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Indiana University-Purdue University Indianapolis (IUPUI) / Because colorectal cancer (CRC) is the fourth most common cancer and the second leading cause of cancer death in the US, identifying biomarkers that might inform disease prevention and early diagnosis is of great public health importance. Mitochondria are key cytoplasmic organelles containing an independent genome, i.e., mitochondrial DNA (mtDNA). It has been increasingly recognized that mtDNA copy number (mtDNAcn) is a biomarker for mitochondrial function and cellular oxidative stress. To date, the few studies that have assessed associations between mtDNAcn and CRC outcomes have yielded inconsistent findings. Further, no epidemiologic study has examined the relationship between insulinemic potential of lifestyle and mtDNAcn. Therefore, in this dissertation, three studies were conducted using data from the Nurses’ Health Study and the Health Professionals Follow-Up Study. First, the association between pre-diagnostic leukocyte mtDNAcn and CRC risk was studied in a nested casecontrol study (324 cases/658 controls). Lower mtDNAcn was significantly associated with increased risk of CRC and proximal colon cancer. That inverse association remained significant among individuals with ≥ 8 years’ follow-up since blood collection, suggesting that mtDNAcn might serve as a long-term predictor of CRC risk. Second, possible associations of pre-diagnostic mtDNAcn with overall and CRC-specific survival were examined among 587 CRC patients. MtDNAcn was not significantly associated with survival overall or in subgroups by cancer location, grade, or stage. Among current smokers, there was an inverse association between one standard deviation (SD) decrease in mtDNAcn and increased overall death risk. Among patients diagnosed at or before 70.5 years of age and those with anti-inflammatory diets, reduced mtDNAcn was associated with lower CRC-specific death risk. Lastly, the cross-sectional association between empirical lifestyle index for hyperinsulinemia (ELIH) and mtDNAcn was investigated among 2,835 subjects without major chronic diseases (cancers, diabetes, and cardiovascular diseases). A significant inverse association was found: least-squares means ± SD of mtDNAcn z-score decreased dramatically across ELIH quintiles. Overall, the findings from this dissertation will contribute to the evaluation of mtDNAcn as a potential biomarker for CRC risk and prognosis, and inform future interventions designed to reduce the insulinemic potential of lifestyle factors to preserve mitochondrial function. / 2022-04-06

The introduction of new genetic information into microbial production organisms is one of the keystones of modern biotechnology, this can be achieved i.a. by the introduction of plasmids. Extrachromosomal plasmids replicate autonomously and show a plasmid-specific plasmid copy number (PCN) per cell. In growing cells, a fine-tuned copy number regulation is achieved by a tightly controlled balance of the initiator of replication and the repressor. In theory, the plasmid copy number can be influenced at will by a targeted manipulation of this balance. In order to allow transient PCN changes in growing cells, an inducible, precisely adjustable strategy is needed. In this thesis, cop, the repressor of plasmid replication of pE194, was over-expressed independently from the native regulatory system. The developed overexpression system was used to force a severe imbalance in favor of the repressor, therefore intentionally driving the loss of replicating plasmid from the population. As a consequence, cells that previously incorporated the plasmid into their genome gain a selective advantage as they stably inherit an antibiotic resistance encoded on the now non-replicative plasmid. In established genomic modification strategies, a forced imbalance is dependent on elevated temperatures. However, the established protocols for genomic modifications are time-consuming and require extensive screening for clones with the desired genotype. The aim of this thesis was to conceive and establish a novel vector for a reliable, faster and more efficient protocol with a minimal screening requirement. The promotor controlling the repF-cop operon was characterized by supplying the repressor Cop in trans, revealing a complete repression at high Cop concentrations. Furthermore, the gradual dose-dependency of this repression could be shown. Over several intermediate steps, the novel All-in-one vector pBAio was designed. With this vector, the PCN in growing cells can be controlled and even fine-tuned. Forcing a complete stop of plasmid replication, resulted in a 100% plasmid integration rate that eliminated the need for integrand screening and increased the efficiency (i.e. mutant to wild type ratio). The new, pBAio-based protocol allows for fast and efficient markerless genomic modification in different, industrially relevant Bacillus sp..

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21300号 / 医博第4389号 / 新制||医||1030(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 戸井 雅和, 教授 小川 修, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Cytochrome P450 2D6 (CYP2D6) is an important drug metabolizing enzyme that is involved in the metabolism of 20-25% of commonly prescribed drugs. There is interindividual variability in CYP2D6 enzyme activity and this leads to compromised metabolism of many drugs. Genetic and environmental factors explain only a part of the interindividual variability; the other factors that contribute to this variability are largely unknown. Hence, it becomes important to study CYP2D6 to understand the endogenous and exogenous factors that control its activity. The specific objective of this research was to determine the contribution of genetic and epigenetic factors in the regulation of CYP2D6 expression and activity. The specific aims were: (1) to identify the common CYP2D6 polymorphisms in Vietnamese and Filipino women with breast cancer and evaluate its association with plasma concentrations of endoxifen (an active metabolite of the breast cancer therapeutic drug, tamoxifen); (2) to identify the CYP2D6 copy number variations (CNVs) in these women and evaluate their association with endoxifen concentration; and (3) to identify microRNAs (miRNAs) that regulate the expression of CYP2D6 directly or indirectly. The results of this study indicated that: (1) in Vietnamese and Filipino women, the reduced function allele CYP2D6*10 was frequent (~55%) and it was significantly associated with reduced endoxifen concentration; (2) in these women, only 39% carried two copies of the CYP2D6 gene, the rest had a genomic imbalance for CYP2D6, primarily involving the CYP2D6(*36)n-*10 allele. However, carrying multiple copies of CYP2D6*36 allele did not significantly affect CYP2D6 activity, suggesting that multiple copies of a gene does not always translate to additive effects; and (3) microRNAs were identified to target HNF4A, a transcriptional factor that regulates CYP2D6 expression. These miRNAs are likely to play an important role in the indirect regulation of CYP2D6. Taken together, these results emphasize on the role of polymorphisms, CNVs and possibly miRNAs in the regulation of CYP2D6. These clinically important biomarkers will help to improve the efficacy and reduce the side effects of many CYP2D6 substrate drugs and thus contribute to personalization of drug therapy.

Mitochondria are essential cellular organelles and the location of the electron transport chain, the site of the majority of energy production in the cell. Mitochondria contain their own circular genome approximately 16,000 base pairs in length. The mitochondrial genome (mtDNA) encodes 11 protein-coding genes essential for the electron transport chain, 22 tRNA genes, and two rRNA genes. Mitochondrial malfunction occurs in many diseases, and changes in the mitochondrial genome lead to numerous disorders. Multiple mitochondrial haplotypes and sequence features are associated with Alzheimer's disease. In this dissertation we utilized TreeScanning, an evolutionary-based haplotype approach to identify haplotypes and sequence variation associated with specific phenotypes: Alzheimer's disease case-control status, mitochondrial copy number, and 16 neuroimaging phenotypes related to Alzheimer's disease neurodegeneration. In the first two studies we utilized 1007 complete mitochondrial genomes from participants in the Cache County Study on Memory Health and Aging. First, individuals with mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD. Our study is the largest to date and the only study with complete mtDNA genome sequence data. Next, each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. Each of these three variants was associated with higher mitochondrial copy number and we suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that lead to changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes. Lastly, we used an endophenotype-based approach to further characterize mitochondrial genetic variation and its relationship to risk markers for Alzheimer's disease. We analyzed longitudinal data from non-demented, mild cognitive impairment, and late onset Alzheimer's disease participants in the Alzheimer's Disease Neuroimaging Initiative with genetic, brain imaging, and behavioral data. Four clades were associated with three different endophenotypes: whole brain volume, percent change in temporal pole thickness, and left hippocampal atrophy over two years. This was the first study of its kind to identify mitochondrial variation associated with brain imaging endophenotypes of Alzheimer's disease. Together, these projects provide evidence of mtDNA involvement in the risk and physiological changes of Alzheimer's disease.

This essay uses different angles to analyze how authentic objects and copies are used in an exhibition context. The survey focuses on four research questions. The questions concern the significance of copies in exhibitions and focuses on the relationship between the information conveyed in exhibitions and the objects used to convey the information. The questions also aim to examine the relationship between authenticity and copies and the museum visitors view on copies. The purpose of the survey is to see if the answers to these questions mean that the authentic museum objects are the most significant or if the information is the most valuable and that the objects in that case could just as easily consist of copies.  The methods used for collecting the empirical material is analyzes of relevant literature and interviews. The empirical material is used to illuminate different perspectives on the use and view of copies in exhibition contexts. Previous research is used to analyze the concepts of authenticity and copies. Classic cases of exhibitions based on copies are used to get an insight into how copies can be used in exhibitions. Part of the empirical material consists of interviews with Swedish museums, the interviews are used to investigate how museums today use copies and to perceive the museums´ views on the use of copies. The analysis of the empirical material provides many interesting aspects on this topic, and it turns out that the researchers, the classical cases, and the interviewed museums are more or less in agreement when it comes to the use of copies in exhibition contexts. The copies are perceived to have many positive qualities that can be used in different ways in an exhibition. Overall, the reasoning leads to copies having a significant role in exhibition contexts and is a somewhat unused resource that could be used more than what it currently does.  This is a two-year master´s thesis in Museum and Cultural Heritage Studies.

This thesis aims to investigate copying in architecture in relation to context. Is it possible to copy a building?With the advances of 3D-scanning and digital fabrication, the possibilities for copying form and material are constantly increasing. However, more so than the artistic object, architecture is always contextual - a building exists on a specific site. Therefore, when investigating copies in architecture, context becomes crucial. How does context change the specifics of the building?While studying the copy in relation to context and the adaptations, the relation between the original and the copy is highlighted. At what point does the copy detach itself from the original? Is there a copy or just multiple originals?In relation to these questions, there are several issues that surface, that might be defined by studying the subject. What is architecture? What is a building? What makes a building a piece of architecture? Who has authorship over a piece of architecture? And most importantly: where in a building lies the originality?

Frequently evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Here, various methods of post PCR purification were evaluated for their effects on the sensitivity of fluophore-based allelic detection. A method of post PCR purification is described which increases the sensitivity of standard 28 cycle PCR such that low copy number DNA templates (<100 pg DNA) can be analyzed. Full profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele drop-out, increased stutter, and contamination (allele drop-in) typical of LCN analysis were observed. A single incident of contamination was observed in a reagent blank (not duplicated upon re-amplification) however, no contamination was observed in negative amplification controls.

This thesis is an investigation of the structural and formal semantic properties of copy raising constructions in English, as well as their expletive counterparts. The first main claim is that contrary to what has been previously assumed, the perceiver of the event (i.e. the Pgoal in Asudeh & Toivonen's 2012 terms) is an obligatory syntactic and semantic argument of the matrix verb. I argue that the identification of the Pgoal is not left to pragmatics, but rather that is represented as a silent pronoun in the structure: one that picks up a logophoric antecedent. The result of this is that the material in the embedded clause is semantically interpreted with respect to the Pgoal's perspective. The second major claim of the thesis is that this perspective-sensitivity is most appropriately captured using a modal semantic framework (Kratzer, 1977, 1981 von Fintel & Heim, 2002). Specifically, I argue that each of the different copy raising verbs encodes a different accessibility relation between possible worlds or situations, while the Pgoal's information state provides the relevant domain of worlds. Using these insights, I propose truth conditions for these constructions, which ultimately are sensitive to a kind of stereotypical ordering, and account for inter-speaker variability. Finally, I discuss the anomalous class of copy raising constructions with non-thematic subjects, and argue that overlapping discourse functions may have resulted in a shift away from modal semantics in these cases. / Thesis / Master of Science (MSc) / This thesis investigates the linguistic meaning associated with the "copy raising" sentence construction, e.g. "Your cat looks like she wants to go outside." I argue that the interpretation of these sentences is dependent on establishing the individual whose perspective is conveyed in the sentence, which does not need to be the speaker. After examining the range of contexts in which various different copy raising constructions can be used, I propose an analysis of their core meaning that draws on the philosophical idea of possible worlds, and the psychological notion of stereotypicality. I also address the question of whether these constructions are related to the phenomenon of evidentiality, a property of certain languages which allows the speaker to linguistically mark the source of evidence for their claim.