The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

A simple method to assess freezing of gait in Parkinson's disease patients

Freezing of gait (FOG) can be assessed by clinical and instrumental methods. Clinical examination has the advantage of being available to most clinicians; however, it requires experience and may not reveal FOG even for cases confirmed by the medical history. Instrumental methods have an advantage in that they may be used for ambulatory monitoring. The aim of the present study was to describe and evaluate a new instrumental method based on a force sensitive resistor and Pearson's correlation coefficient (Pcc) for the assessment of FOG. Nine patients with Parkinson's disease in the "on" state walked through a corridor, passed through a doorway and made a U-turn. We analyzed 24 FOG episodes by computing the Pcc between one "regular/normal" step and the rest of the steps. The Pcc reached ±1 for "normal" locomotion, while correlation diminished due to the lack of periodicity during FOG episodes. Gait was assessed in parallel with video. FOG episodes determined from the video were all detected with the proposed method. The computed duration of the FOG episodes was compared with those estimated from the video. The method was sensitive to various types of freezing; although no differences due to different types of freezing were detected. The study showed that Pcc analysis permitted the computerized detection of FOG in a simple manner analogous to human visual judgment, and its automation may be useful in clinical practice to provide a record of the history of FOG.

Formal development of resilient distributed systems

Resilience is the property of a system to remain trustworthy despite changes. Changes of a different nature, whether due to failures of system components or varying operational conditions, significantly increase the complexity of system development. Therefore, advanced development technologies are required to build robust and flexible system architectures capable of adapting to such changes. Moreover, powerful quantitative techniques are needed to assess the impact of these changes on various system characteristics. Architectural flexibility is achieved by embedding into the system design the mechanisms for identifying changes and reacting on them. Hence a resilient system should have both advanced monitoring and error detection capabilities to recognise changes as well as sophisticated reconfiguration mechanisms to adapt to them. The aim of such reconfiguration is to ensure that the system stays operational, i.e., remains capable of achieving its goals. Design, verification and assessment of the system reconfiguration mechanisms is a challenging and error prone engineering task. In this thesis, we propose and validate a formal framework for development and assessment of resilient systems. Such a framework provides us with the means to specify and verify complex component interactions, model their cooperative behaviour in achieving system goals, and analyse the chosen reconfiguration strategies. Due to the variety of properties to be analysed, such a framework should have an integrated nature. To ensure the system functional correctness, it should rely on formal modelling and verification, while, to assess the impact of changes on such properties as performance and reliability, it should be combined with quantitative analysis. To ensure scalability of the proposed framework, we choose Event-B as the basis for reasoning about functional correctness. Event-B is a statebased formal approach that promotes the correct-by-construction development paradigm and formal verification by theorem proving. Event-B has a mature industrial-strength tool support { the Rodin platform. Proof-based verification as well as the reliance on abstraction and decomposition adopted in Event-B provides the designers with a powerful support for the development of complex systems. Moreover, the top-down system development by refinement allows the developers to explicitly express and verify critical system-level properties. Besides ensuring functional correctness, to achieve resilience we also need to analyse a number of non-functional characteristics, such as reliability and performance. Therefore, in this thesis we also demonstrate how formal development in Event-B can be combined with quantitative analysis. Namely, we experiment with integration of such techniques as probabilistic model checking in PRISM and discrete-event simulation in SimPy with formal development in Event-B. Such an integration allows us to assess how changes and di erent recon guration strategies a ect the overall system resilience. The approach proposed in this thesis is validated by a number of case studies from such areas as robotics, space, healthcare and cloud domain.

Detection of enterotoxins produced by B. cereus through PCR analysis of ground and roasted coffee samples in Rio de Janeiro, Brazil

Coffee is one of the most appreciated drinks in the world. Coffee ground is obtained from the fruit of a small plant that belongs to the genus Coffea. Coffea arabica and Coffea canephora robusta are the two most commercially important species. They are more commonly known as arabica and robusta, respectively. Two-thirds of Coffea arabica plants are grown in South and Central America, and Eastern Africa - the place of origin for this coffee species. Contamination by microorganisms has been a major matter affecting coffee quality in Brazil, mainly due to the harvesting method adopted. Brazilian harvests are based on fruits collected from the ground mixed with those that fall on collection cloths. As the Bacillus cereus bacterium frequently uses the soil as its environmental reservoir, it is easily capable of becoming a contaminant. This study aimed to evaluate the contamination and potential of B. cereus enterotoxin genes encoding the HBL and NHE complexes, which were observed in strains of ground and roasted coffee samples sold in Rio de Janeiro. The PCR (Polymerase Chain Reaction) results revealed high potential of enterotoxin production in the samples. The method described by Speck (1984) was used for the isolation of contaminants. The investigation of the potential production of enterotoxins through isolates of the microorganism was performed using the B. cereus enterotoxin Reverse Passive Latex Agglutination test-kit (BCET-RPLA, Oxoid), according to the manufacturer's instructions. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR) methods for hblA, hblD and hblC genes (encoding hemolysin HBL) and for nheA, nheB and nheC genes (encoding non-hemolytic enterotoxin - NHE). Of all the 17 strains, 100% were positive for at least 1 enterotoxin gene; 52.9% (9/17) were positive for the 3 genes encoding the HBL complex; 35.3% (6/17) were positive for the three NHE encoding genes; and 29.4% (5/17) were positive for all enterotoxic genes.

Formal modelling for digital media distribution

Human beings have always strived to preserve their memories and spread their ideas. In the beginning this was always done through human interpretations, such as telling stories and creating sculptures. Later, technological progress made it possible to create a recording of a phenomenon; first as an analogue recording onto a physical object, and later digitally, as a sequence of bits to be interpreted by a computer. By the end of the 20th century technological advances had made it feasible to distribute media content over a computer network instead of on physical objects, thus enabling the concept of digital media distribution. Many digital media distribution systems already exist, and their continued, and in many cases increasing, usage is an indicator for the high interest in their future enhancements and enriching. By looking at these digital media distribution systems, we have identified three main areas of possible improvement: network structure and coordination, transport of content over the network, and the encoding used for the content. In this thesis, our aim is to show that improvements in performance, efficiency and availability can be done in conjunction with improvements in software quality and reliability through the use of formal methods: mathematical approaches to reasoning about software so that we can prove its correctness, together with the desirable properties. We envision a complete media distribution system based on a distributed architecture, such as peer-to-peer networking, in which different parts of the system have been formally modelled and verified. Starting with the network itself, we show how it can be formally constructed and modularised in the Event-B formalism, such that we can separate the modelling of one node from the modelling of the network itself. We also show how the piece selection algorithm in the BitTorrent peer-to-peer transfer protocol can be adapted for on-demand media streaming, and how this can be modelled in Event-B. Furthermore, we show how modelling one peer in Event-B can give results similar to simulating an entire network of peers. Going further, we introduce a formal specification language for content transfer algorithms, and show that having such a language can make these algorithms easier to understand. We also show how generating Event-B code from this language can result in less complexity compared to creating the models from written specifications. We also consider the decoding part of a media distribution system by showing how video decoding can be done in parallel. This is based on formally defined dependencies between frames and blocks in a video sequence; we have shown that also this step can be performed in a way that is mathematically proven correct. Our modelling and proving in this thesis is, in its majority, tool-based. This provides a demonstration of the advance of formal methods as well as their increased reliability, and thus, advocates for their more wide-spread usage in the future.

Kant and zeno of elea: historical precedents of the "sceptical method"

For Kant's interpretation of Zeno in KrV A502-507/B530-535, scholars have usually referred to Plato's Phaedrus (261d); in reality the sources Kant uses are, on one hand, Brucker (who depends in turn on the pseudo-Aristotelian De Melisso, Xenophane, et Gorgia, 977 b 2-21), and, on the other, Plato's Parmenides (135e6-136b1) and Proclus' commentary on it, as quoted by Gassendi in a popular textbook he wrote on the history of logic.

The application of the structural correlation method to determine the reaction coordinate for a putative ring closure reaction

The cr ystal structure of the compound 2-benzoylethylidene-3-(2,4- dibromophenyl)-2,3-dihydro-5-phenyl-l,3,4-thiadiazole* C23H16Br2NZOS (BRMEO) has been determined by using three dimensiona l x-ray diffraction data. The crys tal form is monoclinic, space group P21/c, a = 17.492(4), o -.t' 0 R 0 b =: 16.979(1), c = 14.962(1) A, "X. =o= 90 ',= 106.46(1) , z = 8, graphite-monochromatized Mo~ rad iation, Jl= 0.710J3~, D = 1.62g/cc and o D = 1.65g/cc. The data were col lected on ~ Nonius CAD-4 c diffractometer. The following atoms were made anisotropic: Br, S, N, 0, C7, and C14-C16 for each i ndependent molecu le ; the rest were left isotropic. For 3112 independent refl ec tions with F > 6G\F), R == 0.057. The compound has two independent molecules within the asymmetric unit. Two different conformers were observed which pack well together. /l The S---O interaction distances of 2.493(6) and 2 . 478(7) A were observed for molecules A and B respectively. These values are consistent with earlier findings for 2-benzoylmethylene-3-(2,4-dibromophenyl)- ~~ 2,3-dihydro-5-phenyl-l,3,4-thiadiazole C22H14Br2N20S (BRPHO) and 2-benzoylpropylidene-3-(2,4-dibromophenyl)-2,3-dihydroiii ,'r 5-phenyl-l,3,4-thiadiazole C24H18Br2N20S (BRPETO ) where S---O distances are l ess than the van der Waals (3.251\) but greater than those expected for () a single bond (1.50A). From the results and the literature it appears obvious that the energy/reaction coordinate pathway has a minimum between the end structures (the mono- and bicyclic compounds). * See reference (21) for nomenclature.

ESL students' off-line and on-line texts : differences and similarities /

This study was undertaken to investigate any textual differences and similarities within essays written with a word processing program and an e-mail editor by non-native writers. It arose from many contradictions and a paucity of empirical research within the field of second language learning and electronic technology. To further explore these contradictory observations, 3 classes of intermediate level ESL (English as a Second Language) students v^ote 6 essays, alternating between a word processing program and an e-mail editor. Prior to the data collection, students read brief texts and responded to questions that focused upon three formal topics: immigration, economics, and multiculturalism. Data were examined for (a) the differences in the frequency counts of 12 cohesive devices, (b) sentence complexity, which focused upon the occurrences of simple and complex sentences, (c) the number of words within the writings, (d) the method of contextualization preferred by writers, and (e) any variations in the final grades of the students' texts that resulted from holistic rating. Results of analysis indicated that there were no statistically significant differences in the frequency counts of the linguistic features. Sentence complexity did not vary within the off-line and on-line essays. The average number of words found within the off-line essays was approximately 20% greater than within on-line essays. Contextualization methods were not different within word-processed or e-mailed essays. Finally, there was no difference in the quality of the texts when holistically rated.

(A) Optimization of solid body phase synthesis of RNA using the Cpep chemistry. (B) Synthesis of BODIPY labeled oligonucleotides

(A) Solid phase synthesis of oligonucleotides are well documented and are extensively studied as the demands continue to rise with the development of antisense, anti-gene, RNA interference, and aptamers. Although synthesis of RNA sequences faces many challenges, most notably the choice of the 2' -hydroxy protecting group, modified 2' -O-Cpep protected ribonucleotides were synthesized as alternitive building blocks. Altering phosphitylation procedures to incorporate 3' -N,N-diethyl phosphoramidites enhanced the overall reactivity, thus, increased the coupling efficiency without loss of integrety. Furthermore, technical optimizations of solid phase synthesis cycles were carried out to allow for successful synthesis of a homo UIO sequences with a stepwise coupling efficiency reaching 99% and a final yield of 91 %. (B) Over the past few decades, dipyrrometheneboron difluoride (BODIPY) has gained recognition as one of the most versatile fluorophores. Currently, BODIPY labeling of oligonucleotides are carried out post-synthetically and to date, there lacks a method that allows for direct incorporation of BODIPY into oligonucleotides during solid phase synthesis. Therefore, synthesis of BODIPY derived phosphoramidites will provide an alternative method in obtaining fluorescently labelled oligonucleotides. A method for the synthesis and incorporation of the BODIPY analogues into oligonucleotides by phosphoramidite chemistry-based solid phase DNA synthesis is reported here. Using this approach, BODIPY-labeled TlO homopolymer and ISIS 5132 were successfully synthesized.

Cholesterol-Independent Effects of Methyl-b- Cyclodextrin on Chemical Synapses

The cholesterol chelating agent, methyl-b-cyclodextrin (MbCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MbCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MbCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MbCD impaired impulse propagation and decreased EJP amplitude by 40% (P,0.05) in preparations from crayfish acclimatized to 14uC but not from those acclimatized to 21uC. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P,0.05). MbCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and coldacclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P,0.05; 50% reduction in warm, P,0.05). MbCD reduced cholesterol in isolated nerve and muscle from cold- and warmacclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P,0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MbCD on glutamatesensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MbCD can affect both presynaptic and postsynaptic properties, and that some effects of MbCD are unrelated to cholesterol chelation.

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Die Wahrnehmung des Fremden im Caoba-Zyklus von B. Traven

Les six romans du cycle de Caoba retracent sur un mode fictif le déroulement de la Révolution mexicaine et montrent les conséquences de la colonisation de même que les injustices sociales et économiques dont souffre tout particulièrement la population indigène. Dans ce contexte de conflit culturel entre colonisés et colonisateurs, le personnage de l’indigène est perçu de façon variée dans les différents romans. L’analyse de la représentation de la population indigène est donc le sujet principal de ce travail qui combine une étude narratologique et une approche postcoloniale. L’examen détaillé d’extraits de texte permettra de vérifier dans quelle mesure l’auteur germanophone B. Traven, dans sa représentation de l'étranger, se détache d’un discours colonial, et s’il peut être considéré comme un auteur postcolonial avant la lettre. Dans ces analyses, les questions suivantes serviront de fil conducteur : dans quelle mesure la représentation du personnage de l’indigène correspond-elle à celle du «bon sauvage» ? Comment les différents groupes sociaux du Mexique sont-ils représentés – par des individus, des descriptions stéréotypées, des allégories? Quels moyens l’auteur utilise-t-il pour familiariser son lectorat européen/occidental avec cette culture étrangère? Ainsi, le travail se penche sur les procédés narratifs employés par l’auteur pour dépeindre la société à partir de perspectives diverses. Dans le but de dénoncer des conditions d’oppression et d’exploitation, Traven écrit à partir du regard du colonisé. Mais lorsqu'il cherche à comprendre le système dictatorial, il écrit dans la perspective du colonisateur. Cette méthode correspond à celle des regards croisés que le théoricien Edward E. Said décrit dans son ouvrage Orientalisme. L’emploi de cette méthode contrapunctique - permet-il d’exercer une critique (post)coloniale? Dans quelle mesure ce texte révèle-t-il l’importance de l’hybridité de la culture telle qu'elle a été théorisée par K. Bhabha? Dans quelle mesure l'accent est-il mis sur les rapports transculturels, sur la façon dont les cultures s'influencent les unes les autres?

Analyse diagrammatique des désintégrations de type B vers PPP sans quarks charmés

Ce mémoire présente l’application de la méthode de décomposition en termes de diagrammes aux désintégrations de mésons B vers trois mésons de type pseudos- calaire ne comportant pas de quarks charmés. La décomposition diagrammatique des désintégrations de types B → Kππ, B → KKK ̄, B → KK ̄π et B → πππ est effectuée de façon systématique. Il est démontré que lorsque l’on néglige les dia- grammes d’échanges et d’annihilations, dont les contributions sont estimées être petites, de nouvelles relations apparaissent entre les amplitudes. Ces relations sont de nouveaux tests du modèle standard qui ne peuvent être obtenus que par la méthode diagrammatique. Lorsque les données nécessaires sont disponibles, nous vérifions ces relations et obtenons un bon accord avec les données expérimentales. Nous démontrons également qu’il est possible d’utiliser le secteur B → Kππ pour mesurer la phase faible γ avec une incertitude théorique que nous estimons être de l’ordre de 5%. Les autres secteurs de désintégrations ne permettent d’extraire des phases faibles que si l’on invoque des approximations de précisions inconnues.

La importancia del género gramatical en la enseñanza de español como lengua extranjera

Le présent travail est encadré dans le domaine de la linguistique appliquée de l'espagnol comme langue étrangère (ELE), et plus spécifiquement dans l'enseignement du genre grammatical en ELE. Notre intérêt en tant que enseignants c’est de pouvoir établir une méthode fiable selon les critères du Plan curricular de l'Institut Cervantès et la technique du consciousness-raising, ainsi que élaborer des activités destinées à l'enseignement du genre grammatical dans la classe d’ELE. L'enseignement d'ELE au Québec suit les mêmes méthodes qu'en Europe. En ce qui concerne l'enseignement du genre grammatical, les études consultées ratifient le manque d'instruction pertinente à propos du genre grammatical, ainsi que la difficulté dans la concordance même dans des niveaux avancés. Cependant, l'analyse de manuels d'ELE utilisés dans les diverses institutions de Montréal permet de conclure que ceux-ci ne suivent pas les règles établies par le Plan curricular en ce qui concerne l'enseignement du genre. Pour vérifier ces faits un travail de champ a été mis en place avec 84 étudiants et étudiantes de six institutions de Montréal pendant deux mois et deux semaines. Les résultats de la recherche et l'analyse d'erreurs nous montrent qu’il y a des problèmes avec le genre grammatical chez les étudiants de niveau intermédiaire et que les erreurs ne disparaissent pas avec les activités de renfort créées. Il est donc nécessaire d’adopter une méthode plus appropriée à l'apprentissage du genre grammatical dans une classe d’ELE et la présence du professeur pour la présenter. En effet, l'exécution d'activités créées n'est pas suffisant, car bien que les résultats montrent un léger progrès dans le cas du groupe B, ou d’expérience, en comparaison au groupe A, ou de control, on a constaté qu’une instruction formelle aurait entrainé un meilleur et plus complet apprentissage du genre grammatical dans le cas de nos étudiants; de là la nécessité d'établir une méthode fiable pour son enseignement.