Compared to making a city solely out of one building, it is rather important for people to understand what roles a building can play in a city; especially within urban condition. How a building could actually react and respond to certain human perceptions and functions as an element of a city has become a significant issue. From place to place, the city mutates in several different phases, such as usage, terrain, orientation and material. Therefore, to embrace the urban situation and to optimize the space emerged as the main element of the whole thesis. The particular character of the site starts this program as a mix-usage building from the beginning. Whether it is the usage or orientation that mutates along the city, they should finally come to a concord of optimization and solution that represents the city either on the street or in the sky. / Master of Architecture

No description available.

HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.

Mutation testing is a powerful, but resource intense technique for asserting software quality. This report investigates two claims about one of the mutation operators on procedural logic, the relation operator replacement (ROR). The constrained ROR mutant operator is a type of constrained mutation, which targets to lower the number of mutants as a “do smarter” approach, making mutation testing more suitable for industrial use. The findings in the report shows that the hypothesis on subsumption is rejected if mutants are to be detected on function return values. The second hypothesis stating that a test case can only detect a single top-level mutant in a subsumption graph is also rejected. The report presents a comprehensive overview on the domain of mutation testing, displays examples of the masking behaviour previously not described in the field of mutation testing, and discusses the importance of the granularity where the mutants should be detected under execution. The contribution is based on literature survey and experiment. The empirical findings as well as the implications are discussed in this master dissertation.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Pheochromocytomas and paragangliomas are neuroendocrine tumors that arise from neural crest-derived cells of the adrenal medulla and the extra-adrenal paraganglia. They cause hypertension due to an abnormally high production of catecholamines (mainly adrenaline and noradrenaline), with symptoms including recurrent episodes of headache, palpitations and sweating, and an increased risk of cardiovascular disease. Malignancy in the form of distant metastases occurs in 10-15% of the patients. The malignant cases are difficult to predict and cure, and have a poor prognosis. About a third of pheochromocytomas and paragangliomas are caused by hereditary mutations in a growing list of known susceptibility genes. However, the cause of the remaining, sporadic, tumors is still largely unknown. The aim of this thesis project has been to further characterize the genetic background of pheochromocytomas and paragangliomas, with a focus on the sporadic tumors. First, we investigated the role of the genes known from the familial tumors in the sporadic form of the disease. By studying mutations, copy number variations, DNA methylation and gene expression, we found that many of the known susceptibility genes harbor somatic alterations in sporadic pheochromocytomas. Particularly, we found that the NF1 gene, which plays an important role in suppressing cell growth and proliferation by regulating the RASMAPK pathway, was inactivated by mutations in more than 20% of the cases. The mutations occurred together with deletions of the normal allele and were associated with a reduced NF1 gene expression and a specific hormone profile. We also detected activating mutations in the gene EPAS1, which encodes HIF-2α, in a subset of sporadic cases. Microarray analysis of gene expression showed that several genes involved in angiogenesis and cell metabolism were upregulated in EPAS1-mutated tumors, which is in agreement with the role of HIF-2α in the cellular response to hypoxia. In order to comprehensively investigate all the known susceptibility genes in a larger patient cohort, we designed a targeted next-generation sequencing approach and could conclude that it was fast and cost-efficient for genetic testing of pheochromocytomas and paragangliomas. The results showed that about 40% of the sporadic cases had mutations in the tested genes. The majority of the mutations were somatic, but some apparently sporadic cases in fact carried germline mutations. Such knowledge of the genetic background can be of importance to facilitate early detection and correct treatment of pheochromocytomas, paragangliomas and potential co-occurring cancers, and also to identify relatives that might be at risk. By sequencing all the coding regions of the genome, the exome, we then identified recurrent activating mutations in a novel gene, in which mutations have previously only been reported in subgroups of brain tumors. The identified mutations are proposed to cause constitutive activation of the encoded receptor tyrosine kinase, resulting in the activation of downstream kinase signaling pathways that promote cell growth and proliferation. In summary, the studies increase our biological understanding of pheochromocytoma and paraganglioma, and possibly also co-occurring cancers in which the same genes and pathways are involved. Together with the findings of other scientific studies, our results may contribute to the development of future treatment options.

Early events occurring during induction of the SOS response in Escherichia coli are poorly understood. In order to understand the early steps in SOS induction more fully, we have isolated several mutations which constitutively express the SOS regulon. Using a Mud(Apᴿ,lac) fusion to the SOS regulated gene sulA, we isolated Lac⁺ colonies as mutants in which RecA protein is constitutively activated for repressor cleavage. The mutations map to four loci: dam, lig, uvrD and recA. The extent of constitutive SOS induction in these mutants varied greatly, indicating different levels or types of signal in the cell. The mutations isolated demonstrate two early steps in SOS induction. The first step in SOS induction is signal generation and includes mutations found in dam, lig and uvrD genes. The mutant gene products presumably alter DNA metabolism to produce an inducing signal. These non-lethal mutations lead to sub-induction and probably generate very specific signals, such as abnormally unwound DNA in the case of DNA helicase II mutants or unsealed DNA nicks that result from deficient ligation in lig mutants. Greater induction may require quantitatively more signal or different types of signal generated by severe defects leading to cell death. These mutations also show that signal is a variable quantity, allowing the cell to fine tune the levels of SOS repair activity according to the amount or type of signal (damage) perceived. In some cases (such as dam mutations), blocking the SOS response by lexA(Ind⁻) alleles leads to cell death. In this type of constitutively activated strain, the increased level of repair from SOS induction is required to allow the cell to tolerate potentially lethal DNA structures generated by the mutant gene product. The second step in induction is the interaction of signal with RecA protein and is shown by isolating 8 recA mutants. Mutant recA alleles caused the strongest SOS induction in any mutants obtained, similar to the level found in strains lacking repressor (lexA(Def) mutants). This full induction in the absence of lethal DNA damage underscores the pivotal role of RecA protein in regulating the SOS response.

In antibiotic therapy design, conventional wisdom holds that higher antibiotic dosages always leads to the observation of fewer bacterial cells, resulting in a monotonic decay in cell number as a function of increasing antibiotic dose; accordingly, throughout this thesis, we will call this phenomenon a monotone dose-response profile. When we analysed the evolution of antibiotic resistance mediated by the multi-drug efflux pump AcrAB-TolC in Escherichia coli to study if such a monotone dose-response is maintained at all times, our analysis showed that higher dosages can, in fact, lead to higher bacterial loads. This is because selection for drug resistance is mediated by the duplication of the genes, AcrAB-TolC, that encode the aforementioned efflux pump. As explained in detail below, our work highlights the idea that Darwinian selection on additional copies of AcrAB-TolC is a non-linear function of antibiotic dose and that the observed transition from monotone to non-monotone dose-response is a consequence of AcrAB-TolC being strongly selected at very specific dosages. We term this phenomenon an ‘evolutionary hotspot’. Next, we extended the above experimental system to solid media to study how selection on resistance mediated by AcrAB-TolC leads to a ‘spatio-genomic patterning’ effect that we call a ‘bullseye’. Using a bespoke culture device developed as part of this PhD, we show that spatial selection on resistance also depends non-linearly on the distance of the cell from an antibiotic source, and that the non-linearity can be multi-modal as a function of distance, and therefore also of antibiotic dose. This result also contradicts the aforementioned principle that higher antibiotic dosages necessarily lead to fewer bacterial cells. Following on from this, we then studied the ability of microbial competitors for resources to modulate the antibiotic sensitivity of a particular strain of E. coli, namely Tets , using a range of multi-species experiments. We measured the sensitivity to antibiotics of Tets both with, and without, one bacterial or fungal competitor. When that competitor was equally sensitive to the antibiotic, we observed that Tets was less sensitive to it, in part due to an ‘antibiotic sinking’ effect carried out by the competitor strain. However, when the competitor was not sensitive to the antibiotic, Tets was, accordingly, more sensitive than in the absence of competition. In this latter case, the competitor seemed to reduce the growth of Tets by carbon theft as part of a phenomenon known as ‘competitive suppression’. Moreover, this ecological effect is one that synergises with the action of the antibiotic. Finally, we turned to a study of an ecological trade-off motivated by ribosome-binding antibiotics. So, by manipulating the content of ribosomal RNA in the E. coli cell, a large and essential molecule that is bound by antibiotics such as tetracycline or erythromycin, we could subsequently manipulate what is known as a metabolic trade-off between growth rate and growth yield. The latter is the number of cells produced per molecule of carbon found in the extracellular environment of the bacterial population. Using glucose as carbon source we therefore constructed an empirical fitness landscape that shows how the optimum number of ribosomal rRNA operons depends on extracellular glucose concentration. Whilst this study does not relate directly to the presence of an antibiotic, it does show that by altering the number of operons in a manner that is known to affect antibiotic susceptibility, we can also mediate important growth parameters like cell yield, aka efficiency, and growth rate.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy