Univ., Diss., 2007--Frankfurt (Main) / Benutzbar nur im Campus-Bereich Frankfurt am Main

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 162-173).

Techn. Universiẗat, Diss., 2005--München.

Thesis (Ph.D.)--The University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography.

Würzburg, Univ., Diss., 2004.

Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.

Methionine sulfoxide reductase (MSR) is an important antioxidant to help mitigate oxidative stress that contributes to age-associated neurodegenerative diseases, such as Alzheimer’s Disease and Parkinson’s Disease. In MSR deficient Drosophila melanogaster (fruit flies), larvae show a developmental delay like that seen when wild-type larvae are reared on nutrient deficit culture medium. These investigators further showed that serotonin levels were depressed in these nutrient deficient larvae. The overarching aim of this study was to better understand the role of serotonin in MSR regulated physiology. Supplementing food with serotonin partially rescued the slower mouth hook movements (MHM) observed in the MSR-deficient flies. However, supplementation with serotonin altering drugs that cross the blood brain barrier (5-hydroxytryptophan, fluoxetine, or paravi chlorophenylalanine) did not rescue MHM and caused impairments to the growth of larvae during development. This study indicates that serotonin regulates feeding behavior partially through the regulation of MSR production but acts independently to regulate development. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Protein degradation poses a significant challenge for the efficient production of recombinant proteins in plants, affecting the stability and yield of the recombinant protein. In this study the E. coli-derived enzyme glutathione reductase (GR) was transiently expressed in transgenic tobacco plants constitutively expressing the cysteine protease inhibitor OC-I and non-transgenic plants. A protein resembling the GR was detected in infiltrated leaves. Transiently expressing GR in transgenic N tabacum plants resulted in almost two fold significant increases in GR activity. Transgenic tobacco plants expressing the rice cysteine protease inhibitor OC-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher GR activity and also higher GR amounts in transgenic plants. The study has demonstrated that OC-I is an effective companion protease inhibitor candidate with the potential to protect other high value proteins such as GR, from cysteine protease degradation. / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

A Thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the Degree of Doctor of Philosophy. / A deficiency of the enzyme NADP dependent xylitol dehydrogenase (L-xylulose reductase) has previously been found to be the cause of chronic essential pentosuria. Essential pentosuria is a recessively inherited condition which is marked by the continual excretion of relatively large amounts of the enzymes substrate, L-xylulose. The major objective of the study described was to find a simple method for the identification of individuals who are heterozygous for the "pentosuria" and normal alleles. The pentosuria allele could then be used as a gene marker in linkage studies aimed at mapping the L-xylulose reductase locus. A L-xylulose reductase assay suitable for the identification of carriers of essential pentosuria was developed and tested on members of a South African Lebanese family in which the inheritance of pentosuria had previously been suggested to be dominant. It was found that family members could, on the basis of their L-xylulose reductase activities, be classified as either normal, heterozygous or homozygous for the pentosuria allele. Measurements of serum L-xylulose concentrations revealed that pentosuria is, contrary to the previous report, . recessively inherited in this family. A sample of the local Ashkenazi Jewish population was screened for pentosuria carriers. Six out of the 237 individuals screened were found (on the basis of their L-xylulose reductase activities and from the results of a loading test), to carry the pentosuria allele. The frequency of the pentosuria allele in this population was estimated from the apparent heterozygote frequency to be 0.0127. Linkage analyses were carried out on the families of the identified heterozygotes and on members of the Lebanese family mentioned above. No evidence of tight linkage was found between the pentosuria allele's locus and those coding for various red cell antigens, red cell enzymes and serum proteins. Kinetic, chromatographic and electrophoretic studies revealed that the red cells of normal individuals contain two distinct L-xylulose reductases, a minor and a major isozyme. Pentosurics lack the major isozyme but appear to have approximately normal amounts of the minor isozyme. The minor isozyme is e1ectrophoretica 1 1 y distinct from the major isozyme, has markedly higher Michael is constants for the substrates L-xylulose and xylitol and shows a lower pH optimum when catalysing the oxidation of xylitol. Electrophoresis also revealed that liver tissue contains two L-xylulose reductases which occur in similar proportions to those of red cells but which migrate at slightly different rates. / WHSLYP2016

<p> Nitrate assimilation is the process by which nitrate is converted into ammonia, and ultimately into organic nitrogenous compounds, which are then made available to organisms which require an exogenous supply of organic nitrogen. Nitrite is an intermediate in this process and the mechanism of its conversion to ammonia, which is catalyzed by the enzyme nitrite reductase, needs clarification. </p> <p> The purpose of this investigation was to find a suitable assay system for nitrite reductase in N. crassa and to examine some aspects of the metabolic control of the enzyme. A new assay system for nitrite reductase is described and evidence suggesting that the enzyme is derepressible is presented. </p> / Thesis / Master of Science (MSc)