Dissecting the role of REEP1 in preventing Tau-mediated neurodegeneration in a D.melanogaster Alzheimer's disease modelAlessio Guglielmi
, 2019, doktorska disertacija
Opis: Tau is natively an unfolded protein that promotes the assembly and the stability of the
axonal microtubules in the central nervous system. Increased formation of Tau protein
aggregates has been causatively implicated in several neurodegenerative diseases called
tauopathies. In the present study, we used the Drosophila melanogaster system to express
the longest isoform of human Tau (2N4R) in the nervous system of adult flies, recreating
the main features of the human pathology. Herein, this Tau-mediated neurodegeneration
model was used as a platform to perform genetic screenings to identify putative modifiers
of Tau toxicity. Our strategy exploited the modulation of genes considered as risk factors of
Alzheimer’s disease (AD), Frontotemporal Dementias and other neurodegenerative
diseases by RNA interference in vivo. This approach allowed us to identify a new gene
which participates in the neuronal response against Tau induced neurotoxicity in
Drosophila: D-Reep1, homologue of human REEP1 gene (h-Reep1). D-Reep1 knockout flies
showed no apparent phenotypes in physiological growing and developmental conditions,
however, they showed peculiar sensitivity to stress conditions. In addition, D-Reep1
knockout enhanced the neurodegeneration mediated by Tau expression in Drosophila eyes.
On the contrary, the overexpression of UAS-D-Reep1 and UAS-h-Reep1 abolished the
typical rough eye phenotype induced by the presence of Tau. The Co-expression of D-Reep1
in Tau backgrounds did not alter the phosphorylation pattern of this protein while, the
presence of D-Reep1 seemed to prevent the formation of Tau aggregates in vivo. Thus, the
data support the idea that D-Reep1 exerts a protective role on Tau induced toxicity which is
independent of its phosphorylation status. In this work, I analysed the mechanisms behind
the neuroprotective role of D-Reep1 and, in particular, I found that REEP1 is involved in the
regulation of the unfolded protein response (UPR) through the PERK-ATF4 cascade within
the ER. By the activation of this pathway, the neurotoxic aggregates of Tau are removed
from Drosophila neuronal tissues rescuing the normal characteristics of the affected
tissues. Evidences also suggest that the activation of autophagy was behind the removal of
Tau aggregates, providing new molecular information about the physiological role of D
Reep1 in the nervous system.
Ključne besede: AD Alzheimer Disease APP Amyloid precursor protein CNS Central Nervous System DM Drosophila melanogaster HSP Hereditary Spastic Paraplegia LN Lewy’s neurite MT Microtubule MAP Microtubule associated protein MT Microtubule/s MTBD Microtubule binding domain NFT Neurofibrillary tangle NP Neuritic plaques PHF Paired helical filament PS1 Presenilin 1 PS2 Presenilin 2 SPG Spastic Paraplegia ThS Thioflavin S
Objavljeno: 06.12.2019; Ogledov: 2574; Prenosov: 108
Polno besedilo (2,59 MB)
Zr/Cu-TiO2 CATALYSTS FOR PHOTOCATALYTIC WATER TREATMENTOlena Pliekhova
, 2019, doktorska disertacija
Opis: This work entitled «Zr/Cu-TiO2 catalysts for photocatalytic water treatment» tackles a problem of tons of dyes discharged everyday mainly from textile industries. This is a huge concern because of dyes persistence, toxicity and potential to the bioaccumulation in living organisms. Here, a small contribution to overall problem is presented.
The research work consists of three main parts: Theoretical background, Experimental and Results and discussion. In the Theoretical background chapter the overall problem is identified and discussed. The main water treatment techniques are presented briefly with their advantages and drawbacks. Photocatalysis assisted with TiO2 is shown as an alternative additional technique with its own pros and cons. Further, a cursory overview of TiO2 modification techniques is made and advantages in using copper and zirconium oxides for TiO2 modification are presented. The reason and possible positive effect of using of two oxides simultaneously for TiO2 modification is indicated. Additionally, a positive impact of solar light for overall rate of dyes degradation assisted with TiO2 is discussed. Within the framework of photosensitization effect, the problem of using dyes for assessment of photocatalytic properties of materials under the visible light is touched on. Photothermal methods as possible beneficial techniques for this purpose are proposed.
In the Experimental chapter, all experimental techniques used in current research work with the technical details specific for the research work are presented. There are methods of materials preparation, photocatalytic tests under different conditions and varying model pollutants, and physico-chemical characterization techniques.
The Results and discussion chapter is divided by three subchapters. Each chapter is dedicated to one hypothesis, which was checked and confirmed or disproved. The first hypothesis is about beneficial loading of copper and zirconium oxides to TiO2. After numerous experiments held, it was concluded that the pair of copper and zirconium oxides is beneficial for simultaneous application on TiO2 surface to promote its performance.
The second subchapter is about zirconium distribution and its impact on the activity of studied materials towards the antraquinone dye Reactive Blue 19. Different techniques such as XRD, TEM, EXAFS and many others were used in order to characterize the materials and to understand the details of the processes taking place. The existence of upper limit of copper oxide loading for its beneficial effect on TiO2 performance was demonstrated. It was observed that zirconium species loading leads to improvement in performance of materials with higher copper loading, which otherwise lowers activity of TiO2 – this is in case when copper only above its optimal level is present. It was concluded that zirconium oxide containing species occupy rutile surface sites in mixed phase TiO2 and this way beneficially influence the material performance towards the dye removal.
The third subchapter is about the visible light TiO2 assisted dye degradation. It is well known that photosensitization mechanism of dye degradation appears under the visible and as a consequence under the solar light irradiation. This leads to the overall enhancement of dyes degradation, which is positive and may be used as a benefit for faster pollutant destruction. However, this fact leads to mishmash in the determination of real photocatalytic activity of materials towards the dye. It was hypothesized that the impact of photosensitization mechanism on the overall material performance against Reactive blue 19 dye (RB19) descends with the dye concentration lowering. Thermal lens spectrometry (TLS) which is sensitive to chemicals at low concentrations was used for hypothesis evaluation.
To summarize the research work achievements, the conclusions of the thesis are given at the end.
Ključne besede: titanium dioxide, photocatalysis, zirconium/copper, surface modification, water treatment, dyes
Objavljeno: 03.12.2019; Ogledov: 4139; Prenosov: 117
Polno besedilo (3,18 MB)
Photoactive nanocomposite thin films on glass and thermosensitive substratesNives Vodišek
, 2019, doktorska disertacija
Opis: Photocatalysis is a well-known process for the last few decades; it is predominantly used for water and air purification, but also for self-cleaning and antibacterial surfaces. The photocatalytic process is one of the advanced oxidation processes, where semiconductors are mainly used as photocatalysts. The most known and used semiconductor is nanosized titania, which can non-selectively decompose organic matters. One of the side effects of nano TiO2 under UV irradiation is photoinduced hydrophilicity, which furthermore improves the self-cleaning effect. The main drawback of TiO2 is that for its activation UV light is needed, which represents only 5 percent of the solar spectrum. Consequently, actinic irradiance power is low and the response of photocatalyst is limited. Novel approaches are being introduced to improve TiO2 response to visible light such as doping, coupling, modification of surface morphology and others.
The thesis consists of five principal chapters. The first chapter is focused on a short literature review and explanation of some basic terms and principles that are related to this thesis.
The aim of the research was to prepare transparent photocatalytically active thin films on glass and thermosensitive substrates. Four thermosensitive substrates were used: polyvinyl chloride (PVC) foil, polymethyl methacrylate (PMMA) sheet and polyester (PES) fabric coated with a polyvinyl and acrylic coating, with (D1) or without (D2) an additional polyvinylidene fluoride topcoat. The synthesis of films was modified with the introduction of zirconium aiming at improving the photocatalytic activity of the TiO2 films, and the final films had a SiO2 phase present for increased mechanical robustness.
The third chapter is dedicated to the experimental part of the thesis. The detailed process of synthesis is described. The sol-gel process was used to prepare initial Ti-Zr sols, colloidal aqueous solutions with TiO2 and ZrO2 nanoparticles derived from titanium and zirconium alkoxides. The four different Ti-Zr sols were prepared with 0, 5, 10 and 20 molar % of Zr according to Ti. The depositing solution was prepared by mixing Ti-Zr sol, SiO2 sol binder, 1-propanol and 2-propoxyethanol, and then thin films were deposited by dip-coating technique. After the deposition, samples did not require high-temperature calcination since the photocatalytic anatase phase was present already after drying the deposited layers. Coated glass slides were put into a furnace at 150 °C, while samples on thermosensitive substrates were just treated by a heat gun. Samples in powder and thin film form were characterized by UV-Vis, FTIR-ATR and laser beam deflection spectroscopy, X-ray diffraction, scanning electron microscopy and thermal analysis. One of the main aspects of the photocatalyst is its activity. Photocatalytic activity of the thin films was determined, either quantitatively by the formation of fluorescent hydroxyterephthalic acid, one of the first degradation products of terephthalic acid deposit, or qualitatively by a visual-based method where degradation of resazurin ink was observed. Two additional methods were used on glass samples, electron paramagnetic resonance, and degradation of methyl stearate by observing the change in water contact angle under UVA irradiation. Mechanical stability of films on various substrates is an essential factor, describing how successful was immobilization of the photocatalytic materials on the substrate. It was determined by the pencil hardness test, i.e., Wolff-Wilborn method.
In the fourth chapter, titled “Results and Discussion,” the most important part of the thesis is placed. Materials characterization and associated discussion of the results are divided into three major parts: i) powder samples characterization; ii) characterization of thin films on a glass substrate, and iii) characterization of films on thermosensitive substrates.
In the conclusions, the main findings of the Ph.D. research work are summarized. One of our initial hypotheses was disapproved, thin films were not more active with a higher content of zirconium. However, they were more durable. All the obtained films were transparent and photoactive, despite the low-temperature synthesis procedure. Besides commonly used glass substrate, immobilization on thermosensitive substrates was also successful.
Ključne besede: glass substrate, immobilization, nanocomposite, nanomaterials, PES, photocatalysis, PMMA, PVC, self-cleaning surfaces, silica, plastic substrates, thin films, titania, zirconia
Objavljeno: 14.10.2019; Ogledov: 2879; Prenosov: 158
Polno besedilo (6,23 MB)
SEARCH FOR NEUTRINOS AT EXTREME ENERGIES WITH THE PIERRE AUGER OBSERVATORYMarta Trini
, 2019, doktorska disertacija
Opis: The detection of Ultra-High-Energy (UHE) neutrinos around and above 10 18 eV (1 EeV) can be the key to
answering the long-standing question of the origin of the UHE cosmic rays. The Pierre Auger Observatory
is the largest experiment that can detect the extensive air showers produced when the cosmic rays and
neutrinos interact in the earth’s atmosphere. In particular, with the Infilled array of the Surface Detector
of the Pierre Auger Observatory we can detect sub-EeV neutrino-induced particle showers. In this thesis
we demonstrate that it is possible to discriminate neutrino-induced showers from the background showers
produced by the more numerous nucleonic cosmic rays. The sensitivity to neutrinos is enhanced in the
inclined directions with respect to the vertical to the ground, where cosmic ray-induced showers starting in
the upper layers of the atmosphere are dominated by the muonic component of the shower, while deeply-
penetrating neutrino showers in contrast exhibit a large electromagnetic component. Based on this idea in
this thesis we have developed a search procedure for UHE neutrinos that consists on selecting inclined
events in the Infilled array of the Pierre Auger Observatory in which the signals in the water-Cherenkov
stations are spread in time, characteristic of the presence of electromagnetic component in the shower. We
have established a complete chain of criteria to first select the inclined events among the sample of all
events triggering the Infilled array, and then identifying those that have a large electromagnetic component
at ground, and hence can be considered as neutrino candidates. We have identified a single variable, the
so-called area-over-peak averaged over all of the stations in each event, as a suitable observable for neutrino
identification purposes. The neutrino selection was established using extensive Monte Carlo simulations of
the neutrino-induced showers in the Infilled array of Auger as well as a fraction of the data assumed to
be totally constituted of background nucleonic cosmic rays. Using these neutrino simulations we have also
computed the exposure of the Infilled array to UHE neutrinos in the period 1 January 04 - 31 December 2017.
Associated systematic uncertainties on the exposure are also described. Expecting no candidate neutrinos in
the period up to 31 December 2017, and adopting a differential neutrino diffuse flux dN ν /dE ν = k E ν −2 in
the energy range from 0.05 to 1 EeV, we have obtained a 90% C.L. upper limit on the all neutrino flavor,
k 90 < 7.97 × 10 −8 GeV cm −2 s −1 sr −1 .
Ključne besede: astroparticles, astrophysical neutrinos, cosmic rays showers, Pierre Auger Observatory, Infilled
Objavljeno: 03.10.2019; Ogledov: 3794; Prenosov: 135
Polno besedilo (6,02 MB)
Factors influencing collected quantities of municipal solid wasteSlavica Schuster
, 2019, doktorska disertacija
Opis: The first part of the survey, using the survey (enclosed with the annexes), tried in various ways to identify samples and facts by which we could predict the amount of waste collected according to some social and economic indicators of all municipalities of the Republic of Slovenia. Data were collected through surveys to determine the way we work and think about the collection of waste and landfills. For my research I have selected the Republic of Slovenia and its 212 municipalities. It is precisely Slovenia that in many respects represents a bright spot in waste management and management.
In the second part of the research we used statistical methods to find the factors that influence the increase of waste. From the results we have proved our hypotheses and one of the most important is how the average age of people influences the increase of waste. We did the research over a period of 6 years (2012-2017). Where, by comparison, we find that they do not change significantly in structure by age, but of course socio-economic indicators such as average age change.
In the last section, we compared the growth of waste collected with GDP growth. We show that with the growth of GDP, the amount of waste collected also increases. The research is also based on the Republic of Slovenia and beyond. In the second part of the last survey we also included data for EU Member States (28 of them).
Ključne besede: waste, waste management, amount of waste, socio-economic impacts, Slovenia, wild-landfills, GDP
Objavljeno: 02.10.2019; Ogledov: 3110; Prenosov: 207
Polno besedilo (3,88 MB)
HOLISTIC GEOMORPHOLOGICAL SPATIAL ANALYSIS OF KARST ON KRK ISLANDEla Šegina
, 2019, doktorska disertacija
Opis: The intriguing spatial variability of surface features on Krk Island has stimulated the research of this karst area located in the coastal zone of the Dinaric karst in Croatia. Field inspection, ortho-rectified aerial photos (0.5m resolution) and a topographic map (1:5,000) were used for the detection and delineation of detectable karst surface features appearing on the island with the area of 405.5 km². This method resulted in the identification of several yet undefined types of surface features occurring on karst, requiring the revision of the existing classification and re-establishment of a new classification system compatible with the particular field reality. Several morphologic and distributive parameters that had been calculated for each re-classified type of surface feature provided insight into the surface features elementary characteristics, their spatial variability and the correlation to the other types of surface features and to the recent karst relief. This analysis based on a large, accurate dataset, contributed to the general knowledge on karstic surface features, the conditions of surface features in Dinaric karst and to the understanding of the karst surface evolution on Krk Island.
Ključne besede: karst geomorphology, GIS, spatial analysis, Krk Island, Dinaric karst, Adriatic Sea
Objavljeno: 13.09.2019; Ogledov: 3433; Prenosov: 201
Polno besedilo (17,12 MB)
DETEKCIJA KOVINSKIH KOMPLEKSOV IN ORGANOKOVINSKIH SPOJIN V VZORCIH IZ OKOLJA S SPEKTROMETRIJO TERMIČNIH LEČLeja Goljat
, 2019, doktorska disertacija
Opis: Environmental pollution is one of the greatest challenges that the world is facing today. Toxic compounds, such as pesticides, allergens, pharmaceuticals, toxins and heavy metals are widely present in the air, water and soil, and can affect the health of people and animals even in small quantities, as well as they may cause long- or short-term damage in plants [Hill, 1997].
Heavy metals (mercury, arsenic, cadmium…) are widely spread in the environment. They derive from a number of sources, including mining, industrial wastes and vehicle emissions [Tchounwou et al., 2012]. They are easily incorporated into biological molecules and exert their toxic effects by displacing essential metals of a lower binding power in biologically active molecules or by acting as noncompetitive inhibitors of enzymes, affecting neurological, reproductive, renal and hematological systems [Sunil D’Souza et al., 2003; Heavy-Metal Pollution, 2018]. Metals form countless compounds (e.g. metal complexes and organometallic compounds) which are essential for living organisms (vitamin B12, hemoglobin, chlorophyll) and/or have a wide range of applications in industry and other areas, including analytical chemistry. Because of the potential risk which toxic metals represent to the living organisms and also because of the importance of some essential metals, different analytical techniques and detection methods have been developed for studies of their occurrence, fate and concentration in the environment and in organisms. However, providing a required sensitivity for determination and speciation of different metals and their compounds, especially in small- volume samples is still a challenge.
Therefore, general objectives of this dissertation were development of novel analytical methods for sensitive, reliable and fast determination of metal species, based on highly sensitive optothermal technique thermal lens spectrometry (TLS), which can be used as detection tool following colorimetric reaction of a selected metal ion or for direct detection of colored organometallic compounds.
This dissertation is composed of the following chapters: introduction, research goals, theoretical background, results and discussion, conclusion and references. The core of this dissertation is presented in the fifth chapter (results and discussion), which is divided into three parts. They separately cover development of methods for determination of iron redox species, pyoverdine and Fe-pyoverdine complexes and mercury. Pyoverdine is a siderophore, excreted by a certain bacteria in order to scavenge iron in the environment and is closely related to the chemistry of iron in such biological systems. Therefore, the first two parts are closely related.
Procedures for batch mode thermal lens microscopy (TLM), flow-injection thermal lens sprectrometry (FIA-TLS) and µFIA-TLM (flow injection and TLS detection in microspace) were developed for Fe(II) and Fe(III) determination, based on colorimetric reaction of Fe(II) with 1,10-Phenanthroline. All these procedures were focused on cloudwater examination with a tendency to minimize sample consumption but at the same time preserve low limits of detection (LOD) and limits of quantification (LOQ). TLM measurements with highly collimated probe beam were performed in a 100 μm optical path length cell (40 µL volume), which resulted in a considerably smaller sample volume requirement (500 µL in total) and consumption, as compared to UV-Vis spectrophotometry, which required at least 25 mL of sample due to large volume (almost 30 mL) of the 10 cm optical path-length sample cell. LODs for mode-mismatched TLM were 0.16 and 0.14 µM for Fe(II) and Fe(total) (sum of Fe(II) and Fe(III) concentrations), respectively, while LODs for UV-Vis spectrophotometry were 0.01 µM for both Fe(II) and Fe(total). By using the mode mismatched TLM we were able to detect concentrations corresponding to absorbances as low as 1.5 × 10-5, while the lowest absorbance detectable on the UV-Vis spectrophotometer corresponded to 1.1 × 10-3, despite the use of the 10 cm optical path-length cell.
Another important step in the development of new methods for Fe(II) and Fe(III) determination was the use of TLS detection in FIA (FIA-TLS). By injecting 50 µL of the sample into the FIA-TLS system, cca. 10 times lower LODs were achieved (1 × 10-3 µM for Fe(II) and 8 × 10-4 µM for Fe(total)), as compared to the UV- Vis spectrophotometry.
Nevertheless, the development of μFIA-TLM method, with on-line colorimetric reaction for Fe(II) and Fe(III) determination is considered as the most important achievement of this study. The results show that despite 100 times shorter optical path length and low sample consumption (3 µL of each sample/injection) compared to UV-Vis spectrophotometry, LODs for µFIA-TLM were 0.10 and 0.07 μM for Fe(II) and Fe(total) respectively, which is sufficiently for cloudwater analysis, since concentrations, lower than 0.1 μM are not expected [Parazols et al., 2006; Deguillaume et al., 2014]. Linear range for Fe(II) and Fe(III) determination by μFIA-TLM was between 0.1 and 70 µM. To test the accuracy of this method, artificial cloudwater was prepared, spiked with different amounts of Fe(II) and Fe(III) and analyzed for iron content by µFIA-TLM and UV-Vis spectrophotometry. Good agreement was observed between the two methods. To ascertain the ruggedness of the method 7 (or more) replicate determinations at two different concentrations for both, Fe(II) and Fe(total) in artificial cloudwater were carried out on day 1 (replicates were measured instantly after fortification), day 2 and day 5. A student’s t-test (p=0.05) was applied to compare 3 sets of obtained data (day 1, day 2 and day 5) and showed that sets are not significantly different from each other. Considering very low sample volume requirement of µFIA-TLM, this should be the method of choice for determination of Fe(II) and Fe(III) in investigations of processes in cloudwater, where multiparameter analysis is desired (determination of other ions, ligands, microbial counts, etc.). When larger sample volumes are available, FIA-TLS can be used for accurate determination of iron species at lowest concentration levels.
High performance liquid chromatography (HPLC) was applied for separation and detection of pyoverdine (PVD), produced by Pseudomonas fluorescens 36b5, a bacterial strain isolated from the aqueous phase of clouds at the Puy de Dôme station (1465 m, France). Reversed-phase (RP) chromatography (RP-18 chromatographic column Hypersil gold), hydrophilic interaction liquid chromatography (HILIC) (ZIC®-Hilic column) and three different detection systems
(diode-array (DAD), spectrofluorimetry (FLD) and TLS) were tested for their performance in separation and determination of pyoverdines and corresponding complexes of pyoverdine with iron (Fe(III)-PVDs).
PVDs and Fe(III)-PVD complexes could not be separated and quantified by applying HILIC technique, therefore it was concluded, that HILIC is not suitable for HPLC-DAD and also not for HPLC-TLS, since the method should offer a simultaneous sensitive detection of free PVDs as well as Fe(III)-PVD complexes in a single chromatographic run.
Since pyoverdine standards were available only as a mixture of several different forms of PVDs, whereby the exact composition was unknown, the quantification of each of the four major specie (two fluorescent PVDs and two nonfluorescent Fe(III)-PVDs) in the standard, which was obtained from Université Clermont Auvergne, Institut de Chimie de Clermont-Ferrand, was performed. When applying Hypersil gold column, a linear correlation between fluorescence intensity and absorbance of each component was observed in a concentration range 3–24 µg/mL, whereby LODs were estimated to be 0.03–0.04 µg/mL for each of the major PVD species (HPLC-DAD). Even though HPLC-FLD method provided cca. 100 times lower LODs, it is not the method of choice for determination of PVD species in cloudwater, because it does not allow detection of PVD complexes with Fe(III). When comparing HPLC-TLS and
HPLC-DAD, LODs were 5 to 8 times lower in case of HPLC-TLS, which was a significant improvement. Furthermore, recoveries (89–111 %) at two concentration levels of four PVD species in two independent samples, showed good reliability of the method.
Almost all mercury in uncontaminated drinking-water is thought to be in the form of Hg2+ [WHO, 2010]. Therefore, the method for Hg2+determination based on colorimetric reaction with triamterene, described originally by Al-Kady and Abdelmonem was further investigated in this study, as well as the possibilities of application of this reaction for Hg2+ determination by TLS. The stoichiometry of the complex formation was determined by the method of continuous variations and saturation experiment, suggesting formation of the complex with the formula Hg2-triamterene. The obtained value of the molar absorption coefficient was 9988 Lmol-1cm-1 at 403 nm, which significantly contradicts the existing data in literature, which reports the molar absorption coefficient of 5.32 × 104 Lmol-1cm-1 [Al-Kady and Abdelmonem, 2013]. Even though the spectrophotometric results were not encouraging for triamterene as colorimetric reagent for Hg2+ determination, it was further investigated for its performance in TLS system. Fe(II)-1,10-phenanthroline (ferroin) was used for comparison, because it was well studied for TLS applications previously. The results showed that Hg2-triamterene in solutions was degraded when it was exposed to the light of the excitation beam. Due to the lower molar absorptivity than reported in literature, fotodegradation and unfavorable complex stoichiometry, triamterene was not confirmed as a suitable colorimetric reagent for highly sensitive Hg2+ determination by TLS.
In summary, this dissertation investigates alternative approaches for analysis of metal complexes and organometallic compounds in small-volume environmental water samples. Methods, which were developed in this research, could potentially serve as improvements of existing technologies, to facilitate analysis of such samples, by offering simple handling of samples and superior sensitivity over the UV-Vis spectrophotometry.
Ključne besede: thermal lens spectrometry, thermal lens microscopy, high performance liquid chromatography, microfluidics, metal complexes, organometallic compounds, iron, pyoverdine, mercury
Objavljeno: 05.09.2019; Ogledov: 3010; Prenosov: 142
Polno besedilo (3,65 MB)
THE RECEPTION OF THE WEST-EUROPEAN AND AMERICAN PLAYWRIGHTS IN SLOVENIAN DRAMA AND THEATRE (1945–1970)Rok Andres
, 2019, doktorska disertacija
Ključne besede: reception, drama, Slovenian drama, theatre history, existentialism, absurd drama, SNG Drama Ljubljana, western playwrights
Objavljeno: 27.08.2019; Ogledov: 3011; Prenosov: 181
Polno besedilo (1,47 MB)
Structural and functional determinants of TDP-43 aggregationSanja Škaro
, 2019, doktorska disertacija
Opis: TDP-43 (TAR DNA-binding protein) is an hnRNP that was identified as the main component of the brain inclusions characteristically found in patients suffering of Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. As an hnRNP protein, TDP-43 fulfills diverse roles in mRNA metabolism, localization and transport. Structurally, TDP-43 is composed of a well conserved N terminal domain (NTD), two RRM domains of which RRM1 is necessary for recognizing and binding to its target, UG rich RNA sequences, and the C-terminal domain (CTD) which is a Glycine rich domain. The CTD also contains a Q/N rich region that plays a key role in protein aggregation and interaction with another hnRNP proteins and polyglutamine repeats. This thesis focus on the structural determinants involved in the different TDP-43 interactions with itself and with other hnRNPs. Both the carboxyl and amino terminal domains are involved in these interactions. We have mapped the regions more relevant for the function of TDP-43 and for the aggregation process characteristic of the pathological pathway leading to neurodegeneration. We have started to further study the N-terminal domain. Previous results in our laboratory using a cellular aggregation model have shown that the N-terminal domain is also necessary for sequestering the endogenous TDP-43 into the aggregates. In particular, the intact NTD, specifically residues 1 to 77, have been shown to be needed to efficiently recruit TDP-43 monomers into these aggregates. We have extended our knowledge of NTD structure and function, by assessing the behavior of a series of proteins in which key structural features (α-helix and β-sheets) were modified and TDP-43 splicing function together with structure via NMR were analyzed. It was found that by disrupting protein secondary structure in the NTD (mutation in α-helix NTD-31V/R-32T/R) the capacity of the aggregates to sequester enough TDP-43 to induce loss of function was lost.In fact, this protein is also unable to recovery TDP-43 functionality when it is disrupted due to sequestration of the endogenous TDP-43 in add back experiments. Disturbing protein stability through substitution of residues in α-helix also affects its ability to form an active conformation. On the other hand, synthesis of hybrid peptides containing certain NTD and CTD segments was performed in order to see if they are capable to bind to the TDP-43 aggregates. However, it has been shown that these synthetic peptides have a greater ability to induce TDP-43 aggregation than to bind to them, probably due to specific functional characteristics of NTD and CTD segments used for their synthesis.The main focus of the thesis was on the C-terminal domain sequences involved in protein-protein interaction, misfolding and aggregation.A comparison of human, mouse, zebrafish, Annelida, flatworms and Drosophila showed a very strong conservation of the NTD and RRMs, but the C terminal regions of human and other TDP-43 orthologues are very different.I have studied Human and Drosophila melanogaster orthologues, because Drosophila orthologue contains different paralogs of TDP-43.Through a series of deletions and mutations it was shown that the shorter paralog of Drosophila TDP-43 (TBPH-RA) is more active than the longer one (TBPH-RC), and that this is due to a combination of two factors: 1. TBPH-RC by itself aggregates more than TBPH-RA, 2. The functionality of TBPH-RC is downregulated by intramolecular interactions in the C terminal domain. Apparently there is a cation-π interaction involving Tryptophan and Arginine in TBPH-RC that has a high relevance to the protein function and is lacking in the TBPH-RA.Overall this data has identified structural features essential for the proper function of TDP-43.In addition, we have also identified sequences that are critical in the pathological aggregation process of TDP-43 that lead to the characteristic brain inclusions in ALS and FTLD and to the loss of functionality
Ključne besede: TDP-43 structural determinants, hybrid peptides, protein-protein interactions, intramolecular interaction, cation-π interaction, Drosophila orthologues.
Objavljeno: 22.07.2019; Ogledov: 2539; Prenosov: 167
Polno besedilo (6,84 MB)
Regional biodiversity and seasonal dynamics of the bacterial communities in karstic springs of SloveniaMaja Opalički Slabe
, 2019, doktorska disertacija
Opis: Information on groundwater’s natural bacterial communities is important for evaluating pristine groundwater’s quality, as environmental changes potentially lead to alterations in bacterial community structures. In four seasons, 15 hypothetically pristine springs across five karst eco-regions in Slovenia (Central Europe) were analysed during their low discharge, and their basic physical and chemical parameters were recorded. The diversity of bacterial community structures was assessed with a terminal restriction fragment length polymorphism fingerprinting method. Total cell counts as bacterial abundance and electron transport system activity as bacterial respiratory activity were used for quantitative evaluation of bacterial communities. To detect anthropogenic pollution, spring water was tested by the cultivation of coliform bacteria and Escherichia coli (E. coli).
Results indicate distinct regional differences in environmental parameters, bacterial respiratory activity, presence of coliforms and E. coli, bacterial abundances, and qualitative bacterial community structures, but there were no regional differences in the numbers of operational taxonomic units and biodiversity indices (Shannon-Wiener’s and Simpson’s diversity index, Buzas and Gibson’s evenness). The analysis of seasonal environmental parameters showed significant differences in dissolved organic carbon and pH, and also significant changes in bacterial respiratory activity and abundances, as well as differences in bacterial community structures, the numbers of operational taxonomic units, and biodiversity indices. No seasonal variations were shown for coliforms and E. coli.
Despite seasonal oscillations, regional differences prevailed among five karst eco-regions and indicate a significant influence on the qualitative and quantitative aspects of groundwater bacterial community structures. The study confirmed both regional and seasonal differences in groundwater bacterial community structures, which should be included in further sustainable management plans of the aquifers.
Ključne besede: karst springs, bacterial community structure (BCS), terminal restriction fragment length polymorphism (T-RFLP), total cell counts (TCC), electron transport system activity (ETSA)
Objavljeno: 26.06.2019; Ogledov: 2848; Prenosov: 151
Polno besedilo (3,76 MB)