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: 1439; Prenosov: 135
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: 2900; Prenosov: 317
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: 2131; Prenosov: 18
Polno besedilo (53,65 MB)
CAPOEIRA: CONCEPTUALIZATION AND PRESENTATION OF THE BODYUrška Stolnik
, 2015, doktorska disertacija
Opis: This thesis is the result of twenty-one months of fieldwork among the capoeiristas in Brazil, primarily in the city of Salvador, between 2003 and 2013. Combining historical accounts with the experiences of the present, the work focuses on changes in the social perception of body and mind in both capoeira and wider society. The intricacies of society and the politics of a particular epoch are clearly reflected in this Brazilian national game: sometimes in terms of incorporation or acceptance of specific social hierarchies, and other times of exclusion or rejection of them. A tension between the black and white worlds is constantly present, as it is between the male and female ones. Even when these separate realms in different historical eras of capoeira seem to come closer, they become separated again through new forms of institutionalization. Perceived sometimes as a martial art or dance, sometimes as a game, and yet other times as a sport through which one can gain health and beauty, capoeira evades a firm and permanent definition. Such evasion resembles capoeira’s three main, cunning characteristics: malandragem (trickery), malícia (deception) and mandinga (seduction).
Cunningness, as a tactic to overplay the opponent, is a means of intentional opening and closing of the body in the game. Although opening and closing might seem exclusive, they are, on the contrary, very much inseparable. They coexist in symbiosis not just at the level of a capoeirista playing the game, but also at the level of roda, academia, a capoeira group or school, at the level of capoeira as a practice, and finally, at the level of wider Brazilian society. All of these bodies are striving to be fechado (closed) and secure, but cannot accomplish that before opening themselves first.
Being dexterous in the simultaneous alternation of opening and closing one’s own body is a skill that can be learnt through a long-term and continually engendered process of practice, experiences and interaction with others and the environment at large. That skill, associated with the capoeirista’s awareness of
constant vulnerability, is closely related to learning the specific way of perceiving and responding. The capoeirista cultivates and embodies capoeiristic habitus, which emphasizes the necessity of being permanently attentive to several things at once. This is possible when all senses work together in synthesis. At that time, the capoeirista uses peripheral sight and is aware of peripheral sounds. The skill of heightened sensitivity and keen perception enables the capoeirista to negotiate between the opening and closing of his body. Opening and closing the body at the right time and taking the advantages of an unstable situation in roda can be accomplished only by simultaneous thinking and performing, which is the outcome of the incorporation of capoeira movements.
The socio-economic situation of Brazilian society based on racial and gender discrimination enhances the cultivation of capoeiristic habitus and dictates the need for the acquisition of capoeiristic skills and attitudes in order to cope with, understand, and live within an unequal world. Capoeiristic habitus can also be learnt and acquired as a by-product of capoeira training. But it remains important whether the environment in which a capoeirista practises tolerates or inhibits the cultivation of capoeiristic skills.
Ključne besede: capoeira, body and mind, game, sport, movement, opened and closed society, perception, historical changes, Salvador, Brazil
Objavljeno: 22.01.2016; Ogledov: 2189; Prenosov: 161
Polno besedilo (4,71 MB)
V očeh razpadajočega imperija: Podoba Slovenije v Sovjetski zvezi in Rusiji v času slovenskega osamosvajanja (1990 – 1992)Andrej Stopar
, 2015, doktorska disertacija
Opis: Za mlado državo je priznanje mednarodne skupnosti ključnega pomena. Tudi za Republiko Slovenijo je bilo. Sovjetska zveza je odločno podpirala enotnost in ozemeljsko celovitost SFR Jugoslavije, katere razpad je obravnavala v kontekstu lastne dezintegracije. Disertacija V očeh razpadajočega imperija: Podoba Slovenije v Sovjetski zvezi in Rusiji v času slovenskega osamosvajanja (1990 – 1992) tematizira sovjetske in ruske poglede na razpad SFR Jugoslavije in jugoslovanskega sistema ter na slovenska prizadevanja za rusko priznanje neodvisnosti in suverenosti republike. Pri tem se opira na gradivo najpomembnejših sovjetskih in ruskih tiskanih medijev v obdobju 1990 – 1992, na sovjetske in ruske diplomatske vire, uradne izjave, komentarje akademskih krogov ter spominske in dnevniške zapise. Disertacija skuša pokazati, da je bila odločitev Moskve posledica kratkotrajnega sovpadanja določenih mednarodnopolitičnih razmer, takrat nove ruske zunanjepolitične strategije, ki se je hitro spremenila in je kot takšna pomenila izjemo v ruski tradiciji zunanjepolitičnih konceptov, ter predvsem notranjepolitičnih napetosti v Ruski federaciji po razpadu Sovjetske zveze.
Ključne besede: Sovjetska zveza, Rusija, Jugoslavija, Slovenija, diplomatski odnosi, mediji, razpad socialističnih federacij
Objavljeno: 22.01.2016; Ogledov: 2232; Prenosov: 170
Polno besedilo (2,02 MB)
Water salinity and the efficiency of constructed wetlandsMilana Karajić
, 2015, doktorska disertacija
Opis: Raziskava teze je bila osredotočena na vpliv slanosti na učinkovitost rastlinske čistilne naprave. Raziskava je bila izvedena na rastlinski čistilni napravi Dragonja ki se nahaja na Jadranski obali, na jugo-zahodu Slovenije in na pilotskem modelu rastlinske čistilne naprave. Rezultati raziskave sugerirajo, da povečanje slanosti negativno vpliva na učinkovitost skoraj vseh spremljanih parametrov (amonijaka, fosfata, nitrata, nitrita, COD in BOD5), ampak korelacija ni bila signifikantna. Majhni koeficienti korelacije pomenijo, da koncentracije kloridov in prevodnost lahko razložijo majhen delež variabilnosti učinkovitosti rastlinske čistilne naprave Dragonja. Mogoče ima prisotnost različnih koncentracij drugih polutantov v izcedni vodi, ki jih nismo spremljali v nalogi, inhibitorni vpliv na mikroorganizme v rastlinski čistilni napravi in tako negativno vpliva na učinkovitost rastlinske čistilne naprave Dragonja.
Za raziskavo procesa čiščenja slane odpadne vode so bili v substratu pilotskega modela rastlinske čistilne naprave inokulirani halotolerantni mikroorganizmi in spremljana učinkovitost čiščenja odpadne vode. Vpliv slanosti na učinkovitost, preživetje in uporabo halotolerantnih mikroorganizmov je bil preverjen pri 0%, 1,5% in 3,0% NaCl v odpadni vodi. Prav tako smo raziskali vpliv aeracije ter vpliv aeracije z dodano saharozo (2g/l) v sintetični odpadni vodi. V substratu pilotskega modela peščenega filtra rastlinske čistilne naprave smo spremljali spremembe v pH, prevodnosti, redoks potencialu, koncentraciji kisika, amonijevih ionov, kloridnih ionov, fosfatnih ionov, KPK, BPK5 in ETS aktivnosti, koncentraciji CO2 v vodi in koncentraciji CO2. Ugotovili smo, da je na učinkovitost pilotskega modela, inokuliranega s halotolerantnimi mikroorganizmi, večji vpliv imela aeracija in prisotnost saharoze (kot organskega izvira ogljika), kot spremembe v slanosti vode.
Ključne besede: rastlinske čistilne naprave, slana odpadna voda, halotolerantni mikroorganizmi
Objavljeno: 22.12.2015; Ogledov: 1829; Prenosov: 45
Polno besedilo (6,15 MB)
Depression between biomedicine and Ayurvedic medicineMaja Kolarević
, 2015, doktorska disertacija
Opis: The present work explores the "major depression", which falls in a psychiatric categorization of mental disorders in the spectrum of mood disorders. The subject of the research was the (de)construction of scientific knowledge of depression in terms of Indian medicine: Ayurveda and biomedicine /psychiatry, and the understanding of treatment, accompanied by an example of these two medical systems from the points of view of the patients in Slovenia, diagnosed with major depression.
This dissertation represents multi-layered content analysis highly relevant to the field of mental health – to cope and deal with the depressive mood disorder, as one of the most common forms of mental health problems. To this end the following was conducted: (1) a comparison between the conceptualization and treatment of major depressive disorder in psychiatric and Ayurvedic theory, (2) an examination of Ayurveda practice transferred to a different cultural environment, i.e. in Slovenia, in comparison with the environment from which it comes, i.e. in India, (3) an identification of key factors that prompt them to undergo Ayurvedic or psychiatric practice, (4) an analysis and a comparison of the course and the outcome of the treatment using both approaches, and (5) a comparison of participants understandings of depression from the psychiatric and the Ayurvedic aspects using narrative approach.
The purpose of this research was to examine the differences between the two approaches of treatment for depression arising from the different concepts of understanding its causes, its course and consequently its therapeutic intervention. The main focus lies on the advantages / disadvantages of compared practices as experienced by patients with depression. This issue is particularly important because, according to health statistics, depression today ranks among one of the most common forms of mental disorders, and its incidence is on the rise. This is not necessarily a reflection of the increased cases as such, but might also be attributed to the better recognition and greater awareness of depression by healthcare professionals and the general public.
The effects of Ayurvedic practice as treatment of mental disorders are still relatively poorly examined, as well as the transfer of Ayurvedic medicine into a different cultural environment. Studies on the treatment of mental disorders with Ayurvedic medicine from the perspective of a European patient experience are few and there is a major gap in this research field. In my opinion, the dissertation would immensely contribute to the enormous research gap on the treatment of major depressive disorders with Ayurvedic practice in Slovenian territory. From the scientific point of view, the results of this research represent a significant development in our current understanding of the still prevailing concepts of medical theories and practices in the field of mental health.
Ključne besede: major depression, psychiatry, Ayurvedic medicine, medicalization, paramedicalization
Objavljeno: 15.12.2015; Ogledov: 1749; Prenosov: 162
Polno besedilo (1,85 MB)
MOLECULAR MECHANISMS REGULATING ATP SIGNALING IN MOUSE SENSORY NEURONSTanja Bele
, 2015, doktorska disertacija
Opis: Coordinated and harmonized neuronal and glial responses to variations in extracellular levels of active soluble mediators such as ATP are essential in controlling neuronal activity. In pathological conditions involving sensory nervous system, elevations in extracellular ATP levels are believed to be one of the main reason for neuronal sensitization.
This notion led us to explore mechanisms of ATP release in sensory ganglia and we found that association among P2X purinergic receptors, their downstream effectors (CASK and CaMKII) and hemichannel Panx1 regulates inhibition of ATP release in basal conditions and that same players are involved in P2X3 receptor evoked-ATP release which globally suggest that even if observed proteins are expressed in different cells, they could be modulated by similar mechanisms and are possibly part of an “ATP-keeper molecular system” that finely regulates extracellular levels of ATP by its sensing and further adjustments of peculiar extracellular concentrations.
Further we showed that P2X3 receptors interact with Panx1 in sensory neurons and that molecular coupling between P2X3, CASK and Panx1 contributes to decoding of the complex purinergic signaling involved in nociception which represents a novel and interesting mechanism of pain regulation that could be precisely targeted in order to alleviate tedious disorders of sensory neurons.
Ključne besede: ATP, ATP release, purinergic signaling, trigeminal ganglion, pain, migraine, P2X3, CASK, Pannexin-1, synaptic strength
Objavljeno: 10.12.2015; Ogledov: 1975; Prenosov: 113
Polno besedilo (3,75 MB)
Ustno izročilo in kulturni spomin v zgornjem ObsoteljuKatarina Šrimpf
, 2015, doktorska disertacija
Opis: Doktorska disertacija z naslovom Ustno izročilo in kolektivni spomin v zgornjem Obsotelju je celovita študija pripovednega izročila zgornjega Obsotelja, območja na slovenskem etničnem ozemlju. V uvodu prinaša predstavitev območja in krajev v treh raziskovanih občinah z zgodovinskega, gospodarskega pa tudi družbenega in kulturnega vidika ter predstavitev gradiva, tako starejšega, ki je bilo zbrano s pomočjo arhivskega dela, kot tudi novejšega, ki je bilo zbrano s terenskim delom. Gradivo je opisano in analizirano skozi žanrsko in tematsko strukturo. Drugi del disertacije je osredotočen na pomen in vlogo, ki jo imajo izbrani elementi pripovednega izročila v družbenem in kulturnem življenju obravnavanega območja. Kot ključni elementi za razumevanje pomena in vloge pripovedništva so se pokazali trije kriteriji: 1) percepcija prostora in časa, 2) konstruiranje identitet, 3) sodobna raba pripovedi in njihovih funkcij. Prostor in čas, kot se kažeta skozi pripovedno izročilo, sta koncipirana s pomočjo liminalnega časa in prostora ter etioloških povedk in zgodovinskih zgodb. Šaljive zgodbe o Lemberžanih ter zbadljivke in druge zgodbe o Hrvatih so v zgornjem Obsotelju priročno sredstvo za ločevanje ene skupine od druge, kar omogoča konstruiranje tako lokalnih kot nacionalnih in drugih identitet. Funkcijo vzbujanja občutka lokalne pripadnosti in vzpostavljanja lokalne identitete ima tudi sodobna raba lokalnega pripovednega izročila, ki je vzeto iz primarnega okolja. Poskus razumevanja pomenov in funkcij, ki jih ima ustno izročilo v lokalnem okolju, je glavna tema te disertacije.
Ključne besede: ustno izročilo, pripovedništvo, zgornje Obsotelje, percepcija prostora in časa, ustvarjanje identitet
Objavljeno: 04.12.2015; Ogledov: 2099; Prenosov: 114
Polno besedilo (5,95 MB)