Bakgrund: Kronisk hosta är ett vanligt medicinskt tillstånd och definieras som hosta som varar mer än åtta veckor och är behandlingsresistent (refraktär) trots noggranna utredningar eller medicinska behandlingar. Det drabbar cirka 10% av den vuxna befolkningen och har en betydande negativ inverkan på livskvalitet. Det finns hittills inga studier som beskriver hur hostan påverkar individernas vardag och möjlighet att genomföra fysisk aktivitet. Syfte: Att undersöka hur individer som har diagnostiserats med kronisk refraktär hosta upplever att hostan påverkar vardagen och hens förmåga att vara fysisk aktiv.  Metod: Kvalitativ metod med semistrukturerade intervjuer tillämpades. Intervjuerna (n=10) transkriberades och tolkades med tematisk analys med induktiv ansats.  Resultat: Analysen resulterade i tre huvudteman 1) Betydande inverkan på vardagen, 2) Kampen om kontrollen och 3) Hostan under fysisk aktivitet. Dessa tre teman och deras åtta underteman belyser komplexiteten och omfattningen av hur hostan påverkar individernas liv. Slutsats: Kronisk hosta har avsevärd påverkan på vardagen och har negativa effekter på individens psykosociala tillstånd. Studien belyser dock att fysisk aktivitet är genomförbart för individer med kronisk hosta men att anpassningar kan behövas avseende intensitet eller typ av aktivitet. En bättre förståelse för innebörden och konsekvenserna av att leva med kronisk hosta kan vara ett bidrag till att ytterligare utveckla framgångsrika behandlingsmetoder. / Background: Chronic cough is a common medical condition and is defined as cough that lasts more than eight weeks and is treatment-resistant (refractory) despite careful investigations or medical treatments. It affects about 10% of the adult population and has a significant negative impact on quality of life. So far, there are no studies describing how the cough affects individuals' everyday lives and ability to carry out physical activity. Aim: To investigate how individuals who have been diagnosed with chronic refractory cough experience that the cough affects everyday life and his/her ability to be physically active. Method: Qualitative method with semi-structured interviews was applied. The interviews (n=10) were transcribed and interpreted with thematic analysis with an inductive approach. Results: The analysis resulted in three main themes: 1) Significant impact on everyday life, 2) The struggle for control and 3) The cough during physical activity. These three themes and their eight subthemes highlight the complexity and scope of how the cough affects the lives of individuals. Conclusion: Chronic cough has a significant impact on everyday life and has negative effects on the individual's psychosocial state. However, the study highlights that physical activity is feasible for individuals with chronic cough but that adaptations may be needed regarding intensity or type of activity. A better understanding of the meaning and consequences of living with chronic cough can be a contribution to further developing successful treatment methods.

Progressive disorders of airway protection, namely cough (dystussia) and swallowing (dysphagia) dysfunction, are highly prevalent in Parkinson’s disease (PD), impacting quality of life and contributing to the development of aspiration pneumonia – a leading cause of death in this population. To date, dysphagia rehabilitation has remained the primary (and often only) treatment target of choice by clinicians managing dysphagia in patients with PD and other neurodegenerative disease. This is a major concern since the progressive nature of PD makes it somewhat unreasonable to expect that treatments can fully rehabilitate swallowing dysfunction and eliminate chronic aspiration. Instead, rehabilitating cough dysfunction can serve as an adjunctive approach to promote pulmonary health. Considering that impairments in motor control and organization are primary features of PD, skill training may have a necessary role in cough rehabilitation. Despite a growing body of research supporting the feasibility and effectiveness of cough skill training, a significant gap remains in our understanding of optimal skill training parameters that maximize treatment outcomes through motor learning. This document addresses this gap in the literature in a series of three research studies. Chapter 1 will begin by reviewing the current body of literature related to normal and disordered mechanisms of airway protection dysfunction in PD, skill training as an efficacious approach to rehabilitate cough dysfunction, and motor learning considerations to maximize treatment outcomes. Chapter 2 will characterize motor performance and learning during a voluntary cough skill training paradigm, and evaluate the contributions of physiologic (i.e., lung volume) and treatment-specific (i.e., biofeedback) factors to treatment response in PD. Chapter 3 will then characterize trajectories of motor performance during multiple sessions of sensorimotor cough skill training and explore the role of task-specific predictors (i.e., variability, motor learning) on motor performance. Chapter 4 will examine the effects of cough skill training with variable practice on motor performance and motor learning and characterize contributions of laryngeal and respiratory subsystems to cough strength. This document will then conclude (Chapter 5) by synthesizing results from these studies and discussing clinical implications, limitations, and potential directions for future research.

Finally, intestinal absorption of compounds A and H were also investigated by Caco-2 monolayer cell model. These results demonstrated, for the first time, that these stemona alkaloids were well absorbed in a gastrointestinal cell culture model. Furthermore, compound A was demonstrated to have a marked preference in the basolateral to apical transport direction, and such efflux (basolateral to apical) transport was inhibited by both verapamil and cyclosporine A, P-glycoprotein (P-gp) inhibitors, but not by probenecid and MK371, multidrug resistant-associated protein (MRP) inhibitors. The results suggested that compound A transported through active efflux mechanisms via P-gp but not MRP pathway. / High performance liquid chromatography (HPLC) coupled with evaporative laser scattering detector (ELSD) was developed to qualitatively and quantitatively determine the chemical profiles of Radix Stemonae. The results demonstrated that the type and quantity of the main bioactive ingredients, stemona alkaloids, present in various herbal samples varied significantly. Compound A (neotuberostemonine) was identified as a predominant alkaloid in two commercial Radix Stemonae samples, whereas compound F (croomine), compound H (tuberostemonine) and compound G (oxoneotuberostemonine) were identified as the major alkaloids present in other three commercial samples, respectively. Chemical variations were observed in several fresh Radix Stemona samples collected in mainland China. These chemical variations might be due to species and/or environmental differences. / In addition to the antitussive activities, it was found that a high dose of compound A caused markedly behavioral changes, including head and body shaking via both intraperitoneal and intracerebroventricular administration. Such adverse effect was abolished by a centrally acting dopamine D2 antagonist haloperidol, suggesting that a central dopaminergic effect might contribute to the behavioral activities produced by compound A. Moreover, compound A was found, for the first time, to dose-dependently and competitively inhibit monoamine oxidase (MAO)-B and compound A was identified to be a weak and non-competitively inhibitor on MAO-A. It was further demonstrated that compound A increased the intercellular concentration of dopamine in the cultured PC12 cells and prevented MPTP-induced cell death in PC12 cells via inhibition of MAO. Therefore, the behavioral changes induced by compound A was suggested to be involved with dopaminergic pathway via reduction of dopamine metabolism caused by inhibition of MAO. / On the other hand, compound F was demonstrated to cause acute lethal toxicity via intraperitoneal but not via oral administration. The results suggested that compound F might have a low oral bioavaiIability. Further absorption study by Caco-2 model demonstrated that this alkaloid had a good intestinal absorption, thus its low oral bioavailability could be due to extensive first-pass effects in the gastrointestinal tract. / Pharmacological properties of stemona alkaloids were studied in vivo using the citric acid-induced guinea pig cough model. The three stemona alkaloids present in different Radix Stemonae samples were all found to be antitussive. Compounds A and H were both orally active and had similar antitussive potencies via both oral and intraperitoneal (i.p.) administrations. Compound F was demonstrated to be antitussive via i.p. administration only. The mechanism of antitussive activity of the representative stemona alkaloid, compound A, was further investigated. However, none of the currently known antitussive pathways were identified to be involved in compound A. Thus, compound A and also other stemona alkaloids are likely to produce their antitussive activity via a novel pathway. / Radix Stemonae is derived from the root tubes of three different species of Stemona genus (Stemonaceae). This herb has been prescribed in traditional Chinese medicine (TCM) as an antitussive agent for over thousands of years. To date, over fifty stemona alkaloids have been identified from various Stemona species. However, there is a lack of direct evidence to link stemona alkaloids to the effectiveness of the herb in the treatment of cough. The aim of the present study is to investigate Stemona species used as plant sources for Radix Stemonae, the chemical and pharmacological properties in relation to antitussive activity of the herb, and the intestinal absorption of the main bioactive constituents, stemona alkaloids, in the herb. / The identity of fresh Radix Stemonae samples was investigated using a DNA based polymorphism assay. 5S-rRNA, ITS-1 and ITS-2 are highly conserved spacer regions; thus, the diversity of these spacer regions was used for the identification of Radix Stemonae samples. The amplified spacer regions of different Radix Stemona samples collected from different geographical locations in Mainland China were sequenced and compared. The result demonstrated that there were at least three different DNA patterns among seven samples examined and this DNA sequential assay could distingue species in Stemona genus from species in other genera. However, the findings suggested that the variation in chemical profiles of different Radix Stemonae samples was not directly related to their DNA sequences. DNA sequential method could be used to authenticate the correct plant sources for Radix Stemonae but it can not to provide information on chemical profiles of the herb. / The overall results demonstrated that the quantities and types of stemona alkaloids varied significantly depending upon plant sources. Furthermore, these stemona alkaloids differed considerably in pharmacological activities, toxicological effects and absorption profiles. Therefore, these variations in different Radix Stemonae samples may lead to different therapeutic outcomes, including efficacies, adverse effects, and potential herb-drug and herb-herb interactions. Nevertheless, the present study provided a scientific basis for the therapeutic use of Radix Stemonae and illustrated a potential for the development of herbal Radix Stemonae or pure stemona alkaloids into a new class of antitussive TCM herbal products or TCM-based agents in the future. / Leung Pak Ho Henry. / "January 2006." / Adviser: Ge Lin. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6328. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 179-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

<p>Uprkos rezultatima postignutih imunizacijom, veliki ka&scaron;alj je i danas vodeći uzrok smrti među zaraznim bolestima protiv kojih se sprovodi imunizacija. Postojanje različitih vrsta nadzora, prisutnih nedostataka laboratorijske dijagnostike oboljenja, slabosti u dostupnim definicijama slučaja velikog ka&scaron;lja i neprepoznavanje oboljenja, otežavaju realno sagledavanje opterećenja velikim ka&scaron;ljem i poređenje postignutih rezultata u različitim zemljama. Postojeće definicije slučaja velikog ka&scaron;lja nisu prihvatljive u svim uzrastima. Cilj istraživanja je bio da se na reprezentativnom uzorku Grada Novog Sada utvrdi: stopa incidencije velikog ka&scaron;lja tokom jedne godine savremenog nadzora; senzitivnost i specifičnost definicija slučaja velikog ka&scaron;lja, datih od strane Globalne pertusis inicijative za tri uzrasne grupe. U istraživanje je uključeno 213 ispitanika pacijenata iz sentinelnog nadzora nad velikim ka&scaron;ljem Doma zdravlja Novi Sad i 107 ispitanika pacijenata iz hospitalnog nadzora sa iste teritorije koji su ispunjavali predložene definicije slučaja za tri uzrasne grupe (0-3 meseca; 4 meseca-9 godina života; 10 godina i stariji). Podaci za istraživanje dobijeni su popunjavanjem anketnog upitnika. Laboratorijsko testiranje sumnji na veliki ka&scaron;alj sprovedeno je u Centru za mikrobiologiju Instituta za javno zdravlje Vojvodine. Kod pacijenata sa ka&scaron;ljem u trajanju do 21 dan testiranje je vr&scaron;eno upotrebom PCR metoda, a kod pacijenata sa ka&scaron;ljem u trajanju vi&scaron;e od 21 dan, dokazivanje oboljenja vr&scaron;eno je upotrebom ELISA IgA i IgG serolo&scaron;kih testova. Potvrđenim slučajem velikog ka&scaron;lja smatran je pacijent sa ispunjenom definicijom slučaja oboljenja i laboratorijskom potvrdom oboljenja, PCR ili ELISA testom. Procenjena stopa incidencije velikog ka&scaron;lja u Novom Sadu je 237,7/100.000, a stopa incidencije hospitalizovanih u Novom Sadu je 16,4/100.000. Najvi&scaron;e stope incidencije velikog ka&scaron;lja u sentinelnom nadzoru registruju se u uzrastu 10-14, a u hospitalnom u uzrastu od 7 do 9 godina. Senzitivnost, specifičnost i stepen verovatnoće pozitivnog rezultata testiranog simptoma/znaka iz predloženih definicija slučaja se razlikuju po uzrastima i po pojedinim simptomima/znacima. Predložene definicije slučaja u uzrastima od 4 meseca do 9 godina i u uzrastu od 10 godina i starijih imaju veću verovatnoću otkrivanja obolelih u hospitalnom u odnosu na sentinelni nadzor za pojedine simptome/znakove. Budući da je tokom istraživanja u sentinelnom nadzoru oboljenje potvrđeno kod svakog petog, a u hospitalnom kod svakog drugog testiranog pacijenta, predložene definicije slučaja se mogu koristiti u nadzoru nad velikim ka&scaron;ljem.</p> / <p>Despite all results achieved by immunization, pertussis is still the leading cause of death among vaccine preventable diseases. Different types of surveillance and laboratory confirmation of pertussis, weakness of existing case definitions for pertussis and broad spectrum clinical manifestation of disease, complicate overview of disease and result comparison of surveillance in different countries. The current pertussis case definition is not acceptable for all age groups of patients. The aim of this research was to determine: the pertussis incidence rate in population of Novi Sad during one year of modern surveillance; the sensitivity and specificity of clinical case definition for pertussis, given by the Global Pertussis Initiative for three age groups, with a representative sample of population in the City of Novi Sad. 213 patients from sentinel surveillance of pertussis Novi Sad Health Centre and 107 hospitalized patients from Novi Sad, who fulfilled criteria of case definition proposed for the three age groups (0-3 months, 4 months-9 years; 10 years of age and older), were included in the research. Research data obtained from a questionnaire. Laboratory testing of suspected cases were conducted at the Centre for Microbiology, Institute of Public Health of Vojvodina. For patients with coughing less than 21 days PCR method was used, and for patients with cough lasting more than 21 days, laboratory confirmation of disease was performed using ELISA IgA and IgG serological tests. Confirmed case of pertussis consider to be a patient with symptoms/signs according to proposed case definition and with laboratory confirmation of the pertussis, PCR or ELISA. Estimated incidence rate of pertussis for population in the city of Novi Sad was 237.7/100,000, and the pertussis incidence rate in hospitalized patient in Novi Sad was 16.4/100,000. The highest incidence rate of pertussis in the sentinel surveillance was registered in the age group 10-14 and in the hospital surveillance in the group 7 to 9 years of age. The values of sensitivity, specificity and positive likelihood ratio of symptoms/signs from the proposed case definition were calculated and they different by age and by certain symptoms/signs from proposed case definition. Certain symptoms/signs of the proposed case definition have a higher probability of detection among patients aged 4 months to nine, and at the age of ten year and older, in the hospital versus those in sentinel surveillance for pertussis. During research from the patients who were tested in sentinel surveillance every fifth was laboratory confirmed case and in the hospital surveillance every second patient was confirmed case, so the proposed case definitions can be used in the surveillance of pertussis.</p>

Bordetella pertussis is a strictly human pathogen colonizing the upper respiratory tract, causing a respiratory disease known as whooping cough or pertussis. The introduction of whole-cell vaccines and acellular vaccines, resulted in a significant reduction in the incidence of disease and reduce the fatalities associated with infection. However, epidemiological data show a significant increase in the incidence of the disease in recent decades. The increasing incidence is mainly attributed to the transition from the whole- cell vaccine to an acellular vaccine. Based on research from recent years has shown that acellular vaccines have many drawbacks, and it is therefore necessary to change the vaccination strategy. One possible solution to the situation is the development of a new generation of whole-cell vaccines with reduced reactogenicity. The new whole-cell vaccine was prepared by a genetically modified B. pertussis strain. B. pertussis was modified using allelic exchange to develop strain encoding enzymatically inactive pertussis toxin, modified lipid A and lacking dermonecrotic toxin. This combination of genetic modifications in mice led to a decrease in reactogenicity test vaccine in vivo. In case of intranasal infection whole-cell vaccine containing genetically modified strain is providing...

Contexte – La stimulation de la muqueuse trachéale peut provoquer une réponse défensive à type de toux. La finalité de cette réponse est la protection des voies aériennes via la clairance du mucus et des particules déposées au sein de l’arbre trachéo-bronchique. La stimulation de la muqueuse nasale est à l’origine de réponses défensives n’incluant toutefois pas la toux. La modulation de la toux suggère de possibles interactions centrales des afférences provenant de localisations anatomiques distinctes. Objectif – Déterminer si une stimulation trachéale mécanique discriminante est capable de provoquer une toux lors d’une apnée provoquée par l’instillation nasale d’eau distillée. Méthodes – Douze lapins anesthésiés et trachéotomisés ont été étudiés. Les stimulations trachéales mécaniques ont été réalisées dans 3 conditions : contrôle, après instillation nasale de sérum physiologique, et lors d’apnée suite à l’instillation d’eau distillée. Résultats – Les paramètres ventilatoires de références ne sont pas différents dans les 3 conditions. Un total de 171 stimulations trachéales a été réalisé. Lors de l’apnée, 81% des réponses sont des réflexes expiratoires et le pic de débit expiré est inférieur (p < 0.0001) à celui observé dans les conditions contrôle et sérum physiologique. L’incidence des réponses comprenant un réflexe de toux est plus faible (p < 0,0001) en cas d’instillation d’eau distillée que dans les 2 autres conditions.Conclusion – La stimulation nasale par l’eau distillée désensibilise les réflexes de défense respiratoire faisant suite à une stimulation trachéale mécanique / Context - Cough may be triggered by irritation of afferents located in the airway mucosa. Primary role is to expel inhaled foreign matter from the lungs or clear the airways of endogenous mucus. Stimulation of the nasal mucosa provokes defensive responses but not cough. The ‘cough center’ can be tuned by various afferent inputs, suggesting possible interactions at a central level of neural pathways originating from distant anatomical sites. Objective - The present study was designed to determine whether brief mechanical stimulation of the trachea could trigger cough during apnoea elicited by nasal instillation of water. Method - Twelve anesthetized, tracheotomized rabbits were studied. Mechanical stimulation of the trachea was performed under 3 conditions: baseline control, after instillation of saline into the nose and during apnoea following instillation of water. Results - Baseline breathing pattern did not differ between the 3 conditions. In a series of 171 stimulations, expiration reflex occurred in 81% of stimulations during apnoea with a significantly (p < 0.0001) lower peak expiratory flow than at baseline or during saline instillation. The incidence of responses comprising a cough reflex was also lower during water instillation than at either baseline or with saline (p < 0.0001). Conclusion - These results indicate that stimulation of nasal afferents with distilled water likely down-regulates cough

Whooping cough (pertussis) is a highly contagious acute respiratory disease caused by the strict human pathogen Bordetella pertussis, a gram-negative coccobacillus. The worldwide mass-vaccination was started in 1940s and to date, a number of whole-cell (Pw) and acellular pertussis vaccine (Pa) formulations were developed. Yet the current vaccines are incapable of providing sustained, lifelong immunity and eliminating subclinical infections, which pose a threat especially for unimmunized infants as well as adolescents and adults. Thus, finding new protein candidates with high immune protective capacities is necessary to enhance the clinical efficacy of current acellular pertussis (Pa) vaccines. In this study, iron-superoxide dismutase (FeSOD) protein was investigated for its capacity of conferring protectivity as well as stimulating humoral and cellular responses against B. pertussis infection in a mouse model. For this purpose, sodB gene, which encodes iron-superoxide dismutase FeSOD protein, was amplified from the genomic DNA of the universal B. pertussis strain &lsquo / Tohama I&rsquo / and sequentially cloned to pGEM&reg / -T subcloning and pET-28a(+) expression vectors. Afterwards sodb/pET28a(+) construct was introduced to E. coli BL21(DE3) cells and the gene was overexpressed therein via IPTG induction. The expressed FeSOD protein was then purified by affinity chromatography and its previously reported immunogenicity was confirmed by Western blot. After filter-sterilization, the protein was adsorbed to alum [Al(OH)3] adjuvant and introduced to BALB/c twice at three weeks intervals intraperitoneally at a concentration of 20 &mu / g purified FeSOD protein/mouse. Another group of mice were immunized in tandem with heat-inactivated whole-cell suspension of B. pertussis. Ten days after the second immunization, mice were intranasally challenged with the local &lsquo / Saadet&rsquo / strain of B. pertussis. Next the lungs of groups of mice were excised, homogenized and plated as serial dilutions on days 5, 8 and 14 post-challenge, and viable lung CFU counts were carried out. Whole cell immunization conferred complete bacterial clearance following B. pertussis intranasal infection while FeSOD immunization failed to attain such protection. In addition to the protectivity assay, ELISA was performed to assess the humoral (i.e. IgG) immune response triggered upon FeSOD- and whole-cell immunizations and a statistically significant increase in anti-FeSOD IgG production was observed in FeSOD-immunized group. Finally, cellular immune response was tested via cytokine (IFN-&gamma / ) assay, in which spleens of mice were excised, splenocytes were cultured and the level of IFN-&gamma / production upon FeSOD addition to the cultures was measured via ELISA. This test showed that whole-cell immunization triggered IFN-&gamma / production at significant levels while FeSOD-immunization did not / indicating the failure of alum-adsorbed FeSOD immunization in inducing cell-mediated immune response.

Bordetella pertussis est l’agent responsable de la coqueluche. Pour coloniser le tractus respiratoire humain, cette bactérie à Gram négatif, aérobie stricte, produit de nombreux facteurs de virulence dont l’expression est sous la dépendance du système à deux composants BvgAS. BvgS est un senseur-kinase dimérique. Chaque monomère est constitué de trois domaines putatifs de perception - deux domaines Venus flytrap périplasmiques et un domaine PAS cytoplasmique -, suivis du domaine enzymatique et deux autres domaines, de phospho-transfert et receveur, impliqués dans la cascade de phosphorylation. L’expression du régulon de virulence est activée suite à la phosphorylation par BvgS du régulateur de réponse BvgA. BvgS est en mode kinase à l’état basal, et la perception de basses températures ou de signaux chimiques comme les ions sulfate ou nicotinate cause son passage en mode phosphatase. L’étude présentée dans ce manuscrit vise à caractériser le senseur-kinase BvgS en analysant les domaines putatifs de perception ainsi que la transduction de signal qui s’effectue au sein de la molécule. L’étude de la portion périplasmique a permis de mettre en évidence, à l’état basal, un gradient de dynamique décroissant. En se fixant au domaine VFT2 proximal à la membrane, le nicotinate induirait une diminution de la dynamique du second lobe du VFT1, causée par la formation d’un bloc compact entre le domaine VFT2 et le deuxième lobe du domaine VFT1. Cette rigidification exercerait une tension sur les hélices α qui précèdent les segments transmembranaires, provoquant une transition de la portion cytoplasmique vers l’état phosphatase. La perception de modulateurs par le domaine VFT2 - ou possiblement la fixation d’un ligand dans la cavité du VFT1- modifierait cette dynamique et causerait le changement d’activité de BvgS. Ainsi, nous proposons un modèle dans lequel le VFT1 est considéré comme le moteur du système, lui impulsant une dynamique qui serait relayée ou atténuée par le domaine VFT2. Une recherche de ligands antagonistes pour le domaine VFT1 a été entreprise, selon l’idée que la fixation d’un ligand réduirait la dynamique de ce dernier. Au sein du dimère, des connecteurs prédits pour former des enroulements d’hélices α (‘coiled coil’) relient entre eux les domaines VFT et PAS, et les domaines PAS et kinase de BvgS. La transduction d’information entre les domaines périplasmiques et le site enzymatique de BvgS a été analysée par mutagénèse dirigée et ‘cysteine scanning’. Des contacts proches sont observés entre les hélices constituant le segment transmembranaire, qui ne semblent pas être modifiés après perception de modulateur. Nous suggérons donc un modèle de piston symétrique pour la transmission d’information au travers de la membrane. Le coiled coil putatif précédant le domaine PAS présente une certaine dynamique rotationnelle à l’état basal. La perception de modulateurs semble induire l’écartement de ces hélices, ce qui pourrait permettre un changement de l’interface des domaines PAS. L’étude de la topologie du domaine PAS confirme une modification de cette interface entre les modes kinase et phosphatase de BvgS. Enfin, le coiled coil reliant les domaines PAS et kinase est sujet à une forte dynamique rotationnelle à l’état basal, en accord avec un modèle de régulation de l’activité kinase proposé dans d’autres systèmes. Suite à la perception de modulateur, une rigidification marquée de ce coiled coil est observée, permettant le passage en mode phosphatase. L’existence de deux états dynamiques différents de ce coiled coil a également été mise en évidence en absence du domaine PAS.Ces études ont permis d’avancer dans la compréhension de BvgS et de proposer un modèle de la signalisation au sein de ce senseur-kinase, qui pourrait s’appliquer aux autres membres de la famille de BvgS. / Bordetella pertussis is the agent of an acute and highly contagious respiratory disease, whooping cough. In order to colonize the human respiratory tract, this strictly aerobic Gram negative bacterium produces many virulence factors, the expression of which is regulated by the BvgAS two-component system. BvgS is a sensor-kinase composed of three putative domains of perception –two periplasmic Venus flytrap domains and a cytoplasmic PAS domain -, followed by the enzymatic domain and two other domains called phosphotransfert and receiver involved in the phophorelay. The expression of the virulence regulon is activated after the phosphorylation by BvgS of the response regulator BvgA. BvgS is in a kinase mode at the basal state, and the perception of low temperatures or chemical signals like sulfate ions or nicotinate causes a shift to the phosphatase state. The study presented in this manuscript has focused on the characterization of the BvgS sensor-kinase. We have analyzed its putative domains of perception and the mechanisms of signal transduction.Investigations into the dynamics of the periplasmic moiety has provided evidence for a decreasing gradient of dynamics from N to C-terminus at the basal state. Nicotinate binding to the membrane-proximal VFT2 domains decreases the dynamics of the second lobe of VFT1. Tighter interactions between the latter and the VFT2 domain cause a tension on the α helices that precede the transmembrane segments, triggering the transition to the phosphatase state of the enzymatic portion. Perception of modulator by the VFT2 domains –or possibly binding of a ligand in the VFT1 cavity- thus appears to modify periplasmic dynamics, which shifts BvgS activity. We propose that the VFT1 domains are the motor for BvgS activity, and their dynamics are relayed or attenuated by the VFT2 domains. A search for antagonistic VFT1 ligands has been undertaken, along the idea that ligand binding may reduce their dynamics.The VFT and PAS domains, and the PAS and kinase domains are joined to each other by long α helices predicted to form coiled coils. We performed directed mutagenesis and cysteine scanning analyses to decipher signal transduction between the periplasmic domains and the enzymatic moiety of BvgS. The close contacts between the helices of the transmembrane segment are not modified after perception of the modulator, suggesting that signal transduction across the membrane is mediated by symmetrical piston motions. The putative coiled coil before the PAS domain shows rotational dynamics at the basal state. Modulator perception causes the helices to splay, and this motion may modify the PAS domains interface. Our topology analyses of the PAS domain confirm that changes occur at this interface between the kinase and phosphatase states of BvgS. Finally, the coiled coil between the PAS and kinase domains presents a strong rotational dynamics at the basal state, which is consistent with the model of regulation of kinase activity proposed for other sensor-kinases. After perception of a modulator, this coiled coil becomes more rigid, allowing the shift to the phosphatase state. The occurrence of two states of dynamics for this coiled coil has also been demonstrated in the absence of the PAS domain.These studies have advanced our understanding of BvgS and allow us to propose a model of signaling by this sensor-kinase, which may apply more broadly to other family members.

The thesis deals with the problem of the occurrence of whooping cough in the South Bohemian Region in the period of 2003-2012. Despite the high level of vaccination the incidence of this illness after a significant fall in the 1980s has been growing constantly since 1993. This trend evidently also applies in other developed countries with a high rate of vaccination. The main aim is to draw up an overview of the occurrence of whooping cough in the South Bohemian Region in the monitored period of the last 10 years, compare this occurrence with other regions in the Czech Republic and map out the changes which have occurred in whooping cough vaccination. Quantitative and qualitative research was chosen to process the empirical part. Data for quantitative research was acquired from the database of infectious diseases (EPIDAT), and consisted of reported cases of whooping cough. Another essential group of data were demographic figures about the number and age structure of the population. The second qualitative part was drawn up on the basis of a literary search. I acquired additional information by communicating with individual territorial workplaces of the Regional Hygiene Station of the South Bohemian Region based in České Budějovice. Using the processed data I discovered that the incidence of whooping cough in the South Bohemian Region does not have an ascending trend in 2003-2012 unlike the trend in the whole of the Czech Republic. The highest occurrence of whooping cough in the Czech Republic in the monitored period was reported in the 10-14 age group. The research also showed that in the period of 2003-2012 individual regions differed greatly in the occurrence and can be divided into two groups ? regions with the highest reached incidence below 10 cases/100,000 people and regions with incidence with the highest reached incidence above 10 cases/100,000 people which includes the South Bohemian Region. There have been many changes in the vaccination of whooping cough and the most important was the transition from whole cell vaccine to acellular in 2007 and adding a 6th booster dose between the 10th and 11th year in 2009. The greatest attention should be paid above all to increase information or test the knowledge of the lay public, especially women preparing for pregnancy and people taking care of newborn babies and infants about the occurrence, potential risks and possibilities of vaccination against this infection even at an adult age. In my opinion a further necessity is the consolidation of diagnostics, definition of a case and procedures of surveillance throughout the Czech Republic, education of paediatricians and general practitioners about the illness and topicality of the problem, an appeal to report the occurrence of this infection to epidemiologists, observing the isolation of the patient, monitoring possible epidemiological connections between cases and making use of the offer of the NRL (National Reference Laboratory) services against whooping cough and diphtheria. This thesis can be used in practice as a summary of information material for the lay and professional public about the occurrence of whooping cough in the South Bohemian Region and throughout the Czech Republic and overview of vaccination against whooping cough. It can be an incentive for further anti-epidemic measures and the need for further research into this problem.

La voie de sécrétion bactérienne de type V permet l’exportation à la surface cellulaire de protéines dont certaines ont été identifiées comme d’importants facteurs de la pathogénicité bactérienne. Le type V regroupe la sécrétion des autotransporteurs et la sécrétion à deux partenaires (TPS). Les autotransporteurs sont constitués d’un domaine en tonneau β; et d’un domaine passager. L’interaction de l’autotransporteur avec le complexe protéique Bam, dont la pièce centrale est le transporteur BamA, permet l’insertion dans la membrane externe du tonneau β; et la sécrétion du passager. En revanche, la sécrétion à deux partenaires fait intervenir deux protéines, l’une appelée TpsA correspondant à la protéine exportée et l’autre, TpsB, formant un tonneau &#946; qui contrôle le transport à travers la membrane externe. Les protéines TpsB sont spécifiques à leur(s) TpsA associée(s), et font partie de la superfamille des transporteurs Omp85 qui effectuent l’insertion de protéines dans la membrane externe bactérienne comme BamA, et dans celles des organites eucaryotes dont les chloroplastes et les mitochondries. Au cours de mon doctorat, je me suis intéressé à la sécrétion de l’hémagglutinine filamenteuse (FHA), qui est l’adhésine majoritaire de Bordetella pertussis, l’agent étiologique de la coqueluche. Cette adhésine qui permet à la bactérie de coloniser le tractus respiratoire de l’hôte est une protéine TpsA de 220 kD. Elle est très efficacement sécrétée par la voie de sécrétion à deux partenaires grâce à son transporteur spécifique TpsB nommé FhaC. L’étude cristallographique de FhaC a révélé un tonneau β; à 16 brins qui forme un canal dans la membrane externe obstrué par l’hélice-α; amino-terminale, H1, partagée par la majorité des TpsB, et par une boucle de surface, L6, conservée dans la superfamille Omp85. Cette conformation suggère un état au repos dans lequel le canal bouché ne pourrait pas transporter son partenaire. Afin de comprendre comment la FHA transite à l’intérieur du pore, il est donc nécessaire de connaître les changements de conformations que subit FhaC. Durant mon travail de thèse, nous avons apporté une vision plus dynamique de la sécrétion à deux partenaires en utilisant le couple FHA/FhaC comme modèle d’étude. Pour cela nous avons utilisé principalement la Résonance Paramagnétique Electronique (RPE). Cette technique de biophysique permet d’étudier FhaC en solution ou réincorporée dans une bicouche lipidique et de rendre compte de la mobilité à un site donné par l’utilisation de sondes paramagnétiques. Ainsi nous avons pu montrer que FhaC est en équilibre entre plusieurs conformations, avec H1 dans le pore ou du côté périplasmique de FhaC. La présence de la FHA déplace cet équilibre, favorisant ainsi la sortie de l’hélice hors du pore. Nous avons, par ailleurs, pu démontrer expérimentalement que la FHA transitait bien à l’intérieur du pore formé par FhaC et que l’hélice H1 se trouvait alors dans le périplasme. L’étude de la boucle L6 nous a permis de montrer que la mobilité de cette boucle était fortement contrainte à l’intérieur du pore même lors de la reconnaissance avec la FHA. Ce ralentissement de mobilité est lié, en autre, à une interaction avec un résidu d’un motif conservé présent sur le brin β13 qui influence la taille du pore. De manière plus générale, cette étude de la dynamique de FhaC contribue à la compréhension des mécanismes moléculaires de la voie TPS et des transporteurs de la superfamille Omp85. / Type V secretion in bacteria mediates the export to the cell surface of proteins, some of which have been identified as important factors of pathogenicity. Type V includes the secretion of autotransporters and the ‘Two-partner Secretion’ (TPS) pathway. Autotransporters consist of a &#946; barrel domain and a passenger domain. The interaction of autotransporters with the Bam complex, of which the BamA transporter is the central component, allows the insertion of the β; barrel in the outer membrane and the secretion of passenger domain. In contrast, the two-partner secretion involves two proteins, the exported ‘TpsA’ protein and its TpsB partner that controls its transport across the outer membrane. TpsB proteins are specific to their associated TpsA(s) and belong to the superfamily of the Omp85 transporters, which carry out the insertion of proteins into the bacterial outer membrane, like BamA, or in the outer membranes of eukaryotic organelles including chloroplasts and mitochondria. For my PhD work, I have been interested in the secretion of filamentous hemagglutinin (FHA), which is the major adhesin of Bordetella pertussis, the causative agent of whooping cough. This adhesin allows the colonization by this bacterium of its host’s respiratory tract. This protein corresponds to a 220kD TpsA protein efficiently secreted by its specific transporter TpsB named FhaC. Crystallographic studies have revealed that FhaC harbours a 16-stranded β;-barrel occluded by both the N-terminal α;-helix, H1, shared by the majority of TpsB proteins, and by a surface loop, L6, that carries a conserved, hallmark motif of the Omp85 superfamilly. This conformation suggests that FhaC is in a resting state in which the channel does not transport its partner. To understand how the FHA passes through the FhaC pore, it is necessary to address the conformational changes undergone by FhaC. During my thesis work, we provided a more dynamic view of the TPS pathway using the FHA/FhaC couple as study model. For this we used Electron Paramagnetic Resonance (EPR). This biophysical technique allows to study of FhaC in solution or reincorporated into a lipid bilayer and it reports the mobility at specific sites of the protein by using paramagnetic probes. Thus we have shown that FhaC is in equilibrium between multiple conformations, with H1 in the pore or at the periplasmic side of FhaC. The presence of FHA displaces the conformational equilibrium, promoting the exit of the helix going from the pore. We have also experimentally demonstrated that FHA does transit through the pore formed by FhaC while helix H1 is then in the periplasm. The study of the L6 loop enabled us to show that the mobility of this loop is highly constrained in the pore and remains so upon the recognition of FHA. Its slow mobility is linked to an interaction between an invariant L6 residue and a conserved motif present on the β; strand 13 of the barrel. This interaction affects the size of the FhaC pore.More generally, the study of the dynamics of FhaC contributes to the understanding the molecular mechanisms of the TPS pathway and of transporters of the Omp85 superfamily.