Protein quality control in the endoplasmic reticulum and cytosol

Metzger, Meredith Boyle The Johns Hopkins University 2009 해외박사(DDOD)

Protein misfolding contributes to a variety of human diseases such as cystic fibrosis, hereditary emphysema, and Huntingdon's disease. Protein quality control is the essential cellular process that monitors the folding status of proteins throughout the cell. This thesis focuses on defining the Saccharomyces cerevisiae protein quality control systems that act on misfolded proteins in the endoplasmic reticulum (ER) and cytosol. Chapter One presents a brief introduction to the transcriptional and degradative protein quality control systems that operate in different cellular compartments, including the ER, cytosol, mitochondria, and nucleus. In Chapter Two, I analyze and compare three distinct transcriptional responses that result from misfolded proteins in the ER lumen, ER membrane, or the cytosol (Metzger, M. B., and Michaelis, S. (2009) Mol Biol Cell 20, 1006-1019). This study defines a novel transcriptional response induced by misfolded membrane proteins, the UPR-M/C response. The UPR-M/C response is dependent upon Rpn4p, a transcriptional activator of the proteasome. My results further demonstrate that the UPR-M/C response is essential for cellular viability in the presence of misfolded membrane proteins. Chapter Three characterizes the machinery required for the degradation of a cytosolic protein, Ura3p, fused to the CL1 degron, a short destabilizing sequence that targets the Ura3p-CL1 fusion for destruction via the ubiquitin-proteasome system (Metzger, M. B., Maurer, M. J., Dancy, B. M., and Michaelis, S. (2008) J Biol Chem 283, 32302-32316). Surprisingly, I find that Ura3p-CL1 is degraded at the ER by the machinery that acts on membrane proteins with misfolded cytosolic domains My results indicate that the cytosolic face of the ER could be serving as a "platform" for the degradation of most cytosolic misfolded proteins. In Chapter Four, I describe a novel destabilizing degron, CL1*, and characterize a role for Rkr1p, an E3 ubiquitin-ligase, in the degradation of a Ura3p-CL1* fusion protein. This is the first study to implicate Rkr1p in cytosolic protein quality control and Ura3pCL1* is the first substrate of Rkr1p to be identified. This thesis has initiated quality control studies of misfolded membrane and cytosolic proteins and will provide a basis for future studies within the protein quality control field.

SOLUBILITY TESTING OF ACTINIDES ON BREATHING-ZONE AND AREA AIR SAMPLES (INHALED RADIONUCLIDES)

METZGER, ROBERT LAWRENCE ARIZONA STATE UNIVERSITY 1997 해외박사

Die Berufungsvereinbarung

Metzger, Stefan Friedrich Wilhelms Universitat 1995 국내석사

Targeted gene therapy using nicks to induce homologous recombination

Metzger, Michael J University of Washington 2010 해외박사(DDOD)

Despite the early promise of gene therapy, monogenic diseases continue to be a significant source of morbidity and mortality. We have investigated the ability of the human retrovirus xenotropic murine leukemia virus-related virus (XMRV) to induce transformation in cell culture and found that it is not acutely oncogenic but can generate rare transformed foci, confirming the potential for transformation by untargeted integration of retroviral vectors and furthering our understanding of a possible human pathogen. Additionally, we investigated contamination of adeno-associated virus (AAV) vectors with cap DNA that can express capsid in vector-transduced cells, and found that capsid-expressing DNA is present in vectors, but it can be prevented through the use of an oversized cap gene in vector production. We next investigated gene targeting using homologous recombination (HR). HR can be induced by double strand breaks (DSBs), but these breaks can be toxic and mutagenic. Using the I-Anil homing endonuclease engineered to produce only nicks we found that nicks induce HR with both plasmid and AAV vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold for plasmid transfection and 4 to 6-fold for AAV vectors), but still represented a significant increase (240-fold and 30-fold, respectively). We observed severe toxicity with the I-Anil 'cleavase,' but no evidence of toxicity with the I-Anil 'nickase,' and we detected 100-fold fewer mutations at the I-AniI site with the nickase than with the cleavase. These results, and the observation that sequence surrounding the target site affects nick-induced but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs. Combining this with the oversized cap AAV production could allow for a targeted gene correction strategy without the toxicity of DSBs that could also have reduced contamination of the AAV vector.

Disentangling the effects of mutation and selection on the evolution of gene expression

Metzger, Brian P. H University of Michigan 2015 해외박사(DDOD)

Mutation is the ultimate source of phenotypic variation. However, little is known about the effects of new mutations in the absence of natural selection and whether these effects can influence the course of evolution. This is particularly true for changes in gene expression and regulation. In this thesis I measure the effects of new cis- and trans-regulatory mutations on the expression of the Saccharomyces cerevisiae TDH3 gene. Using these measurements, I show that cis- and trans-regulatory mutations have fundamentally different effects on gene expression. In particular, I find that cis-regulatory mutations are on average larger than trans-regulatory mutations and skewed towards decreases in TDH3 expression, while trans-regulatory mutations are often, but not always, more common than cis-regulatory mutations and skewed towards increases in TDH3 expression. To determine how natural selection has acted on these differences, I generate genome sequences and genetically tractable versions of over 60 diverse S. cerevisiae strains previously isolated from a range of environments. I use these strains to determine the effects of cis- and trans-regulatory polymorphism on TDH3 expression. Comparing these effects to the effects of new mutations, I find that natural selection has acted on both cis- and trans-regulatory variants. Interestingly, the effects of selection varies between cis- and trans-regulatory changes due to differences in the effects of new mutations. Using the same approach, I also identify differences in the action of natural selection on cis- and trans-regulatory changes for the variability in expression amongst genetically identical individuals, i.e. gene expression noise. Finally, I determine the evolution of regulatory changes over long evolutionary timescales in Saccharomyces. I find widespread evidence for compensatory changes in regulation, particularly for trans-regulatory changes that act in opposite directions. Consistent with this finding, I identify hundreds of trans-acting QTL affecting TDH3 expression amongst four strains of S. cerevisiae. Together these results suggest that trans -regulatory changes are a common, but individually small, source of regulatory variation. In total, this thesis shows that understanding the effects of new mutations and comparing these effects to observed differences in natural populations can be a powerful approach for elucidating the underlying molecular mechanisms governing evolution.

POST-MELT CHEWINESS AND WHITENESS OF LOWFAT MOZZARELLA CHEESE (LOW FAT CHEESE)

METZGER, LLOYD ERNST CORNELL UNIVERSITY 1999 해외박사(DDOD)

Baking characteristics and post-bake properties of Mozzarella cheese are important because it is used on pizza. In response to consumer demand, low fat Mozzarella cheese has been developed and extensive research has been aimed at improving its quality. However, the post-melt properties of low fat Mozzarella are still not equivalent to full fat Mozzarella. The goal of this research was to improve the post-melt properties of low fat Mozzarella cheese. An objective test to measure post-melt chewiness of Mozzarella cheese was developed. The results of the post-melt chewiness test were well correlated with sensory results. Thermally reversible whiteness changes in low fat and low moisture part skim Mozzarella during heating and cooling were quantified. Changes in whiteness of low fat Mozzarella during heating and cooling were larger than changes in whiteness of low moisture part skim Mozzarella. Reversible whiteness changes in low fat and low moisture part skim Mozzarella were caused in part by hydrophobic protein-protein interactions among the caseins and peptides derived from casein in the serum phase of cheese. A model of the contribution of protein and fat to whiteness during heating and cooling was proposed. The effect of milk preacidification on cheese composition, yield, manufacturing characteristics, unmelted and melted functional properties, and post-melt properties of low fat Mozzarella cheese was determined. The type of acid used and the level of preacidification influenced cheese calcium content. Milk preacidification to pH 5.8 with citric acid caused about a 40% reduction in cheese calcium content and cheese yield decreased (about 5%). Reduced calcium content decreased cheese hardness, unmelted whiteness, apparent viscosity, expressible serum, and post-melt chewiness. Reduction in hardness, apparent viscosity and post-melt chewiness are quality improvements. The proportion of total calcium that was soluble in the serum phase of the cheese increased with decreasing cheese pH during storage. At low levels of water insoluble calcium and high levels of proteolysis the post-melt chewiness and whiteness of low fat Mozzarella cheese are reduced. The lower level of pre and post-melt whiteness caused by calcium reduction are undesirable. It may be possible to use homogenization of the fat to increase cheese whiteness in cheese made from preacidified milk.

Auger recombination in low-bandgap n-type indium gallium arsenide

Metzger, Wyatt Keith University of Colorado at Boulder 2001 해외박사(DDOD)

The recombination lifetime of n-In_xGa_1−xAs was determined for three different compositions that correspond to bandgaps of 0.74, 0.60, and 0.50 eV (x = 0.53, 0.66, and 0.78, respectively) over the doping range of 3 × 10<super>18</super> to 5 × 10<super>19</super> carriers/cm<super>3</super>. Picosecond up-conversion time-resolved photoluminescence measurements, together with external quantum efficiency measurements and previous lifetime studies of lightly doped samples, clearly indicate that Auger recombination is the dominant recombination mechanism in this doping range. The Auger recombination rate is far less than expected by interpolation of the experimental results for InAs and In_0.53Ga_0.47As, and it matches the behavior predicted for the phonon-assisted process. This makes low-bandgap (0.53 < x < 1) In_xGa_1−xAs a more attractive material for use in infrared detectors, thermophotovoltaic converters, laser diodes, and other applications. Theoretical treatments of Auger recombination are very sensitive to the band model. Plasma reflectance and Raman spectroscopy on n-In_xGa_1−xAs and n-InAs_yP_1−y were used to determine the effective electron mass as a function of carrier concentration for different compostions of degenerate n-In_xGa_1−xAs and n-InAs_yP_1−y. The predictions of the Kane band model agree with the experimental results. However, at large carrier concentrations (>5 × 10<super>19 </super>carriers/cm<super>3</super>), the model becomes unreliable if the T symmetry point is used as the initial k-value, as it frequently is in Auger recombination theory.

Understanding Preservice Teachers' Spatial Reasoning and How It Affects Their Work with Elementary Students

Metzger, Michelle R ProQuest Dissertations & Theses The University of 2019 해외박사(DDOD)

Spatial reasoning involves those skills that allow one to mentally picture and manipulate objects which plays a unique role in learning and succeeding in science, technology, engineering, and mathematics fields (STEM). Despite the urgent need for strong spatial reasoning skills, our current education system spends little time fostering elementary students' visual and spatial reasoning skills. This is becoming increasingly problematic as the need to become literate in the STEM fields has never been greater.The purpose of this qualitative study was to examine the spatial reasoning skills that preservice teachers demonstrated and how their spatial reasoning skills were used in the enactment of the tasks of teaching. Thirty-two preservice teachers completed a spatial reasoning task. Each preservice teacher then teamed with their practicum partner, created an adapted plan using the same spatial reasoning task, and enacted their plan with an elementary student in Grades K-5. Finding from this study indicate that the spatial reasoning skills of preservice teachers are weak, which hinders flexible thinking when observing elementary students engaged in a spatial reasoning task. How learners represent and connect pieces of knowledge is a critical factor in whether they will understand it deeply and can use it in problem solving.

Convergence of Senior Administrators and Professional Employees: Case Studies of Institutional Transformation Via Convergent Hybrid Planned and Emergent Change

Metzger, Michael C University of Massachusetts Boston ProQuest Disser 2020 해외박사(DDOD)

Establishing Normative Data for the Fuld Object Memory Evaluation on a Younger Population

Metzger, Christopher T Indiana University of Pennsylvania ProQuest Disser 2020 해외박사(DDOD)

The Fuld Object Memory Evaluation (FOME), developed by Pamela Fuld in 1980, is a measurement of memory that was specifically designed for use with older adults. The measure was uniquely designed to help mitigate potentially confounding factors found in neuropsychological assessment with older adults such as sensory impairment, anxiety, and inattention. Accounting for these factors is accomplished through examinees processing memory stimuli through multiple sensory modalities (e.g., touch, vision). The FOME has been demonstrated to be both sensitive and specific in differentiating individuals diagnosed with dementia, mild cognitive impairment, and those who are cognitively healthy, in individuals aged 70 years and older. Although there has been considerable research on the FOME for individuals over the age of 70 , and adequate normative data exists for adults in this age range, there is a dearth of normative data available for younger adults. The current study collecting normative data on the FOME for 49 participants between the ages of 60 and 69 years old yielded ceiling effects. To establish convergent and divergent validity the Wechsler Memory Scale-4th edition, Controlled Oral Word Association Test, Trails A, and Trails B tests were administered. Within this sample, the FOME demonstrated convergent and divergent construct validity. Further, FOME demonstrated no relationship with level of education, which is consistent with the literature. Results suggest limited utility of the FOME in those under age 70.