Lam Ching-wan. / "May 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 91-101). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

<div>Vocal fold scarring is the fibrotic manifestation of most common pathological voice disorders. Voice disorders lead to direct healthcare costs of over $200 million annually and significantly reduce quality of life for patients. Despite advances in understanding the pathophysiology of vocal fold scarring, effective treatments for scarring and fibrosis remain elusive. The wound-healing cascade associated with vocal fold injury involves complex signaling interactions between cells and their extracellular matrix (ECM), which remain largely unexplored due to the lack of a physiologically relevant preclinical model to study them. Traditional preclinical models do not capture the complex 3D microenvironment of the vocal folds, and the use of stem cells or fibroblasts alone in models has resulted in poor reproducibility and predictability of in vitro models. Toward this end, this work describes the development of a preclinical model that strives to take into account cellular interactions between fibroblasts and epithelial cells and achieve a balance in the native vocal fold 3D environment to function as an in vitro model.</div><div><br></div><div>Since a major shortcoming of current in vitro models is the lack of a standardized epithelial fibroblast coculture, initial work focused on developing a coculture system between commercially available tracheal epithelial cells and vocal fold fibroblasts in an in vitro setting that would provide more accurate information about the disease pathophysiology and help design better targeted treatments. We designed a healthy and disease state coculture model that can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. We also demonstrated that both cell types maintained phenotype in the healthy and disease state coculture models.</div><div><br></div><div>To further transfer this model in a physiologically relevant 3D system, follow-up research characterized 3D matrices to mimic the native ECM of the vocal folds by using natural biomimetic materials found in the vocal folds such as hyaluronic acid, type I collagen, and type III collagen. We hypothesized that the ability to control the viscoelastic and structuralcharacteristics of the scaffold in combination with presenting relevant biological cues to cells will result in a better biomimetic scaffold. This research is expected to lay effective groundwork for developing a functional tissue engineered 3D coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.</div>

Esse estudo tem como objetivo caracterizar a presença de células globosas alveolares ou pneumócitos tipo II e o início da produção de lipoproteína surfactante, em bovinos, correlacionado com a idade gestacional. Para tanto, foram utilizados 28 fetos em idades gestacionais compreendidas entre quatro e oito meses de gestação e dois animais recém - nascido. Após a coleta os fetos foram dissecados, pesados e medidos, utilizando-se o método de Crow - Rump (CR). Para a descrição morfológica, foram coletados fragmentos pulmonares com cerca de 2 cm² cada e, em seguida, foram fixados em Liquido de Bouin e formalina a 10% para microscopia de luz e em glutaraldeído 2,5% para microscopia eletrônica de transmissão. Para o estudo bioquímico, foram feitas incisões na região da traquéia e introdução de cateter e, através deste, solução fisiológica 0,9% e assim obter o lavado pulmonar. O pulmão de fetos com idade gestacional de 4 meses encontravam-se em fase canalicular do desenvolvimento, sem presença de células globosas e nem aparecimento de bandas eletroforéticas compatíveis com presença de proteínas surfactantes. Nos fetos com 5 meses de idade gestacional, foi observado que o pulmão encontra-se em fase de saco terminal, com aparecimento de alvéolo primitivo formado por epitélio cúbico, com áreas formada por células pavimentosas e células globosas . Nas análises de eletroforese não foram identificadas proteínas surfactantes. No pulmão de fetos com 6 meses de idade gestacional, foram observados desenvolvimento na fase de saco terminal com a presença de pneumócitos tipo I e pneumócito tipo II, nesta fase foi possível observar, na análise eletroforética, o aparecimento de bandas em torno de 28 kDa, demonstrando a presença de SP - A, que é a proteína surfactante encontrada em maior quantidade. A partir dos 7 meses de idade gestacional a fase de saco terminal desenvolve-se ainda mais no pulmão fetal com o surgimento de uma vascularização mais intensa, os pneumócitos tipo I estão com aspecto mais pavimentoso e os pneumócitos tipo II, mais globosos. Na análise eletroforética do lavado brônquio - alveolar foram observadas bandas de proteínas surfactantes, com perfil eletroforético semelhante ao animal recém - nascido. No recém - nascido observou-se o pulmão na fase alveolar, com os pneumócitos tipo I e tipo II bem definidos. O perfil eletroforético do lavado brônquio - alveolar desse animal foi igual ao perfil do lavado de um animal adulto, apresentando as mesmas bandas no triplet. / The objective of this study is to characterize the presence of alveolar type II and the beginning of the surfactant lipoprotein production, in bovines, correlated with the gestacional age. For that to happen, 28 embryos in gestacional age, between four and eight months, and two newborn animals had been used. After the collection, the embryos were dismembered, weighed and measured, using the method of Crow - Rump (CR). For the morphologic description, pulmonary fragments of about 2cm2 each were collected. After that, they had been set in Bouin Liquid for light microscopy and in glutaraldeyde 2.5% for electronic microscopy transmission. For the biochemist study , incisions and the introduction of catheter were made in trachea area and, through this, physiological solution 0.9% were introduced, in order to obtain a pulmonary wash. The lung of embryos with gestacional age of 4 months, were found in the canalicular development phase, without presence of globule cells, nor appearance of compatible eletroforetic bands compatible with the presence of surfactant protein. In the 5 months gestacional age embryos, were observed that the lung was in theterminal sac fase, with the presence of primitive alveolus formed by cubical epithelium, with areas formed by pavimented cells and globule cells. In the analyses of eletroforese surfactants, proteins had not been identified. In the lung of embryos at 6 months of gestacional age, was observed development in the phase of terminal sac, with the presence of type I and alveolar type II. In this phase, was possible to observe, in the SDS -PAGE analysis, the appearance of bands around 28 kDa, demonstrating the SP-A presence -, which is the surfactant protein found in greater amount. From 7 months of gestacional age and up, the phase of terminal sac developed even more in the fetal lung, with the sprouting of a more intense vascularization. The alveolar type I had a more pavimented aspect and the alveolar type II, were globules. In the SDS-PAGE analysis of the bronchial - alveolar wash, surfactant protein bands had been observed, with similar profile from the newborn.In the newborn animal, lung in the alveolar phase was observed, with the alveolar type I and II well defined.The profile of the bronchial-alveolar wash of this animal was the same as the profile of the washed one of an adult animal, presenting the same bands in the triplet.

The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.

Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

RSV is the most common cause of infant bronchiolitis, leading to morbidity and mortality in both infants and the elderly. The relationship between RSV and asthma development further highlights the need to fully understand the immune responses involved in order to develop effective vaccines and therapeutics to aid prevention and treatment of RSV infection respectively. Helminths have long been studied both as a major pathogen of humans, infecting approximately 3 billion people worldwide, and also their ability to modulate the host immune response to allow survival and chronic infection to ensue. Specifically, helminth infections are thought to modulate the host immune response through regulatory mechanisms which are not fully understood. This not only confers protection and survival of the parasites themselves, but also modulates the immune response to unrelated antigens and pathogens. In this thesis, the potential role of a strictly enteric helminth infection, with Heligmosomoides polygyrus, in the modulation of respiratory syncytial virus (RSV) infection was investigated and the associated immune mechanisms were investigated. Firstly, the effects of prior H. polygyrus infection on RSV infection and immune responses in the lung were analysed. H. polygyrus significantly reduced the number of natural killer cells, CD8+ T cells, B cells and conventional dendritic cells in the lung following RSV infection. Co-infection also reduced the production of pro-inflammatory cytokines IL-6 and TNF-α in the lungs. All of these reductions were associated with significantly lower viral titres on day 4 of RSV infection. Interestingly, this attenuation of immune responses and viral titres, correlated with reduced severity of clinical disease, as assessed by weight loss and lung function. H. polygyrus excretory secretory product (HES) was not found to be the immune-modulatory factor in this system, as HES failed to suppress viral titres and reduce immune cell responses to RSV infection. However, irradiated larvae with stunted maturation to adult worms, revealed that larval stages were sufficient to suppress viral titres. Next, the role of type 2 signalling for H. polygyrus effects on RSV infection were examined, using IL-4Rα-/- mice. H. polygyrus infection maintained the ability to attenuate RSV infection and subsequent immune responses in IL-4Rα-/- mice. Furthermore, the presence of the adaptive immune response was not required for H. polygyrus-induced attenuation of RSV infection, as demonstrated in recombinase-activating gene (RAG-/-) deficient mice. H. polygyrus induces innate type 2 immune responses indicating the release of the innate alarmin, IL-33, in the lung and consequently an accumulation of group 2 innate lymphoid cells (ILC2). Their contribution to H. polygyrus effects remain to be fully elucidated. Finally, the role of antiviral responses was explored in H. polygyrus and RSV co-infection. H. polygyrus infection alone induced expression of antiviral genes, IFN-β, OAS1A, Viperin and the antimicrobial peptide CRAMP, in both the duodenum and the lung. Expression of these genes was still higher in the lung 1 hour after RSV in H. polygyrus co-infected mice compared to controls without co-infection. The importance of type I IFN signalling pathway was demonstrated using mice deficient in the type I IFN receptor in H. polygyrus co-infection, which failed to suppress RSV titres and subsequent lung immune cell infiltration. These data highlight the ability of the strictly enteric helminth H. polygyrus to attenuate RSV infection and subsequent immune responses in the lung through the potentiation of type I IFN signalling and consequent upregulation of antiviral immune responses in the lung.

A mucopolissacaridose tipo I (MPS I) é uma doença autossômica recessiva causada pela deficiência da enzima α-L-iduronidade (IDUA). Essa deficiência resulta no acúmulo de glicosaminoglicanos levando a diversas manifestações clínicas. A microencapsulação de células recombinantes que superexpressam IDUA tem sido considerada uma estratégia promissora para o tratamento de MPS I. Neste contexto, o presente estudo teve por objetivo a otimização da encapsulação de células BHK (Baby Hamster Kidney) superexpressando IDUA em microcápsulas de alginato revestidas com poli-L-lisina (PLL) utilizando-se um extrusor eletrostático. Em uma primeira etapa, um estudo de otimização das microcápsulas de alginato (MC-A) foi realizado por meio de um desenho experimental do tipo Box-Behnken (software Mini-Tab®) que permitiu avaliar simultaneamente a influência da voltagem (kV), fluxo alginato/células (mL/h) e concentração de alginato (%) sobre o tamanho das microcápsulas e a atividade de IDUA. Após, as microcápsulas foram revestidas sequencialmente com PLL e alginato (MC-APA) com o objetivo de aumentar a sua estabilidade. Nas condições experimentais empregadas, MC-A e MC-APA apresentaram-se monodispersas (span < 1,22) com um diâmetro médio inferior a 350 μm, determinado por difração a laser. O revestimento alterou a morfologia das microcápsulas (microscopia eletrônica de varredura) e a sua resistência mecânica (analisador de textura), sendo observado um aumento de cerca de 6 vezes na força necessária para compressão das mesmas. O revestimento final pelo alginato (MC-APA) parece ter sido parcial de acordo com as análises de infravermelho por transformada de Fourier com refletância atenuada. Em uma última etapa, a atividade enzimática foi avaliada em modelo murino MPS I após implante subcutâneo de MC-APA. Foi observado um aumento significativo da atividade de IDUA na pele, após 30 dias de tratamento. Nas análises histológicas foi observado um infiltrado inflamatório no local da aplicação que não impediu a liberação da enzima nas condições avaliadas. No seu conjunto, esse estudo demonstra a potencialidade das MC-APA para a liberação local de IDUA. / Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA). This deficiency results in the accumulation of glycosaminoglycans leading to various clinical manifestations. The microencapsulation of recombinant cells overexpressing IDUA has been considered as a promising strategy for the treatment of MPS I. In this context, the present study aimed to optimize the encapsulation of BHK cells overexpressing IDUA in poly-L-lysine (PLL) coated alginate microcapsules using an electrostatic extruder. In a first step, a Box-Behnken experimental design (Mini-Tab® software) was carried out for the optimization of the alginate microcapsules (MC-A), which allowed to evaluate simultaneously the influence of voltage (kV), alginate/cell flow (mL/h) and alginate concentration (%) on the size of the microcapsules and IDUA activity. Thereafter, the microcapsules were sequentially coated with PLL and alginate (MC-APA) in order to increase their stability. In the experimental conditions used, MC-A and MC-APA were monodisperse (span <1.22) with an average diameter of less than 350 μm, determined by laser diffraction. The coating modified microcapsules morphology (scanning electron microscopy) and their mechanical resistance (texture analyzer), being observed a six-fold increase in the required force for their compression. The final alginate coating (MC-APA) appears to have only partially coated the microcapsules, according to the attenuated total reflectance Fourier transform infrared spectroscopy analyses. In a final step, the enzymatic activity was evaluated in a MPS I murine model after subcutaneous implantation of MC-APA. A significant increase in IDUA activity was observed in the skin at 30 days after treatment. Histological analszes revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the evaluated conditions. Overall, this study demonstrates the potentiality of MC-APA for the local release of IDUA.

Lu, Jing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 119-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Trend scoring is often used in large-scale assessments to monitor for rater drift when the same constructed response items are administered in multiple test administrations. In trend scoring, a set of responses from Time A are rescored by raters at Time B. The purpose of this study is to examine the ability of trend-monitoring statistics to detect rater effects in the context of trend scoring. The present study examines the percent of exact agreement and Cohen’s kappa as interrater agreement measures, and the paired t-test and Stuart’s Q as marginal homogeneity measures. Data that contains specific rater effects is simulated under two frameworks: the generalized partial credit model and the latent-class signal detection theory model. The findings indicate that the percent of exact agreement, the paired t-test, and Stuart’s Q showed high Type I error rates under a rescore design in which half of the rescore papers have a uniform score distribution and the other half have a score distribution proportional to the population papers at Time A. All these Type I errors were reduced when using a rescore design in which all rescore papers have a score distribution proportional to the population papers at Time A. For the second rescore design, results indicate that the ability of the percent of exact agreement, Cohen’s kappa, and the paired t-test in detecting various effects varied across items, sample sizes, and type of rater effect. The only statistic that always detected every level of rater effect across items and frameworks was Stuart’s Q. Although advances have been made in the automated scoring field, the fact is that many testing programs require humans to score constructed response items. Previous research indicates that rater effects are common in constructed response scoring. In testing programs that keep trends in data across time, changes in scoring across time confound the measurement of change in student performance. Therefore, the study of methods to ensure rating consistency across time, such as trend scoring, is important and needed to ensure fairness and validity.

The main purpose of this thesis is to compare the performance of the score test and the likelihood ratio test by computing type I errors and type II errors when the tests are applied to the geometric distribution and inflated binomial distribution. We first derive test statistics of the score test and the likelihood ratio test for both distributions. We then use the software package R to perform a simulation to study the behavior of the two tests. We derive the R codes to calculate the two types of error for each distribution. We create lots of samples to approximate the likelihood of type I error and type II error by changing the values of parameters. In the first chapter, we discuss the motivation behind the work presented in this thesis. Also, we introduce the definitions used throughout the paper. In the second chapter, we derive test statistics for the likelihood ratio test and the score test for the geometric distribution. For the score test, we consider the score test using both the observed information matrix and the expected information matrix, and obtain the score test statistic zO and zI . Chapter 3 discusses the likelihood ratio test and the score test for the inflated binomial distribution. The main parameter of interest is w, so p is a nuisance parameter in this case. We derive the likelihood ratio test statistics and the score test statistics to test w. In both tests, the nuisance parameter p is estimated using maximum likelihood estimator pˆ. We also consider the score test using both the observed and the expected information matrices. Chapter 4 focuses on the score test in the inflated binomial distribution. We generate data to follow the zero inflated binomial distribution by using the package R. We plot the graph of the ratio of the two score test statistics for the sample data, zI /zO , in terms of different values of n0, the number of zero values in the sample. In chapter 5, we discuss and compare the use of the score test using two types of information matrices. We perform a simulation study to estimate the two types of errors when applying the test to the geometric distribution and the inflated binomial distribution. We plot the percentage of the two errors by fixing different parameters, such as the probability p and the number of trials m. Finally, we conclude by briefly summarizing the results in chapter 6.