Le transport de Na+ par ENaC et de Cl- par CFTR, dans l’épithélium des voies aériennes (VA), contrôle la clairance mucociliaire en modulant le volume du liquide de surface (ASL). L’expression et la fonction de ces canaux peuvent être modifiées au cours des maladies inflammatoires chroniques des VA. Dans un modèle de culture primaire de cellules épithéliales nasales humaines (CENH), nous avons démontré l’existence, au cours de la polypose nasosinusienne, d’une chute de la sécrétion de Cl- par CFTR dont l’expression est diminuée par le TGFß1, cytokine de l’inflammation. Nous avons ensuite étudié l’effet de l’élastase, et de son inhibiteur l’EPIhNE4 sur la fonction d’ENaC dans des CENH de patients atteints ou non de mucoviscidose (CF) où l’hyperactivité d’ENaC provoque une déshydratation de l’ASL. Nous avons démontré l’existence d’une activité sérine protéase endogène dans les CENH (patients CF ou non CF) qui, après inhibition, permet de révéler une augmentation de la fonction d’ENaC induite par l’élastase alors inhibable par l’EPI-hNE4. Enfin, nous avons analysé l’effet du 4 phenylbutyrate (4PBA), traitement proposé chez les patients CF, sur le canal ENaC. Le 4- PBA entraîne une augmentation de la fonction d’ENaC dans les CENH de patients CF ou non en adressant des sous-unités du canal à la membrane apicale via la régulation d’une molécule chaperone Hsc70. Le 4-PBA pourrait donc être utilisé dans des pathologies respiratoires avec défaut d’expression d’ENaC mais son usage pourrait être délétère chez les patients CF. La régulation des transports ioniques au cours des maladies inflammatoires chroniques des VA représente donc une cible thérapeutique intéressante / Na+ and Cl- transport by ENaC and CFTR respectively in airway epithelium (AE) control mucociliary clearance by regulating the airway surface liquid (ASL). Ion channel expression and function could be altered by chronic inflammation in AE. In primary cultures from human nasal epithelial cells (HNEC) we demonstrated, in nasal polyposis, a decrease of CFTR expression and Cl- secretion induced by TGFß1, an inflammatory cytokine. We also evaluated the effects of elastase, and its inhibitor EPI-hNE4, on ENaC function in HNEC from cystic fibrosis (CF where ENaC hyperactivity contributes to ASL dehydration) and non CF patients. We detected an endogenous serine protease activity in HNEC (CF or non CF) which, after inhibition, unmask an increase in ENaC function induced by elastase, this increase being then inhibited by EPI-hNE4. Finally, we investigated the effects of a drug now tested in CF patients treatment, the 4-phenylbutyrate (4PBA). 4-PBA promoted ENaC cell surface expression and activity in CF or non CF HNEC by increasing ENaC subunits translocation to plasma membrane via the regulation of Heat Shock Protein 70. 4-PBA treatment could therefore be of interest in the treatment of airway diseases when ENaC trafficking is disrupted but should be used with caution when treating CF patients where ENaC function is already upregulated. All together these results highlight the role of ion transport regulation during chronic inflammatory airway diseases and could lead to new therapeutic strategies

Certains virus respiratoires comme rhinovirus semblent favoriser la colonisation par staphylococcus aureus. Cependant, les détails des mécanismes impliqués dans cette synergie n’ont pas été suffisamment élucidés. Le but de cette thèse a été de développer et valider un modèle in vitro mimant la colonisation du vestibule nasal par s. aureus en utilisant les kératinocytes humains hacat. Ce modèle a permis d’étudier (i) les pouvoirs d’adhésion et d’internalisation d’une collection de souche clinique de s. aureus, (ii) l’efficacité intracellulaire des molécules antimicrobiennes utilisées dans le cadre de la décolonisation nasale de s. aureus, (iii) l’effet de la clarithromycine sur l’infection par rhinovirus et (iv) l’impact de l’infection par rhinovirus ou de l’inflammation non spécifique sur la colonisation par s. aureus. ce travail a principalement permis d’identifier un nouveau mécanisme alternatif de l’internalisation de s. aureus à travers la liaison entre la protéine bactérienne eap (extracellular adherence protein) et le récepteur cellulaire icam-1 (intracellular adhesion molecule 1). Cette voie alternative est favorisée en cas d’infection par rhinovirus ou d’inflammation, ce qui pourrait expliquer les observations cliniques de l’augmentation de la charge de s. aureus ou du risque d’infection par cette bactérie lors des infections virales respiratoires ou d’inflammation post-traumatique. Les résultats de cette thèse illustrent la complexité des interactions entre les cellules épithéliales de la muqueuse, s. aureus et les pathogènes viraux et ouvrent les perspectives sur d’autres études nécessaires afin de proposer des stratégies préventives ou thérapeutiques adaptées. / Some respiratory viruses such as rhinoviruses seem to promote staphylococcus aureus colonization. However, the details of the bacterial and cellular mechanisms involved in this synergy have not been sufficiently elucidated. The aim of this thesis was to develop and validate an in vitro model mimicking s. aureus colonization of the nasal vestibule by using hacat human keratinocytes. This model allowed to study (i) the adhesion and internalization capacities of various clinical s. aureus strains, (ii) the intracellular efficiency of the antimicrobial molecules used for s. aureus nasal decolonization, (iii) the effect of clarithromycin on rhinovirus infection, and (iv) the impact of rhinovirus infection and non-specific inflammation on s. aureus colonization. This work has mainly identified a new alternative mechanism for the internalization of s. aureus through the binding between the bacterial protein eap (extracellular adherence protein) and the cell receptor icam-1 (intracellular adhesion molecule 1). This alternative pathway is favored in case of rhinovirus infection or inflammation; which could explain the clinical observations of the increase of the load of s. aureus or the risk of infection by this bacterium during respiratory viral infections or post-traumatic inflammations. The results of this thesis illustrate the complexity of the interactions between the mucosal epithelial cells, s. aureus and viral pathogens and suggest that other studies are needed to propose appropriate preventive or therapeutic strategies.

Introdução: A rinossinusite crônica com polipose nasal constitui um subgrupo particular da rinossinusite crônica, caracterizado por uma inflamação da mucosa que leva a um espessamento da mesma e à formação de pólipos, podendo ser especialmente difícil de tratar. A Doxiciclina é um antibiótico bacteriostático, de largo espectro, que também tem uma ação antiinflamatória, e tem se mostrado útil no controle dos sintomas das recidivas desta doença, promovendo inclusive uma diminuição do pólipo. Objetivo: avaliar se o uso de baixas doses de Doxiciclina, por períodos prolongados, pode melhorar o controle clínico da rinossinusite crônica com polipose nasal, de difícil tratamento. Métodos: este é um estudo prospectivo, aberto, realizado em 60 pacientes com rinossinusite crônica de difícil tratamento que se submeteram a cirurgia endoscópica nasal. Os pacientes foram divididos em 2 grupos: 28 pacientes receberam corticóide nasal, lavagem nasal com soro fisiológico, e Doxiciclina (200mg no primeiro dia, seguido por 100mg uma vez ao dia) por 12 semanas, enquanto 30 pacientes receberam apenas corticóide nasal e lavagem nasal com soro fisiológico. O principal resultado avaliado foi a existência de uma melhora, dose efeito, clinicamente significativa do SNOT-20 após o tratamento com a Doxiciclina. Outros resultados avaliados foram os valores do SNOT-20, NOSE e do Lund-Kennedy. Os seguintes parâmetros também foram analisados: asma, rinite, doença respiratória exacerbada pela Aspirina (DREA), níveis séricos de IgG, IgA, IgE, IgM, ANCA e contagem de eosinófilos. Resultados: oy tratamento com a Doxiciclina promoveu uma melhora, dose efeito, clinicamente significativa do SNOT-20. Pacientes que receberam a Doxiciclina também tiveram resultados significativamente melhores do SNOT-20, NOSE e Lund-Kennedy. Houve uma associação negativa entre a melhora clinicamente significativa do SNOT-20 e a presença de asma, DREA e níveis séricos elevados de IgE pré-tratamento. Conclusão: os achados sugerem que a doxiciclina pode ter uma ação benéfica nos pacientes com rinossinusite crônica com polipose nasal, especialmente naqueles pacientes sem asma, DREA ou níveis séricos elevados de IgE prétratamento / Introduction: Chronic rhinosinusitis with nasal polyps is a particular subset of chronic rhinosinusitis characterized by a mucosal inflammation that leads to mucosal thickening and polyp formation, and can be especially difficult to treat. Doxycycline is an oral, available, broad-spectrum bacteriostatic antibiotic which also has anti-inflammatory action, that has been used to treat this disease and has shown a successful control of symptoms even reducing the volume of polyps. Objective: Evaluate if long-term low-dose doxycycline is effective in controlling clinical symptoms of difficult-to-treat chronic rhinosinusitis with nasal polyps. Methods: This was a prospective, open-label study of 60 patients with difficultto- treat chronic rhinosinusitis with nasal polyps who had undergone endoscopic sinus surgery. Patients were divided into two groups: 28 received nasal steroids, saline irrigation, and doxycycline (200 mg on the first day, followed by 100 mg daily) for 12 weeks, while 30 received only nasal steroids and saline irrigation. The main outcome measure was an adequate effect size of doxycycline treatment on clinically meaningful significant improvement of SNOT-20. Other outcome measures were the SNOT-20, NOSE, and Lund-Kennedy scores. The following parameters were also analyzed: asthma, rhinitis, non-steroidal-exacerbated respiratory disease (NERD), and baseline serum IgG, IgA, IgE, IgM, ANCA, and eosinophil count. Results: There was an adequate effect size of doxycycline treatment on clinically meaningful significant improvement of SNOT-20. Patients who received doxycycline also had significantly better outcomes regarding SNOT-20, NOSE, and Lund-Kennedy scores. There was a negative association among a clinically significant improvement of SNOT-20 and presence of asthma, NERD, and elevated serum IgE levels before treatment. Conclusion: These findings suggest that doxycycline may have a beneficial role for chronic rhinosinusitis with nasal polyps patients, especially for those without asthma, NERD or high levels of serum IgE before treatment

As proteínas de classe 3 são candidatas na preparação de uma vacina contra a doença meningocócica. O objetivo deste estudo é determinar a imunogenicidade da proteína de classe 3 purificada da cepa de Neisseria meningitidis do sorogrupo B juntamente com a capacidade adjuvante de whole cells de Bordetella pertussis. Foram imunizados camundongos BALB/c neonatos em um intervalo de 3 a 12 dias entre 1 e 4 doses da proteína de classe 3 mais adjuvante, pela via intranasal e no 21º dia pela via intramuscular com a proteína de classe 3 emulsificada com hidróxido de alumínio. Os resultados demonstraram que após 2 doses pela via intranasal e 1 dose pela via intramuscular houve rápido estímulo das células imunes nos camundongos adultos BALB/c e neonatos BALB/c e outbred. Todos os soros foram analisados por ELISA e immunoblot. O adjuvante B. pertussis administrado pelas vias intranasal ou intramuscular, aumentou a resposta imune comparada com os controles. Anticorpos bactericidas e de alta afinidade foram produzidos. / Proteins of class 3 sound candidates in the preparation of vaccine against meningococcal illness. The aim of this study was to determine the immunogenicity of class 3 proteins purified of Neisseria meningitidis of the serogroup B along with whole cells of Bordetella pertussis as adjuvant. BALB/c and outbred neonate mice between 3 and 12 days old were immunized with 1 to 4 doses of the purified class 3 proteins with or without adjuvant given by the intranasal route, and on the 21st day the animals received an intramuscular dose of the class 3 proteins with or without aluminum hydroxide. The results demonstrated that after 2 doses by the intranasal route and 1 dose intramuscular there was a rapid stimulation of the immune cells in BALB/c adult mice as well as BALB/c and outbred neonates mice. All sera were analyzed by ELISA and immunoblot. The adjuvant B. pertussis used in the present investigation and given via the intranasal or intramuscular route increased the immune response compared with the controls. High affinity and bactericidal antibodies were produced.

Die antioxidativen Enzyme Katalase (KAT), Glutathion-Peroxidase (GPX), Glutathion-Reduktase (GR), Superoxid-Dismutase (SOD) und Glutathion-S-Transferase (GST) sind an der intrazellulären Abwehr von oxidativem Stress beteiligt. Diverse Arbeitsgruppen fanden eine Hochregulation der antioxidativen Enzyme (AOEs) nach Exposition auf Ozon. In der vorliegenden Studie sollte an einem von Schierhorn und Mitarbeitern entwickelten Kulturmodell der nasalen Mukosa des Menschen untersucht werden, ob Aktivitätsänderungen der AOEs nach Ozonexposition in vitro zu verzeichnen sind. Zu diesem Zweck wurde die nasale Mukosa von 67 Patienten, die sich wegen nasaler Atmungsbehinderung einer Conchotomie unterzogen hatten, 24 Stunden bei 37 °C und 5% CO2 kultiviert und parallel unter den selben Bedingungen einer zusätzlichen Ozonkonzentration von 120 ppb ausgesetzt. Tendenzielle Aktivitätsänderungen durch Ozon ließen sich bei der GPX (13.8 auf 17.7 mU/mg Protein, 28% Steigerung) und der SOD (8.4 auf 9.7 U/mg Protein, 15% Steigerung) feststellen. Diese Aktivitätszunahmen wiesen jedoch keine Signifikanz auf. Aktivtätsänderungen bei KAT, GR und GST durch die Ozonexposition wurden nicht gefunden. Alter der Patienten, Geschlecht und Zigarettenrauchen nahmen den Ergebnissen dieser Studie nach keinen Einfluß auf die Regulation der AOEs nach Ozonexposition. Die Deletion der Glutathion-S-Transferase M1, die bei etwa 50% der mitteleuropäischen Bevölkerung zu finden ist, veränderte die Regulation der SOD nach in vitro Ozonexposition. Die GST-defizienten Patienten dieser Studie beantworteten die Ozonexposition mit einer signifikanten Hochregulation der SOD (p / Antioxidant enzymes as catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD) and glutathione S-transferase (GST) are thought the primary cellular defense mechanism against reactive oxygen species. Ozone, a highly reactive oxidant, is known to cause respiratory symptoms at ambiental doses. A number of studies have shown the mucosa of the respiratory tract to be the first target site of ozone toxicity. Other animal studies demonstrated an upregulation of mucosal antioxidant enzymes after ozone exposition. Concerning to the antioxidant defense mechanisms of the human nasal mucosa no studies are found so far. The purpose of this study was to determine if in vitro ozone exposure of human nasal mucosa results in changes in the activity of CAT, GPX, SOD, GST and glutathione reductase (GR). Nasal mucosa from 67 patients was cultivated in a specially designed in vitro organ culture and exposed to 120 ppb ozone for 24 hours. The results were compared with the histamin release which is known to be upregulated from human nasal mucosa after ozone exposition (60-200 ppb).

Made available in DSpace on 2016-04-28T19:33:34Z (GMT). No. of bitstreams: 1 VANESSA CARRASCO SANTOS.pdf: 568112 bytes, checksum: 7d6197c30bcd8602e15b311c046c0af8 (MD5) Previous issue date: 2011-11-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This dissertation is situated in the area of Portuguese Language Description and limited aspects of nasal vowels articulation pre, post and tonic in Brazilian Portuguese. There has been concern in the quantitative and qualitative description of the standardized pronunciation, by TV Globo, the nasal vowels of Brazilian Portuguese. The aim of this work is to contribute to studies of identity of a standard pronunciation of Brazilian Portuguese, in order to provide the "desestrangeiração" students, speakers of other languages who are learning Brazilian Portuguese. It has specific goals for: 1 - The qualitative and quantitative description of the pronunciation of nasal vowels pre-stress; 2 - The qualitative and quantitative description of the pronunciation of nasal vowels post-tonic; 3 - The qualitative and quantitative description of the pronunciation of nasal vowels tonic; The procedure of analysis was descriptive and explanatory materials being collected in the database of pronunciation stander, file this one from Prof. Dr. Regina Celia Pagliuchi da Silveira. The results enabled the formulation of rules for the pronunciation of Brazilian Portuguese idiom: 1-The pre-stress nasal vowels are articulated following the control of the letter; 2-The vowels post-tonic is made by nasal vowel or the vowel reduction or desnasalação; 3-Vowels tonic is made by nasal vowels monotongações or ditongações, depending on the number of syllables in the word, The 4-ditongações preferably are increasing; We conclude that there is a standardized pronunciation for the nasal vowels of Brazilian Portuguese and this is controlled by phonological rules, phonetic and morfofonológicas / Esta dissertação situa-se na área da Descrição de Língua Portuguesa e delimitada aspectos articulatórios das vogais nasais pré, pós e tônicas do português brasileiro. Tem-se por tema, a descrição quantitativa e qualitativa da pronúncia standardizada, pela TV Globo, das vogais nasais do português brasileiro. O objetivo geral deste trabalho é contribuir com os estudos identitários de uma pronúncia standar do português brasileiro, a fim de propiciar a desestrangeiração de alunos, falantes de outras línguas que estão aprendendo o português brasileiro. Tem-se por objetivos específicos: 1- A descrição qualitativa e quantitativa da pronúncia das vogais nasais prétônicas; 2- A descrição qualitativa e quantitativa da pronúncia das vogais nasais pós tônicas; 3- A descrição qualitativa e quantitativa da pronúncia das vogais nasais tônicas; O procedimento de análise foi descritivo e explicativo, sendo os materiais coletados no banco de dados da pronúncia standar, arquivo este da Profª Drª Regina Célia Pagliuchi da Silveira. Os resultados obtidos possibilitaram a formulação de regras para a pronúncia idiomática do português brasileiro: 1-As vogais pré-tônicas nasais são articuladas seguindo o controle da letra; 2-As vogais pós-tônicas são realizadas pela vogal nasal ou pela redução vocálica ou desnasalação; 3-As vogais tônicas são realizadas por vogais nasais monotongações ou ditongações, dependendo do nº de sílabas da palavra; 4-As ditongações preferencialmente são crescentes; Conclui-se que há uma pronúncia standardizada para as vogais nasais do português brasileiro e esta é controlada por regras fonológicas, fonéticas e morfofonológicas

Esta tese investiga a aquisição fonológica variável da nasal velar em coda silábica final (como em living ‘vivendo’ e singing ‘cantando’, por exemplo) por aprendizes brasileiros de inglês como segunda língua. O estudo é motivado pela diferença de status da nasal velar em português (L1, primeira língua) e inglês (L2, segunda língua): enquanto na L1 o segmento é realização fonética resultante de assimilação de ponto de articulação (como em atum, em que a nasal bilabial assimila o traço [+dorsal] da vogal precedente realizando-se como velar), na L2 a nasal velar é fonema (como em sin ‘pecado’ x sing ‘cantar’, realizados com a nasal alveolar e velar, respectivamente). Em algumas comunidades de falantes nativos de inglês, palavras derivadas pelo sufixo {ing} (como read.ing ‘lendo’ e study.ing ‘estudando’, por exemplo) constituem um fenômeno de variação linguística estável, em que os falantes alternam a produção das nasais velar e alveolar no final das palavras. Essa variação é estrutural e socialmente condicionada (LABOV, 1994; 2001). Os dados empíricos deste estudo provêm da fala coletada através de gravações com aprendizes de inglês, que pertecem a dois níveis de proficiência: básico e pré-intermediário. São realizadas duas análises formais: uma que explica a variação sistemática na interlíngua, observável no desempenho dos aprendizes, através do software Goldvarb (SANKOFF, TAGLIAMONTE e SMITH, 2015), e outra que formaliza a organização interna dessa gramática variável, através de algoritmos de aprendizagem vinculados à Teoria da Otimidade Estocástica, o Algoritmo de Aprendizagem Gradual (GLA) (BOERSMA e HAYES, 2001) e o ORTO Ajuste Paramétrico (DORNELLES FILHO, 2014). A Análise de Regra Variável demonstrou que a interlíngua é um sistema linguístico sujeito à variação ordenada como as demais línguas naturais, condicionada por aspectos linguísticos (classe morfológica) e extralinguísticos (nível de proficiência). A variação é verificada pela produção oral dos aprendizes, examinada de oitiva, que alterna as nasais palatal (63,6%) e velar (36,4%) em coda silábica final. A análise estocástica revelou um sistema de interlíngua dominado por restrições de Marcação, cuja variação é decorrente do aumento dos valores de ponto de seleção de restrições de Fidelidade. As restrições diretamente envolvidas na aquisição fonológica variável da nasal velar são AGREEplaceVN#, que exige que a sequência Vogal+Nasal em coda final partilhem ponto de articulação, e IDENTnasal, que requer identidade de traço nasal entre as formas presentes no input e no output. As análises realizadas, que representam a língua dos aprendizes em seus aspectos interno e externo, ou, conforme a terminologia de Chomsky ([1965]1 1975), a competência e o desempenho linguísticos, comprovaram a natureza variável da interlíngua e proporcionaram uma reflexão teórica acerca da possibilidade de diálogo entre o estrutural e o social para a explicação de fenômenos linguísticos variáveis. / This thesis investigates the variable phonological acquisition of the velar nasal in final syllabic coda (such as in living ‘vivendo’ and singing ‘cantando’, for example) by Brazilian learners of English as a second language. The study is motivated by the different status of the velar nasal in Portuguese (L1, first language) and in English (L2, second language): while the segment is a phonetic realization of assimilation of place of articulation in the L1 (‘atum’, where the bilabial nasal assimilates the feature [+dorsal] of the preceding vowel, being realized as velar), the velar nasal is a phoneme in the L2 (sin ‘pecado’ x sing ‘cantar’, realized as the alveolar and velar nasal, respectively). In some native English-speaking communities, words derived from the suffix {ing} (such as read.ing ‘lendo’, study.ing ‘estudando’, for example) are in linguistic stable variation, in which the speakers alternate the production of the alveolar and velar nasal the velar nasal at the end of words. This variation is both structurally and socially conditioned (LABOV, 1994; 2001). The empirical data of this study were collected through recordings with the informants, who belong to two levels of proficiency: basic and pre-intermediate. Two formal analysis are made: one that explains the observable systematic variation in the Interlanguage, through the software Goldvarb (SANKOFF, TAGLIAMONTE and SMITH, 2015) and another that formalizes the internal organization of this variable grammar, through gradual learning algorithms associated to the Stochastic Optimality Theory, the Gradual Learning Algorithm (GLA) (BOERSMA and HAYES, 2001) and the ORTO Ajuste Paramétrico (DORNELLES FILHO, 2014). The Variable Rule Analysis has demonstrated that interlanguage is a linguistic system subject to ordered variation as in all the other natural languages, conditioned by linguistic aspects (morphological class) and extralinguistic ones (level of proficiency). The variation is verified by the oral production of the learners, examined by hearing analysis, which alternates the palatal nasal (63,6%) The stochastic analysis has revealed an Interlanguage system dominated by Markedness constraints, where variation is due to the increase on the values of selection points for Faithfulness constraints. The constraints directly involved in the variable phonological acquisition of the velar nasal are AGREEplaceVN#, which demands the sequence Vowel+Nasal in final coda to share place of articulation, and IDENTnasal, which demands feature identity [+nasal] between input and output forms. The analyses made, which represent the learners’ language in its internal and external aspects, or, according to Chomsky’s terminology ([1965] 1975), the linguistic competence and performance, have proved the variable nature of the Interlanguage and have enabled a theoretical reflection upon the possibility of dialogue between the structural and the social to the explanation of variable linguistic phenomena.

<p>Drug delivery to the nasal and ocular mucosa faces several obstacles. One of these is from the effective clearance mechanisms present in the nose and eye. Polymer gels with suitable rheological properties can facilitate the absorption of poorly absorbed drugs by increasing the contact time of the drug with the mucosa. This has been attributed to the rheological and mucoadhesive properties of the gel. The main objective of this thesis was to investigate the importance of these features for the anticipated in vivo contact time, here exemplified by the ocular and nasal routes of administration.</p><p>The in situ gelling polymer gellan gum was found to have a favourable rheological and in vivo performance. When administered in the nasal cavity of rats, a gel was formed that could remain at the site of administration for up to 4 hours. In addition, the epithelial uptake and transfer of a 3 kDa fluorescein dextran was higher than for a mannitol solution. Therefore, it was concluded that a gellan gum formulation should be a promising strategy for nasal drug delivery.</p><p>The potential mucoadhesive properties of a variety of polymer gels were investigated using a rheological method and by measuring the tensile force required to detach the gel from a mucosa. With both methods the rheological properties of the gel were a determining factor for the results obtained. The rheological method was found to have several limitations. One of these was that a positive response, interpreted as mucoadhesion, was only seen with weak gels. The tensile method could, in contrast, detect strengthening of the mucus only for strong gels. However, this method reflects the in vivo performance of the gel better than the rheological method.</p><p>Finally, dielectric spectroscopy was explored as a tool for investigating the likelihood of intimate surface contact between the gel and the mucus layer. This novel approach involved determining the ease with which a charged particle can pass the gel-mucus interface layer, and may enable the study of the events at the interface closer to the molecular level, than is possible with the rheological and tensile strength methods.</p>

<p>The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO₂) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO₂) and the reasons for the strikingly different toxicity were investigated. </p><p>A strong bioactivation and protein adduction of 2,6-diClPh-MeSO₂ in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO₂ was observed and the bioactivation in the liver for both chlorinated isomers was negligible. <i>In vitro</i> studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO₂. The 2,6-diClPh-MeSO₂-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated <i>in vivo</i>, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. </p><p>Based on these results, the proposed mechanism for 2,6-diClPh-MeSO₂-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO₂.</p>

Drug delivery to the nasal and ocular mucosa faces several obstacles. One of these is from the effective clearance mechanisms present in the nose and eye. Polymer gels with suitable rheological properties can facilitate the absorption of poorly absorbed drugs by increasing the contact time of the drug with the mucosa. This has been attributed to the rheological and mucoadhesive properties of the gel. The main objective of this thesis was to investigate the importance of these features for the anticipated in vivo contact time, here exemplified by the ocular and nasal routes of administration. The in situ gelling polymer gellan gum was found to have a favourable rheological and in vivo performance. When administered in the nasal cavity of rats, a gel was formed that could remain at the site of administration for up to 4 hours. In addition, the epithelial uptake and transfer of a 3 kDa fluorescein dextran was higher than for a mannitol solution. Therefore, it was concluded that a gellan gum formulation should be a promising strategy for nasal drug delivery. The potential mucoadhesive properties of a variety of polymer gels were investigated using a rheological method and by measuring the tensile force required to detach the gel from a mucosa. With both methods the rheological properties of the gel were a determining factor for the results obtained. The rheological method was found to have several limitations. One of these was that a positive response, interpreted as mucoadhesion, was only seen with weak gels. The tensile method could, in contrast, detect strengthening of the mucus only for strong gels. However, this method reflects the in vivo performance of the gel better than the rheological method. Finally, dielectric spectroscopy was explored as a tool for investigating the likelihood of intimate surface contact between the gel and the mucus layer. This novel approach involved determining the ease with which a charged particle can pass the gel-mucus interface layer, and may enable the study of the events at the interface closer to the molecular level, than is possible with the rheological and tensile strength methods.