RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosomal biogenesis, and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of some of the most fundamental processes in mammalian cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Dry age-related macular degeneration (AMD) is a progressive neurodegenerative disorder characterized by geographical atrophy of the retinal pigment epithelium (RPE), causing irreversible central vision loss. Systemically-delivered bone marrow-derived cells (BMDCs), programmed to RPE-like cells via expression of human RPE65, regenerate damaged RPE and preserve vision in murine models of retinal degeneration. RPE65 rapidly activates adenylate cyclase (AC), which then activates endogenous Rpe65 and RPE-associated marker Cralbp. Previous studies expressed RPE65 from an integrating lentiviral vector (ILV), which is an unnecessary safety risk due to the potential for insertional mutagenesis, as long- term expression of RPE65 is not required for BMDC programming. Here, we developed a 3rd generation integrase-defective lentiviral vector (IDLV) for programming both murine and human BMDCs to RPE-like cells, reducing insertional mutagenesis risk and expanding the protocol to include human cells. We enhanced IDLV3-RPE65 infection of murine and human BMDCs by preloading concentrated vector on RetroNectin at MOI 50, and infecting with low-speed centrifugation, increasing RPE65 mRNA levels from ~12-fold to ~25-fold (p<0.05). IDLV3-RPE65 infection initiates expression of endogenous Rpe65 mRNA expression in murine BMDC and Cralbp/CRALBP mRNA in both murine and human BMDCs, indicating programming to RPE-like cells. Inhibiting AC in RPE65infected BMDCs abrogated expression of the endogenous genes, confirming the role of AC activation in programming. Critically, IDLV3-RPE65-infected murine BMDCs are recruited to and incorporate into to the RPE layer, and preserve vision in murine models of retinal degeneration. We conclude that BMDCs programmed with IDLV3-RPE65 successfully prevent retinal degeneration progression and are appropriate for testing in human cells, with a view to move into human clinical trial for the treatment of dry AMD. This approach significantly increases the safety of the therapy and is, to the best of our knowledge, the first application of a single IDLV in the generation of therapeutic cells from adult stem cells.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12843号 / 論医博第2083号 / 新制||医||1006(附属図書館) / 31426 / (主査)教授 朝長 啓造, 教授 小柳 義夫, 教授 小川 誠司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Identification of an appropriate material capable of enamel remineralization to fill a cavity after the removal of dental tissue is a new field of dental research. Elucidation of the associated factors involved in enamel defects remain in question including the mechanisms of enamel hypoplasia as well as improvement needed in our current treatment models. The purpose of this thesis is to evaluate the factors associated with enamel defects, to understand the mechanism by which they evolve, as well as look into a new method of treatment called, fluorapatite cement. This new treatment has shown promising affects to improve our current treatment to restore enamel defects, however, to find the best treatment we must understand the foundations of enamel and how defects arise. Enamel is the strongest tissue in the body because it must maintain resistance to factors such as bacterial adhesion, acidity, and temperature. Enamel defects can present as white spots, discoloration, or deep fissures in the enamel due to a disturbance that occurred during tooth oogenesis. Enamel hypoplasia provides favorable conditions for the early development of caries and the retention of plaque, which can progress and reach deep into the enamel and the dentin and cause sensitivity. Our current treatment is efficient and has been used for decades, however, requires the surrounding health enamel to be removed. This is a disadvantage and has encouraged scientists to continue researching for an effective way to remineralize enamel and avoid the loss of healthy enamel. Diseases such as coeliac disease have an influence on the formation of enamel that lead to defects. A recent study investigated if dentists can play a role in early diagnosis of diseases including coeliac disease by examining patients for enamel defects. The study discovered that there was not a significant difference in oral evaluations to determine upon early diagnosis, however, they did conclude that there was pattern in the types of defects that were found in coeliac patients. Specifically, grade I and II defects were found on the anterior teeth of coeliac patients, therefore, the study still recommends seeing the dentist for oral exams as well as supports further study. To understand the mechanism of how defects arise, scientists studied first permanent molars in children during their first three years of life with factors including, institutionalization, gender, medications, and diseases. Studies showed that females have a two-fold risk for enamel defects over males and institutionalized children have a three-to-four-fold risk of enamel defects. To improve our current treatment and avoid loss of healthy enamel and a costly procedure, scientists are looking into the use of hydroxyapatite (HA), a natural occurring mineral in our body, to create a possible new synthetic enamel called fluorapatite (FA) cement. Research analyzed fluorapatite cement compared to natural enamel and found that FA cement has a stronger resistance to acidity with a weight loss of 0.75% wt% compared to enamel with 1.2% and greater resistance to bacterial adhesion than natural enamel by three times. FA cement was also found to have more stability and higher cellular activity than hydroxyapatite. As for safety for dental application, FA cement and HA were placed in simulated body fluid to test for cytotoxicity levels, and none were present. Therefore, FA cement has a promising approach to restore enamel defects effectively and conveniently. Further research is recommended to understand the association of females and institutionalized children with enamel defects versus all children in a larger study group. The current reported results from fluorapatite cement are very promising. This cement can allow an inexpensive and more efficient method of treatment due to its abilities to remineralize enamel that even stronger than natural enamel. The recent hydroxyapatite and fluoride toothpaste products show the growing research that is being put into this idea of creating a synthetic enamel with our current mineral hydroxyapatite. This thesis supports the idea that FA cement has the potential as an alternative to current treatment for enamel defects and hopefully will be ready to undergo a clinical trial in the near future.

Scope and Contents: Virus specific RNA components of the cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis virus were examined. Studies were carried out both in the presence and absence of defective particle interference. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24841号 / 医博第5009号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長, 啓造, 教授 波多野, 悦朗, 教授 竹内, 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The main goal of this thesis is to identify and analyze problematic aspects of rights arising from defective performance in B2B sale. The analysis is set into a complex description of rights arising from defective performance. The work reflects relevant decisions of courts, national, European and international legal regulations and other texts. The thesis is divided into nine chapters including introduction and ending. The chapters are divided systematically in order to complexly introduce rights arising from defective performance to a reader, while problematic aspects of mentioned legal institute are continuously presented to a reader. In such cases those aspects are always analyzed, relevant propositions of a professional public are discussed, the author critically examines them and presents his own propositions and possible interpretation points of departure. After the introductory chapter the author defines criteria, which are deciding for a legal regime of B2B sale. At the beginning of the third chapter the author explains reasons, why he is using term obligations and rights arising from defective performance instead of a traditional term liability for defects. Then this chapter analyzes sole defective performance, a dispositive nature of obligations arising from defective performance, a...

Manufacture testing of digital integrated circuits is essential for high quality. However, exhaustive testing is impractical, and only a small subset of all possible test patterns (or test pattern pairs) may be applied. Thus, it is crucial to choose a subset that detects a high percentage of the defective parts and produces a low defective part level. Historically, test pattern generation has often been seen as a deterministic endeavor. Test sets are generated to deterministically ensure that a large percentage of the targeted faults are detected. However, many real defects do not behave like these faults, and a test set that detects them all may still miss many defects. Unfortunately, modeling all possible defects as faults is impractical. Thus, it is important to fortuitously detect unmodeled defects using high quality test sets. To maximize fortuitous detection, we do not assume a high correlation between faults and actual defects. Instead, we look at the common requirements for all defect detection. We deterministically maximize the observations of the leastobserved sites while randomly exciting the defects that may be present. The resulting decrease in defective part level is estimated using the MPGD model. This dissertation describes the MPGD defective part level model and shows how it can be used to predict defective part levels resulting from static defect detection. Unlike many other predictors, its predictions are a function of site observations, not fault coverage, and thus it is generally more accurate at high fault coverages. Furthermore, its components model the physical realities of site observation and defect excitation, and thus it can be used to give insight into better test generation strategies. Next, we investigate the effect of additional constraints on the fortuitous detection of defects-specifically, as we focus on detecting dynamic defects instead of static ones. We show that the quality of the randomness of excitation becomes increasingly important as defect complexity increases. We introduce a new metric, called excitation balance, to estimate the quality of the excitation, and we show how excitation balance relates to the constant τ in the MPGD model.

Manufacture testing of digital integrated circuits is essential for high quality. However, exhaustive testing is impractical, and only a small subset of all possible test patterns (or test pattern pairs) may be applied. Thus, it is crucial to choose a subset that detects a high percentage of the defective parts and produces a low defective part level. Historically, test pattern generation has often been seen as a deterministic endeavor. Test sets are generated to deterministically ensure that a large percentage of the targeted faults are detected. However, many real defects do not behave like these faults, and a test set that detects them all may still miss many defects. Unfortunately, modeling all possible defects as faults is impractical. Thus, it is important to fortuitously detect unmodeled defects using high quality test sets. To maximize fortuitous detection, we do not assume a high correlation between faults and actual defects. Instead, we look at the common requirements for all defect detection. We deterministically maximize the observations of the leastobserved sites while randomly exciting the defects that may be present. The resulting decrease in defective part level is estimated using the MPGD model. This dissertation describes the MPGD defective part level model and shows how it can be used to predict defective part levels resulting from static defect detection. Unlike many other predictors, its predictions are a function of site observations, not fault coverage, and thus it is generally more accurate at high fault coverages. Furthermore, its components model the physical realities of site observation and defect excitation, and thus it can be used to give insight into better test generation strategies. Next, we investigate the effect of additional constraints on the fortuitous detection of defects-specifically, as we focus on detecting dynamic defects instead of static ones. We show that the quality of the randomness of excitation becomes increasingly important as defect complexity increases. We introduce a new metric, called excitation balance, to estimate the quality of the excitation, and we show how excitation balance relates to the constant τ in the MPGD model.

Consider a large population containing a small number of defective items. A commonly encountered goal is to identify the defective items, for example, to isolate them. In the classical non-adaptive group testing (NAGT) approach, one groups the items into subsets, or pools, and runs tests for the presence of a defective itemon each pool. Using the outcomes the tests, a fundamental goal of group testing is to reliably identify the complete set of defective items with as few tests as possible. In contrast, this thesis studies a non-defective subset identification problem, where the primary goal is to identify a “subset” of “non-defective” items given the test outcomes. The main contributions of this thesis are: We derive upper and lower bounds on the number of nonadaptive group tests required to identify a given number of non-defective items with arbitrarily small probability of incorrect identification as the population size goes to infinity. We show that an impressive reduction in the number of tests is achievable compared to the approach of first identifying all the defective items and then picking the required number of non-defective items from the complement set. For example, in the asymptotic regime with the population size N → ∞, to identify L nondefective items out of a population containing K defective items, when the tests are reliable, our results show that O _ K logK L N _ measurements are sufficient when L ≪ N − K and K is fixed. In contrast, the necessary number of tests using the conventional approach grows with N as O _ K logK log N K_ measurements. Our results are derived using a general sparse signal model, by virtue of which, they are also applicable to other important sparse signal based applications such as compressive sensing. We present a bouquet of computationally efficient and analytically tractable nondefective subset recovery algorithms. By analyzing the probability of error of the algorithms, we obtain bounds on the number of tests required for non-defective subset recovery with arbitrarily small probability of error. By comparing with the information theoretic lower bounds, we show that the upper bounds bounds on the number of tests are order-wise tight up to a log(K) factor, where K is the number of defective items. Our analysis accounts for the impact of both the additive noise (false positives) and dilution noise (false negatives). We also provide extensive simulation results that compare the relative performance of the different algorithms and provide further insights into their practical utility. The proposed algorithms significantly outperform the straightforward approaches of testing items one-by-one, and of first identifying the defective set and then choosing the non-defective items from the complement set, in terms of the number of measurements required to ensure a given success rate. We investigate the use of adaptive group testing in the application of finding a spectrum hole of a specified bandwidth in a given wideband of interest. We propose a group testing based spectrum hole search algorithm that exploits sparsity in the primary spectral occupancy by testing a group of adjacent sub-bands in a single test. This is enabled by a simple and easily implementable sub-Nyquist sampling scheme for signal acquisition by the cognitive radios. Energy-based hypothesis tests are used to provide an occupancy decision over the group of sub-bands, and this forms the basis of the proposed algorithm to find contiguous spectrum holes of a specified bandwidth. We extend this framework to a multistage sensing algorithm that can be employed in a variety of spectrum sensing scenarios, including non-contiguous spectrum hole search. Our analysis allows one to identify the sparsity and SNR regimes where group testing can lead to significantly lower detection delays compared to a conventional bin-by-bin energy detection scheme. We illustrate the performance of the proposed algorithms via Monte Carlo simulations.