EVALUATION OF MARINE SEDIMENTS FROM THE PORT OF LUKA KOPER FROM THE ENVIRONMENTAL PERSPECTIVE AND IN TERMS OF THEIR USABILITY IN THE BRICK INDUSTRYPatrik Baksa
, 2016, magistrsko delo
Opis: The majority of the world’s goods are transported over water and dredging is essential for the development of harbors and ports. Therefore, the management of dredged material is a worldwide issue. Due to its chemical and petrographic, mineralogical and homogeneity composition, marine sediments are an appropriate raw material to use in the brick industry. Marine sediments can serve as raw material for the production of clay blocks, roofing and ceramic tiles.
Different analyses were carried out in order to determine if the dredged material from the Port of Koper is environmentally friendly and suitable to use in the brick industry. These analyses included: a chemical analysis, a mineralogical analysis, a particle size analysis and a chloride content (Cl-) analysis, and tests of firing in a gradient furnace. Furthermore, tests of mechanical properties, as well as tests of frost-resistance of the samples were carried out.
On the basis of primary analyses and samples prepared in a lab, it was established that marine sediments from the Port of Koper without any additives are only conditionally suitable as a source material for producing brick products. In collaboration with Gorica brickworks (Goriške opekarne), a pilot production from a mixture of 60% component B from Gorica brickworks and 40% component A (marine sediments) from the Port of Koper was prepared. Different tests showed the mixture could be appropriate for brick production.
Ključne besede: marine sediments, recycling, clay bricks, chemical analysis, mechanical properties, brick production.
Objavljeno: 05.09.2016; Ogledov: 1473; Prenosov: 122
Polno besedilo (3,30 MB)
THE STUDY OF OPTIMAL TECHNOLOGICAL PROCEDURES OF INTERNAL PLUMBING SYSTEM DISINFECTION FACILITIES IN USE BY THE SENSITIVE HUMAN POPULATIONSJanez Škarja
, 2016, magistrsko delo
Opis: In a developed world, water is used in a variety of installations and devices for the improvement of life standard. It is important for these elements to be suitably managed and maintained, otherwise they can present a risk to people's health. Although potable water from a public plumbing system coming via the water supply into an internal plumbing system normally is compliant with the regulations, the quality of water in an internal plumbing system often changes – water gets contaminated. There are several types of microorganisms that can grow in water. They carry a great potential for the growth and proliferation of bacteria from the genus Legionella that cause Legionnaires' disease and Pontiac Fever. Even the fact that Legionella infection has for several years been mentioned in the accident insurance conditions of insurance companies in relation to receiving the insurance fee as compensation for bed day, shows the extent and foremost the seriousness of the disease (5–15% mortality rate).
The research part of the master thesis focused mainly on determining the presence of bacteria from the genus Legionella in the water of internal plumbing systems of healthcare facilities. During the entire research period of 8 years, 2,676 samples of cold and hot water were acquired from 23 healthcare facilities.
The main purpose of the research was to determine the efficiency or adequacy of applied approaches, i.e. physical disinfection with heat and chemical disinfection with chlorine, in eliminating or reducing the amount of Legionella present in the water of selected healthcare facilities. Another aim was to evaluate the impact of softening potable water with polyphosphates on the proliferation of certain microorganisms in water, and to determine the by-products of chemical disinfection.
Upon sampling, an organoleptic examination of water (appearance, odour), electrometric measurements of water temperature, and colorimetric measurements of free chlorine concentrations in water were performed. The data for the analysis of Legionella presence were acquired by water sampling and the isolation of bacteria found in the water. In order to acquire laboratory results of specific physical and chemical parameters, ion chromatography, gas chromatography, spectrophotometry, titration, and inductively coupled plasma – mass detector were used.
Although water samples from only two healthcare facilities showed no presence of Legionella, the number of water samples with the presence of Legionella decreased for at least 25% from the beginning to the end of period set. Also, after certain general sanitary and technical measures a noticeable improvement within healthcare facilities after 2008 could be observed. Regarding the initial part of the set period, a more favourable relationship between positive and negative samples, a lower number of samples with highest concentrations of present Legionella, and a higher number of samples with lower concentrations of present Legionella were determined. The research has shown that the existing system of ensuring health-compliant water is fairly efficient; however, the results could be improved additionally by investing more into the preparation and renovation of systems. Due to a limited number of samples, a direct impact of adding polyphosphates to potable water could not be linked to the occurrence of Legionella. None of the samples showed an increased concentration of by-products of potable water disinfection.
In its conclusion, the research has shown that the procedures after overheating and after chlorine disinfection result in a similar success. While filtration proved to be most efficient, from the perspective of Legionella infections the use of medical bathtubs can present a high risk to people's health, therefore such devices should be subject to more frequent maintenance and supervision.
Ključne besede: potable water, internal plumbing system, biofilm, health facilities, disinfection, Legionella
Objavljeno: 05.09.2016; Ogledov: 1852; Prenosov: 120
Polno besedilo (1,70 MB)
STRUCTURAL PROPERTIES OF MANGANESE-FUNCTIONALIZED SILICA AEROGELSTatjana Kobal
, 2016, magistrsko delo
Opis: Manganese-functionalized silica with interparticle mesoporosity and isolated Mn sites (Mn/Si = 0.01) is an excellent Fenton catalyst for water cleaning. However, there is a problem that needs to be solved, i.e., Mn leaching from the silica support during the reaction. The solution may lie in using aerogels as the silicate supports for the manganese. The goal of this master thesis is the synthesis and characterization of manganese-functionalized silica aerogels with different Mn/Si molar ratios and a determination of their structural properties. The emphasis is on the preparation of manganese-functionalized silica aerogels with isolated manganese sites.
Firstly, manganese-functionalized microporous and mesoporous silicates with a molar ratio Mn/Si = 0.005–0.02 were synthesized according to the literature and characterized as reference materials for manganese-functionalized aerogels. Manganese silicalite-1 (MnS-1) as a microporous zeolite-type silicate and manganese-functionalized silica (MnKIL-2) as a mesoporous silicate were prepared by the sol-gel process with a hydrothermal and solvothermal treatment, respectively. MnS-1 and MnKIL-2 with a molar ratio of Mn/Si ≤ 0.01 contain manganese as isolated sites in the silica framework and with a molar ratio of Mn/Si > 0.01 contain manganese as manganese oxides.
Secondly, manganese-functionalized aerogels (MnAEG) with a molar ratio Mn/Si = 0.005–0.02, were prepared according to the acid-base of the sol-gel polymerization of a tetraethylorthosilicate precursor, which is followed in combination with a supercritical drying using CO2. The prepared materials were characterized using the following techniques: X-ray powder diffraction (XRD), energy-dispersive X-ray elemental analysis (EDX), nitrogen physisorption, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The characterization results on manganese-functionalized aerogels (MnAEG) showed Mn oxide nanoparticles incorporated into the aerogel already at a low Mn concentration (Mn/Si = 0.005), which means that Mn aerogels are not promising silica supports for Mn catalysts applied for water cleaning, because they do not contain isolated Mn sites.
Ključne besede: : SiO2 aerogels, manganese-functionalized SiO2 aerogels, sol–gel process, CO2 supercritical drying
Objavljeno: 02.09.2016; Ogledov: 2011; Prenosov: 108
Polno besedilo (4,37 MB)
ECOLOGICAL STUDIES OF EPIKARST COMMUNITIES IN ALPINE AND PRE-ALPINE CAVESFederica Papi
, 2016, doktorska disertacija
Opis: The karst ecosystem shows heterogeneity and variability of geology, hydrology, morphology and ecology in space and time. Chemical composition of water in karst plays a crucial role not only in dissolution of karst rocks and deposition but also in ecological processes. The heterogeneous nature of karst aquifers leads to difficulties in predicting groundwater flow and contaminant transport direction and travel times. For its position at the top of karst, epikarst represents the interface between surface and underground. Epikarst ecology, biodiversity and fauna have rarely been systematically studied. The typical absence of enterable passages makes necessary an indirect approach.
In this research, epikarst biodiversity in relation to environmental conditions was studied in seven Alpine and Pre-Alpine caves, located at different altitudes, in Slovenia and in Italy: Snežna jama na planini Arto (1556 m a.s.l.), Jama pod Babjim zobom (860 m a.s.l.), Zadlaška jama (300 m a.s.l.) and Pološka jama (500 m a.s.l.), in Slovenian Alps and Pre-alps, and Grotta A del Ponte di Veja (600 m a.s.l.), Covolo della Croce (875 m a.s.l.) and Grotta di Roverè Mille (1005 m a.s.l.) in Lessinian Massif. In these caves, percolating water was monthly monitored for fauna in the period of one year. Temperature, discharge, conductivity and pH were measured at the same time, and water samples for the laboratory analyses of total hardness, concentrations of various ions (calcium, chlorine, nitrate, sulphate and phosphate) and dissolved organic carbon were taken.
66 aquatic and terrestrial, mostly hypogean, species were found. Aquatic fauna was dominant in all caves, with the exception of Covolo della Croce. Caves located at higher elevations harbour less diverse communities. No animals were found when discharge was very low and in correspondence of moonmilk formations. The most abundant and diverse group was Copepoda, with thirteen different species and 776 individuals at different maturity stadia, totally representing 61% of the community (between two and three copepod species per cave in Slovenia and between one and six per cave in Italy).
The 62% of copepod species were stygobionts. Some copepod species (e.g.
Speocyclops infernus) have wide ecological tolerance to environmental variables, other have more restricted tolerances (e.g. Lessinocamptus insoletus). With regard of copepods, there was a good agreement between the number of observed species and the total number of predicted species, confirming that the sampling method was appropriate and sampling complete. Correlations between the copepod community structure and measured parameters, obtained with Pearson correlation coefficient and principal component analysis (PCA) show different patterns. Presence of copepods was positively correlated with conductivity and dissolved organic carbon in Grotta di Roverè Mille, and with carbonate and nitrate in Pološka jama. In other caves not so high correlations were found between copepod species and measured parameters and between pairs of species.
Investigations of the Alpine and Pre-alpine epikarst fauna can help to understand better the ecology of the epikarst fauna and its roles within the large range of different shallow subterranean habitats.
Ključne besede: Epikarst, Alpine and Pre-Alpine caves, percolating water, speleobiology, fauna.
Objavljeno: 31.08.2016; Ogledov: 1841; Prenosov: 129
Polno besedilo (2,00 MB)
SINGLE NUCLEOTIDE POLYMORPHISMS OF THE CHROMOSOME SEGREGATION GENES INVOLVED IN THE DEVELOPMENT OF GASTRIC CANCERMarija Rogar
, 2016, doktorska disertacija
Opis: INTRODUCTION. Gastric cancer represents the fifth most frequently diagnosed cancer in the world. Despite numerous research studies, mechanisms leading to disease are poorly known and unclear. At the molecular level, many changes are involved in the development of gastric cancer, including malfunction of chromosome segregation genes. These abnormalities can lead to chromosomal instability (CIN). Segregation gene function can be affected by the low penetrance errors which include polymorphisms.
AIM. The aim of this study is to examine the effect of selected polymorphisms in specific segregation genes on gastric cancer development.
HYPOTHESIS. The study focused on exploring genotypes of selected polymorphisms in specific mitotic segregation genes. Those that differ significantly between the subjects and the healthy control population, may be associated with higher risk for developing gastric cancer or with certain clinical and histopathological characteristics, and may have effect on the survival of gastric cancer patients.
METHODS. 30 polymorphisms in genes BUB1B, CASC5, ESPL1, PTTG1, SMC1A, TPX2, TTK and ZWINT were included in the study. Subjects were compared with the control group. Polymorphisms were determined using quantitative real-time PCR (qPCR), restriction fragment length polymorphism (RFLP) and DNA sequencing.
RESULTS. The association between polymorphisms rs2277559 (BUB1B), rs2241666 (ZWINT), rs11858113 (CASC5) and rs11855334 (CASC5) and increased risk of developing gastric cancer in male population was determined. As concerning rs11855334, statistically significant difference was also observed in the genotype distribution between the whole population of subjects and controls. The association between the genotypes of polymorphisms (in gene BUB1B) rs2277559, rs2290551, rs1801376, rs1047130, rs1565866, rs2277560 and Lauren classification was recognized. Genotypes of polymorphisms rs1801376 (BUB1B), rs11855334 (CASC5), rs2241666 (ZWINT), rs2910101 (PTTG1) and rs1047130 (BUB1B) are linked to different tumour differentiation grades. Survival analysis revealed association between the lymph node involvement and perineural invasion. Statistically higher frequencies of haplotypes G-A-G-T-G-G-A, G-G-A-G-A-A-G and A-G-G-T-A-G-A in gene BUB1B and of haplotypes A-A-A-C and C-C-G-T in gene ESPL1 were observed in gastric cancer patients, whereas haplotypes A-C-A-T and C-A-G-T in gene ESPL1 were significantly more frequent in the control group. Association with gastric cancer was not noted with other polymorphisms.
CONCLUSIONS. The association between specific polymorphisms of selected chromosome segregation genes and gastric cancer was recognized. Findings could provide guidelines for further research and polymorphisms linked to gastric cancer could serve as potential diagnostic and prognostic biomarkers.
Ključne besede: gastric cancer, chromosomal instability, polymorphisms, segregation genes
Objavljeno: 27.07.2016; Ogledov: 2137; Prenosov: 159
Polno besedilo (2,87 MB)
THE EFFECTS OF MIXED COMMUNAL WASTE RECYCLING MANAGEMENT IN SLOVENIA - CASE STUDYPeter Bohinec
, 2016, magistrsko delo
Opis: With the adjustment of the Slovenian legislation with the European one we have an obligation to increase the reuse and recycling target for municipal solid waste by up to 65% by 2030 and gradual limitation of the landfilling of municipal solid waste to 10% by 2030 (Directive 2008/98 on waste, 2015). This represents a major challenge for municipalities in Slovenia. This study contains a plan for establishing new waste treatment facility in the Municipality of XY. As all new constructions in Slovenia, the presented facility needs to provide all the environmental protective measures predicted by the legislation. Beside the legislation, it is also important to ensure that new waste treatment facilities will have a low or no effect on the environment. Based on the Environmental Protection Act and the Construction Act of Slovenia, measures to ensure long term monitoring of the waste treatment facility as a case study in the Municipality of XY are presented. Before constructing the new facility, the track of “baseline” – a one-year monitoring of the following environmental parameters was crucial: groundwater, air dust deposit, condition of the soil on the area where new facility will operate in the future.
The thesis points out the importance of the so-called track “baseline” monitoring of environmental parameters before new waste treatment facility will operate. This will be the basis for the introduction of operating monitoring for the new waste treatment facility in the future. Data for the analysis of presence of pollutants were acquired from groundwater, air dust deposit, soil sampling and containment of chemical pollutants. The following methods of analysis were employed in the acquisition of laboratory results of specific physical and chemical parameters: ion chromatography, gas chromatography, spectrophotometry, titration, inductive coupled plasma – mass detector, measurement with an electrode. It was determined that in the period between 2011 and 2012, the groundwater samples taken from three drilled wells containing pollutants as for example in the P-2 well there were adsorbable organic halogens - AOX, boron and mineral oils found. The results of the dust deposit show increased value of lead and zinc. More concerning are analysis of the soil samples where the quantity of copper at the MM2 measuring point were 530mg/kg, on 20 April 2012 the present critical level was 300 mg/kg. At the same measuring point (MM2) on the same day, the value of zinc was 650 mg/kg and almost reached the critical level of 720 mg/kg. The levels of heavy metals as cadmium, copper, lead and zinc in the soil samples taken at the MM2 and MM3 measuring points were at the warning level. All the detected parameters were evaluated and present a good basis for future operational monitoring of the environment in the area of the new waste treatment facility in the future.
This case study has shown that planned steps in the improvement of the waste management in the Municipality of XY are crucial to protect the environment. The analysis and interpretation present an important basis for planning and monitoring a new waste treatment facility which will help to improve our goals and reduce landfilling to 10% and increace recycling by up to 65% by 2030 as predicted by the Directive on waste (Directive 2008/98 on waste, 2015).
Ključne besede: odpadki, krožno gospodarjenje z odpadki, podtalnica, zemljine, prašni delci v zraku, spremljanje stanja okolja.
Objavljeno: 20.07.2016; Ogledov: 1268; Prenosov: 139
Polno besedilo (3,32 MB)
ANALYSIS OF ALTERNATIVE CHANCES FOR SLUDGE TREATMENT AT NEW CENTRAL WASTE WATER TREATMENT PLANTRenata Janja Slovša
, 2016, magistrsko delo
Opis: The purpose of this master’s thesis is to present the operation of a large municipal waste water treatment plant in Slovenia with over 300.000 PE and its efficiency, and through a theoretical analysis to present the current practice and alternative options of sewage sludge treatment in Slovenia and around the globe. The main objective of master's thesis is to present the most possible alternative scenarios for the final managing of sewage sludge from WWTPs and at this objective we take into consideration the impact on Carbon Foot Print (CFP).
Due to scenarios (thermal utilisation of dried sludge (90% d.m.) in a cement plant, co-incineration of dehydrated sludge (25% d.m.) in a local power plant, thermal utilisation of dried sludge (90% d.m.) in a local power plant, incineration of dehydrated sludge (25% d.m.) in own plant, incineration of dried sludge (90% d.m.) in own plant, and also prohibited scenarios such as sludge utilisation in the soil -fertilization, sludge disposal in the landfill) we calculated CFP and we find out that CFP of various methods of thermal sludge treatment are more or less similar but absolutely smaller than convetional sewage sludge treatment. The CFP is more favourable for dehydrated sludge than for pre-dried sludge, and it is also more favourable for treatment in industrial furnaces (heat power plant, cement plant) than in individual waste incineration facilities.
Ključne besede: odpadne vode, odpadno blato iz čistilnih naprav, čistilne naprave, primerjalne analize, analiza življenjskega cikla, ogljični odtis, toplogredni plini
Objavljeno: 20.07.2016; Ogledov: 1434; Prenosov: 125
Polno besedilo (2,33 MB)
A REVERSE GENETIC SYSTEM FOR ROTAVIRUSGiuditta De Lorenzo
, 2016, doktorska disertacija
Opis: Studies on rotavirus biology have always suffered for the lack of a reverse genetics system applicable to all genome segments and independent from the use of helper virus. One of the main reasons proposed to explain the difficulties encountered is the poor expression of the transfected cDNAs.
In this work we investigated the role of 5’UTRs in rotavirus cDNAs expression in un-infected cells. We showed that in the 5’UTR of several (but not all) genome segments of rotavirus is present a 5’-terminal inhibitory motif (IM = 5’-GGY(U/A)UY-3’) that, independently from the ORF or the 3’UTR, downregulates both transcription and translation of rotavirus cDNAs when expressed exploiting T7 polymerase-encoding recombinant vaccinia virus. We mapped two mutations (insertion of a G upstream 5’UTR and the U to A mutation of the fifth nucleotide of IM) that are capable of making the inhibitory motif non-functional restoring a satisfying yield of protein synthesis. After the analysis of all genome segment 5’UTR of SA11 strain, we created two distinct sets of mutated rotavirus genome segments containing non-functional IM. We planned to employ these sets in the development of a plasmid-based reverse genetics system that exploit vaccinia virus-encoded T7 polymerase.
In an attempt to produce in vivo biotinylated Tripled-Layered Particles (TLPs), we created a recombinant VP4 fused to the Biotin Acceptor Peptide (BAP) that, when co-expressed with the biotin ligase BirA, is efficiently in vivo biotinylated. We exploited recombinant vaccinia virus system to achieve high levels of VP4-BAP and the enzyme BirA in cells. Upon infection with rotavirus, VP4-BAP would be incorporated in the newly forming viral particles. In addition, we constructed a full-length genome segment 4 cDNA encoding the recombinant VP4-BAP to be used with a VP4 temperature sensitive rotavirus in order to generate a recombinant rotavirus encoding VP4-BAP.
Thus, the results obtained allowed us to design two distinct possible ways of generating recombinant rotavirus. The first was a genetic strategy for the incorporation of an exogenous genomic segment, with the characteristic of being helper-virus free and applicable to all genomic segments. This was the consequence of the description of an inhibitory motif present in many viral mRNAs and the identification of two mutations that abrogate its inhibitory activity allowing expression of viral proteins. The second focused on the expression of a recombinant viral structural protein expressed in virus-infected cells for the consequent incorporation into newly made virions.
Ključne besede: rotavirus, UTR, reverse genetics, T7 polymerase, in vivo biotinylation
Objavljeno: 08.06.2016; Ogledov: 1381; Prenosov: 128
Polno besedilo (2,87 MB)
CHARACTERIZATION OF SLOVENIAN APPLE JUICE WITH RESPECT TO ITS GEOGRAPHICAL ORIGIN AND AGRICULTURAL PRODUCTION PRACTICEKarmen Bizjak Bat
, 2016, doktorska disertacija
Opis: Determination of food authenticity is an important issue in quality control and food safety. Recent studies predict a growing demand for natural and more authentic food and beverage products. The quality and authenticity of apple juice is also of a great economic importance since the popularity and demand for apple juice consumption has increased. The growth of the market for organically produced apples and apple juice is due to the increasing demand for healthy food requirements, protection of the environment and the promotion of biotic diversity. Organic foods have a higher nutritional and health value, but they are more expensive, because their production is more difficult and less profitable. In addition to how food is produced, consumers are increasingly placing emphasis on food products of specific region, which are known for their unique natural flavours and taste.
The presented thesis is based on four separate but closely interrelated studies, in which a combination of different isotopic ratios of bioelements (2H/1H, 13C/12C, 15N/14N, 18O/16O), multi-element analysis, and major primary and secondary metabolite profiles were exploited to differentiate the geographical origin and agricultural production practice (organic vs integrated/conventional) of Slovenian apples. These parameters were used to establish the first database of authentic Slovenian apple juice, which can be used to verify the authenticity of commercially available apple juice in Slovenia.
The first preliminary study was entitled “Organic Cultivation ~ Geographical Origin (OCGO)” and was performed using apples from the 2009 growing season. Its aim was to examine the use of stable isotope and multi-element data for determining the geographical origin and agricultural production practice of fresh apple juices. Fruits of six apple (Malus domestica Borkh) cultivars (Topaz, Idared, Golden Delicious, Goldrush, Gala, Gloster) were collected from four different geographical regions of Slovenia (Alpine, Dinaric, Pannonian and Mediterranean) grown under organic and integrated/conventional orchard management systems. The results revealed that stable isotope parameters in sugar, pulp and water were the most significant variables for differentiating between the regions. Good separation was achieved between the geographical regions in Slovenia based on the δ18O and δ2H values in water and Rb and S levels in the apple fruit juice. The most significant variables that distinguished between organically and integrated/conventionally cultivated apples were the 15N/14N ratio and antioxidant activity of the apple juice. Significant differences were also observed in the ascorbic acid content of the juice. Based on these results the number and types of apples and the minimum number of samples needed from the same region for determining geographical origin were determined.
The second study was called “Organic ~ Conventional Apple Cultivation” (OCAC) and was performed in 2010 and 2011 in a Gala apple orchard. The aim was to determine the effect of different fertilizers allowed either in organic or conventional/integrated agricultural regimes on different parameters. Quality parameters, isotopic composition of C in sugars and in pulp together with N and elemental analysis were investigated. The following five fertilizers were applied: Biosol and Plantella organic (organic) and Ca cyanamide, KAN and UREA (mineral) at a rate of 60 and 120 kg of nitrogen per hectare. From the obtained data it was possible to differentiate between organic and integrated/conventional apple production when taking into account the following parameters: mass, skin and flesh firmness (SFF), total soluble solids (TSS), and the content of Cl as well as δ15N and δ13C in the pulp.
The “Organic Cultivation ~ Geographical Origin” (OCGO) study, which took place during the 2011 and 2012 growing seasons included a greater number of samples and samples from five different geographical regions: Alpine, Dinaric,
Ključne besede: apple juice, geographical origin, agricultural production practice, biomarkers, phenol compounds, elements, stable isotopes, Slovenia
Objavljeno: 07.06.2016; Ogledov: 2794; Prenosov: 306
Polno besedilo (3,97 MB)
ROLE OF TDP-43 AGGREGATION IN NEURODEGENERATION: A DROSOPHILA MELANOGASTER DISEASE MODEL AND INNOVATIVE THERAPEUTIC APPROACHESLucía Cragnaz
, 2016, doktorska disertacija
Opis: TDP-43 inclusions are important histopathological features of various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. Various hypotheses have been proposed. For instance, it was suggested that the inclusions are (1) primary toxic species, (2) part of the normal cellular protective response to toxic intermediates and (3) responsible for the nuclear depletion of TDP-43. Understanding the relationship between TDP-43 aggregation and neurodegeneration is crucial for the eventual management of the disease.
TDP-43 is a protein that has a marked tendency to unfold and become insoluble. In particular, its C-terminal end has a so-called “prion-like domain”, a sequence rich in Glutamine (Q) and Asparagine (N) that is involved in both the interactions with other proteins and the self-aggregation process. A cellular model of aggregation has been previously developed by our group, using the TDP-43 Q/N rich amino acid sequence repeated 12 times (12xQ/N) fused to EGFP reporter. The EGFP-12xQ/N cellular inclusions are capable of sequestering wild type TDP-43 both in non-neuronal and neuronal cells.
In this study we went further with ALS modeling, creating a Drosophila model with EGFP-12xQ/N-induced aggregates. We show here that Drosophila melanogaster TDP-43 ortholog (TBPH) overexpression in Drosophila eye using GMR-Gal4 driver, is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP-12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue.
Notwithstanding the fact that aggregation is protective in presence of an excess of TBPH, we wanted to further understand the role of the aggregates in an environment where just the endogenous TBPH is present. For this purpose, we induced EGFP-12xQ/N transgene constitutively in CNS using ELAV-Gal4 driver. The flies were born and went through the larval stage without differing from control flies in any significant feature of their
phenotype. However, during aging the locomotion ability and survival rate of EGFP-12xQ/N flies were impaired. Interestingly, the climbing deficit was correlated with a physiological reduction in the endogenous TBPH levels. Thus, the aggregation, when coupled with low TDP-43 levels generates phenotypic consequences in our Drosophila model, probably due to a TDP- 43 loss of function.
In sum, these data suggest that although the aggregates may be a result of neuroprotection in a context where TBPH is in excess, at a certain stage they become responsible for the pathology, likely due to the TBPH loss of function. If we consider that TDP-43/TBPH inclusions act as a sink for the newly formed soluble TDP-43/TBPH, the modulation of these inclusions could be used as a potential therapeutic approach, as this would restore the normal levels of TDP-43/TBPH and its function. Consequently, in the last part of the study we were interested to understand if the clearance of TDP- 43/TBPH aggregates could be an effective strategy to treat ALS, by recovering TDP-43/TBPH function. For this purpose, using the previously established cell-based TDP-43 aggregation models we analyzed aggregate clearance after treatment with several FDA approved drugs. Three of these drugs were found to significantly reduce aggregation through the proteasome pathway. Furthermore, one of the drugs (nortriptyline) was shown to rescue EGFP-12xQ/N dependent locomotor dysfunction in the Drosophila model. Altogether these data indicate that the clearance of TDP-43 aggregates may be a novel therapeutic strategy for ALS treatment.
Ključne besede: TDP-43/TBPH, aggregation, Drosophila melanogaster, ALS
Objavljeno: 03.03.2016; Ogledov: 2066; Prenosov: 18
Polno besedilo (53,65 MB)