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21.
General framework for the conservation of historical rural landscape. Case study of Qadisha Valley in Lebanon.
Bachir Zarif Keyrouz, 2016, doktorska disertacija

Opis: The international community has reacted to the challenges faced by historic cities and cultural landscapes. In 2011, UNESCO adopted the international recommendation on the Historic Urban Landscape in order to draw the attention of governments and local communities to the need to improve the management of historic urban areas. The aim of the present dissertation is to expand the UNESCO definition to Historic Rural Landscapes, and to explore how the proposed methodology could be applied to the management of rural areas. The dissertation takes the Qadisha Valley in Lebanon as an example, where some elements have already been recognised for their outstanding universal value. It is considered however that these historic-cultural elements are part of the larger context of the Qadisha Valley, which has its specificity and its qualities that merit being recognised. It is also considered that the management of the elements already recognised by UNESCO can only be successfully safeguarded if the management is understood in the context of the entire valley. Consequently, the thesis is articulated taking into account the following parameters: 1. UNESCO has already set the restoration plan to solve the problems of Qadisha Valley. Based on this plan, the question can be raised whether or not the rural planning in Qadisha is well managed at the present? 2. In case Qadisha Zone is not well conserved, what improvements could be brought about and implemented in order to safeguard the cultural heritage and historical rural landscape, and what should be the guiding policies of restoration? 3. Once a restoration plan is implemented in Qadisha Zone, what management plan should be brought up and implemented to safeguard its cultural heritage and historical rural landscape? 4. How can conservation of cultural heritage in Qadisha Zone be done culturally and in a socially acceptable way? 5. What sectors of the nation are to be involved in the Management plan? How can the MP preserve and restore Qadisha Zone? The aim of the dissertation has been to examine the possibilities of involvement of the local community and relevant authorities in a more structured management of their properties within the context of the entire valley and its exceptional historic and landscape qualities. It is hoped that the study can generate more interest in the management of the rural context within the increasingly globalising society, and draw attention to the qualities of historic rural landscapes as the setting for an improved quality of life.
Ključne besede: Historic Rural Landscape, Qadisha World Heritage, Heritage Community, Sustainable Tourism, Strategic Urban Management, Outstanding Universal Values & Conservation Process.
Objavljeno: 07.10.2016; Ogledov: 1257; Prenosov: 27
.pdf Polno besedilo (58,03 MB)

22.
Building the shared dimension of the rural landscape: tools, principles and methods
Caterina Groli, 2016, doktorska disertacija

Opis: This research investigates the rural landscape of the lagoon of Venice and its surrounding territory focusing on the forms of exploitation of its natural resources, the transformations that occurred throughout the course of history and the consequent formation of its primary economical sector. New economic sectors, new agricultural policies and other spatial and sectorial drivers transformed the primary sector itself which changed and adapted its productive schemes to the main economic patterns, heavily affecting the former rural asset of the landscape of the lagoon by changing its demographic, economic activities and land use practices. The study carries out a systematic and detailed analysis of all economic, cultural and social features that have interacted and impacted this specific landscape, relying mainly on the comparison of historical and current land-use maps through three different historical spans: 1823-1840 (pre-industrialization phase), 1933-1966 (industrializing process, considered as a determining factor of the transformation of this landscape) and 2013 (present post-industrialized condition). The maps, combined with relative data, are compared with the aid of the Geographical Information System (GIS) computer software. As a result, a set of new maps has been obtained, which point out the historical landscape permanencies, defined through in situ surveys on the territory. A multitude of other archival documents from direct and indirect sources have been examined in order to build up a sound and detailed socio-economic context and the overall historical background. Resources, goods and services provided by the rural landscape and its development are an important part of regional and EU policy instruments and also an essential factor of identity and growth for local communities. The consumption of resources, their availability and maintenance over time and accessibility through property regulations are all factors that shape the common and public dimension of the rural landscape, which represents another study challenge of this work. This study proposes the establishment of the rural park of the lagoon of Venice, through the implementation of the multi-functional role of agriculture, for the fulfilment of two fundamental conditions of the peri-urban rurality: the need of safeguarding the landscape and its significance, by preserving the authenticity and integrity of the rural characters and the dynamism of the primary sector, which is strictly related to the survival of the rural landscape.
Ključne besede: Rural landscape of the lagoon of Venice, UNESCO Management Plan 2012–2018, European Landscape Convention, Common Agricultural Policy (CAP), Italian Constitution, Cultural Heritage and Landscape Code, authenticity and integrity of the rural heritage, intangible heritage, landscape evaluation, land-use cartography, Geographical Information System (GIS), fish-farming, multifunctional agriculture, new rural community, common pool resources, public goods, club goods, property rights, rural park
Objavljeno: 04.10.2016; Ogledov: 901; Prenosov: 22
.pdf Polno besedilo (21,33 MB)

23.
SYMBIOSIS ECOLOGY OF SELECTED SCYPHOZOA
Lucija Raspor Dall'Olio, 2016, doktorska disertacija

Opis: Scyphozoa with symbionts have an advantage in oligotrophic environments due to the additional source of nutrients provided by their symbiontic algae, just as corals have benefits from their symbionts. The literature, however, has thus far devoted far less attention to the association between scyphozoan hosts and Symbiodinium sp. than it does to corals. This thesis investigated the identity of symbionts from scyphozoan medusae (Cotylorhiza tuberculata, Phyllorhiza punctata and Cassiopea xamachana) using a phylogenetic approach. Two scyphozoan species, P. punctata and C. tuberculata, were sampled over the Mediterranean Sea, while Cassiopea xamachana was sampled in the Atlantic Ocean. Symbionts were identified from live medusae and their identity and phylogenetic relationships were determined by analysing two nuclear markers, ITS2 and 28S rDNA, from symbionts. Symbiodinium sp. sequences belong to clades A, B, and C based on markers ITS2 and 28S rDNA. Moreover, individual medusae host only one type Symbiodinium (A, B or C). Host species from the Mediterranean Sea hosted Symbiodinium from clade A and B (C. tuberculata) or only from clade A (Phyllorhiza punctata), while the host from the Atlantic Ocean (C. xamachana) hosted Symbiodinium from clade B or C. The phylogeography of C. tuberculata medusae was analysed using mtCO1. All the haplotypes sampled over the Mediterranean Sea were clustered together without any sign of phylogeographic structuring.
Ključne besede: Scyphozoa, Cotylorhiza tuberculata, Symbiodinium sp., symbiosis, Mediterranean Sea, phylogeography, CO1, 28S rDNA, ITS regions
Objavljeno: 03.10.2016; Ogledov: 1216; Prenosov: 135
.pdf Polno besedilo (3,20 MB)
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24.
Intelektualci in institucije v slovenski zgodovini 17. stoletja
Andrej Koritnik, 2016, doktorska disertacija

Opis: Disertacija želi razkriti in sintetično povezati v kompleksno historiografsko sliko zgodbo 17. stoletja, ki jo razume kot integralni del novega veka, zato je vpogled v 16. stoletje, ko so delovali protestanti (kot posebna forma intelektualcev), in v 18. stoletje, ko so se stare institucije umikale novim in je razsvetljenska filozofija preplavila intelektualne kroge kakor tudi ljudsko kulturo, nujen, da lahko razgrnemo socialno, kulturno in intelektualno zgodovino na Slovenskem v 17. stoletju. Tembolj zato, ker za tedanji čas še ne moremo govoriti o konceptu »nacionalnega«, ki se je povezal z ljudskimi jeziki – pri nas seveda s slovenščino, temveč o počasnem, toda »revolucionarnem« spreminjanju starih družbenih in kulturnih vzorcev, v katerih sta védnost in intelekt legitimno izhodišče za historiografsko, a vseeno interdisciplinarno analizo in sintezo obravnavanega časa. V luči spora med Descartesom in Vicom ter njunima nasprotnima umevanjema zgodovine bo treba premisliti tudi, kako so ideje iz zgodovinopisja vplivale na splošno zgodovino idej, ki se je »dogajala« tudi med Alpami in Jadranom, ter kakšno težo in vpliv imajo »predrazsvetljenski« časi, o katerih pravzaprav ne vemo veliko, na našo sodobnost. Prikazali bomo njeno raznotero, zapleteno podobo kot preplet vseh mogočih silnic – v šolstvu, državni upravi, v fevdalnih razmerjih, v verskih sporih, jezikovni ideologiji, v gospodarstvu in drugod –, skozi katere sledimo liku intelektualca, bodisi kot člana neke institucije védnosti ali pa »osamelca«.
Ključne besede: Intelektualec, institucija, védnost, zgodovina védnosti, 17. stoletje, intelektualna zgodovina, kulturna zgodovina, literarna zgodovina, protestantizem, protireformacija, katoliška obnova, znanstvena revolucija, univerze, osnovne šole, akademije
Objavljeno: 14.09.2016; Ogledov: 993; Prenosov: 90
.pdf Polno besedilo (1,74 MB)

25.
ECOLOGICAL STUDIES OF EPIKARST COMMUNITIES IN ALPINE AND PRE-ALPINE CAVES
Federica 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: 1306; Prenosov: 84
.pdf Polno besedilo (2,00 MB)

26.
SINGLE NUCLEOTIDE POLYMORPHISMS OF THE CHROMOSOME SEGREGATION GENES INVOLVED IN THE DEVELOPMENT OF GASTRIC CANCER
Marija 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: 1575; Prenosov: 104
.pdf Polno besedilo (2,87 MB)

27.
A REVERSE GENETIC SYSTEM FOR ROTAVIRUS
Giuditta 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: 926; Prenosov: 80
.pdf Polno besedilo (2,87 MB)

28.
CHARACTERIZATION OF SLOVENIAN APPLE JUICE WITH RESPECT TO ITS GEOGRAPHICAL ORIGIN AND AGRICULTURAL PRODUCTION PRACTICE
Karmen 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: 1777; Prenosov: 218
.pdf Polno besedilo (3,97 MB)

29.
Poezija Franceta Prešerna v srednješolskih učbenikih in njena recepcija
Zoran Božič, 2010, doktorska disertacija

Opis: Disertacija obravnava dva problemska sklopa: poezijo Franceta Prešerna v srednješolskih učbenikih za pouk književnosti od leta 1850 do leta 2010 ter recepcijo Prešernovih pesmi skozi čas in v sodobnosti. V prvem problemskem sklopu razkriva dejavnike in postopke šolske kanonizacije Prešerna ter razvoj didaktizacije beril v smeri učne knjige za samostojno delo z besedilom. V drugem problemskem sklopu raziskuje posebnosti pri razumevanju, doživljanju in vrednotenju Prešernovih pesmi ter načine za njihovo čimbolj učinkovito šolsko predstavitev. Glede na to, da se je kanonizacija Prešerna začela že za časa njegovega življenja, je bil seveda vseh 160 let kanonski avtor, vendar pa kljub nenehnemu označevanju Prešerna za "največjega slovenskega pesnika" po številu besedilnih enot v berilih ni bil vedno prvi avtor, pač pa so ga večkrat prehitevali mlajši književniki, ki so bolj ustrezali trenutnim afinitetam sestavljavcev srednješolskih beril. Predvsem v drugi polovici 19. stoletja in tudi kasneje je bil zaradi negativnega odnosa šolskih oblasti do ljubezenske poezije izbor Prešernovih pesmi praviloma manj primeren (na nižji stopnji je bil največkrat objavljen sonet Memento mori, na višji stopnji pa cikel Soneti nesreče), nekatere ljubezenske pesmi pa so celo cenzurirali. Od druge polovice 19. stoletja do danes se je razvijal didaktični instrumentarij srednješolskih beril (najprej so začeli dodajati stvarne opombe, kasneje pa še vprašanja in naloge za dejavno razčlembo leposlovnega besedila), vendar so še zlasti v obdobju po drugi svetovni vojni nekateri sestavi beril didaktično pomenili izrazito nazadovanje. Od sedemdesetih let dalje berila vključujejo tako literarnovedne vsebine kot vprašanja in naloge, sodobna berila pa se praviloma (izjema je npr. antološki sestav Krakar 2003) razvijajo v smeri vezanega berila z relativno majhno ponudbo leposlovnih besedil. Zgodovinski pregled je pokazal, da so imeli težave z razumevanjem Prešernovih pesmi že njegovi sodobniki, kasneje pa tudi izobraženci, celo poznavalci Prešerna. Empirični preizkusi med slovenskimi srednješolci potrjujejo visoko recepcijsko zahtevnost Prešernove poezije. Nekatere osrednje Prešernove pesmi (npr. Krst pri Savici) zahtevajo izjemno gostoto dodanih opomb, zato je najustreznejše didaktično sredstvo za boljše razumevanje in doživljanje oblikovno in slogovno zahtevne klasične poezije dodana prozifikacija, kar so v celoti potrdili tudi rezultati pedagoškega eksperimenta.
Ključne besede: poezija, Prešeren, didaktika, recepcija, kanonizacija, srednja šola, berilo, kanonizacija, prozifikacija, pedagoški eksperiment
Objavljeno: 12.05.2016; Ogledov: 946; Prenosov: 19
.pdf Polno besedilo (10,63 MB)

30.
ROLE OF TDP-43 AGGREGATION IN NEURODEGENERATION: A DROSOPHILA MELANOGASTER DISEASE MODEL AND INNOVATIVE THERAPEUTIC APPROACHES
Lucía 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 8 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: 1503; Prenosov: 11
.pdf Polno besedilo (53,65 MB)

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