Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated Mycobacterium bovis

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research.

Sparse hierarchical Bayesian models for detecting relevant antigenic sites in virus evolution

Understanding how virus strains offer protection against closely related emerging strains is vital for creating effective vaccines. For many viruses, including Foot-and-Mouth Disease Virus (FMDV) and the Influenza virus where multiple serotypes often co-circulate, in vitro testing of large numbers of vaccines can be infeasible. Therefore the development of an in silico predictor of cross-protection between strains is important to help optimise vaccine choice. Vaccines will offer cross-protection against closely related strains, but not against those that are antigenically distinct. To be able to predict cross-protection we must understand the antigenic variability within a virus serotype, distinct lineages of a virus, and identify the antigenic residues and evolutionary changes that cause the variability. In this thesis we present a family of sparse hierarchical Bayesian models for detecting relevant antigenic sites in virus evolution (SABRE), as well as an extended version of the method, the extended SABRE (eSABRE) method, which better takes into account the data collection process. The SABRE methods are a family of sparse Bayesian hierarchical models that use spike and slab priors to identify sites in the viral protein which are important for the neutralisation of the virus. In this thesis we demonstrate how the SABRE methods can be used to identify antigenic residues within different serotypes and show how the SABRE method outperforms established methods, mixed-effects models based on forward variable selection or l1 regularisation, on both synthetic and viral datasets. In addition we also test a number of different versions of the SABRE method, compare conjugate and semi-conjugate prior specifications and an alternative to the spike and slab prior; the binary mask model. We also propose novel proposal mechanisms for the Markov chain Monte Carlo (MCMC) simulations, which improve mixing and convergence over that of the established component-wise Gibbs sampler. The SABRE method is then applied to datasets from FMDV and the Influenza virus in order to identify a number of known antigenic residue and to provide hypotheses of other potentially antigenic residues. We also demonstrate how the SABRE methods can be used to create accurate predictions of the important evolutionary changes of the FMDV serotypes. In this thesis we provide an extended version of the SABRE method, the eSABRE method, based on a latent variable model. The eSABRE method takes further into account the structure of the datasets for FMDV and the Influenza virus through the latent variable model and gives an improvement in the modelling of the error. We show how the eSABRE method outperforms the SABRE methods in simulation studies and propose a new information criterion for selecting the random effects factors that should be included in the eSABRE method; block integrated Widely Applicable Information Criterion (biWAIC). We demonstrate how biWAIC performs equally to two other methods for selecting the random effects factors and combine it with the eSABRE method to apply it to two large Influenza datasets. Inference in these large datasets is computationally infeasible with the SABRE methods, but as a result of the improved structure of the likelihood, we are able to show how the eSABRE method offers a computational improvement, leading it to be used on these datasets. The results of the eSABRE method show that we can use the method in a fully automatic manner to identify a large number of antigenic residues on a variety of the antigenic sites of two Influenza serotypes, as well as making predictions of a number of nearby sites that may also be antigenic and are worthy of further experiment investigation.

Towards in silico IBV vaccine design: defining the role of polymorphism in viral attenuation

The gammacoronavirus, Infectious Bronchitis Virus (IBV), is a respiratory pathogen of chickens. IBV is a constant threat to poultry production as established vaccines are often ineffective against emerging strains. This requires constant and rapid vaccine production by a process of viral attenuation by egg passage, but the essential forces leading to attenuation in the virus have not yet been characterised. Knowledge of these factors will lead to the development of more effective, rationally attenuated, live vaccines and reduction of the mortality and morbidity caused by this pathogen. M41 CK strain was egg passaged four times many years ago at Houghton Poultry Research Station and stored as M41-CK EP4 (stock virus at The Pirbright Institute since 1992). It was the first egg passage to have its genome pyrosequenced and was therefore used as the baseline reference. The overall aim of this project was to analyse deep sequence data obtained from four IBV isolates (called A, A1, C and D) each originating from the common M41-CK EP4 (ep4) and independently passaged multiple times in embryonated chicken eggs (figure 1.1). Highly polymorphic encoding regions of the IBV genome were then identified which are likely involved in the attenuation process through the formation of independent SNPs and/or SNP clusters. This was then used to direct targeted investigation of SNPs during the attenuation process of the four IBV passages. A previously generated deep sequence dataset was used as a preliminary map of attenuation for one virulent strain of IBV. This investigation showed the nucleocapsid and spike as two highly polymorphic encoding regions within the IBV genome with the highest proportion of SNPs compared to encoding region size. This analysis then led to more focussed studies of the nucleocapsid and spike encoding region with the ultimate aim of mapping key attenuating regions and nucleotide positions. The 454 pyrosequencing data and further investigation of nucleocapsid and spike encoding regions have identified the SNPs present at the same nucleotide positions within analysed A, A1, C and D isolates. These SNPs probably play a crucial role in viral attenuation and universal vaccine production but it is not clear if independent SNPs are also involved in loss of virulence. The majority of SNPs accumulated at different nucleotide positions without further continuation in Sanger sequenced egg passages presenting S2 subunit (spike) and nucleocapsid as polymorphic encoding regions which in nature remain highly conserved.

Los mecanismos de influencia en el liderazgo: dos estudios de caso

Este trabajo investigativo busca aportar a la literatura sobre las tácticas de influencia en el liderazgo. Surge como una aplicación, a dos casos específicos, del proyecto de investigación “Los mecanismos de influencia en la relación de liderazgo”, desarrollado por el profesor Juan Javier Saavedra Mayorga e inscrito en la línea de investigación en Estudios Organizacionales del Grupo de Investigación en Dirección y Gerencia. La investigación tiene como objetivo fundamental identificar las tácticas de influencia que utilizan dos líderes organizacionales en su trato cotidiano con sus colaboradores, así como la reacción de estos últimos ante dichas tácticas. El proyecto parte de una revisión teórica sobre tres elementos: el liderazgo, la influencia y el poder, y las reacciones de los colaboradores frente a las tácticas de influencia utilizadas por el líder. La estrategia metodológica empleada es el estudio de caso. El trabajo de campo se desarrolló en dos organizaciones: Microscopios y Equipos Especiales S.A.S. y Tecniespectro S.A.S. La técnica de recolección de información es la entrevista semi estructurada, y el método de análisis de información es el análisis de contenido temático.

Los mecanismos de influencia en el liderazgo

Este trabajo tiene como objetivo fundamental identificar las tácticas de influencia que utiliza el señor Carlos Pérez, gerente y socio principal de G. & M., en su trato cotidiano con sus colaboradores, así como la reacción de estos últimos ante dichas tácticas.

Rapid Control Prototyping of an L2+ Highway Assist System

Advanced Driver Assistance Systems (ADAS) are proving to have huge potential in road safety, comfort, and efficiency. In recent years, car manufacturers have equipped their high-end vehicles with Level 2 ADAS, which are, according to SAE International, systems that combine both longitudinal and lateral active motion control. These automated driving features, while only available in highway scenarios, appear to be very promising towards the introduction of hands-free driving. However, as they rely only on an on-board sensor suite, their continuative operation may be affected by the current environmental conditions: this prevents certain functionalities such as the automated lane change, other than requiring the driver to keep constantly the hands on the steering wheel. The enabling factor for hands-free highway driving proposed by Mobileye is the integration of high-definition maps, thus leading to the so-called Level 2+. This thesis was carried out during an internship in Maserati's Virtual Engineering team. The activity consisted of the design of an L2+ Highway Assist System following the Rapid Control Prototyping approach, starting from the definition of the requirements up to the real-time implementation and testing on a simulator of the brand new compact SUV Maserati Grecale. The objective was to enhance the current Level 2 highway driving assistance system with hands-free driving capability; for this purpose an Autonomous Lane Change functionality has been designed, proposing a Model Predictive Control-based decision-maker, in charge of assessing both the feasibility and convenience of performing a lane-change maneuver. The result is a Highway Assist System capable of driving the vehicle in a traffic scenario safely and efficiently, never requiring driver intervention.

Stability issues in racing motorcycles: an in-depth analysis of the chatter vibration

Racing motorcycles are prone to an unstable oscillatory motion of the swingarm and rear wheel, commonly known as ‘chatter’. This vibration mode typically has a frequency of 17 Hz to 22 Hz and typically occurs during heavy braking manoeuvres. The appearance of chatter can cause reduced rider confidence, and therefore lead to longer lap times during races and the increased risk of crashing. This thesis looks to further the understanding of this mode. It includes the development of a simplified model to explore the effects roll angle and lateral dynamics have on the chatter mode using linear analysis. The mechanisms of instability and parameter sensitivities are also examined. The effects of the nonlinearities present in the minimal model equations of motion are examined, including the identification of limit cycles and their stability, inspecting individual nonlinear terms and their effects, and introducing tyre relaxation and determining the effect it has on the dynamics. Finally, an exploratory study of the mid-corner region of a typical racing manoeuvre is performed in hopes to better understand if any high frequency tyre induced instabilities like chatter can occur.

Search for same-sign top quark pairs with the ATLAS detector at the LHC

In this thesis, a search for same-sign top quark pairs produced according to the Standard Model Effective Field Theory (SMEFT) is presented. The analysis is carried out within the ATLAS Collaboration using collision data at a center-of-mass energy of $\sqrt{s} = 13$ TeV, collected by the ATLAS detector during the Run 2 of the Large Hadron Collider, corresponding to an integrated luminosity of $140$ fb$^{-1}$. Three SMEFT operators are considered in the analysis, namely $\mathcal{O}_{RR}$, $\mathcal{O}_{LR}^{(1)}$, and $\mathcal{O}_{LR}^{(8)}$. The signal associated to same-sign top pairs is searched in the dilepton channel, with the top quarks decaying via $t \longrightarrow W^+ b \longrightarrow \ell^+ \nu b$, leading to a final state signature composed of a pair of high-transverse momentum same-sign leptons and $b$-jets. Deep Neural Networks are employed in the analysis to enhance sensitivity to the different SMEFT operators and to perform signal-background discrimination. This is the first result of the ATLAS Collaboration concerning the search for same-sign top quark pairs production in proton-proton collision data at $\sqrt{s} = 13$ TeV, in the framework of the SMEFT.

Wind analysis of the Messina Strait Bridge design project proposal with stabilizing cables

A new study on suspension bridges has been prompted by the big disaster of the Tacoma Narrow Bridge at half its design speed. The aerodynamic instability of long-span bridges has been studied using wind tunnel tests. As a result of improved aerodynamic performance from the geometrical configuration of the bridge deck, the aerodynamic criteria for suspension and cable-stayed bridges have become well established in recent years, thereby allowing longer bridge spans to be developed. Although the Messina Strait Bridge has yet to be constructed, we are looking forward to evaluating the impact of different deck cross-sections on both aerodynamic stability and cost reduction. To further improve the aerodynamic characteristics of long-span suspension bridges, an optimized multi-box bridge deck model with two side decks for traffic lanes, two middle railway decks, and three gaps separating them has been proposed aerodynamic performance has been experimentally verified. 1:80 scale wind tunnel tests have been conducted. According to the current MIDAS Model, the first torsional and the first vertical frequency ratios are 1.27787 and 1.36[1] respectively. It is the torsional/vertical frequency ratio, combined with the deck aerodynamic properties, that determines the wind response properties of the bridge for the most dangerous possible form of aeroelastic instability. The classic flutter is caused by the coupling of torsional and vertical modes. Stabilizing cables to the deck could be a solution to this classic flutter by reducing lateral displacement of the deck and increasing frequency ratios. Stabilizing cables will be installed on the deck in three different orientations: vertical, inclined, and horizontal, with diameters of 80 cm, 60 cm, and 40 cm in each orientation respectively. An overview of the research undertaken on this topic will be presented, as well as the most important findings.

Independent roles of calcium and voltage-dependent potassium currents in controlling spike frequency adaptation in lateral amygdala pyramidal neurons

The calcium-dependent afterhyperpolarization (AHP) that follows trains of action potentials is responsible for controlling action potential firing patterns in many neuronal cell types. We have previously shown that the slow AHP contributes to spike frequency adaptation in pyramidal neurons in the rat lateral amygdala. In addition, a dendritic voltage-gated potassium current mediated by Kv1.2-containing channels also suppresses action potential firing in these neurons. In this paper we show that this voltage-gated potassium current and the slow AHP act together to control spike frequency adaptation in lateral amygdala pyramidal neurons. The two currents have similar effects on action potential number when firing is evoked either by depolarizing current injections or by synaptic stimulation. However, they differ in their control of firing frequency, with the voltage-gated potassium current but not the slow AHP determining the initial frequency of action potential firing. This dual mechanism of controlling firing patterns is unique to lateral amygdala neurons and is likely to contribute to the very low levels of firing seen in lateral amygdala neurons in vivo.

Morphological and electrophysiological properties of principal neurons in the rat lateral amygdala in vitro

In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic conductances rather than through mechanisms that rely on cell morphology.

Firing properties and connectivity of neurons in the rat lateral central nucleus of the amygdala

Using whole cell recordings from acute slices of the rat amygdala, we have examined the physiological properties of and synaptic connectivity to neurons in the lateral sector of the central amygdala (CeA). Based on their response to depolarizing current injections, CeA neurons could be divided into three types. Adapting neurons fired action potentials at the start of the current injections at high frequency and then showed complete spike-frequency adaptation with only six to seven action potentials evoked with suprathreshold current injections. Late-firing neurons fired action potentials with a prolonged delay at threshold but then discharged continuously with larger current injections. Repetitive firers discharged at the start of the current injection at threshold and then discharged continuously with larger current injections. All three cells showed prolonged afterhyperpolarizations (AHPs) that followed trains of action potentials. The AHP was longer lasting with a larger slow component in adapting neurons. The AHP in all cell types contained a fast component that was inhibited by the SK channel blocker UCL1848. The slow component, not blocked by UCL1848, was blocked by isoprenaline and was significantly larger in adapting neurons. Blockade of SK channels increased the discharge frequency in late firers and regular-spiking neurons but had no effect on adapting neurons. Blockade of the slow AHP with isoprenaline had no effect on any cell type. All cells received a mixed glutamatergic and GABAergic input from a medial pathway. Electrical stimulation of the lateral (LA) and basolateral (BLA)nuclei evoked a large monosynaptic glutamatergic response followed by a disynaptic inhibitory postsynaptic potential. Activation of neurons in the LA and BLA by puffer application of glutamate evoked a small monosynaptic response in 13 of 55 CeA neurons. Local application of glutamate to the CeL evoked a GABAergic response in all cells. These results show that at least three types of neurons are present in the CeA that can be distinguished on their firing properties. The firing frequency of two of these cell types is determined by activation of SK channels. Cells receive a small input from the LA and BLA but may receive inputs that course through these nuclei en route to the CeA.

Childhood epilepsy with neuropsychological regression and continuous spike waves during sleep: epilepsy surgery in a young adult.

We describe the case of a man with a history of complex partial seizures and severe language, cognitive and behavioural regression during early childhood (3.5 years), who underwent epilepsy surgery at the age of 25 years. His early epilepsy had clinical and electroencephalogram features of the syndromes of epilepsy with continuous spike waves during sleep and acquired epileptic aphasia (Landau-Kleffner syndrome), which we considered initially to be of idiopathic origin. Seizures recurred at 19 years and presurgical investigations at 25 years showed a lateral frontal epileptic focus with spread to Broca's area and the frontal orbital regions. Histopathology revealed a focal cortical dysplasia, not visible on magnetic resonance imaging. The prolonged but reversible early regression and the residual neuropsychological disorders during adulthood were probably the result of an active left frontal epilepsy, which interfered with language and behaviour during development. Our findings raise the question of the role of focal cortical dysplasia as an aetiology in the syndromes of epilepsy with continuous spike waves during sleep and acquired epileptic aphasia.

Ca2+-activated K+(BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala

In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K+ currents that contribute to action potential repolarisation. In different neuronal cell types these K+ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K+ currents. Recent findings show that inactivation of a Ca2+-dependent K+ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca2+ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or alpha-dendrotoxin. We conclude that while inactivation of BK-type Ca2+-activated K+ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.