No description available.

No description available.

No description available.

No description available.

Selective protein degradation by the ubiquitin-proteasome system is essential for cellular homeostasis and the regulation of diverse biological processes. The selectivity of this system is imparted by hundreds of ubiquitin ligases that specifically recognise substrates and catalyse their ubiquitination, thereby targeting them for degradation. Among ubiquitin ligases, multisubunit cullin-RING ubiquitin ligases constitute the largest group. However, despite significant advances in understanding their assembly, regulation, and molecular architecture, the substrates and functions of most of them remain unknown. This thesis focuses on two ubiquitin ligases from the cullin-RING ubiquitin ligase 4 (CRL4) subfamily: CRL4DCAF4 and CRL4DCAF12 . To identify their candidate substrates and to address their biological roles, several different approaches have been employed. First, proteomic screening revealed a wide range of candidate substrates. Next, detailed characterisation of the identified interactions and exploration of the condition under which candidate substrates undergo degradation was performed. Finally, knockout human cell lines and mice with a targeted disruption of genes encoding DCAF4 and DCAF12 were generated to explore the physiological roles of CRL4DCAF4 and CRL4DCAF12 . In summary, the herein...

Purpose: To determine the effect of four postexercise beverages, differing in macronutrient content, on metabolic response to an acute resistance exercise bout. Methods: Forty male subjects performed five sets of eight repetitions at 80% 1RM for leg press and leg extension, and then consumed one of four postexercise beverages (Placebo, PL: a carbohydrate-electrolyte beverage, CE; or one of two milk-based beverages, MILK 1: 1% chocolate milk; MILK 2: a high protein milk beverage). Indicators of muscle protein synthesis (MPS) were assessed before and 1-hr after consuming a postexercise beverage. Muscle protein degradation (MPD) was examined the day before and the day of exercise. Results: No significant differences were found among groups in MPS. The resistance exercise bout increased the amount of eIF4E-eIF4G by 4.5% 1-hr postexercise (p<0.05) without affecting the amount of eIF4E-4E-BP1. One hour after beverage consumption, serum total amino acid concentration increased for MILK 1 (p=0.003) and MILK 2 (p<0.001) but decreased for CE (p=0.028) and PL (p=0.276). Consumption of MILK 1, MILK 2, and CE significantly increased circulating levels of serum insulin (p<0.001). Serum growth hormone increased 3-fold as a result of the exercise bout but fell to baseline for all groups by 60 min (p<0.001). Conclusion: The resistance exercise bout was anabolic as shown by the increase in the active eIF4E-eIF4G complex and serum growth hormone. Consumption of MILK 2 led to the most optimal environment for muscle anabolism; however, none of the experimental beverages influenced the measured indicators of muscle protein translation 1-hr after ingestion. / Master of Science

No description available.

No description available.

Fungi, diverse and impactful organisms, exert both beneficial and harmful effects on plants, animals, and humans. Certain fungi produce auxin or indole-3-acetic acid (IAA), a crucial plant growth hormone that influences various aspects of plant growth and defense mechanisms. Conversely, pathogenic fungi can produce auxin and manipulate auxin signaling in their host plant to promote fungal virulence and infection progression. Targeting the auxin signaling pathway in pathogenic fungi offers a novel strategy for combating fungal infections in both plants and humans. Nevertheless, the auxin biosynthesis pathway and the role of auxin in fungal symbioses is not fully understood, in part, due to the lack of a tool for measuring intracellular auxin with high spatial and temporal resolution. This dissertation presents the first genetically encoded biosensor engineered from the E3 ubiquitin ligase to detect and quantify intracellular auxin in a Saccharomyces cerevisiae model. The biosensor has been applied to begin studying auxin metabolism and biosynthesis in yeast as well as better understand the plant auxin co-receptor proteins from which it is built. Additionally, the biosensor is re-engineered for application in inducible protein degradation, controlled by auxin. This tool could be applied to identify novel protein targets for disrupting pathogenic fungal species. Overall, this research offers valuable tool and platform for studying auxin biosynthesis pathway, plant protein and auxin signaling as well as intracellular proteins in fungi. / Doctor of Philosophy / Fungi affect plants, animals, and humans, in both beneficial and harmful ways. Some fungi aid other organisms, while others cause illness. Certain fungi produce a hormone called auxin, or indole-3-acetic acid (IAA), which is essential for plant growth and many environmental responses. Auxin can also assist plants in defending against harmful fungi. Conversely, fungi that infect plants can utilize auxin to promote their own growth and spread. Some fungi even produce auxin, possibly aiding in their colonization of plants. In human fungal infection, it is suggested that auxin may be involved in virulent traits and disease progression. Targeting the auxin signaling pathway in harmful fungi presents an innovative approach to combat fungal infections in both plants and humans. However, our understanding of fungal auxin biosynthesis pathways and their role in fungal infections are not fully understood due to the lack of tools to measure auxin in cells efficiently and accurately. This study introduces the first biological tool, called a biosensor, engineered from auxin responsive proteins from plants, to detect and measure intracellular auxin in Baker's yeast. The biosensor has been used to investigate auxin production by yeast. Additionally, the biosensor has been re-engineered for application in inducible protein degradation, controlled by auxin. This tool could be applied to identify novel protein targets for disrupting pathogenic fungal species. Overall, this research provides useful tool and platform to study auxin production, plant protein function and particular proteins in fungi.

The rate limiting step in isoprenoid biosynthesis is catalyzed by 3-hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34). In plants, HMGR is encoded by small gene families whose members are differentially expressed. In tomato, hmg2 was previously isolated and sequenced. We report the isolation and sequence analysis of a clone (pCD4) encompassing exon I of tomato hmg1 which encodes the putative membrane domain. Sequence comparisons of plant HMGR proteins reveal two hydrophobic stretches within the amino terminus which are highly conserved among species. Using in vitro transcription and translation systems, the membrane domain structure of two tomato HMGR isoforms, HMG1 and HMG2, were analyzed. Results from these experiments reveal that tomato HMGRs are targeted to microsomal membranes in a cotranslational fashion that does not involve cleavage of an N-terminal targeting peptide. Membrane topography of HMGR was revealed by protease protection studies, indicating that both tomato HMGRs span the membrane two times such that both the C- and N-termini are located within the cytosol. HMG2 but not HMG1 was glycosylated in the in vitro system. Deletion of the hmg1 5' untranslated regions and sequences encoding the first six highly charged amino acids resulted in inefficient translation in vitro. However, targeting to microsomes was unchanged. HMG1 membrane domain was tagged with a FLAG epitope to facilitate in vivo studies. Agrobacterium-mediated transformation was used to introduce the tagged hmg1 gene into two Nicotiana tabacum cell lines, BY-2 and KY-14. The slow growth kinetics of KY-14 prevented effective recovery of transformed lines, however, Northern analyses of BY-2 showed that the hmg1 transgene was expressed. Comparisons of BY-2 and KY-14 revealed differences in defense responses to elicitor treatment. BY-2 cells showed minimal defense capabilities, whereas KY-14 cells were rapidly induced as indicated by increased HMGR enzyme activity and browning of the cells. HMGR enzyme activity was decreased in both KY-14 and BY-2 cells following sterol treatment, but the reduction was more pronounced in KY-14 cells. Thus transgenic BY-2 cells may be useful in future in vivo immunolocalization studies, but analyses of HMGR transcriptional regulation and regulated degradation will require use of the more responsive KY-14 cells.. / Ph. D.