Prion diseases or transmissible spongiform encephalopathies (TSE) are characterised by the accumulation of a misfolded conformer (PrPSc) of a host encoded protein (PrPC). The misfolding event that leads to the formation PrPSc can be replicated in the in vitro amplification technique, protein misfolding cyclic amplification (PMCA). This thesis focuses on the application PMCA to study multiple aspects of prion misfolding in relation to ruminant prion diseases, specifically developing techniques to detect and characterise PrPSc in scrapie and BSE infections. Utilising recombinant hamster PrP (rPrP) as substrate in PMCA, multiple genotypes of scrapie were successfully amplified in an attempt to describe a quantifiable technique applicable to a wide range of scrapie isolates. Observations of non-specific protease resistant rPrP formation was investigated with modifications to the PMCA methodology, which ultimately proved unsuccessful in reducing non-specific protease resistant rPrP. Using brain PrPC as substrate, the quantitative PMCA technique was piloted with BSE to correlate in vitro replication efficiency with infectious titre in mouse bioassay, but no correlation was identified. Atypical forms of BSE occur primarily in older cattle, are asymptomatic and thought to be spontaneous diseases. None the less, infection models in rodents and primates have identified the zoonotic potential of H-type and L-type BSE. Therefore PMCA methods were developed which were able to successfully amplify both atypical forms of BSE. In particular, sensitive detection and discrimination from classical BSE was demonstrated for H-type BSE, which has not previously been amplified in PMCA. H-type BSE could be detected in 1x10¬-12 g brain material and was discriminated from classical BSE by increased protease sensitivity, relatively high molecular weight and antibody reactivity. Evidence exists for co-infection of TSE strains, yet scrapie and BSE co-infection in an ovine host remains unaddressed. To study the disease progression and tissue dissemination of co-infections a PMCA assay capable of specifically amplifying BSE PrPSc in the presence of excess scrapie was applied to artificially mixed brain homogenates containing BSE and scrapie, and compared to current statutory strain typing methods. The PMCA was found to have sensitivity and specificity of 100% in mixes containing 0.1% BSE and 99.9% scrapie brain material, which was more effective than conventional strain typing methods. The assay was then applied to the brain, spleen and lymph of scrapie and BSE experimental co-infections in two genotypes of sheep, and to animals which belonged to a flock with endemic natural scrapie and that also received experimental BSE infections. The PMCA data demonstrated that sheep with PRNP genotype ARQ/ARQ (at amino acid positions 134, 154 and 171) were resistant to BSE in a co-infection scenario. In sheep with PRNP genotype of VRQ/ARQ, mixed infections could occur, and animals with scrapie PrPSc only in the brain could harbour BSE PrPSc in peripheral tissues. Co-infection was also possible in sheep with natural scrapie infections. The assay was compared to conventional testing methods of western blotting, PrPd profiling and immunohistochemistry and displayed superior sensitivity in BSE detection. PMCA amplification of bovine BSE isolates in ovine substrates identified several instances in which the molecular characteristics of the PrPSc was scrapie-like in terms of molecular weight, antibody reactivity and glycoform profile, and in some cases PrPSc characteristic of BSE could no longer be recovered. This occurred in a genotype specific manner, ‘molecular switching’ was only apparent in ovine substrate VRQ/VRQ in accordance with previous findings. These results raise the possibility of such an event occurring in in vivo ovine BSE infections and the zoonotic potential of these scrapie like conformers are yet to be fully addressed.

Understanding both the mechanistic basis of virulence and the evolutionary processes under which it can arise, is fundamental if we are to increase our knowledge of disease causing bacteria in an era of ever increasing antibiotic resistance. To date, there has been a substantial effort to understand virulence evolution both theoretically and experimentally. However, comparatively little experimental work has focussed on the evolution of virulence in opportunistic pathogens, and how virulence varies across multiple host organisms. In this thesis, the opportunist Pseudomonas aeruginosa was used to study virulence and its evolution in hosts. The virulence levels of different strains of P. aeruginosa, both laboratory and clinical, were tested in the nematode Caenorhabditis elegans. It was found that virulence of P. aeruginosa was lower in clinical strains isolated from chronic infections, and laboratory strains initially isolated from chronic infections, while a strain isolated from an acute infection was substantially more virulent. Using the C. elegans host, a selection experiment was carried out to test the effect of host density on virulence evolution. This resulted in a varied array of P. aeruginosa virulence phenotypes, but the worm evolved bacteria did not exhibit drastically different phenotypes from the worm negative control experiments. However, the degree of evolution observed across all treatment groups, compared with the wild type, highlighted the changes a bacterial population can undergo in vitro, even from a relatively short period of sub culture. Lastly, P. aeruginosa virulence was tested in alternative host organisms, Galleria mellonella (waxmoth larvae) and Vigna radiate (mung bean seedlings). Virulence varied according to the host, with virulence in G. mellonella being universally high, while no virulence was observed in V. radiate. This work highlights that high or low virulence cannot automatically be assumed in different hosts for opportunistic bacteria such as P. aeruginosa. Furthermore, the results highlight the unpredictable nature of virulence evolution, and how dependent the emergence of virulence is on the interaction between the pathogen and the host. A more complete understanding of the principles underlying bacterial evolution and virulence could allow for the construction of more specific and accurate studies, with experimental conditions carefully engineered to best replicate the clinical conditions of interest.

The aim of the work described here was to examine the antiprotozoal activity of the pericarp from Sapindus rarak DC (Sapindaceae) for its potential to defaunate the rumen, either partially or completely, without having a detrimental effect on the bacterial population and on rumen fermentation, and hence enhancing rumen productivity. An approach to improving microbial efficiency in the rumen is to eliminate protozoa (defaunation). Elimination of protozoa by chemical means is potentially the most convenient method. However, it is likely that some chemicals are not toxic specifically to the protozoa and probably kill other microorganisms and host cells in the rumen, therefore novel natural antiprotozoal agents are being sought. A possible role for saponins has been of interest to many researchers as saponins induce marked reductions in rumen protozoa numbers, particularly when the animal is fed on high-concentrate diets. The susceptibility of rumen protozoa and lack of susceptibility of rumen bacteria to saponins is explained by the reaction of saponins with membrane sterols, which are present only in eukaryotic membranes and not in prokaryotic bacterial cells. Recent in vitro results suggest that S. rarak exhibited higher immobilising activity and produce higher activity to lyse the cells compared to other saponin-containing plants. A preliminary study on the antiprotozoal activity of the active agent present in the pericarp of fruits of S. rarak, well known for the high saponin content of its fruits, was carried out. An antiprotozoal assay was developed. The results were consistent within experiments, but not between experiments, possibly due to the number of protozoa fluctuating widely with time. A study on the effect of Sapindus rarak on rumen bacteria, using a quantitative plating method, showed that there was no effect on rumen bacteria. Raw material and extract of pericarp of S. rarak were screened for their effects on rumen protozoa and bacteria (with a view to predicting its safety as a feed supplement and for its potential to defaunate the rumen, either partially or completely). The pericarp of S. rarak was extracted using selective extraction of saponins with CIS support. The fractions were collected and monitored by Thin Layer Chromatography (TLC). The active compounds were tested in vitro for toxicity to rumen protozoa by visual assessment of protozoal viability (Nottingham studies) and by measuring the degradation of labelled bacterial protein by rumen protozoa (Aberdeen studies). The influence of methanol extract of S. rarak on growth of pure cultures of rumen bacteria was also examined (Aberdeen studies). The results showed that the saponin fractions of S. rarak exhibit antiprotozoal activity as confirmed by visual assessment as well as by measurement of the breakdown of 14C-Ieucine-Iabelled Prevotella bryantti in rumen fluid incubated in vitro. No evidence was found of protozoal resistance to S. rarak. Inclusion of methanol extract of S. rarak in the growth medium of pure cultures of rumen bacteria had no effect, except that cellulolytic bacteria showed susceptibility. Extracts appeared in this study to prolong the lag phase following inoculation of Streptococcus bovis or inhibit the growth of Butyrifibrio fibrisolvens, Ruminococcos albus, and Ruminococcos flavefaciens. It seems that antibacterial properties were more pronounced against gram-positive bacteria, similar to the action ofionophores (McGuffey et al., 2001). A study on the effects of S. rarak on ruminal digestion, fermentation and ammonia concentration, using ruminally and duodenally cannulated dairy cows, showed that direct administration of S. rarak into the rumen did not decrease protozoal numbers in vivo. However, there were some indicators of selective activity against holotrichs. Changes in propionate concentration in the rumen and lack of change in microbial flow parameters suggest no adverse effects on other fermentation measures. Increased production of propionate is beneficial to the animal by affecting the capture of fermentation energy in the rumen. S. rarak caused a decrease in ammonia concentration. The observed ammonia levels represent a balance between the processes of degradation of feed protein and uptake of ammonia for synthesis of microbial protein. The lower ammonia levels could be due to higher incorporation of ammonia, peptide, or amino acids into microbial protein. In conclusion, S. rarak tested in this study has a great potential for suppressing rumen ciliate populations, but exerted negligible general effects on ruminal fermentation. Therefore, controlling rumen ciliate protozoa would be expected to lessen the dependence on protein supplementation under high-production conditions and would also be beneficial under conditions where the quantity of protein absorbed from the post-ruminal gut limits animal productivity, which occurs frequently in animals receiving low-quality tropical forages.

Bovine mastitis is recognised worldwide as the most important and costly disease affecting dairy cattle. The reduction of herd mastitis rates is crucially needed to improve animal welfare and profitability, and lessen the reliance on antibiotics. Single nucleotide polymorphisms (SNPs) within genes that have a critical role in the innate immune response, such as Toll-like receptors (TLRs) and the chemokine receptors CXCR1 and CXCR2, could impact on establishment and progression of intramammary infection, and therefore influence an animal’s susceptibility to disease. The genetic selection of animals with favourable TLR and CXCR1/2 mutations, with no impact on production traits, could be incorporated into dairy breeding programmes. In order to investigate any associations with clinical mastitis (CM) incidence and milk quality and quantity, this study identified and analysed SNPs alongside actual CM and production data from a Holstein-Friesian herd. This revealed 46 SNPs, 9 of which are novel, within boTLR1/4/5, boCD14, boCXCR1 and boCXCR2; selected SNPs were then tested for association with CM. This is the first report of boTLR1 SNPs and a non-coding boCXCR1 SNP that associate significantly with susceptibility to CM. Favourable linkage of reduced CM with increased milk fat and protein was observed, indicating selection for these markers would not be detrimental to milk quality. Furthermore, this study provides evidence that some of these SNPs underpin functional variation in bovine TLR1 and CXCR1, and possibly underlie an immunological mechanism for disease susceptibility. SNPs in boTLR1 and boCXCR1 were significantly associated with impaired transcript levels in milk somatic cells. In addition boTLR1 SNPs associated with impaired cytokine responses from cell populations when exposed to ligand or heat-killed mastitis-causing bacteria. The potential impact of boTLR1 variation on the immune response to Staphylococcus aureus is demonstrated, and this has implications for boTLR1-mediated immune responses to other pathogens.

In order to develop novel antibacterial therapies that combine anti-adhesion, anti-quorum sensing and the delivery of conventional antibiotics, the effects of polymers on bacterial aggregation and quorum sensing (QS) were studied. QS is a term used to describe method by which bacteria use chemical signal molecules to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the signal molecules used for QS are therefore a potential means to control bacterial population responses. In this thesis, the ability of the cationic polymers poly (N-[3-(dimethylamino) propyl] methacrylamide) (p(DMAPMAm), P1) and poly (N-dopamine methacrylamide-co-N-[3-(dimethylamino) propyl] methacrylamide) (p(DMAm-co-DMAPMAm), P2) to cluster a range of bacteria, such as Staphylococcus aureus(Gram-positive), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa(Gram-negative) under conditions of varying pH and polymer concentration was investigated. It was identified that clustering ability was strongly dependent on the balance between charge and hydrophobicity. The results also suggested that catechol moieties might have a positive effect on adhesive properties. Moreover, the potency of polymers against QS of Vibrio harveyi was assayed via testing bioluminescence. P1 which was able to bind to the surface of bacteria through electrostatic interactions enhanced the expression of QS and P2 which could bind to both the bacteria and QS signals showed the ability to both enhance and reduce light production. Furthermore, polymeric vesicles made of copolymers containing poly (3,4-dihydroxy-L-phenylalanine methacrylamide) (p(L-DMAm)) which displayed similar dual affinity compared toP2 were prepared and their ability to modulate QS responses in Vibrio harveyi was demonstrated. All the vesicles showed higher potency in quenching bioluminescence than their linear polymer analogues. To explore the feasibility of using self-assembled polymers for anti-microbial drug delivery, silver loaded DOPG lipid vesicles were made and were found to interfere with QSwhile reducing bacterial viability when the concentration of Ag+ was above the MIC (0.1 μg/mL). The results overall suggested that combined antimicrobial therapies might be possible using polymers and both QS and cytostatic or cytotoxic agents.

Bacteria communicate with each other by releasing chemicals called quorum sensing molecules. A common gram-negative bacteria, Pseudomonas aeruginosa, releases a number of such molecules including, N-3-(oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and Pseudomonas quinolone signal (2-heptyl-3,4-dihydroxyquinoline, PQS). The quorum sensing molecules have immunomodulatory effects on mammalian cells in addition to their role in bacteria. There has been very little study of the vascular effects of quorum sensing molecules on the host. The aim of this thesis was; therefore, to investigate the effect of Pseudomonas aeruginosa quorum sensing molecules, 3OC12-HSL and PQS, on vascular function and endothelial cell permeability and to study the potential mechanisms involved. Both 3OC12-HSL and PQS caused slowly developing vasorelaxation in arterial and venous preparations isolated from pigs. PQS was slightly more potent (pIC50) as a vasorelaxant than 3OC12-HSL. The endothelium was not a prerequisite for 3OC12-HSL-induced vasorelaxation since removal of endothelium and the nitric oxide synthase inhibitor (L-NAME) did not attenuate responses. Indeed, the opposite is true since 3OC12-HSL vasorelaxation responses were larger in the absence of the endothelium and nitric oxide. The mechanism of 3OC12-HSL does not involve either prostanoids, cyclic GMP, mitochondrial targets, calcium channels, chloride channels, PPAR-γ receptors, pannexin receptors, gap junctions or cystic fibrosis transmembrane conductance regulator. However, ouabain and depolarisation induced by potassium chloride decreased 3OC12-HSL responses suggest an impact on membrane potential. Overnight exposure of the porcine coronary artery to wild type Pseudomonas aeruginosa (PAO-L), produced a selective reduction in the magnitude of contractions to potassium chloride. However, neither 3OC12-HSL nor PQS produced an inflammatory response with prolonged exposure since they did not modify tumor necrosis factor alpha or vascular tone under these conditions. Both 3OC12-HSL and PQS increased endothelial cell permeability. Both caused a fall in endothelial resistance and an increase in FITC-dextran transport across human brain microvascular endothelial cells (HBMECs). The immunoblotting data showed that the levels of adherens junction and tight junction protein expression were decreased in HBMECs exposed to 3OC12-HSL, but not PQS. Rather PQS, but not 3OC12-HSL, caused a concentration-dependent phosphorylation of p38 MAPK, suggesting these molecules may modify vascular permeability by different mechanisms. In summary, these data show that 3OC12-HSL and PQS affect mammalian vascular function by decreasing vascular tone and increasing endothelial permeability. The potential advantage of these effects of quorum sensing molecules on host cells allowing bacteria to promote increase local circulation or produce oedema or allow access to promote systemic infection.

The detection and control of microorganisms such as bacteria is important in a wide range of industries and clinical settings. Detection, binding and removal of such pathogenic contaminants can be achieved through judicious consideration of the targets which are available at or in the bacterial cell. Polymers have the ability to present a number of binding ligands for cell targeting on one macromolecule and so avidity of interaction can be greatly increased. The goal of the project was to test whether polymers generated with bacteria in situ would have their composition significantly altered to determine if a templating process was occurring. It was also anticipated that the templated polymers would have better re-binding properties than those produced in the absence of bacteria. A series of chemical functionalities were analysed for their binding properties to bacteria. The functionalities were chosen with consideration to the cell surface characteristics. Further to identification of the most binding and least binding functionalities the polymers were tested for their cytotoxicity against bacteria and human epithelial cells. Concentration ranges were determined which could facilitate bacterial binding and templating yet minimise the lethality of the processes. Templated polymers of the bacteria were generated using a novel method of atom transfer radical polymerisation (ATRP) which we have termed bacterial activated atom transfer radical polymerisation (b-ATRP). This polymerisation method has maximised the potential for templating processes to occur during the polymerisation. Templated polymers differed in both their composition and their binding behaviour to non-templated polymers. The bacterial organic reduction process has also been demonstrated to have greater scope for use within the organic chemistry field as demonstrated by the use of this system to enable in "click-chemistry" via the reduction of copper.

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No. of bitstreams: 1 simão_br_dr_jabo.pdf: 4039244 bytes, checksum: 1f0f7b13d757bcb95d5e59f2107310ea (MD5) Previous issue date: 2017-12-05 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A fim de utilizar a modelagem tridimensional (3D) para estimar área, volume e massa, bem como avaliar o impacto dos desvios envolvidos nas equações empíricas disponíveis na literatura, em animais, foram utilizados seis ovinos Corriedale tosqueados com massa entre 10 e 105 kg e cinco bovinos Nelore com massa entre 600 a 800 kg. Os animais foram treinados utilizando os princípios de habituação e condicionamento, a fim de reduzir a reatividade dos animais. Para medição da área de superfície, utilizaram-se basicamente três procedimentos: equação empírica (EE) para cada espécie, instrumento manual de medição (IMM) e modelagem 3D do animal por fotogrametria (AR) e escaneamento com sensor kinect (AS_KS). Foi realizada uma análise de covariância, sendo a massa a covariável e uma análise de regressão não linear foi ajustada como uma função potência (alometria). Analisando as médias de área de superfície, para ovinos, em relação aos procedimentos utilizados não houve diferença entre IMM e modelos 3D, bem como entre os modelos 3D, enquanto entre IMM e EE houve diferença significativa. Para os bovinos, observou-se que os procedimentos 3D foram, em média, superiores às estimativas de área de superfície pela EE (a=5%). Quando se plotou os dados de área de superfície, de ovinos, dos métodos testados em função da massa corporal, obteve-se aproximações empíricas distintas daquelas estimadas pela EE em mais de 11% para animais acima de 100kg, enquanto que para os bovinos a diferença média entre os modelos 3D e EE foi de mais de 18%. Como os ajustes das equações com IMM e por modelos 3D tiveram taxas de crescimento muito próximas e os desvios entre elas foram inferiores a 2%, constatou-se que o uso de modelos 3D foi ratificado. Quando se plotou os dados de ambas as espécies, observou-se excelentes ajustes (R²=0.99) e estimativas consistentes com aquelas obtidas pelas equações específicas para cada raça, com desvios inferiores a 3,2%. Outras relações, como volume em função da área de superfície, foram estabelecidas, resultando em aproximações consistentes quando utilizada para estimar o volume do animal. Quando se considerou o animal como um Cilindro Horizontal Padrão (CHP), sua área de superfície foi estimada, em média, 36% abaixo daquelas observadas para os modelos 3D e IMM. Por meio da simulação da transferência de calor por convecção, obteve-se uma diferença de mais 56% na taxa de convecção quando se considerou o animal como um CHP em relação aos modelos 3D e cilindro horizontal completo (CHC). Quando se relacionou as informações das imagens bidimensionais (2D) com as informações dos modelos 3D, foi possível encontrar equações para estimar área, volume e massa dos animais. Outra abordagem que ficou constatada foi a utilização de modelos 3D para avaliação em melhoramento genético, como pelo método EPMURAS. O uso de modelos 3D para estimar a área de superfície e volume em animais foi validado. O método parece ser o melhor meio para estimar a área de superfície e volume, e certamente aparece como uma ferramenta para melhorar as pesquisas envolvendo transferência de calor e massa, bem como o melhoramento genético. / In order to use three-dimensional (3D) modeling to estimate area, volume and mass, as well as to evaluate the impact of the deviations involved in the empirical equations available in the literature, in livestock, six shorn Corriedale sheep were weighed with mass between 10 and 105 kg and five Nelore cattle with mass between 600 and 800 kg. The animals were trained using the principles of habituation and conditioning in order to reduce the reactivity of the animals. For the measurement of the surface area, three procedures were used: empirical equation (EE) for each species, manual measurement instrument (IMM) and 3D animal modeling by photogrammetry (AR) and kinect sensor (AS_KS) scanning. A covariance analysis was performed, with the mass being covariable and a non-linear regression analysis was adjusted as a power function (allometry). Analyzing the surface area averages for sheep, in relation to the procedures used there was no difference between IMM and 3D models, as well as between 3D models, while between IMM and EE there was a significant difference. For cattle, it was observed that the 3D procedures were, on average, higher than the surface area estimates by EE (a=5%). When data were plotted on the surface area of sheep and the methods tested for body mass, empirical approximations were obtained, different from those estimated by EE in more than 11% for animals over 100 kg, while for cattle the difference between 3D and EE models was over 18%. As the adjustments of the equations with IMM and 3D models had very close growth rates and deviations between them were lower than 2%, it was verified that the use of 3D models was ratified. When plotting the data of both species, we observed excellent adjustments (R² = 0.99) and estimates consistent with those obtained by the specific equations for each breed, with deviations lower than 3.2%. Other relationships, such as volume versus surface area, were established, resulting in consistent approximations when used to estimate the volume of the animal. When the animal was considered as a Standard Horizontal Cylinder (CHP), its surface area was estimated, on average, 36% lower than those observed for the 3D and IMM models. Simulation of the heat transfer by convection gave a difference of 56% in the convection rate when the animal was considered as a CHP in relation to the 3D models and the full horizontal cylinder (HCC). When the information of the two-dimensional images (2D) was related to the information of the 3D models, it was possible to find equations to estimate area, volume and mass of the animals. Another approach that was verified was the use of 3D models for evaluation in genetic improvement, as by the EPMURAS method. The use of 3D models to estimate surface area and volume in animals was validated. The method seems to be the best medium for estimating surface area and volume, and it certainly appears as a tool to improve research involving heat and mass transfer as well as genetic improvement. / CAPES 1367458 / CAPES 1443570 / CAPES 1707637 / CNPq 166449/2017-2

Diss. Uppsala : Uppsala universitet, 2009.

Interroger la figure du monstre, paradoxalement, permet de questionner notre condition humaine. Si le corps est parfois envisagé sous son aspect tératologique dans l'art contemporain, déformé, défiguré, altéré de multiples façons, manipulé, métamorphosé, décomposé, etc., c'est parce que le monstre a le don de provoquer le regard, en générant tout à la fois fascination et répulsion. Tantôt réminiscences d'êtres fabuleux de civilisations anciennes, tantôt mutants de l'ère industrielle et génétique, les hybrides créés dans l'art d'aujourd'hui sont le miroir des rêves et des cauchemars, des fantasmes et des peurs de notre société. Ainsi, monstres, hybrides, androïdes forment un inquiétant bestiaire qui témoigne des phobies et obsessions corporelles de notre époque, alors que le corps entre dans « l'ère de sa reproductibilité technique » à travers la chirurgie esthétique ou la biotechnologie, qu’il tend à devenir obsolète tant la communication tend à se faire virtuelle. Le monstre possède le pouvoir de se faire métaphore, de devenir un double qui nous montre, (qui nous monstre), l'envers de nous-même, tout en nous permettant d’appréhender l’autre. / Funnily enough, analysing the monster allows us to question the human condition. In contemporary art, the body is often considered as a technological entity, meaning deformed, disfigured, distorted in many ways, manipulated, metamorphosed, decomposed etc. Monsters force us to look at them with both fascination and repulsion. They can be either hybrids – echoes of those fabulous creatures belonging to ancient civilizations – or industrial and genetic mutants. As a matter of fact, monsters in contemporary art represent the dreams, nightmares, fantasies and fears of our modern society. Monsters, hybrids and androids compose an unsettling bestiary displaying the physical fears and obsessions of our present times where bodies can be technically reproduced (either by plastic surgery or biotechnology) or tend to vanish with communication becoming more and more virtual. They are metaphors, doubles revealing ourselves or the way we should approach others. Contemporary art transforms the human body into a phantasmagorical, marvellous, frightening or obscene memento mori that reminds us of our mortality.