Wang, Shu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 119-139). / Abstracts in English and Chinese. / Table of contents --- p.i / Abstract --- p.v / 摘要 --- p.viii / Acknowledgements --- p.x / List of tables --- p.xi / List of figures --- p.xiii / List of abbreviations --- p.xvi / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Overview of nasal drug delivery --- p.2 / Chapter 1.1.1 --- Structure and permeability of the nasal mucosa --- p.3 / Chapter 1.1.2 --- Pathways of drug permeation across nasal mucosa --- p.6 / Chapter 1.1.3 --- Models for nasal drug permeation studies --- p.7 / Chapter 1.1.3.1 --- In vitro models --- p.7 / Chapter 1.1.3.2 --- In situ models --- p.13 / Chapter 1.1.3.3 --- In vivo animal models --- p.16 / Chapter 1.1.4 --- Factors affecting drug absorption across nasal mucosa --- p.19 / Chapter 1.1.4.1 --- Biological factors --- p.20 / Chapter 1.1.4.2 --- Physicochemical properties of drugs --- p.25 / Chapter 1.1.4.3 --- Formulation factors --- p.29 / Chapter 1.1.5 --- Profile of a suitable drug candidate for nasal delivery --- p.33 / Chapter 1.2 --- Physicochemical properties and human pharmacokinetics of the four drug candidates --- p.35 / Chapter 1.2.1 --- Rizatriptan --- p.35 / Chapter 1.2.2 --- Meloxicam --- p.37 / Chapter 1.2.3 --- Lomoxicam --- p.39 / Chapter 1.2.4 --- Nebivolol --- p.40 / Chapter 1.3 --- Scope of the current study --- p.44 / Chapter Chapter Two. --- Preliminary validation of Calu-3 cell line model as an in vitro model for nasal drug permeation screening --- p.45 / Chapter 2.1 --- Introduction --- p.45 / Chapter 2.2 --- Materials --- p.46 / Chapter 2.2.1 --- Chemicals --- p.46 / Chapter 2.2.2 --- Materials for cell culture --- p.46 / Chapter 2.2.3 --- Instruments --- p.47 / Chapter 2.3 --- Methods --- p.47 / Chapter 2.3.1 --- Cell culture --- p.47 / Chapter 2.3.2 --- Cytotoxicity studies by MTS/PES assay --- p.48 / Chapter 2.3.2.1 --- Optimization of MTS/PES assay for the initial cell seeding density and the incubation time --- p.49 / Chapter 2.3.2.2 --- Cytotoxicity studies of non-physiological pH and osmolarity on Calu-3 cells by MTS/PES assay --- p.49 / Chapter 2.3.3 --- Integrity of Calu-3 cell monolayers --- p.50 / Chapter 2.3.3.1 --- Transepithelial electrical resistance (TEER) --- p.50 / Chapter 2.3.3.2 --- Permeabilities of marker compounds --- p.51 / Chapter 2.3.3.3 --- Effect of osmolarity on the Calu-3 cell monolayers --- p.53 / Chapter 2.3.4 --- Inter-passage variation --- p.53 / Chapter 2.3.5 --- Statistical analysis --- p.54 / Chapter 2.4 --- Results and discussions --- p.54 / Chapter 2.4.1 --- Cell culture --- p.54 / Chapter 2.4.2 --- Cytotoxicity studies by MTS/PES assay --- p.55 / Chapter 2.4.2.1 --- Optimization of MTS/PES assay for the initial cell seeding density and the incubation time --- p.55 / Chapter 2.4.2.2 --- Cytotoxicity studies of non-physiological pH and osmolarity on Calu-3 cells by MTS/PES assay --- p.57 / Chapter 2.4.3 --- Integrity of Calu-3 cell monolayers --- p.58 / Chapter 2.4.3.1 --- Transepithelial electrical resistance (TEER) --- p.59 / Chapter 2.4.3.2 --- Permeabilities of marker compounds --- p.60 / Chapter 2.4.3.3 --- Effect of osmolarity on the Calu-3 cell monolayer --- p.63 / Chapter 2.4.4 --- Inter-passage variation --- p.65 / Chapter 2.5 --- Conclusion --- p.66 / Chapter Chapter Three. --- Permeation studies of selected drug candidates using the Calu-3 cell line model --- p.68 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.2 --- Materials --- p.69 / Chapter 3.2.1 --- Chemicals --- p.69 / Chapter 3.2.2 --- Materials for cell culture --- p.69 / Chapter 3.2.3 --- Instruments --- p.69 / Chapter 3.3 --- Methods --- p.70 / Chapter 3.3.1 --- HPLC assay development and validation for the drug candidates --- p.70 / Chapter 3.3.2 --- Stabilities of the drug candidates in loading solutions at different pHs --- p.71 / Chapter 3.3.3 --- Cell culture --- p.71 / Chapter 3.3.4 --- Cytotoxic effects of the drug candidates on Calu-3 cells by MTS/PES assay --- p.71 / Chapter 3.3.5 --- Permeation studies of drug candidates using Calu-3 cell line model --- p.72 / Chapter 3.3.5.1 --- Effect of concentration on the permeabilities of drug candidates across Calu-3 cell line model --- p.72 / Chapter 3.3.5.2 --- Effect of pH on the permeabilities of drug candidates across Calu-3 cell line model --- p.73 / Chapter 3.3.5.3 --- Effect of osmolarity on the permeabilities of drug candidates across Calu-3 cell line model --- p.73 / Chapter 3.3.6 --- Permeation studies of drug candidates in artificial membrane model at different pHs --- p.73 / Chapter 3.3.7 --- Correlation of the permeabilities of drug candidates between Calu-3 cell line model and artificial membrane model --- p.74 / Chapter 3.3.8 --- Statistical analysis --- p.75 / Chapter 3.4 --- Results and discussions --- p.75 / Chapter 3.4.1 --- HPLC methods for the drug candidates --- p.75 / Chapter 3.4.2 --- Stabilities of the drug candidates in loading solutions at different pHs --- p.75 / Chapter 3.4.3 --- Cytotoxic effects of the drug candidates on Calu-3 cells by MTS/PES assay --- p.76 / Chapter 3.4.4 --- Permeation studies of drug candidates in Calu-3 cell line model --- p.81 / Chapter 3.4.4.1 --- Effect of concentration on the permeabilities of drug candidates across Calu-3 cell line model --- p.81 / Chapter 3.4.4.2 --- Effect of pH on the permeabilities of drug candidates across Calu-3 cell line model --- p.84 / Chapter 3.4.4.3 --- Effect of osmolarity on the permeabilities of drug candidates across Calu-3 cell line model --- p.87 / Chapter 3.4.5 --- Permeation studies of drug candidates in artificial membrane model at different pHs --- p.88 / Chapter 3.4.6 --- Correlation of the permeabilities of drug candidates between Calu-3 cell line model and the artificial membrane model --- p.92 / Chapter 3.5 --- Selection of drug candidate for further in vivo studies --- p.93 / Chapter 3.6 --- Conclusion --- p.93 / Chapter Chapter Four. --- In vivo absorption studies of the most promising drug candidate --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials --- p.96 / Chapter 4.2.1 --- Chemicals --- p.96 / Chapter 4.2.2 --- Instruments --- p.96 / Chapter 4.3 --- Methods --- p.97 / Chapter 4.3.1 --- HPLC conditions --- p.97 / Chapter 4.3.2 --- Preparation of standard solutions --- p.97 / Chapter 4.3.3 --- Calibration curves --- p.98 / Chapter 4.3.4 --- Sample preparations --- p.98 / Chapter 4.3.5 --- Validation of the assay method --- p.98 / Chapter 4.3.5.1 --- Specificity --- p.98 / Chapter 4.3.5.2 --- Precision and accuracy --- p.99 / Chapter 4.3.5.3 --- Recovery --- p.99 / Chapter 4.3.5.4 --- Sensitivity --- p.99 / Chapter 4.3.5.5 --- Stability --- p.99 / Chapter 4.3.6 --- Animals --- p.100 / Chapter 4.3.7 --- Drug administration --- p.102 / Chapter 4.3.8 --- Data analysis --- p.102 / Chapter 4.4 --- Results and discussions --- p.103 / Chapter 4.4.1 --- Validation of the assay method --- p.103 / Chapter 4.4.1.1 --- Specificity --- p.103 / Chapter 4.4.1.2 --- "Precision, accuracy and linearity" --- p.105 / Chapter 4.4.1.3 --- Recovery --- p.106 / Chapter 4.4.1.4 --- Sensitivity --- p.107 / Chapter 4.4.1.5 --- Stability --- p.108 / Chapter 4.4.2 --- "In vivo absorption studies through the nasal, intravenous and oral routes in rat model" --- p.108 / Chapter 4.4.3 --- Preliminary correlation between permeabilities of compounds in Calu-3 cell line model and their nasal bioavailabilities in animal models --- p.111 / Chapter 4.5 --- Conclusion --- p.113 / Chapter Chapter Five. --- Overall conclusion --- p.114 / Chapter Chapter Six. --- Future studies --- p.117 / References --- p.119

Le virus de la grippe infecte les muqueuses du tractus respiratoire. Un vaccin intranasal induit une réponse immunitaire proche de celle faisant suite à une infection naturelle en bloquant le virus directement sur le site de l'infection et permet une vaccination sans aiguille. Par ailleurs, les vaccins à base de pseudo-particules virales ou Virus-like Particles (VLP) produites sur cellules représentent une alternative intéressante au vaccin classique produit sur oeufs. Les VLP sont des particules non-réplicatives qui ressemblent au virus et qui peuvent être immunogènes même sans adjuvant, en particulier par voie intranasale. Au cours de ma thèse, une plateforme de production de VLP grippales composées d'hémagglutinine, de neuraminidase et de protéine de matrice M1 a été développée par transfection transitoire des cellules de mammifères. Des immunisations de souris BALB/c ont montré que les VLP de type A et B, purifiées et caractérisées, étaient immunogènes à de faibles doses par voie intramusculaire. L'administration par voie intranasale de VLP avec la sous-unité B de la toxine cholérique, comme adjuvant muqueux, a permis d'obtenir des taux d'anticorps sériques comparables à ceux obtenus par immunisation en intramusculaire mais également une forte réponse IgA au niveau des muqueuses. Par ailleurs, le rendement des VLP s'est révélé souche-dépendant et lié aux protéines HA et NA à la surface de la particule. Pour contourner ce problème, un vaccin quadrivalent composé de deux VLP bivalentes exprimant chacune deux HA et NA différentes à la surface a été produit montrant ainsi la flexibilité de cette plateforme / The influenza virus infects the mucous membranes of the respiratory tract. An intranasal vaccine induces an immune response close to the one induced by the natural infection by blocking the virus directly at the site of infection and allows needle-free vaccination. In addition, vaccines based on Virus-like Particles (VLP) produced in cells represent an interesting alternative to the traditional egg-based vaccine. VLPs are non-replicative particles that mimic the virus. Studies on influenza VLPs have shown protection by the intranasal route without adding an adjuvant. During my thesis, a platform for the production of influenza VLPs composed of the hemagglutinin, the neuraminidase and the M1 matrix proteins was developed by transient transfection of mammalian cells. Immunizations of BALB/c mice showed that the purified and characterized type A and B VLPs were immunogenic at low doses by the intramuscular route. The intranasal administration of VLPs with the B subunit of cholera toxin as a mucosal adjuvant resulted in serum antibody levels comparable to those obtained by intramuscular immunization but also a strong IgA response in the mucosal secretions. In addition, VLP yield was found to be strain-dependent and linked to the HA and NA proteins on the surface of the particle. To overcome this problem, a quadrivalent vaccine based on two bivalent VLPs each expressing two different HAs and NAs at the surface was produced, demonstrating the flexibility of this platform

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No. of bitstreams: 2 Dissertação - Joji Sado Filho - 2017.pdf: 5994690 bytes, checksum: 2a60faf14b700ecba0cd47970099e8f8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-13 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / During dental treatment of children, basic or advanced behavior guidance techniques can be used. Advanced techniques, non-pharmacological (protective stabilization) or pharmacological (sedation and general anesthesia), are indicated when there is no success with the basic techniques. Regarding advanced techniques, sedation has been increasingly widespread in pediatric dentistry, although the evidence is still weak about which sedative regime provides the greatest comfort and the lowest risk. In view of this need to identify the best sedative regimen, the objective of this randomized, masked, controlled, parallel-design trial was to evaluate the effectiveness of intranasal sedation with Midazolam and Ketamine in behavioral control of preschool children undergoing dental treatment. Eighty-four preschoolers, aged between 1.5 and 6 years old, with dental caries and non-cooperative behavior in previous dental treatment, were randomized into three groups: (1) Midazolam and Ketamine intranasally (MKI); (2) Midazolam and oral ketamine (MKO); (3) Oral midazolam. In all groups, the sedative was administered by a pediatrician or anesthesiologist, and the children received restorative treatment under local anesthesia and rubber dam isolation. Dental treatment was performed by pediatric dentists who, in the end, classified child behavior through the Frankl scale. The training sessions were videotaped, and the videos were then analyzed by trained and calibrated researchers to assess behavior using the Ohio State University Behavioral Rating Scale (OSUBRS) scale. The need to discontinue care and to use protective stabilization was recorded. The data obtained were organized and analyzed in the software Statistical Package for Social Sciences (SPSS). The statistical analysis involved the description of the data and bivariate tests, considering a level of significance of 5%. Considering as a parameter for sedation success the "positive" or "definitely positive" behavior verified with the Frankl scale, the following rates were observed: MKI - 50.0%; MKO - 64.3% and MO - 28.6%. Success rates differed significantly between MKO compared to MKI and MO (P = 0.03). When comparing the groups in relation to the behavior evaluated by the OSUBRS scale, no statistically significant difference was observed in the frequency of "quiet" (P = 0.22), "movement without crying" (P = 0.69) and "combative (P = 0.30). The groups did not differ in assessing the number of cases in which there was a "quiet" behavior in at least 51.7% (median) of the session duration (P = 0.27) and in the number of cases in which the child was "combative" "In at least 24.5% (median) of the session duration (P = 0.65). The need to suspend treatment (P = 0.69) and use protective stabilization (P = 0.14) did not differ significantly among the three groups. The results of behavior evaluation using the Frankl and OSUBRS scale were strongly correlated (rho = -0.84, P0.01). From these results, it was concluded that the sedative protocol MKO presented a higher success rate in behavioral control when compared to the other groups (MKI and MO). The combination of Midazolam and Ketamine, regardless of route of administration, was more effective in controlling behavior than Midazolam given alone. Therefore, the use of the combination of Midazolam and Ketamine, both orally and intranasally, is an effective alternative for controlling the behavior of non-collaborating preschoolers. / Durante o atendimento odontológico de crianças, podem ser utilizadas técnicas básicas ou avançadas de controle do comportamento. As técnicas avançadas, não farmacológicas (estabilização protetora) ou farmacológicas (sedação e anestesia geral), são indicadas quando não há sucesso com as técnicas básicas. Em relação às técnicas avançadas, a sedação tem sido cada vez mais difundida na odontopediatria, embora as evidências ainda sejam fracas sobre qual regime sedativo proporciona o maior conforto e menor risco. Diante dessa necessidade de se identificar o melhor regime sedativo, o objetivo deste ensaio clínico randomizado, mascarado, controlado e de delineamento paralelo foi avaliar a eficácia da sedação intranasal com Midazolam e Cetamina no controle comportamental de crianças pré-escolares submetidas a atendimento odontológico. Oitenta e quatro pré-escolares, com idade entre 1,5 e 6 anos, com cárie dentária e comportamento não colaborador em atendimentos odontológicos anteriores, foram randomizados em três grupos: (1) Midazolam e Cetamina por via intranasal (MKI); (2) Midazolam e Cetamina por via oral (MKO); (3) Midazolam por via oral. Em todos os grupos, o sedativo foi administrado por um médico pediatra ou anestesiologista, e as crianças receberam tratamento restaurador sob anestesia local e isolamento absoluto. O tratamento odontológico foi realizado por odontopediatras que, ao final, classificaram o comportamento infantil por meio da escala de Frankl. As sessões de atendimento foram filmadas e, posteriormente, os vídeos foram analisados por pesquisadores treinados e calibrados a fim de se avaliar o comportamento usando a escala Ohio State University Behavioral Rating Scale (OSUBRS). A necessidade de suspender o atendimento e de utilizar estabilização protetora foi registrada. Os dados obtidos foram organizados e analisados no software Statistical Package for Social Sciences (SPSS). A análise estatística envolveu a descrição dos dados e testes bivariados, considerando-se um nível de significância de 5%. Considerando-se como parâmetro para sucesso da sedação o comportamento “positivo” ou “definitivamente positivo” verificado com a escala de Frankl, foram observadas as seguintes taxas: MKI – 50,0%; MKO – 64,3% e MO – 28,6%. As taxas de sucesso diferiram significativamente entre MKO em comparação com MKI e MO (P = 0,03). Ao se comparar os grupos em relação ao comportamento avaliado pela escala OSUBRS, não foi observada diferença estatisticamente significativa na frequência de comportamentos “quieto” (P=0,22), “movimento sem choro” (P=0,69) e “combativo” (P=0,30). Os grupos não diferiram ao se avaliar o número de casos em que houve comportamento “quieto” em pelo menos 51,7% (mediana) da duração da sessão (P=0,27) e número de casos em que a criança ficou “combativa” em, pelo menos, 24,5% (mediana) da duração da sessão (P=0,65). A necessidade de suspender o atendimento (P=0,69) e de utilizar estabilização protetora (P=0,14) não diferiu significativamente entre os três grupos. Os resultados da avaliação do comportamento por meio da escala de Frankl e da OSUBRS foram fortemente correlacionados (rho = -0,84, P≤0,01). A partir destes resultados conclui-se que o protocolo sedativo MKO apresentou maior taxa de sucesso no controle comportamental, quando comparado aos outros grupos (MKI e MO). A combinação de Midazolam e Cetamina, independentemente da via de administração, foi mais eficaz para o controle do comportamento que o Midazolam administrado isoladamente. Sendo assim, o uso da associação Midazolam e Cetamina, tanto por via oral, quanto intranasal, é uma alternativa eficaz para o controle do comportamento de pré-escolares não colaboradores.

Bakgrund: Ett vanligt symtom inom ambulanssjukvården är smärta, vilken ofta kan förvärra den smärta som redan upplevs av patienten, då de nödvändiga förflyttningarna genomförs av besättningen till bår och ambulans. Det är sjuksköterskorna i ambulansbesättningen som gör bedömningen huruvida smärtlindring krävs akut eller inte under transporten till sjukhus. Syfte: Syftet med studien var att studera patienters upplevelser av intranasal (i.n) smärtlindring jämfört med intravenös (i.v) smärtlindring inom ambulanssjukvården i Stockholm. Metod: Studien är en empirisk studie med deskriptiv design. Huvudresultat: Studien visade endast signifikant skillnad i området där upplevelsen av oro och/eller rädsla mättes i samband med i.n kontra i.v smärtlindringsmetod. Det var fler som upplevde en negativ aspekt i form av oro och eller rädsla i den grupp som fått i.n smärtlindring. Inga andra signifikanta skillnader i upplevelsen av varken, nöjdhet av smärtlindringens effekt, själva sättet att erhålla läkemedlet eller upplevelse av biverkningar påvisades. En jämförelse mellan de rapporterade biverkningarna visade att illamående var den vanligaste biverkningen och den var vanligast i den grupp som erhållit i.n smärtlindring. Slutsats: Med bakgrund av detta resultat kan man ifrågasätta om administreringssättet är avgörande kring effekten och upplevelsen av positiva eller negativa aspekter av smärtbehandlingen. / Background: A common symptom of ambulance care is pain, which can often exacerbate the pain already experienced by the patient, as the necessary movements are carried out by the crew to the stern and ambulance. It is the nurses in the ambulance crew who assess whether pain relief is required urgently or not during transport to hospitals.   Objective: The purpose of this study was to study patient´s experiences of intranasal (i.n) pain relief compared to intravenous (i.v) pain relief within the ambulance service in Stockholm area.    Method: The study is an empirical study with a descriptive design.   Main results: The study showed that the only significant difference in the area where the experience of anxiety and/or fear was measured in connection with i. n versus i. v. pain relief method. It was more that experienced a negative aspect in the form of anxiety and or fear in the group that received in n pain relief. No other significant differences in the experience of pain relief, satisfaction of neither the ring's power, the way to obtain the medicine or experience side effects was demonstrated. A comparison of the reported showed that the most common side effect was nausea, and it was most common in the group that received in n pain relief.   Conclusion: Based on this result, one can question whether the mode of administration is crucial for the effect and experience of positive or negative aspects of pain management.

A bactéria Neisseria meningitidis é um diplococo Gram-negativo. Dentre os sorogrupos de N. meningitidis, o sorogrupo B é um dos principais causadores da doença meningocócica invasiva (DMI), de ocorrência mundial. Recém-nascidos e crianças são especialmente susceptíveis a este tipo de infecção, uma vez que os seus sistemas imunitários ainda estão em desenvolvimento, assim mais estudos sobre a utilização da imunização materna para a proteção contra esta doença, são necessários. O objetivo desse estudo foi avaliar o potencial imunogênico dos antígenos de membrana externa de N. meningitidis sorogrupo B em camundongos Swiss quanto ao efeito da imunização passiva na prole, além de analisar o efeito de diferentes adjuvantes na produção de anticorpos e transferencia materno-fetal destes. O hidróxido de alumínio (HA) e fragmentos de bicamada de brometo de dioctadecildimetilamônio (DODAB-BF) foram utilizados como adjuvantes junto às vesículas de membrana externa (OMVs) para a imunização de camundongos adultos fêmeas, o que resultou em um aumento dos títulos de anticorpos do isótipo IgG quando comparado a imunização apenas com o antígeno, tanto pela via de imunização subcutânea com booster intramuscular (SC/IM), quanto pela via intranasal com booster subcutâneo (IN/SC). Esses anticorpos foram transferidos para a prole, que apresentou títulos de anticorpos significantes até o período no qual os camundongos apresentavam cinco semanas de vida. A imunização com OMVs + DODAB-BF foi capaz de gerar o reconhecimento específico de um maior número de antígenos que OMVs + HA, nos camundongos adultos fêmeas imunizados pela via SC/IM. A imunização com OMVs + DODAB-BF pela via SC/IM também resultou em anticorpos na prole que foram capazes de reconhecer antígenos proteicos da cepa homóloga e de cepas heterólogas, o que não ocorre quando essa imunização é realizada pela via IN/ SC. Apesar da presença de títulos de anticorpos na prole, estes irão apresentar funcionalidade diferente em comparação aos anticorpos da prole de mães imunizadas pela via SC/IM, sugerindo que a via SC/IM é melhor para a imunização materna com OMVs + DODAB-BF. / The Neisseria meningitidis bacterium is a Gram-negative diplococcus. Among the serogroups of N. meningitidis, serogroup B is one of the main causes of invasive meningococcal disease (IMD), worldwide occurrence. Newborns and children are particularly susceptible to this type of infection, since their immune systems are still developing, so further studies about the use of the maternal immunization to protect against this disease, are needed. The aim of this study was to evaluate the potential immunogenic antigens from outer membrane of N. meningitidis serogroup B in Swiss mice as to the effect of passive immunization in the offspring, in addition to analyze the effect of different adjuvants in the production of antibodies and maternal-fetal transfer of these. The aluminium hydroxide (HA) and bilayer fragments from dioctadecyldimethylammonium bromide (DODAB-BF) were used as adjuvants with the outer membrane vesicles (OMVs) for immunization of adult female mice, which resulted in an increase of the antibodies titers of IgG isotype when compared to immunization with only the antigen, either by subcutaneous route of immunization with intramuscular booster (SC/IM), as by the intranasal route with subcutaneous booster (IN/SC). These antibodies were transferred to the offspring, which presented significant antibodies titers to the period in which the mice had five weeks of live. Immunization with OMVs + DODAB-BF was able to generate the specific recognition of a greater number of antigens than OMVs + HA, in adult female mice immunized by SC/IM route. Immunization with OMVs + DODAB-BF SC/IM route also resulted in antibodies in offspring that were able to recognize protein antigens of the homologous strain and heterologous strains, which does not occur when this immunization is carried out by IN/SC route. Despite the presence of antibodies in the offspring, they will present different functionality compared to antibodies of the offspring of mothers immunized by SC/IM route, suggesting that the SC/IM route is better for maternal immunization with OMVs + DODAB-BF.

Este trabalho teve por objetivo avaliar as alterações respiratórias e neurológicas resultantes da infecção por via intranasal das diferentes estirpes nacionais do herpesvírus equino tipo 1 (EHV-1) em hamsters comparando sua susceptibilidade com estudos sobre infecção do EHV-1 em camundongos e equinos. Para isso, hamsters sírios machos, três semanas de idade, foram infectados por via intranasal com as estirpes do EHV-1 obtidas a partir de fetos abortados e potro neonato infectados (A4/72, A9/92, A3/97 e Iso/72). Os animais foram pesados e examinados diariamente em busca de sinais clínicos e neurológicos. Conforme os sintomas neurológicos apareceram, grupos de cinco hamsters foram eutanasiados por overdose de isoflurano na primeira etapa do projeto, e, de cetamina/xilazina por via intraperitoneal na segunda etapa. Na necropsia, sistema nervoso central (SNC), pulmão, fígado, baço e timo foram coletados para isolamento viral em cultura de células E-dermal e para análise histopatológica. Na segunda etapa do projeto, a lavagem broncoalveolar (LBA) com 3 ml de PBS por hamsters foi realizada para determinar a resposta inflamatória pulmonar através da contagem total e diferencial de leucócitos. Similar aos experimentos com camundongos, hamsters desafiados com as estirpes virais A4/72 e A9/92 apresentaram manifestações clínicas severas no terceiro dia pós-inoculação (dpi), tais como perda de peso aguda, dispnéia, desidratação, decúbito e morte. Observou-se também sinais neurológicos como hiperexcitabilidade, paralisia, espasmos, perda de propriocepção, andar em círculos e convulsões. Ao contrário dos camundongos, que não desenvolveram a doença; hamsters inoculados com as estirpes virais A3/97 e Iso/72 manifestaram sintomas respiratórios e neurológicos agudos entre quarto e quinto dpi, sendo as alterações respiratórias as mais evidentes, principalmente hemoptise e conjuntivite. O isolamento do vírus a partir do SNC foi positivo em todos os animais infectados; no entanto, todas as amostras de pulmões foram positivas apenas nos grupos infectado pelas estirpes virais A9/92 e A4/72. Todas as estirpes do EHV-1 foram isoladas a partir do baço, no entanto, a partir do timo foram isoladas apenas as estirpes A9/92 e A4/72, e, de fígado somente a estirpe viral Iso/72 não foi isolada. No LBA, a contagem total de leucócitos não demonstrou diferenças de valores significativas entre os grupos experimentais, sendo apenas evidente a presença de eritrócitos, macrófagos e neutrófilos na maioria dos esfregaços dos hamsters infectados. Porém, na contagem diferencial de leucócitos observou-se um aumento significativo de neutrófilo com consequente diminuição significativa de macrófagos nos hamsters infectados pelas estirpes virais A3/97, Iso/72 e A4/72 quando comparados ao grupo controle. Os hamsters infectados pela estirpe viral A9/92 mantiveram valores próximos aos do grupo controle. Ao avaliar microscopicamente o SNC, fígado e pulmão dos hamsters infectados foi observado infiltrado inflamatório, necrose e manguito perivascular em SNC; leucocitose e necrose no parênquima hepático com discreta pericolangite e proliferação de ducto biliar; e em pulmão observou inflamação, necrose, edema e hemorragia em bronquíolos e alvéolos. Concluindo, os resultados apontaram o hamster como a espécie mais susceptível à infecção pelo EHV-1 servindo como um modelo experimental complementar para estudos de doenças respiratória e neurológica provocadas por este agente em equinos / This study aimed to evaluate the respiratory and neurological disorders resulting from intranasal infection with different national strains of equine herpesvirus type 1 (EHV-1) in hamsters comparing their susceptibility to studies about EHV-1 infection in mice and horses. Therefore, male Syrian hamsters, three weeks of age, were infected via intransal with EHV-1 strains obtained from aborted fetuses and neonatal foal infected (A4/72, A9/92, A3/97 and Iso/72). The animals were weighed and examined daily for clinical search and neurological signs. As neurological symptoms appeared five hamsters groups were euthanized by isoflurane overdose in the first stage of the project, and ketamine/xylazine via intraperitoneal in the second stage. At necropsy, central nervous system (CNS), lung, liver, spleen and thymus were collected for virus isolation in E-dermal cell culture and histopathological analysis. In the second stage of the project, bronchoalveolar lavage (BAL) with 3 ml of PBS per hamsters was performed to determine the pulmonary inflammatory response through the total and differential leukocyte count. Similar to the experiments with mice, hamsters challenged with viral strains A4/72 and A9/92 had severe clinical manifestations in the third day post-inoculation (dpi), such as acute weight loss, dyspnea, dehydration, recumbency, and death. It was also observed neurological signs such as hyperexcitability, paralysis, spasms, loss of proprioception, circling and convulsions. Unlike mice, which did not develop disease; hamsters inoculated with viral strains A3/97 and Iso/72 showed acute respiratory and neurological symptoms between fourth and fifth dpi, and the most evident respiratory distress was hemoptysis and conjunctivitis. The virus isolation from CNS was positive in all infected animals; however, all lung samples were positive only in groups infected by A9/92 and A4/72 viral strains. All EHV-1 strains were recovered from spleen, however, only A9/92 and A4/72 viral strains were recovered from thymus, but only Iso/72 viral strain was not recovered from liver. BAL total leukocyte count showed no significant differences in values between the experimental groups but the presence of erythrocytes, macrophages and neutrophils were evident in most smears of infected hamsters. However, in the leukocyte differential count it was observed a significant increase in neutrophils with consequent significant reduction of macrophages in hamsters infected by viral strains A3/97, Iso/72 and A9/92 when compared with the control group. Hamsters infected with the A9/92 viral strain maintained values similar to the control group. When evaluating microscopically the CNS, liver and lungs of infected hamsters it was observed inflammatory infiltrate, necrosis and perivascular cuff in CNS; leukocytosis and necrosis in the liver parenchyma with discrete pericholangitis and bile duct proliferation; and inflammation, necrosis, edema and hemorrhage in the bronchioles and alveoli. Concluding, the results showed the hamster as the most susceptible species to EHV-1 infection serving as a complementary experimental model for studies of respiratory and neurological diseases caused by this agent in horses

Este trabalho teve por objetivo avaliar as alterações respiratórias e neurológicas resultantes da infecção por via intranasal das diferentes estirpes nacionais do herpesvírus equino tipo 1 (EHV-1) em hamsters comparando sua susceptibilidade com estudos sobre infecção do EHV-1 em camundongos e equinos. Para isso, hamsters sírios machos, três semanas de idade, foram infectados por via intranasal com as estirpes do EHV-1 obtidas a partir de fetos abortados e potro neonato infectados (A4/72, A9/92, A3/97 e Iso/72). Os animais foram pesados e examinados diariamente em busca de sinais clínicos e neurológicos. Conforme os sintomas neurológicos apareceram, grupos de cinco hamsters foram eutanasiados por overdose de isoflurano na primeira etapa do projeto, e, de cetamina/xilazina por via intraperitoneal na segunda etapa. Na necropsia, sistema nervoso central (SNC), pulmão, fígado, baço e timo foram coletados para isolamento viral em cultura de células E-dermal e para análise histopatológica. Na segunda etapa do projeto, a lavagem broncoalveolar (LBA) com 3 ml de PBS por hamsters foi realizada para determinar a resposta inflamatória pulmonar através da contagem total e diferencial de leucócitos. Similar aos experimentos com camundongos, hamsters desafiados com as estirpes virais A4/72 e A9/92 apresentaram manifestações clínicas severas no terceiro dia pós-inoculação (dpi), tais como perda de peso aguda, dispnéia, desidratação, decúbito e morte. Observou-se também sinais neurológicos como hiperexcitabilidade, paralisia, espasmos, perda de propriocepção, andar em círculos e convulsões. Ao contrário dos camundongos, que não desenvolveram a doença; hamsters inoculados com as estirpes virais A3/97 e Iso/72 manifestaram sintomas respiratórios e neurológicos agudos entre quarto e quinto dpi, sendo as alterações respiratórias as mais evidentes, principalmente hemoptise e conjuntivite. O isolamento do vírus a partir do SNC foi positivo em todos os animais infectados; no entanto, todas as amostras de pulmões foram positivas apenas nos grupos infectado pelas estirpes virais A9/92 e A4/72. Todas as estirpes do EHV-1 foram isoladas a partir do baço, no entanto, a partir do timo foram isoladas apenas as estirpes A9/92 e A4/72, e, de fígado somente a estirpe viral Iso/72 não foi isolada. No LBA, a contagem total de leucócitos não demonstrou diferenças de valores significativas entre os grupos experimentais, sendo apenas evidente a presença de eritrócitos, macrófagos e neutrófilos na maioria dos esfregaços dos hamsters infectados. Porém, na contagem diferencial de leucócitos observou-se um aumento significativo de neutrófilo com consequente diminuição significativa de macrófagos nos hamsters infectados pelas estirpes virais A3/97, Iso/72 e A4/72 quando comparados ao grupo controle. Os hamsters infectados pela estirpe viral A9/92 mantiveram valores próximos aos do grupo controle. Ao avaliar microscopicamente o SNC, fígado e pulmão dos hamsters infectados foi observado infiltrado inflamatório, necrose e manguito perivascular em SNC; leucocitose e necrose no parênquima hepático com discreta pericolangite e proliferação de ducto biliar; e em pulmão observou inflamação, necrose, edema e hemorragia em bronquíolos e alvéolos. Concluindo, os resultados apontaram o hamster como a espécie mais susceptível à infecção pelo EHV-1 servindo como um modelo experimental complementar para estudos de doenças respiratória e neurológica provocadas por este agente em equinos / This study aimed to evaluate the respiratory and neurological disorders resulting from intranasal infection with different national strains of equine herpesvirus type 1 (EHV-1) in hamsters comparing their susceptibility to studies about EHV-1 infection in mice and horses. Therefore, male Syrian hamsters, three weeks of age, were infected via intransal with EHV-1 strains obtained from aborted fetuses and neonatal foal infected (A4/72, A9/92, A3/97 and Iso/72). The animals were weighed and examined daily for clinical search and neurological signs. As neurological symptoms appeared five hamsters groups were euthanized by isoflurane overdose in the first stage of the project, and ketamine/xylazine via intraperitoneal in the second stage. At necropsy, central nervous system (CNS), lung, liver, spleen and thymus were collected for virus isolation in E-dermal cell culture and histopathological analysis. In the second stage of the project, bronchoalveolar lavage (BAL) with 3 ml of PBS per hamsters was performed to determine the pulmonary inflammatory response through the total and differential leukocyte count. Similar to the experiments with mice, hamsters challenged with viral strains A4/72 and A9/92 had severe clinical manifestations in the third day post-inoculation (dpi), such as acute weight loss, dyspnea, dehydration, recumbency, and death. It was also observed neurological signs such as hyperexcitability, paralysis, spasms, loss of proprioception, circling and convulsions. Unlike mice, which did not develop disease; hamsters inoculated with viral strains A3/97 and Iso/72 showed acute respiratory and neurological symptoms between fourth and fifth dpi, and the most evident respiratory distress was hemoptysis and conjunctivitis. The virus isolation from CNS was positive in all infected animals; however, all lung samples were positive only in groups infected by A9/92 and A4/72 viral strains. All EHV-1 strains were recovered from spleen, however, only A9/92 and A4/72 viral strains were recovered from thymus, but only Iso/72 viral strain was not recovered from liver. BAL total leukocyte count showed no significant differences in values between the experimental groups but the presence of erythrocytes, macrophages and neutrophils were evident in most smears of infected hamsters. However, in the leukocyte differential count it was observed a significant increase in neutrophils with consequent significant reduction of macrophages in hamsters infected by viral strains A3/97, Iso/72 and A9/92 when compared with the control group. Hamsters infected with the A9/92 viral strain maintained values similar to the control group. When evaluating microscopically the CNS, liver and lungs of infected hamsters it was observed inflammatory infiltrate, necrosis and perivascular cuff in CNS; leukocytosis and necrosis in the liver parenchyma with discrete pericholangitis and bile duct proliferation; and inflammation, necrosis, edema and hemorrhage in the bronchioles and alveoli. Concluding, the results showed the hamster as the most susceptible species to EHV-1 infection serving as a complementary experimental model for studies of respiratory and neurological diseases caused by this agent in horses

Le glioblastome (GB), tumeur primitive du cerveau, la plus agressive, et la plus fréquente chez l’adulte, présente une prolifération vasculaire importante. Des agents thérapeutiques innovants ciblant à la fois l'angiogenèse et les cellules tumorales sont recherchés, ainsi que des systèmes pour augmenter leur délivrance dans la tumeur cérébrale. Un de ces agents est le sorafénib (SFN), un inhibiteur de tyrosine kinase. Sa mauvaise solubilité aqueuse et ses effets secondaires indésirables limitent son utilisation. Le premier objectif de cette thèse était d'encapsuler cet agent dans des nanocapsules lipidiques (NCL) pour contrer ces inconvénients. Nous avons développé des NCL avec une haute efficacité d'encapsulation du SFN qui inhibaient in vitro l'angiogenèse et la viabilité de la lignée de GB humain U87MG. La délivrance intratumorale de SFN-NCL chez des souris porteuses d’une tumeur intracérébrale U87MG induit une normalisation vasculaire tumorale précoce qui pourrait améliorer l'efficacité de la chimiothérapie et de la radiothérapie. Le second objectif était de définir si la délivrance intranasale de NCL pouvait constituer une voie non-invasive alternative. Nous avons étudié via le transfert d'énergie par résonance de type Förster, le devenir des NCL chargées d’un fluorochrome à travers des monocouches de cellules Calu-3, un modèle de l'épithélium nasal. L'utilisation de NCL augmente le passage du fluorochrome à travers les cellules Calu-3, mais les particules sont rapidement dégradées après leur capture. Ces données mettent en évidence que les NCL sont appropriées pour la délivrance locale du SFN mais doivent être modifiées pour une délivrance intranasale. / Glioblastoma (GB), the most aggressive, and the most frequent primary tumor of the brain in adults, present a prominent vascular proliferation. Innovative therapeutic agents targeting both angiogenesis and tumor cells are urgently required, along with competent systems for their delivery to the brain tumor. One such agent is sorafenib (SFN), a tyrosine kinase inhibitor. However, poor aqueoussolubility and undesirable side effects limit its clinical application. The first objective of this thesis was to encapsulate this drug inside lipid nanocapsules(LNCs) to overcome these drawbacks. We developed LNCs with a high SFN encapsulation efficiency (>90%) that inhibited in vitro angiogenesis and the viability of the human U87MG GB cell line. Intratumoral delivery of SFN-LNCs in mice bearing intracerebral U87MG tumors induced early tumor vascular normalization which could be used to improve the efficacy of chemotherapy and radiotherapy in the treatment of GB. The second objective was to define whether intranasal delivery of LNCs could be an alternative non-invasive route. In this regard, we investigated through Förster resonance energy transfer, the fate of dye-loaded LNCs across Calu-3 cell monolayers, a model of the nasal mucosa. We showed that employment of LNCs dramatically increased the delivery of the dye acrossCalu-3 cell monolayer but they were rapidly degraded after their uptake. These data highlight that LNCs are suitable nanocarriers for the local delivery of SFN but must be redesigned for enhancing their nose-to-brain delivery.

<p>No</p> / Dissertation

"Gaseous nitric oxide, in concentrations between 0 and 20 ppm, is currently being used to treat patients with post-surgical complications and respiratory disorders. Currently available instruments are expensive and have problems that limit their usefulness for this application. This thesis discusses the development of an inexpensive, direct and continuous sensor for the measurement of inhaled nitric oxide. The prototype sensor incorporates a 0.125 cm, gas permeable, flow-thru liquid cell into a probe that can be incorporated into a ventilator circuit. Sensor operation is based on the complexation reaction of NO with cytochrome-c (Fe III), a biologically derived heme. The complex is monitored spectrophotometrically in the visible region of the spectrum at 563 nm by an optical spectrograph card. LabVIEW is used for all hardware control, signal acquisition, data processing, display and storage. The sensor has a sensitivity of 2x10-4 Abs/ppm, where Abs denotes absorbance units, a minimum detectable limit of 1.5 ppm, resolution of 0.5 ppm, is stable over the course of 8 hours, has less than 1 ppm error and a response time of less than 2 minutes. All aspects of sensor design and development will be discussed."