Transmissible spongiform encephalopathies (TSEs), such as scrapie and variant Creutzfeldt-Jakob disease are infectious, fatal, neurodegenerative diseases. Following peripheral infection TSE agents usually accumulate in lymhoid tissues before spreading to the central nervous system. In mice, follicular dendritic cells (FDCs) expressing the host prion protein (PrPC) are essential for scrapie agent accumulation in lymphoid tissues. The accumulation of the scrapie agent on FDCs is critical for the efficient spread of infection to the brain. However, it is unknown whether FDCs themselves actively replicate the scrapie agent, or simply accumulate it following production by other cells types such as neurones, lymphocytes or other stromal cell populations. To definitively address this issue a transgenic mouse model was created in which PrPC is switched on or off exclusively on FDCs. Expression of cre-recombinase (Cre) under the action of cell-specific gene promoters can be used to induce or delete the expression of a target gene in specific cell populations. In this model, Cre expression is driven by the complement receptor type 2 gene (Cr2/CD21) which is expressed by FDCs and mature B lymphocytes. Characterisation of the CD21-cre mouse line was achieved by crossing with a ROSA26 reporter strain. The CD21-cre mouse line was subsequently crossed with floxed-PrP mouse lines to produce compound transgenic mouse lines in which PrPC expression was switched on or off, only in FDCs. Cre expression by B lymphocytes was eliminated by γ-irradiation and grafting recipient mice with Cre-deficient bone marrow. Immunohistochemical analysis confirmed the expression PrPC had been switched on or off exclusively on FDCs. Subsequently, the mice were challenged with scrapie by intra-peritoneal injection to determine the precise role of FDCs in the accumulation of scrapie in lymphoid tissues. Switching off PrPC expression exclusively on FDCs prevented the accumulation of TSE agent specific disease-associated PrPSc in the spleen after i.p inoculation. Conversely, in mice in which PrPC was expressed only on FDC, successful replication of the agent occurred on the FDC network in the spleen. Taken together, these data show PrPC-expressing FDCs alone are sufficient to support the accumulation of the scrapie agent within lymphoid tissues. Furthermore, these data suggest FDC replicate the TSE agent and do not simply accumulate it following synthesis by other cell types.

Transmissible spongiform encephalopathies (TSEs) are a group of fatal, neurodegenerative diseases that can affect both humans and animals. TSEs can be sporadic, familial, or acquired diseases. The prion hypothesis states that a misfolded form of the host glycoprotein, PrPC, acts as the infectious agent in TSE disease. The misfolded form, PrPSc, is increased in β-sheet content, detergent insoluble and partially resistant to proteinase K (PK) digestion. Based on the prion hypothesis, most current post-mortem diagnostic tests rely on the presence of PrPSc as indicative of TSE disease. However, recently experimental cases of TSE disease have been identified where no PrPSc deposition is evident. One example of this is a murine transgenic model of Gerstmann Sträussler Scheinker (GSS) disease. GSS is a familial TSE disease, caused by a number of different mutations in human PrP including a point mutation from proline to leucine at residue 102. A murine model of GSS disease, produced through gene-targeting, contains the same point mutation at the equivalent residue, 101, in murine PrP. These mice do not develop spontaneous disease during their lifespan, but when inoculated intra-cerebrally with either human P102L GSS (101LL/GSS) or hamster 263K scrapie (101LL/263K); develop a clinical disease and vacuolar TSE-related pathology. Upon biochemical and immunohistochemical analysis, the brain tissues of these clinically ill mice contain little or no detectable PrPSc. However titration experiments have previously shown infectivity titres of 107-109IU/g of brain tissue. Standard PK digestion (at 37°C), NaPTA precipitation and isolation of PrPSc through detergent insolubility and differential centrifugation all confirmed the observation of little or no detectable PK-resistant PrP (PrP-res) in the 101LL/GSS and 101LL/263K brain tissues, despite the high levels of TSE infectivity. The presence of PrPSc and/or TSE infectivity in the spleen during disease pathogenesis is dependent upon TSE agent strain and host species. Previous studies utilising wild-type mice infected with ME7, have shown that the levels of infectivity observed in spleen tissue are 2- 3log10 lower than those observed in the brain tissue of the same mice. However, experiments conducted as part of this thesis showed that sub-passage of both the brain and spleen tissue from clinically ill 101LL/GSS and 101LL/263K mice into 101LL mice by intra-cerebral inoculation result in short incubation periods, indicating that infectivity levels were similarly high in both tissues. Biochemical analysis of the primary spleen tissue identified the presence of PrP-res, albeit at lower levels than those observed in wild-type spleens infected with a standard laboratory TSE strain, ME7 or 79A. However, the presence of PrP-res indicates that the spleen has a role in disease pathogenesis, which will require further investigation. Additionally, the spleen tissue maintains the discrepancy between PrP-res and TSE infectivity that is observed in the brain tissue of these models and further questions the prion hypothesis. As little or no PrP-res was detectable in the brain tissues of 101LL/GSS and 101LL/263K mice by standard biochemical and immunohistochemical techniques, it was hypothesised that an in vitro amplification technique, protein misfolding cyclic amplification (PMCA) could amplify PrPSc to detectable levels. A series of optimisation experiments were performed to produce a reliable positive control for amplification of mouse PrPSc from a standard laboratory mouse TSE strain, 79A or ME7, with a normal wild-type mouse brain homogenate substrate. While a wide range of technical and experimental conditions were investigated, consistent and reproducible amplification of mouse PrPSc was not achieved and therefore amplification of PrPSc from 101LL/GSS and 101LL/263K tissues could not be performed as interpretation of results would be complicated without the presence of a positive control. Previous research has shown that while other commercial assays, e.g. TeSeE (BioRad), identified tissues from these models as borderline positive or negative for TSE disease, one TSE diagnostic assay, the IDEXX HerdChek kit, that utilises the Seprion ligand, identified both the brain and spleen tissue from 101LL/GSS and 101LL/263K clinical mice as positive for TSE disease. In order to identify if TSE infectivity is associated with the target of the Seprion ligand, brain tissue homogenates from 101LL/GSS, 101LL/263K and a positive control wild-type/79A homogenate were depleted of the Seprion ligand target utilising a PAD-beads kit (Microsens Biotechnologies), which incorporates the Seprion ligand as the capture agent, in combination with magnetic beads. Upon inoculation, a single depletion of the homogenates produced no significant reduction in incubation period to clinical disease in either the depleted homogenates or the wash buffers produced, in comparison to a non-depleted brain homogenate. This result indicates that a single depletion with the Seprion ligand, did not remove enough of the aggregated protein to significantly alter the level of infectivity in the depleted homogenate and that any infectious agent, which was initially bound to the Seprion ligand due to non-specific interactions, was then released during the wash steps of the procedure. Proteomic differences between all components produced during a single depletion of an infected brain homogenate, wild-type/79A, or a normal uninfected brain homogenate were assessed to potentially identify the target of the Seprion ligand. In conclusion, these murine models of TSE disease, 101LL/GSS and 101LL/263K, which contain both high infectivity levels with little or no PrP-res in the brain tissue and similar high levels of infectivity with low levels of PrP-res in the spleen, questions the accepted correlation between levels of infectivity and PrP-res or PrPSc as proposed by the prion hypothesis. It is hypothesised that either an alternative form of PrP, which has not yet been identified is the infectious agent in these disease models, or that the TSE infectious agent is a component which associates with PrPSc rather than being PrPSc itself. The eventual identification of the infectious agent present in these unusual disease models will increase our understanding of these diseases, potentially offer improved diagnostics for infectivity, and perhaps identify novel therapeutic targets.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that affect several species, such as scrapie in sheep or goats and CJD in humans. In several species, neurological disease is preceded by TSE agent accumulation in lymphoid tissues prior to neuroinvasion. While oral transmission is considered the most common route for scrapie, transmission can also occur through lesions to the skin or mucosa, for example in the mouth or gastrointestinal tract due to rough feed, or birth associated skin damage. Scrapie has also been experimentally transmitted through skin scarification in mice. Following scrapie infection via skin scarification, PrPSc accumulates in the draining lymph node (LN) before spreading to other organs in the lymphoreticular system. It is not yet known by what means the scrapie agent is transported from the skin to the draining LN. Dendritic cells (DCs) in the skin have been found to transport viruses, such as HIV or Dengue, from the skin, thereby raising the question whether DCs or Langerhans cells (LCs), located within the epidermis, play a role in the uptake and transport of the TSE agent from the skin to the draining LN. CD11c is a cell surface marker traditionally used to identify or isolate DCs from other cell types. Mice and rats are naturally resistant to Diphtheria toxin (DTX). A transgenic mouse line was created where the Diphtheria toxin receptor (DTR) was expressed on CD11c+ cells. The presence of this receptor on CD11c+ cells allowed for the temporary conditional depletion of CD11c+ cells following a single injection of DTX. The cells repopulate the tissues within a time frame specific to the tissues the cells are located in. These mice were used to determine whether the absence of CD11c+ cells at the time of scrapie infection via the skin had an effect on the early accumulation of PrPSc within the lymphoid tissues and on disease progression. Immunohistochemical analysis demonstrated that early PrPSc accumulation in the draining LNs was delayed following depletion of CD11c+ cells, indicating that their potential role in the transport of the scrapie agent from the skin. Scrapie incubation period was not affected by the absence of the CD11c+ cells at the time of infection. Recent findings show that CD11c is not exclusive to DCs and is also expressed on macrophage populations. Following DTX-mediated depletion, DCs repopulate the tissues much faster than CD11c+ macrophages. Scrapie infection was carried out in the skin in DTX treated mice after DCs had repopulated the tissues but before macrophage numbers had returned, to determine whether macrophages rather than DCs played a role in the early accumulation of PrPSc in the draining LNs. No differences in PrPSc accumulation were observed in mice depleted of macrophages compared to controls and there was no effect on disease incubation period. Another transgenic mouse line was used, where DTX expression on langerin+ cells (LCs and langerin+ DCs in the dermis), allowed for their temporary depletion through DTX treatment. Following langerin+ cell depletion, increased PrPSc accumulation was observed in the draining LNs 7 weeks post infection, but did not affect the incubation period of disease. These results indicate that the absence of LCs somehow accelerated PrPSc accumulation, and that LCs might play a preventative role in early stages after infection. Histopathological analysis was used to complement microarray studies aimed to determine what immune responses were associated with scarification and DTXmediated depletion of cells within the skin and whether these responses might be linked to disease transmission. DCs and LCs in the skin appear to play different roles in the early stages following scrapie infection via the skin, but the lack of effect on incubation period does not rule out the involvement of other cell types or cell-free mechanisms of scrapie agent spread from the skin.

A feature of prion disease and other protein misfolding neurodegenerative disease is the formation of amyloid plaques. Amyloid is commonly found in the brain of individuals who have died from prion disease and Alzheimer’s disease. The formation and purpose of amyloid in such diseases is poorly understood and it is not currently known whether the material is neurotoxic, neuroprotective or an artefact. Several methods are used to investigate the formation of amyloid both in vitro and in vivo. A cell free protein conversion assay has been optimised to gain insight into the protein misfolding pathway and prion infection has been introduced to a newly characterised whole brain organotypic slice culture model. Fibrillar, but not oligomeric, recombinant PrP species induce a seeding effect on amyloid formation in the protein conversion assay. Brain homogenate containing amyloid from a β-amyloid aggregation mouse model is demonstrated to have a similar effect to recombinant fibril seeds with a PrP substrate indicating a cross-seeding effect. A whole brain organotypic slice culture (BOSC) model has been developed and slices maintained in culture for up to 8 months. During this time slices remain viable with low levels of stress and thin down from 400μm to 30-50μm with morphological consequences. A prominent glial scar forms on the surface of the slice as a result of astrocyte activation and proliferation. The neuronal population decreases while the microglia have a consistent presence throughout time in culture. Replication of misfolded prion protein has been successfully demonstrated within whole BOSC following prion infection after 2 months in culture. The BOSC model represents an accessible short term in vitro model of the brain which can offer insights into protein misfolding in a complex multicellular context. Amyloid formation has been investigated in vivo using a β-amyloid misfolding mouse model following seeding with a range of recombinant protein and brain homogenate seeds. No seeding effect was observed in animals which had received intracerebral inoculations compared to control animals within the time frame of the experiment. A lack of overall amyloid within all animals at the final time point investigated suggests later time points are required for observation of seeding. The functional role of amyloid in protein misfolding neurodegenerative diseases remains unclear. From the cell free protein conversion assay oligomers do not form on the direct pathway towards amyloid in prion misfolding. BOSC provide an accessible and useful short term in vitro model which retains multiple characteristics of the brain. BOSC support replication of misfolded protein and amyloid formation therefore this model can now be utilised to investigate plaque growth and the effect of amyloid formation on surrounding cells. Results from these assays provide important information to guide future in vivo studies and aid the search for therapeutic intervention in these devastating diseases.

本文以意識形態途徑，研究中共建國後毛澤東探索後革命時期理論與實踐 統一的過程。第一章導論，說明研究方法；第二章論一九四九至一九五三 年中共內政外交情勢與毛澤東之態度；第三章論一九五三年至一九五五年 底一五計畫及社會主義過渡時期總路線之初期實踐經驗，置重點於工業化 產生的問題，農業合作化的超速完成，以及毛澤東在這些實踐經驗中的地 位及其反省；第四章論一九五六年以後蘇共二十大及波蘭、匈牙利事件如 何促使中共公開反省蘇聯模式，即毛澤東提出「論十大關係」的過程及其 意義，並由此導出中共為另尋發展模式，必須借重本國 識精英，因此才 有由毛澤東提倡的百花齊放、百家爭鳴的寬鬆政策；但由於知識精英出發 點與毛澤東不同，終使雙百失控，導致反右鬥爭，使毛澤東帶領中共走向 極「左」思潮。綜觀建國後至一九五七年，中共及毛澤東所面對的客觀情 境，實為後革命時期的必然發展，包括城鄉分離、工農剪刀差危機、知識 精英與群眾對立等；毛澤東通過個人認知結構（本文以民族主義、民粹主 義、實踐性格及矛盾世界觀概括），企圖找出能指導後革命時期的理論， 但由於後革命時期客觀情境之本質，及毛本人認知框架之限制，終使毛走 上以革命手段企圖達成後革命任務的極「左」思潮。

<p>Idag är det för en stark stat i västvärlden inte svårt att vinna ett konventionellt krig. Dock har man svårt med konflikter där en stat möter en icke-stat, till exempel USA i Afghanistan idag, trots att gerillan nästan uteslutande är sämre rustad och tränad jämfört med sin motståndare. Sverige är ett förhållandevis litet land och frågan finns om vi skulle klara en eventuell invasion. Det har tidigare existerat en debatt om gerillakrig är något för de svenska stridskrafterna och det torde vara aktuellt än idag. Principer för hur striden ska föras benämns i Sverige taktiska grundprinciper. Man kan fråga sig vart dessa taktiska grundprinciper, som fastställs i och med doktrinupprättandet, är hämtade från. Dessa ska förmedla beprövad erfarenhet, men Sverige har inte varit i krig på 200 år.</p><p>I konflikter där gerillan visat upp brukande av principer har man också varit framgångsrik. Ernesto Che Guevara och Mao Tse-tung teoretiserade båda om kriget i skrift och båda var mycket framgångsrika. Därför torde det vara intressant att undersöka hur dessa principer i teorin stämmer överens med våra svenska taktiska grundprinciper, då dessa taktiska grundprinciper ska vara tillämpbara i alla situationer.</p><p>Syftet är att undersöka om de svenska taktiska grundprinciperna stämmer överens, eller liknar de principer framgångsrika gerillakrigare förordar i sina litterära verk och om vissa principer är mer allmängiltiga än andra inom gerillakrigföringens taktik. Om Sverige som liten nation ska förhindra en invasion, kommer principerna för krigföring från gerillakrigare som också fått agera underlägsen styrka?</p><p>Frågan som ska besvaras i denna uppsats lyder: finns det likheter mellan Ernesto Che Guevaras och Mao Tse-tungs grundprinciper för gerillakrigföring på taktiskt nivå och svenska taktikens grundprinciper på markarenan? Om likheter finns, vilka är dessa?</p>

Idag är det för en stark stat i västvärlden inte svårt att vinna ett konventionellt krig. Dock har man svårt med konflikter där en stat möter en icke-stat, till exempel USA i Afghanistan idag, trots att gerillan nästan uteslutande är sämre rustad och tränad jämfört med sin motståndare. Sverige är ett förhållandevis litet land och frågan finns om vi skulle klara en eventuell invasion. Det har tidigare existerat en debatt om gerillakrig är något för de svenska stridskrafterna och det torde vara aktuellt än idag. Principer för hur striden ska föras benämns i Sverige taktiska grundprinciper. Man kan fråga sig vart dessa taktiska grundprinciper, som fastställs i och med doktrinupprättandet, är hämtade från. Dessa ska förmedla beprövad erfarenhet, men Sverige har inte varit i krig på 200 år. I konflikter där gerillan visat upp brukande av principer har man också varit framgångsrik. Ernesto Che Guevara och Mao Tse-tung teoretiserade båda om kriget i skrift och båda var mycket framgångsrika. Därför torde det vara intressant att undersöka hur dessa principer i teorin stämmer överens med våra svenska taktiska grundprinciper, då dessa taktiska grundprinciper ska vara tillämpbara i alla situationer. Syftet är att undersöka om de svenska taktiska grundprinciperna stämmer överens, eller liknar de principer framgångsrika gerillakrigare förordar i sina litterära verk och om vissa principer är mer allmängiltiga än andra inom gerillakrigföringens taktik. Om Sverige som liten nation ska förhindra en invasion, kommer principerna för krigföring från gerillakrigare som också fått agera underlägsen styrka? Frågan som ska besvaras i denna uppsats lyder: finns det likheter mellan Ernesto Che Guevaras och Mao Tse-tungs grundprinciper för gerillakrigföring på taktiskt nivå och svenska taktikens grundprinciper på markarenan? Om likheter finns, vilka är dessa?

Les Encéphalopathies Spongiformes transmissibles (EST) sont des maladies neurodégénératives fatales caractérisées par l’accumulation d’un conformère anormal (PrPSc) d’une protéine de l’hôte (PrP). Chez l’homme, plusieurs centaines de cas de transmissions iatrogènes de la maladie de Creutzfeldt Jakob (MCJ) ont été identifiées, notamment chez des patients ayant fait l’objetd’ une greffe de dure-mère, de cornée ou des injections d’hormones de croissance extractives. Plus récemment, plusieurs cas du variant de la maladie de Creutzfeldt Jakob (vMCJ) ont été observés chez des patients transfusés avec des produits sanguins issus de donneurs en incubation de la maladie. D’un point de vue sanitaire, l’évaluation du risque de contamination interindividuelle par des tissus ou des fluides biologiques issus de patients atteints représente un enjeu important en matière de santé publique. La première partie de notre travail a consisté à comparer la sensibilité relative de modèles de souris transgéniques sur-exprimant la PrP à celle de l’hôte conventionnel exprimant la même séquence. Les résultats obtenus ont validé le concept d’une absence d’impact du niveau d’expression de la PrP ou du fond génétique de l’hôte sur la sensibilité finale du modèle à l’infection. A l’aide de ces modèles de souris transgéniques, nous avons alors mesuré les niveaux d’infectiosité dans le sang de patients atteints de différentes formes d’’EST. Chez un patient atteint de vMCJ, nous avons mis en évidence de faibles niveaux d’infectiosité dans les concentrés de globules rouges, les leucocytes et le plasma. Nous avons également pu détecter de l’infectiosité dans le plasma issu de 2 patients atteints de sMCJ sur 4 testés. Parallèlement à ces expériences, nous avons démontré dans un modèle expérimental d’infection chez le mouton, que l’administration de 104 à 105 leucocytes suffisent à transmettre par voire transfusionnelle la maladie. Ces résultats soulignent l’intérêt et les limites de la leuco-déplétion appliquée de manière standard en médicine transfusionnelle, pour limiter les risques de transmission du vMCJ. Enfin, nous avons testé la capacité de différents outils in vitro à détecter la présence des Prions dans le sang. / Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders occurring in a wide spectrum of animals. They are characterized by accumulation of abnormally folded conformers (PrPSc) derived from normal cellular PrP protein (PrP) of the host. In human, many iatrogenic transmissions of Creutzfeldt Jakob disease (CJD) have been reported after dura mater graft, corneal graft or extractive growth hormone injections, prepared from affected donors. More recently, several cases of vCJD transmissions were reported in individuals that were transfused with blood from asymptomatic donors that subsequently developed vCJD. Risk assessment of interindividual transmission with contaminated tissues or body fluids remains a major public health issue. In a first part, we validated the final pertinence of infectious titers as measured in mice overexpressing PrP to the risk of transmitting the disease in the natural host species. In a second time, we used this model to evaluate the presence of infectivity in blood from TSE affected patients. We were able to detect the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with vCJD and in the plasma of 2 out of 4 persons whose tests were positive for sporadic CJD. We then demonstrated in a sheep TSE model, that intravenous administration of 104 to 105 leucocytes was sufficient to cause disease in recipient sheep, underlying the efficacy and potential limits of leuko-reduction processes that are currently applied in transfusion medicine to mitigate the TSE transmission risk. Finally, using the same model, we tested different in vitro methods to detect prions in blood.

Abstracts in English, Afrikaans and Sesotho / As one of the renowned anomalies in modern investment theory, the low idiosyncratic volatility anomaly may be the most bewildering and captivating of them all. The anomaly defies the traditional asset pricing theories of modern portfolio theory, which state the fundamental principle that high-risk portfolios are compensated for with higher expected returns. This study determined if the low idiosyncratic volatility premium is present on the cross-section of share returns of the JSE. 12-, 36- and 60-month volatility estimation periods were used in this study to determine if this has any significant effect on share returns. A relevant 26-year sample period from January 1994 to December 2019 was employed. In examining the CAPM OLS regression results utilising the 60-month idiosyncratic volatility estimation period, statistically significant evidence was found to support the alternative hypothesis of a low idiosyncratic volatility anomaly on the cross-sectional returns on the JSE. These findings are supported by a statistically significant alpha for five of the six portfolios examined and clearly indicate the superior performance of the low volatility portfolio in contrast to the high idiosyncratic volatility portfolios. These findings of the 60-month CAPM regression analysis provide clear evidence of a low idiosyncratic volatility anomaly and reject the null hypothesis that there is no statistically significant evidence in favour of a low idiosyncratic volatility anomaly on the cross-section of share returns on the JSE after estimating volatility utilising a 60-month volatility estimation period. / As een van die bekendste anomalieë in moderne beleggingsteorie, is die lae idiosinkratiese gestadigheidsanomalie moontlik die mees verbysterende en boeiende anomalie van almal. Hierdie besondere anomalie bied ʼn uitdaging aan die tradisionele bateprysingsteorie van moderne portefeuljeteorie, die grondbeginsel waarvolgens daar vir hoërisiko-portefeuljes vergoed word deur hoër verwagte opbrengste. Die doel van hierdie studie is om te bepaal of die lae idiosinkratiese gestadigheidspremie aanwesig is by die deursnee-aandeleopbrengste op die JSE. In hierdie studie, is gestadigheidsramingstydperke van 12, 36 en 60 maande gebruik om te bepaal of dit enige beduidende uitwerking op aandeleopbrengste het. ʼn Relevante steekproeftydperk van 26 jaar van Januarie 1994 tot Desember 2019 is gebruik. Deur ondersoek van regressieresultate van die kapitaalbateprysingsmodel (KBPM) kleinste-kwadratemetode aan die hand van ʼn idiosinkratiese gestadigheidsramingstydperk van 60 maande is statisties-beduidende bewyse gevind om die alternatiewe hipotese van ʼn lae idiosinkratiese gestadigheidsanomalie in die deursnee-opbrengste op die JSE te ondersteun. Hierdie bevindings word ondersteun deur ʼn statisties-beduidende alfa vir vyf van die ses portefeuljes wat ondersoek is en dit dui duidelik op die superieure prestasie van die laegestadigheidsportefeulje in kontras met die hoë idiosinkratiese gestadigheidsportefeuljes. Die bevindings van die KBPM-regressie-analise van 60 maande voorsien duidelike bewyse van ʼn lae idiosinkratiese gestadigheidsanomalie en verwerp die nulhipotese dat daar nie statisties-beduidende bewyse is ten gunste van ʼn lae idiosinkratiese gestadigheidsanomalie in die deursnee-aandeleopbrengste op die JSE nie nadat gestadigheid geraam is aan die hand van ʼn gestadigheidsramingstydperk van 60 maande. / E le e nngwe ya diphoso tse tummeng kgopolong ya sejwale-jwale ya matsete, bothata bo tlase ba ho hloka botsitso e ka ba ntho e makatsang le e hohelang ka ho fetisisa. Phoso e ikgethileng ha e latele dikgopolo tsa ditheko tsa thekiso ya thepa ya sejwale-jwale, e hlalosang molao-theo wa hore dipotefoliyo tse kotsing e kgolo di lefellwa bakeng sa dikgutliso tse phahameng tse lebelletsweng. Phuputso ena e ne e ikemiseditse ho fumana hore na tefo e tlase ya botsitso e teng dikarolong tse sa tshwaneng tsa dikgutliso tsa dikabelo ho JSE. Phuputsong ena ho sebedisitswe dinako tsa tekanyetso ya ho hloka botsitso ya dikgwedi tse 12, 36 le tse 60 ho fumana hore na sena se na le phello e kgolo ho dikgutliso tsa dikabelo. Nako ya sampole e loketseng ya dilemo tse 26 ho tloha ka Pherekgong 1994 ho isa ho Tshitwe 2019 e ile ya sebediswa. Ha ho hlahlojwa sephetho sa tekanyo ya CAPM OLS ho sebediswa nako ya dikgakanyo tsa ho hloka botsitso ha dikgwedi tse 60, ho fumanwe bopaki ba bohlokwa ho tshehetsa mohopolo o mong wa phokotso dikgutlisong tsa dikarolo tse fapaneng ho JSE. Diphumano tsena di tsheheditswe ke qaleho ya dipalo bakeng sa dipotefoliyo tse hlano ho tse tsheletseng tse hlahlobilweng mme di bontsha tshebetso e phahameng ya potefolio e tlase ya ho hloka botsitso ho fapana le dipotefoliyo tse phahameng tsa ho hloka botsitso. Diphumano tsena tsa tlhahlobo ya tekanyo ya CAPM ya dikgwedi tse 60 di fana ka bopaki bo hlakileng ba phokotso e sa tlwaelehang ya ho hloka botsitso le ho hanyetsa kgopolo-taba ya hore ha ho na bopaki ba dipalo-palo bo tshehetsang boemo bo tlase ba ho hloka botsitso bo sa tlwaelehang dikarolong tse sa tshwaneng tsa dikabelo ho JSE kamora ho lekanyetsa ho hloka botsitso ho sebedisang nako ya dikgakanyo tsa ho hloka botsitso ya dikgwedi tse 60. / Business Management / M. Com. (Business Management)

Scrapie und die Bovine Spongiforme Enzephalopathie (BSE) sind stets tödlich verlaufende transmissible spongiforme Enzephalopathien (TSE) bei kleinen Wiederkäuern und Rindern. Nach der „Prion-Theorie“ ist die pathologische Isoform eines zellulären Proteins, des Prion-Proteins, der Hauptbestandteil, wenn nicht sogar die einzige Komponente der TSE-Erreger. Gemäß dieser Theorie lagert sich die pathologische Isoform, PrPSc, an die zelluläre Form, PrPC, an und führt so zu einer Konformationsänderung von PrPC. Bisher lassen sich TSE-Erreger nur im Tierversuch sowie in wenigen überwiegend von Nagetieren stammenden Zelllinien vermehren. Das Ziel der vorliegenden Arbeit war deshalb die Identifikation und Charakterisierung neuer TSE-empfänglicher Zelllinien, wobei vor allem die Infektion oviner und boviner Zelllinien mit Scrapie- bzw. BSE-Feldisolaten im Vordergrund stand. Hierzu wurde zunächst der Einfluss unterschiedlicher Kultur- und Infektionsbedingungen (Nährmedien, Umsetzraten, Temperatur, Herstellung der Inokulate, Inokulationsprotokoll) auf den Infektionserfolg studiert. Basierend auf den dabei gewonnenen Daten wurde ein Infektionsprotokoll erstellt, das im weiteren Verlauf für alle in dieser Studie untersuchten Zelllinien verwendet wurde. Durch die Zellbank des Friedrich-Loeffler-Instituts stand eine Vielzahl unterschiedlicher eukaryotischer Zelllinien zur Verfügung, von denen 53 aus geeigneten Organen und Geweben und größtenteils vom Wiederkäuer stammende Zelllinien ausgewählt wurden. Anschließend wurden diese Zelllinien kultiviert und wiederholt hinsichtlich ihrer PrPC-Expression analysiert. Dabei zeigte sich, dass das PrPC-Expressionniveau für jede Zelllinie sehr individuell und weder spezies- noch gewebespezifisch ist. 34 der 53 Zelllinien exprimierten PrPC in detektierbarer Menge und wurden in den weiteren Infektionsstudien verwendet. Dabei wurden sie mit verschiedenen TSE-Stämmen bzw. -Isolaten (bovines BSE-Material, ovines und caprines Scrapie-Material, mausadaptierte BSE- und Scrapie-Stämme) inokuliert. Der Großteil dieser Zelllinien (30 von 34) zeigte sich gegen alle eingesetzten Prion-Erreger resistent. Zwei Zelllinien konnten transient für 10 (MGbov900) bzw. 32 (Bov11) Passagen mit bovinem BSE-Material infiziert werden. Damit war die prinzipielle Möglichkeit einer BSE-Infektion dieser Zellen gezeigt. Aus bisher ungeklärten Gründen wurde die Prion-Infektion jedoch von beiden Zelllinien wieder verloren und erneute Infektionsversuche blieben erfolglos. Zwei weitere Zelllinien konnten persistent infiziert werden. Die Zelllinie N2a229, eine Sublinie der in der Prion-Forschung weit verbreiteten Neuroblastomzelllinie N2a, war für den mauspassagierten Scrapie-Stamm RML empfänglich. Des Weiteren wurde eine bovine Zelllinie (Bov5; 154PES) identifiziert, die für zwei ovine Scrapie-Feldisolate empfänglich war. Es handelt sich dabei um die erste nicht transgene Zelllinie, die mit einem Scrapie-Feldisolat infiziert werden konnte. Die beiden verwendeten Isolate (S71/04 und S95/04) stammten von Schafen aus einem klassischen Scrapie-Ausbruch aus dem Jahr 2004. In den infizierten Bov5Sc-Zellen steigerte sich die initial schwache PrPSc-Akkumulation über mehrere Passagen zu einem starken Signal, das durch verschiedene Prion-spezifische Antikörper im Dot-Blot, im Western-Blot und mit dem „Zell-ELISA“ nachgewiesen werden konnte. Die Infektion ist bereits seit über 200 Passagen stabil. Sie war mit den besagten Scrapie-Isolaten mehrfach wiederholbar und durch die Selektion von infizierten Einzelzellen konnten hochpositive Sublinien erhalten werden. Infizierte Bov5Sc-Zellen führten nach Inokulation in transgene Rinder- oder Schaf-PrPC überexprimierende Mäuse zu Scrapie-Erkrankungen und der Bildung von PrPSc im Gehirn der Mäuse. Die Infektion von Rinderzellen mit einem ovinen TSE-Isolat bedeutete die Überwindung einer Speziesbarriere. In weiteren Untersuchungen konnte gezeigt werden, dass eine Adaptation des Erregers an die Zellen stattgefunden hatte, welche sich z. B. in einem veränderten Glykosylierungs-profil darstellt. Verglichen mit zwei murinen TSE-infizierten Zelllinien zeigten die Bov5Sc-Zellen ähnliche Eigenschaften hinsichtlich ihrer Proteinase K-Resistenz, aber eine deutlich verlängerte Reaktionszeit gegenüber PrPSc inhibierenden Substanzen. Neben den Infektionsstudien an den „Zellbank-Zelllinien“ wurden transgene Zelllinien hergestellt, die auf der Basis von RK13-Zellen (Nierenzellen aus dem Kaninchen) und Hpl3-4-Zellen (neuronale Zelle aus PrP-defizienten-Mäusen) das PrPC von Maus, Schaf, Nerz, Hund sowie zwei chimären Konstrukten aus Maus/Rind/Maus bzw. Maus/Schaf/Maus exprimierten. Ein Großteil dieser transgenen Zelllinien war gegenüber einer Infektion und insbesondere einer Infektion mit TSE-Feldisolaten resistent. Durch Infektionsstudien mit dem mausadaptierten murinen Scrapie-Stamm RML konnten jedoch interessante Einblicke in die Hintergründe der zellulären TSE-Empfänglichkeit gewonnen werden. So zeigte sich, dass die Expression eines chimären PrP-Konstruktes aus Maus und Schaf (Mushp) nur in RK13-Zellen, nicht aber in Hpl3-4-Zellen zu einer für den Scrapie-Stamm RML empfänglichen Zelllinie führte. Dagegen konnten murines PrPC exprimierende Hpl3-4-Zellen erfolgreich mit RML, nicht aber mit dem Stamm Me7 infiziert werden. Diese Versuche unterstützen die Annahme, dass für eine Zellinfektion weitere zelluläre Komponenten eine Rolle spielen und zeigen, dass nur die richtige Kombination aus exprimiertem PrPC und zellulärem Hintergrund die Empfänglichkeit einer Zelllinie für einen speziellen TSE-Erreger bestimmen kann. Weitere Studien mit empfänglichen bzw. infizierten bovinen Zelllinien werden zu einem besseren Verständnis der zellulären Pathogenese bei BSE und Scrapie führen, woraus sich möglicherweise auch ein therapeutischer und diagnostischer Nutzen ziehen lässt.