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Tissues arising late in evolutionary time, such as lung alveoli that are unique to air breathing organisms, have been challenging to generate in vitro from pluripotent stem cells (PSCs), in part because there are limited lower organism model systems available to provide the necessary developmental roadmaps to guide in vitro differentiation. Furthermore, pulmonary alveolar epithelial type II cell (AEC2) dysfunction has been implicated as a primary cause of pathogenesis in many poorly understood lung diseases that lack effective therapies, including interstitial lung disease (ILD) and emphysema. Here we report the successful directed differentiation in vitro of human PSCs into AEC2s, the facultative progenitors of lung alveoli. Using gene editing to engineer multicolored fluorescent reporter PSC lines (NKX2-1GFP;SFTPCtdTomato), we track and purify human SFTPC+ alveolar progenitors as they emerge from NKX2-1+ endodermal developmental precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells are able to form monolayered epithelial spheres (“alveolospheres”) in 3D cultures without the need for mesenchymal co-culture support, exhibit extensive self-renewal capacity, and display additional canonical AEC2 functional capacities, including innate immune responsiveness, the production of lamellar bodies able to package surfactant, and the ability to undergo squamous cell differentiation while upregulating type 1 alveolar cell markers. Guided by time-series global transcriptomic profiling we find that AEC2 maturation involves downregulation of Wnt signaling activity, and the highest differentially expressed transcripts in the resulting SFTPC+ cells encode genes associated with lamellar body and surfactant biogenesis. Finally, we apply this novel model system to generate patient-specific AEC2s from induced PSCs (iPSCs) carrying homozygous surfactant mutations (SFTPB121ins2), and we employ footprint-free CRISPR-based gene editing to observe that correction of this genetic lesion restores surfactant processing in the cells responsible for their disease. Thus we provide an approach for disease modeling and future functional regeneration of a cell type unique to air-breathing organisms.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23952号 / 農博第2501号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5387(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This research work is focused on the balanced and partially balanced incomplete block designs when observations within blocks are correlated. The topic for this dissertation was motivated by a problem in pharmaceutical research, when several treatments are allocated to individuals, and repeated measurements are taken on each individual. In that case, there is correlation among the observations taken on the same individual. Typically, it is reasonable to assume that the observations within individual close to each other are highly correlated than observations that are far away from each other. It is also reasonable to assume that the correlation between any two observations within each individual is same. We have characterized balanced and partially balanced incomplete block designs when observations within blocks are autocorrelated. In Chapter 3, we have provided an explicit expression for the average variance of estimated elementary treatment contrasts for designs obtained by Type I and II series of orthogonal arrays, under autocorrelated errors, and compared them with the corresponding balanced incomplete block designs with uncorrelated errors. The relative efficiency of balanced incomplete block design compared to the corresponding balanced incomplete block design obtained by Types I and II series of orthogonal array under autocorrelated errors does not depend on the number of treatments (v) and is an increasing function of the block size (k). When orthogonal arrays of Type I or Type II do not exist for a given number of treatments, we provided alternative partially balanced designs with autocorrelated errors. In Chapter 4, we rearranged the treatments in each block of symmetric balanced incomplete block designs and used them with autocorrelated error structure of the plots in a block. The C-matrix of estimated treatment effects under autocorrelation was given and the relative efficiency of symmetric balanced incomplete block designs with independent errors compared to the autocorrelated designs is given. In Chapter 5, we discussed the compound symmetry correlation structure within blocks. An explicit expression of the average variance of designs obtained by Type I and II series of orthogonal arrays and symmetric balanced incomplete block designs under compound symmetric errors has been provided and compared them with the corresponding balanced incomplete block designs with uncorrelated errors. Finally, the relative efficiencies of these designs with autocorrelated errors vs. compound symmetric error structure are given / Statistics

<p> In this work, optimal censoring schemes are investigated for the non-parametric confidence intervals of population quantiles under progressive Type-II right censoring. The proposed inference can be universally applied to any probability distributions for continuous random variables. By using the interval mass as an optimality criterion, the optimization process is also independent of the actual observed values from a sample as long as the initial sample size n and the number of observations m are predetermined. This study is based on the fact that each (uncensored) order statistic observed from progressive Type-II censoring can be represented as a mixture of underlying ordinary order statistics with exactly known weights [11, 12]. Using several sample sizes combined with various degrees of censoring, the results of the optimization are tabulated here for a wide range of quantiles with selected levels of significance (i.e., α = 0.01, 0.05, 0.10). With the optimality criterion under consideration, the efficiencies of the worst progressive Type-II censoring scheme and ordinary Type-II censoring scheme are also examined in comparison with the best censoring scheme obtained for a given quantile with fixed n and m.</p> / Thesis / Master of Science (MSc)

This dissertation illustrates how to detect the rater centrality effect in a simulation study that approximates data collected in large scale performance assessment settings. It addresses three research questions that: (1) which of several centrality-detection indices are most sensitive to the difference between effect raters and non-effect raters; (2) how accurate (and inaccurate), in terms of Type I error rate and statistical power, each centrality-detection index is in flagging effect raters; and (3) how the features of the data collection design (i.e., the independent variables including the level of centrality strength, the double-scoring rate, and the number of raters and ratees) influence the accuracy of rater classifications by these centrality-detection indices. The results reveal that the measure-residual correlation, the expected-residual correlation, and the standardized deviation of assigned scores perform better than the point-measure correlation. The mean-square fit statistics, traditionally viewed as potential indicators of rater centrality, perform poorly in terms of differentiating central raters from normal raters. Along with the rater slope index, the mean-square fit statistics did not appear to be sensitive to the rater centrality effect. All of these indices provided reasonable protection against Type I errors when all responses were double scored, and that higher statistical power was achieved when responses were 100% double scored in comparison to only 10% being double scored. With a consideration on balancing both Type I error and statistical power, I recommend the measure-residual correlation and the expected-residual correlation for detecting the centrality effect. I suggest using the point-measure correlation only when responses are 100% double scored. The four parameters evaluated in the experimental simulations had different impact on the accuracy of rater classification. The results show that improving the classification accuracy for non-effect raters may come at a cost of reducing the classification accuracy for effect raters. Some simple guidelines for the expected impact of classification accuracy when a higher-order interaction exists summarized from the analyses offer a glimpse of the "pros" and "cons" in adjusting the magnitude of the parameters when we evaluate the impact of the four experimental parameters on the outcomes of rater classification. / Ph. D.

Yes / Type II congenitalLong QT syndrome (LQT2) is due to genetic mutations in hERG channel. Genetic or pharmacological factors could potentially affect hERG channel biogenesis and contributes to LQTS, for example, disease mutations G601S and T473P result in hERG trafficking deficiency [1,2]. Various rescue strategies for hERG dysfuction are being developed. Some correctors for CFTR channel have been reported to act indirectly on proteostasis pathways to promote folding and correction on hERG trafficking deficiency [3]. In this study, we tested the hypothesis that the CFTR corrector KM-11060 and the potentiator PG-01 may correct hERG mutation trafficking diseases. We use HEK293 cell line expressing a well-studied trafficking disease mutation G601S-hERG channel [4]. We treated cells with CFTR potentiator PG-01and corrector KM-11060, which function through different cellular mechanisms, and assessed whether correction occurred via immunoblotting. Whole cell proteins from HEK 293 cells expressing hERG channels were used for analysis [5]. Proteins were separated on 8% SDS-polyacrylamide electrophoresis gels for 1 hour, transferred onto PVDF membrane, and blocked for 1 h with 5% nonfat milk. The blots were incubated with the primary antibody (Santa Cruz Biotechnology) for 12-16 h at 4C temperature and then incubated with a donkey antigoat horseradish peroxidase-conjugated secondary antibody( Santa Cruz Biotechnology). Actin expression was used for loading controls. The blots were visualized using the ECL detection kit (Genshare).Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Our results show that both KM-11060 (5, 10, 20uM) and PG-01(5, 15 uM) can correct G601S mutant alleles of hERG protein trafficking (Fig 1, 2). KM-11060 (20uM) but not PG-01(15 uM) enhance protein expression of wild type hERG channel (Fig 2). Further treatment on cells at low temperature with different drug concentration will be tested. Functional studies are also needed to test whether the drugs can correct the function of hERG mutation channel. These results could potentially provide novel insight into the correction mechanism of CFTR potentiator and also help to develop new treatment for LQT2.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

O estabelecimento de uma resposta TH1 com a produção de IL-12, IFN-gama e de óxido nítrico é crucial no controle do Trypanosoma cruzi, o qual pode colonizar o SNC de crianças e pacientes imunossuprimidos. A inflamação exacerbada em decorrência da persistência de um estímulo antigênico gera o acúmulo de substâncias potencialmente citotóxicas, como mediadores pró-inflamatórios e radicais livres. A partir da infecção de camundongos imunodeficientes (IL-12p40KO) com T. cruzi Sylvio X10/4, avaliamos os danos causados à medula espinal com enfoque na inflamação e neurodegeneração. Além da desmielinização, alta reatividade glial e morte de neurônios no ponto mais tardio da doença, constatamos uma baixa produção de mediadores inflamatórios nas primeiras semanas após a infecção, acompanhada pela proliferação ascendente do parasita no tecido nervoso. Acreditamos que um atraso na produção de IFN-gama seja responsável pela ativação tardia ou ineficiente dos fagócitos da medula espinal, favorecendo a disseminação descontrolada do protozoário e subsequentes danos teciduais. / The establishment of a TH1 response with IL-12, IFN-gamma and nitric oxide production is crucial for controlling the proliferation of Trypanosoma cruzi, which may colonize the CNS of children and immunosuppressed hosts. The exacerbated inflammation due to the persistence of an antigenic stimulus results on the accumulation of potentially cytotoxic substances, such as pro-inflammatory mediators and free radicals. By the infection of immunodeficient mice (IL-12p40KO) with T. cruzi Sylvio X10/4 parasites we evaluated the spinal cord damages, focusing on the inflammation and neurodegeneration. Besides demyelization, high glial reactivity and neuron death at the latest stage of the disease, we noticed low production of inflammatory mediators during the first weeks of the infection, accompanied by an ascendant parasite proliferation in the nervous tissue. We believe that a delay on IFN-gamma production is responsible for the late or inefficient phagocyte activation in the spinal cord, contributing to the uncontrolled protozoan proliferation and subsequent tissue injury.