases with different atmospheric conditions (long range mineral dust transport and local biomass burning) and different expected the dominant presence of specific aerosol types (mineral dust and soot) were investigated in more detail. They revealed significantly different aerosol properties and distributions within the valley, affecting radiative heat exchange. A more detailed investigation of aerosol properties throughout the troposphere in different atmospheric conditions was made possible by the two-wavelength polarization Raman lidar system, deployed in Ajdovščina (town of Vipava valley) from September 2017. Using its aerosol identification capabilities, based on particle depolarization ratio and lidar ratio measurements, it was possible to identify predominant aerosol types in the observed atmospheric structures, for example in different atmospheric layers in the case of the stratified atmosphere. Primary anthropogenic aerosols within the valley were found to be mainly emitted from two sources: individual domestic heating systems, which mostly use biomass fuel and traffic. Natural aerosols, transported over large distances, such as mineral dust and sea salt, were observed both above and entering into the planetary boundary layer. Backscatter contribution of each aerosol type was separated and the corresponding extinction contribution was derived from lidar observations.
Ključne besede: Vipava valley, aerosol distribution, aerosol characterization, lidar remote sensing, in-situ measurements, aerosol loading.
Objavljeno: 23.10.2018; Ogledov: 492; Prenosov: 17
.pdf Polno besedilo (29,39 MB)

14.
PHOTO-EXCITATION ENERGY INFLUENCE ON THE PHOTOCONDUCTIVITY OF ORGANIC SEMICONDUCTORS
Nadiia Pastukhova, 2018, doktorska disertacija

Opis: In this work, we experimentally studied the influence of photoexcitation energy influence on the charge transport in organic semiconductors. Organic semiconductors were small molecules like corannulene, perylene and pentacene derivatives, polymers such as polythiophene and benzothiophene derivatives, and graphene, along with combinations of these materials in heterojunctions or composites. The first part of this study is focused on the photoexcitation energy influence on the transient photoconductivity of non-crystalline curved π-conjugated corannulene layers. The enhanced photoconductivity, in the energy range where optical absorption is absent, is deduced from theoretical predictions of corannulene gas-phase excited state spectra. Theoretical analysis reveals a consistent contribution involving transitions to Super Atomic Molecular Orbitals (SAMOs), a unique set of diffuse orbitals typical of curved π-conjugated molecules. More, the photoconductivity of the curved corannulene was compared to the π-conjugated planar N,N′-1H,1H- perfluorobutyldicyanoperylene-carboxydi-imide (PDIF-CN2), where the photoexcitation energy dependence of photocurrent closely follows the optical absorption spectrum. We next characterized charge transport in poly(3-hexylthiophene) (P3HT) layers deposited from solution. Our results indicate that time-of-flight (TOF) mobility depends on the photoexcitation energy. It is 0.4× 10 −3 cm 2 /Vs at 2.3 eV (530 nm) and doubles at 4.8 eV (260 nm). TOF mobility was compared to field-effect (FET) mobility of P3HT field-effect transistors (OFETs). The FET mobility was similar to the 2.3 eV excitation TOF mobility. In order to improve charge mobility, graphene nanoparticles were blended within a P3HT solution before the deposition. We found that the mobility significantly improves upon the addition of graphene nanoparticles of a weight ratio as low as 0.2 %. FET mobility increases with graphene concentration up to a value of 2.3× 10 −2 cm 2 /Vs at 3.2 %. The results demonstrate that phase segregation starts to influence charge transport at graphene concentration of 0.8 % and above. Hence, the graphene cannot form a bridged conduction channel between electrodes, which would cancel the semiconducting effect of the polymer composite. An alternative approach to enhance mobility is to optimize the molecular ordering of organic semiconductors. For that purpose, we studied an innovative nanomesh device. Free-standing nanomesh devices were used to form nanojunctions of N,N′- iiDioctyl-3,4,9,10-perylenedicarboximide (PTCDI-C8) nanowires and crystalline bis(triisopropylsilylethinyl)pentacene (TIPS-PEN). We characterized the photocurrent response time of this novel nanomesh scaffold device. The photoresponse time depends on the photon energy. It is between 4.5 − 5.6 ns at 500 nm excitation wavelength and between 6.7 − 7.7 ns at 700 nm excitation wavelength. In addition, we found that thermal annealing reduces charge carrier trapping in crystalline nanowires. This confirms that the structural defects are crucial to obtaining high photon-to-charge conversion efficiency and subsequent transport from pn junction in heterostructured materials. Structural defects also influence the power conversion efficiency of organic heterostructured photovoltaics (OPVs). Anticipating that polymers with different backbone lengths produce different level of structural defects, we examined charge transport dependence on the molecular weight of poly[4,8-bis(5-(2- ethylhexyl)thiophen-2-yl)benzo[1,2-b;4,5-b']dithiophene-2,6-diyl-alt-(4-(2- ethylhexyl)-3-fluorothieno[3,4-b]thiophene-)-2-carboxylate-2-6-diyl] (PTB7-Th) from 50 kDa to 300 kDa. We found p-type hopping transport in PTB7-Th, characterized by 0.1 – 3× 10 −2 cm 2 /Vs mobility, which increases with temperature and electric field. The polymer molecular weight exhibits a non-trivial influence on charge transport. FET mobility in the saturation regime increases with molecular weight. A similar trend is observed in TOF mobility and FET mobility in the linear regime, except for the 100kDa polymer, which manifests in the highest mobility due to reduced charge trapping. The lowest trapping at the dielectric interface of OFET is observed at 200 kDa. In addition, the 200 kDa polymer exhibits the lowest activation energy of the charge transport. Although the 100 kDa polymer indicates the highest mobility, OPVs using the 200 kDa polymer exhibit the best performance in terms of power conversion efficiency.
Ključne besede: organic semiconductors, optical absorption spectroscopy, time-of-flight photoconductivity, transient photocurrent spectroscopy, organic thin film transistors, atomic force microscopy, superatomic molecular orbitals, pn heterojunction, organic nanowires, graphene, composites, charge mobility, charge trapping, temperature dependence, photodetector, photovoltaic, solar cell, organic electronics
Objavljeno: 08.10.2018; Ogledov: 512; Prenosov: 30
.pdf Polno besedilo (4,56 MB)

15.
CONTRIBUTION TO DEVELOPMENT OF MESHLESS METHODS FOR FREE AND MOVING BOUNDARY PROBLEMS
NAZIA TALAT, 2018, doktorska disertacija

Ključne besede: Two-phase flow, free and moving boundaries, computational fluid dynamics, phasefield formulation, 2D problems, axisymmetric problems, diffuse approximate meshless method, Rayleigh-Taylor instability, Boussinesq approximation, variable density and viscosity, flow focusing, dripping, jetting
Objavljeno: 11.09.2018; Ogledov: 416; Prenosov: 28 
(1 glas)
.pdf Polno besedilo (4,24 MB)

16.
POLYMORPHISMS IN GENES FOR ENDOTHELIN 1, ENDOTHELIN RECEPTORS AND NITRIC OXIDE SYNTHASE 3 IN PATIENTS WITH DIABETIC RETINOPATHY AND DIABETES MELLITUS TYPE 2
Dejan Bregar, 2018, doktorska disertacija

Opis: The major causes of Diabetes Mellitus Type 2 (T2DM) are multi-factorial consequences of complex interactions between environmental, social and genetic factors. We investigated the genetic risk factors in Slovene patients with T2DM on a model of microvascular complication – Diabetic Retinopathy (DR). Retrospective case-control study includes a T2DM Slovene population with clinical risk factors for T2DM and DR. Only some of the candidate genes with selected single nucleotid polymorphisms (SNPs) were included: (EDN1 (rs5370, rs3087459, rs1476046), EDNRA (rs5335, rs1801708), EDNRB (rs10507875, rs4885493), NOS3 (rs869109213). By genotyping with either real-time polymerase chain reaction or standard polymerase chain reaction (PCR) we successfully identified the contribution of variable number of tandem repeats rs869109213 in DR progression (Proliferative Diabetic Retinopathy (PDR)) in Slovene patients with T2DM. The joint effect of individual genotypes of rs10507875 in EDNRB and rs869109213 in NOS3 on DR onset (DR) and DR progression (PDR) was demonstrated as well. The joint effect of the two polymorphisms on DR onset (DR) and DR progression (PDR) was greater than the individual effect of each polymorphism separately in the analyzed genetic models. Despite genetic research contributions in DR, linkage studies, and Genome-wide association studies the identification of susceptible loci through candidate gene approaches still remains in its early stages. The frequent approach with an ultimate focus on SNP associations with phenotype is likely to underestimate the roles of genetics in human diseases by disregarding not only the joint effect of multiple loci but the complex interaction network between them. By identifying polymorphisms in genetic disorders in a more systematic way, we will be able to deepen our understanding of the regulatory mechanisms and disease etiology which should lead to a more effective development of mechanism-based therapies as well.
Ključne besede: endothelin 1, EDN1, endothelin receptor A, EDNRA, endothelin receptor B, EDNRB, nitric oxide synthase 3, NOS3, diabetic retinopathy, DR, diabetes mellitus type 2, T2DM, polymorphism, SNP, genetic model
Objavljeno: 07.09.2018; Ogledov: 347; Prenosov: 37
.pdf Polno besedilo (3,06 MB)

17.
Amorphous nanocomposite of polycarbosilanes and aluminum oxide
Andraž Mavrič, 2018, doktorska disertacija

Opis: This work presents a paradigm for high temperature stabilization of bulk amorphous aluminium oxide. The thermodynamic stabilization is achieved by preparing a nanocomposite, where polymethylsilane dendritic molecules are dispersed in an aluminium hydroxide gel. Upon heat-treatment the gel transforms to the amorphous aluminium oxide that is stable up to 900°C. The dispersion of the macromolecules and their covalent bonding to the alumina matrix induce homogeneously distributed strain fields that keep the alumina amorphous. The first part of the thesis focuses on the synthesis, characterization and solubility properties of the dendritic polymethylsilane. The polymethylsilane is synthetized by electrochemical polymerization from trichloromethylsilane monomer. The polymerization mechanism, involving a single polymerization pathway, is identified. The polymer growth proceeds through reduction of the monomers to the silyl anions and their addition to the growing polymer. The solubility of three chemically related but topologically different polysilanes (linear, dendritic and network) were studied by dynamic light scattering. At room temperature the agglomerates in a range from 500 to 1300 nm are present. They undergo de-agglomeration at slightly elevated temperatures of around 40°C. The de-agglomeration results in formation of stable solutions, where a hydrodynamic diameter of the individual polymer molecules was measured to be in a range from 20 to 40 nm. The obtained diameters of two dendritic polymethylsilane macromolecules, synthesized under different electrolysis conditions, are much larger than the theoretical size estimated for an ideal dendrimer. We determined by 29Si NMR that the reason for this is in a large number of branching irregularities (defects) contained in the molecular structure. Combining the experimental values obtained by DLS and density measurements with a structural model that considers the branching irregularities, it is shown that the inclusion of the defects allows the dendritic polymer to exceed the sterical limitations and form the hyperbranched dendritic structure. The final size depends on a relative amount of the branching defects. In the second part, the synthetized polymethylsilane molecules were successfully used for the nanocomposite formation. The aluminium hydroxide gel with the dispersed polymethylsilane molecules was prepared as a precursor. Upon heat-treatment it gives the amorphous aluminium oxide stable up to 900°C. The dispersed macromolecules induce homogeneously distributed strain fields that keep the aluminium oxide amorphous during the thermal treatment the dispersed macromolecules covalently bind to the matrix, inducing the interface strain. The amorphous state was confirmed by the presence of penta-coordinated aluminium detected by 27Al NMR and a low bandgap measured by UV-vis absorption spectroscopy.
Ključne besede: amorphous aluminium oxide, polymethylsilane, nanocomposite, electropolymerization, solubility, agglomeration, de-agglomeration, dendrimer, hyperbranched dendritic structure, dynamic light scattering, thermal analysis, transmission electron microscopy, scanning electron microscopy, X-ray diffraction, infrared spectroscopy, UV-Vis spectroscopy
Objavljeno: 19.07.2018; Ogledov: 1055; Prenosov: 65
.pdf Polno besedilo (5,07 MB)

18.
DISTRIBUTION OF ENTERIC VIRUSES IN THE GULF OF TRIESTE AND THEIR INTERACTIONS WITH ENVIRONMENTAL AND BIOLOGICAL PARAMETERS
José Manuel Carita Gonçalves, 2018, doktorska disertacija

Opis: The available classical diagnostic methods, due to many disadvantages, do not allow effective detection of pathogenic enteric viruses in environmental samples. Due to low concentrations of pathogenic viruses in the sea, it is important to develop an effective concentration procedure for their successful detection. In the first part of the doctoral thesis, we focused primarily on the development of a protocol for an effective concentration of pathogenic enteric viruses in coastal water samples. Monolithic chromatographic columns (BIAseparations) were used for the concentration of rotaviruses and noroviruses, prior to the detection with reverse transcription quantitative PCR in real time (RT-qPCR). We tested the efficiency of concentration using columns of various chemical properties and selected pathogenic enteric viruses (rotavirus and norovirus). Among them, hydrophobic interaction monolithic column (CIM® C4) was the most effective. CIM C4 was used to optimize the concentration step and tested in waters with different salinities. The presence of concentrated viruses was confirmed by RT-qPCR and transmission electron microscope. We have developed a protocol that enables rapid concentration of viruses in coastal waters of various salinities and can be used on-site. The presence of RoV and NoV was surveyed, using the developed concentration protocol, prior to one-step RT-qPCR molecular detection, in the inner part of the Bay of Koper, in mussel farming areas and a swimming area. Rotaviruses, noroviruses and fecal indicator bacteria were frequently detected in the inner part of the Bay of Koper. Rotaviruses and noroviruses were detected in the studied area, with higher rates close to the outfall of the wastewater treatment plant in the estuary of river Rižana and were also detected in the middle of the Bay of Koper and in areas used for recreation and mussel farming. The results show that water bodies, which are otherwise defined as suitable for bathing or mussel farming, based on the results of fecal indicator bacteria, still contain low concentrations of pathogenic enteric viruses. In addition to human pathogenic enteric viruses and faecal coliforms, changes in abundance of bacteria and virus particles were studied in relation to temperature, salinity, inorganic and organic nutrient concentrations in the organically polluted Rižana estuary. Preliminary results showed spatially and seasonally changes in bacterial and viral particles abundance, and bacterial composition spatially and seasonally. However, seasonality plays a greater role in bacterial dynamics.
Ključne besede: Concentration of viruses, Enteric viruses, Rotavirus, Norovirus, Feacal coliforms, Feacal contamination, qPCR, RT-qPCR, Monolithic columns, Sewage, Seasonal dynamics, Concentration, Coastal environment, Gulf of Trieste
Objavljeno: 02.07.2018; Ogledov: 904; Prenosov: 75
.pdf Polno besedilo (1,95 MB)

19.
STRUCTURE AND FUNCTIONING OF THE HYPORHEIC ZONES IN THE GRAVEL-BEDS OF FIVE RIVERS IN RELATION TO CATCHMENT LAND USE
Barbara Debeljak, 2018, doktorska disertacija

Opis: The hyporheic zone has been recognised as a functionally important component of streams and rivers. Due to increasing human impact on ecosystems, studies that assess ecological responses of the hyporheic zone are of great importance. The objectives of the thesis are to study the responses of abiotic parameters, sediment biofilm characteristics (the in situ respiration (R), potential respiratory activity (ETSA), protein content), and invertebrate assemblages (analysed as total assemblages, EPT assemblages and Copepoda assemblages) in the hyporheic zones related to different land use patterns (forest, agricultural and urban areas). The focus of study also includes the impact of clogging. This research is comprised of three sampling campaigns conducted in summer (2013), winter (2013) and spring (2014) in five pre-Alpine Slovenian rivers. For each river, three or six sampling locations were chosen in the downwelling hyporheic sections of three dominant land uses (forest, agriculture and urban) within a 250 m wide impact zone. Three sampling points per location were sampled from two depths (5 –15 cm and 20 – 40 cm) using PVC tube and Bou-Rouch method. The results indicate a significant spatial and temporal heterogeneity of measured hyporheic zone components. Physical and chemical parameters of water revealed moderate response to land use. The effect of land use on the amounts of suspendable fine sediments in the hyporheic zone was observed only in the spring season. The effect of land use was significant for in situ R during summer and spring and for ETSA and protein content in all seasons, indicating that land use, such as agriculture, near a stream can affect biogeochemical processes. A relatively high diversity of invertebrates was recognised in the hyporheic zone. Groups such as Nematoda, Oligochaeta, Copepoda and Chironomidae were found in all samples. Within EPT taxa as representatives of occasional hyporheic invertebrates, 35 taxa were identified. Within Copepoda, as representatives of permanent hyporheic invertebrates, 14 Cyclopoida and 19 Harpacticida taxa were identified. Both assemblages were characterised by high abundance of widespread taxa, such as Baetis sp., Leuctra sp., Diacylops cladestinus and Acanthocyclops hispanicus. Statistically significant differences were observed among land uses in Copopoda assemblages but not for EPT assemblages. Calculated metrics on EPT assemblages showed statistically significant differences in land use in the number of EPT taxa, Simpson index and Shannon-Wiener index. Within Copepoda assemblages, the numbers of Copepoda taxa were significantly higher at forest sites. The hyporheic assemblages were relatively well explained by environmental parameters. This thesis presents a comprehensive study of the hyporheic zone, where both structural and functional measures reflected the ecological integrity of the hyporheic zone. The in situ R and ETSA were generally higher in agriculture stream reaches, indicating that hyporheic functioning responded to nutrients and carbon runoff-derived inputs from agricultural activities within the 250 m impact zone. Thus functional parameters in situ R and ETSA were more efficient indicators of land use impacts in the hyporheic zone than physical and chemical parameters. The potential of using hyporheic invertebrates as bioindicators of environmental conditions was confirmed but should be tested with wider gradients of environmental parameters. The results obtained by this study underlined that the hyporheic zone should be integrated as an additional ecological element by assessing the ecological conditions of surface water bodies.
Ključne besede: hyporheic zone, ecosystem functioning, land use, biofilm characteristics, invertebrates
Objavljeno: 02.07.2018; Ogledov: 747; Prenosov: 14
.pdf Polno besedilo (5,31 MB)

20.
MODIFIED U1 RNAs AS SPLICING CORRECTORS IN HUMAN GENETIC DISORDERS
Katarzyna Rajkowska, 2018, doktorska disertacija

Opis: 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.
Ključne besede: Familial Dysautonomia, IKBKAP, IKAP, splicing, splicing defects, ExSpeU1, U1 snRNA, mouse model, AAV
Objavljeno: 26.03.2018; Ogledov: 1100; Prenosov: 15
.pdf Polno besedilo (13,96 MB)

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