Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. It becomes widespread among adolescent and adults as well as infants. Although availability of effective pertussis vaccines seems to decrease the incidence of the disease, B. pertussis circulation in population has not been eliminated. It is thought that the antigenic drifts in major protective antigens and continued circulation of B. pertussis strains will result in gradual loss of the efficacy of the current pertussis vaccines. Therefore, development of more effective acellular pertussis vaccines with conserved protective proteins is a convenient strategy to provide a better protection against whooping cough. In this study, immune responses against putative peptidyl-prolyl cis-trans isomerase (PPIase) which was shown to be immunogenic in B. pertussis for the first time by our immunoproteome group and FimX whose expression was found higher in our local Saadet strain were determined in mice. The genes encoding FimX and putative PPIase were amplified by PCR, cloned into pGEM&reg / -T Easy vector and sequenced. The genes were then introduced into pET-28a (+) vector and they were expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins were purified by His-tag affinity chromatography and dialyzed. After Western blot analyses, 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase were used to immunize BALB/c mice (16-18 g) at day 0 and 21. The mice were challenged intranasally with 2.5 x 109 live B. pertussis Saadet cells. Before second immunization and challenge, the sera were collected to carry out ELISA for measurement of serum-specific IgG levels. According to ELISA results, IgG levels in the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX were found significantly higher than in control groups at both first and second vaccinations (p&lt / 0.01). On the other hand, immunization with 160 &micro / g recombinant putative PPIase provided a significant increase in IgG level (p&lt / 0.05) only at second vaccination. The lungs of the mice were removed at day 2, 5, 8 after challenge and bacterial colonization was determined. No significant decrease in bacterial colonization was observed in the lungs of the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase with respect to control groups. After respiratory challenge and second immunization (at day 30) with 20 &micro / g and 80 &micro / g recombinant FimX, the spleens of the mice were removed and a spleen cell culture was obtained. Supernatants were collected after induction of the cells with the recombinant protein and cytokine ELISA was carried out to measure IFN-&gamma / level. No significant difference was observed between control and vaccinated mice in terms of IFN-&gamma / production.

In radiation therapy, a treatment plan is designed to make the delivery of radiation to a target more accurate, effective, and less damaging to surrounding healthy tissues. In lung sites, the tumor is affected by the patient’s respiratory motion. Despite tumor motion, current practice still uses a static delivery plan. Unexpected changes due to coughs and sneezes are not taken into account and as a result, the tumor is not treated accurately and healthy tissues are damaged. In this thesis we detail a framework of using an accelerometer device to detect and forecast coughs. The accelerometer measurements are modeled as a ARMA process to make forecasts. We draw from studies in cough physiology and use amplitudes and durations of the forecasted breathing cycles as features to estimate parameters of Gaussian Mixture Models for cough and normal breathing classes. The system was tested on 10 volunteers, where each data set consisted of one 3-5 minute accelerometer measurements to train the system, and two 1-3 minute accelerometer measurements for testing.

In radiation therapy, a treatment plan is designed to make the delivery of radiation to a target more accurate, effective, and less damaging to surrounding healthy tissues. In lung sites, the tumor is affected by the patient’s respiratory motion. Despite tumor motion, current practice still uses a static delivery plan. Unexpected changes due to coughs and sneezes are not taken into account and as a result, the tumor is not treated accurately and healthy tissues are damaged. In this thesis we detail a framework of using an accelerometer device to detect and forecast coughs. The accelerometer measurements are modeled as a ARMA process to make forecasts. We draw from studies in cough physiology and use amplitudes and durations of the forecasted breathing cycles as features to estimate parameters of Gaussian Mixture Models for cough and normal breathing classes. The system was tested on 10 volunteers, where each data set consisted of one 3-5 minute accelerometer measurements to train the system, and two 1-3 minute accelerometer measurements for testing.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Introdução: As complicações respiratórias, somadas a baixos volumes pulmonares e tosse ineficiente, decorrentes da fraqueza da musculatura respiratória nas doenças neuromusculares (DNM), são as principais causas de morbidade e mortalidade. Objetivo: Verificar os efeitos do treinamento de empilhamento de ar na função respiratória de pacientes com DNM. Métodos: Estudo prospectivo em 21 pacientes com DNM, idade entre 7 e 23 anos. Todos foram submetidos a avaliações respiratórias a cada 4 e 6 meses. Realizou-se espirometria e medida do pico de fluxo de tosse não assistido e assistido (PFTNA e PFTASS) com insuflações e empilhamento de ar com ressuscitador manual. Os pacientes e cuidadores foram treinados e orientados a realizar o treinamento das manobras de empilhamento de ar diariamente no domicílio. A análise estatística utilizou o pacote estatístico como médias ± desvios-padrão, foram submetidas ao teste de normalidade de D\'Agostino-Pearson. Utilizou-se ANOVA para medidas repetidas, seguidas do teste Post Hoc de Tukey. O pico de fluxo expiratório (PFE) não exibiu distribuição normal e, por isso, foi submetido ao teste de Friedman seguido do teste Post Hoc de Dunn. Os coeficientes de correlação de Pearson foram calculados e nível de significância estabelecido foi p < 0,05. Resultados: Houve aumento na estatura média dos pacientes de 2,5 cm (p < 0,0001). A média da capacidade de insuflação máxima (CIM) foi maior que a capacidade vital forçada (CVF) basal em todas as avaliações (p < 0,0001). Houve aumento na média da CVF e CIM (p < 0,01), PFTNA (p < 0,05) e no PFTASS após período de treinamento nos pacientes com escoliose não estruturada ou ausente. Conclusão: O treinamento domiciliar com insuflações e empilhamento de ar deve ser enfatizado nas DNM, pois aumenta o PFT. Tal treinamento aumenta a CVF basal e o PFTNA nos pacientes sem deformidades torácicas. / Introduction: Respiratory complications, low lung volumes and inefficient cough, resulting from weakness of respiratory muscles are the major causes of morbidity and mortality in neuromuscular patients (NMD). Objective: To assess the effects of air stacking training on lung function in patients with NMD. Methods: Prospective study in 21 patients with NMD aged 7 to 23 years. Al patients underwent respiratory evaluations every 4 to 6 months. Was performed spirometry and measurement of unassisted peak cough flow (UPCF) and assisted peak cough flow (APCF) with insufflations and air stacking with manual resuscitator. The patients and caregivers were trained and were prescribed lung insufflations by air stacking three times each day at home. The statistical analysis used the statistical package GraphPad Prism 5.0 for Windows. Spirometric variables were expressed as means ± standard deviations, were subject to normality test D\'Agostino-Pearson. We used ANOVA for repeated measures followed by post hoc Tukey test. The peak expiratory flow (PEF) did not exhibit normal distribution and therefore was subjected to the Friedman test followed by Dunn´s post hoc test. The Pearson correlation coefficients were calculated and significance level was set at p < 0.05. Results: There was in increase in the average height of 2.5 cm, of the patients (p < 0.0001). The mean maximum insufflation capacity (MIC) was greater than forced vital capacity (FVC) baseline for all evaluations (p < 0.0001). There was increase in mean FVC and MIC (p < 0.001), UPCF (p < 0.05) and APCF (p < 0.01) after air stacking training period in patients without scoliosis or unstructured. Conclusion: The air stacking training home should be emphasized in NMD. This training increases the FVC and UPCF in patients without scoliosis or unstructured.

This thesis presents novel techniques for the reduction of audio recordings and signal processing techniques as part of cough recognition. Evidence collected shows the reduction technique to be effective and the recognition techniques to give consistent performance across different patients. Cough is one of the commonest symptoms reported by patients to GPs. Despite this, it remains a significantly unmet medical need. At present, there exists no practical and validated technique for assessing the efficacy of therapies to treat cough on a large enough scale. Research that is presently undertaken requires fitting a patient with a recording system which will record their coughing and all other sound for a predefined period, usually 24 hours or less. This audio is then counted manually by trained cough counters to produce counts for each record which can be used as data for cough studies. Research in this field is relatively new, but a number of attempts have been made to automate this process. None so far have shown sufficient reliability or precision to be of sufficient use. The aim of this research is to analyse from the ground up signal processing techniques which can aid cough research. Specifically, the research will look into data minimisation techniques to improve the efficiency of manual counting techniques and recognition algorithmsThe research has produced a published record reduction system which can reduce 24 hour cough records down to around 10% of their original size without compromising the statistics of subsequent manual counts. Additionally, a review of signal processing techniques for cough recognition has produced a robust event detection technique and measurement techniques which have shown remarkable consistency between patients and conditions. Throughout the research a clear understanding of the limitations and possible solutions are pursued and reported on to aid further progress on what is a young and developing research field.

Bordetella pertussis e B. parapertussis são agentes etiológicos da coqueluche. A IL-12 liga a imunidade inata e adaptativa. Investigamos alguns mecanismos que controlam a síntese de IL-12 em macrófagos medulares murinos (MfDM) ativados in vitro com estas duas espécies de bactérias. Demonstramos que IL-12p40 e TNF-a foram produzidos pelos MfDM ativados com qualquer uma das bactérias. A síntese de IL-12p40 foi dependente de TNF-a, MyD88 e NFkB e independente de MAPK p38 e ERK 1/2. Durante a estimulação com B. pertussis a produção de IL-12p40 foi dependente de TLR-4, mas com B. parapertussis envolveu outras vias independentes de MyD88 e TLR-4. Estas bactérias não induziram a síntese de IL-12p70, necessitando de sinais moleculares adicionais de IFN-g, que aumentou a síntese desta citocina. A produção de IL-12 p70 aumentou após o bloqueio das vias PI3K, MAPK p38 e ERK1/2 assim como após a adição exógena de PT sobre MfDM ativados com B. parapertussis. Portanto, diversas vias de sinais dependentes e independentes de TLR-4 controlam a produção de IL-12 neste modelo. / Bordetella pertussis and B. parapertussis are etiological agents of whooping cough. IL-12 links the innate and adaptive immunity. We investigated the ability of both bacteria to modulate IL-12 by in vitro activation of bone marrow derived macrophages (MfDM). We demonstrated that IL-12p40 and TNF-a were produced after stimulation of cells with either bacterium. IL-12p40 production was dependent on TNF-a, MyD88 and NFkB but independent of MAPK p38 and ERK 1/2. During B. pertussis activation the production of IL-12p40 was dependent on TLR-4, while B. parapertussis activation was MyD88 and TLR-4 independent. However, the bacteria alone did not induce IL-12p70 synthesis, requiring IFN-g as an additional signal. Evidences indicated MAPK p38, ERK1/2 and PI3K during B. pertussis and B. parapertussis activation, as well as the exogenous addition of PT to B. parapertussis activated MfDM, was critical for the up regulation of IL-12p70. This finding indicates that different TLR-4 dependent and independent signaling pathways may control the production of IL-12 in this model.

As vacinas pertussis celulares apresentam certa reatogenicidade e as acelulares, menos reatogênicas, têm seu uso restrito, devido a seu alto custo. O Instituto Butantan desenvolveu uma vacina pertussis celular (Plow), com baixo teor de lipopolissacarídeo e outra acelular (Pa), por metodologia simples e econômica. Essas preparações, combinadas aos toxóides diftérico e tetânico (DTPlow e DTPa, respectivamente), foram comparadas à DTP tradicional, com diferentes adjuvantes: vitamina A, surfactante pulmonar, BCG, monofosforil lipídeo A (MPL) e Al(OH)3. A resposta humoral em camundongos foi semelhante para as diferentes formulações e independente do adjuvante utilizado. As vacinas induziram níveis equilibrados de IgG1/IgG2a anti-pertussis e a DTPlow mostrou-se menos reatogênica, induzindo níveis significativamente menores de IL-6 sérica. A adição de MPL sugeriu tendência de proteção contra a colonização nasotraqueal no grupo imunizado com DTPa e levou à indução de IFN-g nos grupos imunizados com DTP e DTPa, sugerindo possível atividade imunomodulatória para Th1. / The whole cell pertussis vaccines present some reactogenicity and the acellular, less reactogenic, have prohibitive use due to its high cost. Instituto Butantan developed a less reactogenic whole cell pertussis vaccine (Plow), with low lipopolysaccharide content and an acellular vaccine (Pa), by simple and economic methodology. These preparations, combined to diphtheria and tetanus toxoids (DTPlow and DTPa, respectively), were compared with the traditional DTP, with different adjuvants: vitamin A, pulmonary surfactant, BCG, monophosphoryl lipid A (MPL) and Al(OH)3. The humoral immune response induced in mice by the different vaccine formulations, was similar and independent of the adjuvant used. The vaccines induced balanced levels of IgG1/IgG2a anti-pertussis and DTPlow showed to be less rectogenic, inducing lower levels of serum IL-6. The use of MPL suggested to induce higher protection against nasotracheal colonization in DTPa group and induced IFN-g in the DTP and DTPa groups, suggesting a possible immunemodulatory activity for Th1.

Objetivos: Determinar si existe asociación entre la suplementación con vitamina A y la presencia de síntomas de infecciones respiratorias agudas, en niños de 6 a 59 meses. Métodos: Análisis de datos secundarios de la Encuesta Demográfica y de Salud Familiar (ENDES), realizada en los años 2016 y 2017, tipo de estudio transversal. La variable de resultado fue la presencia de infecciones respiratorias en los últimos 14 días, reportado por la madre, y la variable de exposición fue la suplementación de al menos una dosis de Vitamina A, de acuerdo a la información del carnet de vacunación, o reporte de la madre. Resultados: Un total de 17668 registros fueron analizados, constituida por mujeres con una edad promedio de 31,6 (DE 10,4) y sus hijos menores con una edad promedio de 30,1 (DE 16,9). La proporción de niños que recibieron suplementación con Vitamina A fue de 40,7% (IC 95%: 39,9%- 41,4%), mientras que, la proporción de niños que reportaron síntomas compatibles con infecciones respiratorias agudas, fue de 33,8% (IC 95%: 33,1%- 34,5%). Ajustado por potenciales confusoras, no se encontró asociación significativa entre suplementación con Vitamina A y síntomas de Infecciones respiratorias (RP = 0,99, IC 95% 0,95- 1,04). Conclusión: Recibir la suplementación de vitamina A no estuvo asociado con la presencia de infecciones respiratorias agudas. / Objectives: To determine if there is an association between vitamin A supplementation and the presence of symptoms of acute respiratory infections among children aged 6 to 59 months. Methods: Secondary data analysis from the Demographic and Health Survey (DHS), from 2016 and 2017; cross-sectional study. The outcome of interest was the presence of acute respiratory infections in the last 14 days, reported by the mother, and the exposure variable was the supplementation of at least one dose of vitamin A, according the vaccination card´s information or mother´s report. Results: Data of 17668 records was analyzed; information include women with a mean age of 31.6 (SD: 10.4) and their children had a mean of 30.1 (SD: 16.9) months. The proportion of children who received Vitamin A supplementation was 40.7% (95% CI: 39.9% - 41.4%), whereas the proportion of children who reported symptoms compatible with acute respiratory infections was 33.8% (95% CI: 33.1% - 34.5%). Adjusted for potential confounders, no significant association was found between Vitamin A supplementation and symptoms of acute respiratory infections (PR = 0.99; 95% CI 0.95 – 1.04). Conclusion: Receiving vitamin A supplementation was not associated with acute respiratory infections among children aged 5 to 59 months. / Tesis

La coqueluche est une pathologie due à la bactérie Bordetella pertussis qui touche les voies respiratoires des patients infectés causant toux, leucocytose, fièvre, et dont les symptômes peuvent aller jusqu’au décès chez les individus les plus à risque (nouveau-nés et enfants immunodéprimés en particulier). Ciblée par différents programmes vaccinaux depuis de nombreuses années, cette pathologie sévit à nouveau dans de nombreux pays développés où le nombre de cas augmente fortement depuis la fin des années 2000. Cette résurgence montre la nécessité de développer de nouvelles stratégies afin de comprendre les mécanismes de l’infection par B. pertussis. Dans ce contexte, la recherche préclinique apparaît comme essentielle pour comprendre la physiopathologie de la coqueluche. De nombreux modèles animaux ont été décrits pour l’étude de la coqueluche mais aucun de ces modèles ne permet de reproduire l’ensemble du spectre des symptômes cliniques de la pathologie, notamment la toux. Cependant, au cours des dernières années un modèle d’infection par Bordetella pertussis chez le jeune babouin a été développé aux Etats-Unis et permet de reproduire la pathologie observée chez l’homme, notamment concernant la toux et la transmission. Ce modèle semble ainsi très prometteur pour l’étude de la physiopathologie de la coqueluche.Cependant, de nombreuses inconnues subsistent dans ce modèle, notamment concernant la colonisation bactérienne et les interactions entre la bactérie et l’hôte. Nous avons ainsi cherché dans cette étude à évaluer d’une part l’impact de différents facteurs comme l’âge des animaux, la dose d’infection ainsi que la voie d’exposition sur la pathologie déclarée par les babouins suite à l’infection par la souche B1917 de B. pertussis afin de pouvoir proposer un parallèle avec les données cliniques disponibles. Nous avons également développé l’utilisation de techniques d’imagerie in vivo comme l’endomicroscopie confocale couplée à la bronchoscopie afin d’étudier la localisation et la cinétique de colonisation et certaines interactions du pathogène dans le tractus respiratoire inférieur au cours de la pathologie. Cette étude nous a ainsi permis d’approfondir les connaissances de physiopathologie de la coqueluche dans ce modèle babouin et consolidera cet outil précieux pour l’évaluation des futures stratégies de prévention contre cette pathologie. / Whooping cough, or pertussis, is a respiratory disease caused by Bordetella pertussis bacterial colonization of human airways. Main symptoms are cough, leukocytosis, fever and may even be lethal for some patients (e.g. newborn infants and immuno-deficient patients). Despite a good vaccination coverage worldwide against pertussis, whooping cough cases have been re-increasing in several developed countries in the past twenty years. This resurgence points out the crucial need to develop new control strategies and to better understand pertussis pathophysiology, notably using appropriate animal models. Numerous preclinical models including mice, rats, rabbits and swine have been described for B. pertussis infection studies. However, none of these models reproduce the full spectrum of clinical pertussis symptoms, especially cough. The recent baboon model of whooping cough described in the last few years in the US appears to be a very relevant model for pertussis pathophysiology studies as these animals reproduced all clinical symptoms as observed in humans including cough.However, many aspects of bacterial colonization and interactions with the host have yet to be described in this model.We have then evaluated diverse parameters such as animal age, the inoculum dose and the exposition route on the pathology symptoms and immune responses developed by baboons following B. pertussis B1917 strain inoculation in order to draw a parallel with human clinical data. We also developed in this model in vivo imaging techniques like confocal endomicroscopy coupled with bronchoscopy in order to evaluate bacterial colonization kinetics, localization and some interactions in the lower respiratory tract of infected baboons. Then, this study brought additional data on whooping cough physiopathology in this baboon model, which will be crucial for evaluating future prevention strategies against pertussis disease