Structural characterization of interaction of K11 and K63 mixed-linkage polyubiquitin chains with the tUIMs of epsin1

Ubiquitylation or covalent attachment of ubiquitin (Ub) to a variety of substrate proteins in cells is a versatile post-translational modification involved in the regulation of numerous cellular processes. The distinct messages that polyubiquitylation encodes are attributed to the multitude of conformations possible through attachment of ubiquitin monomers within a polyubiquitin chain via a specific lysine residue. Thus the hypothesis is that linkage defines polyubiquitin conformation which in turn determines specific recognition by cellular receptors. Ubiquitylation of membrane surface receptor proteins plays a very important role in regulating receptor-mediated endocytosis as well as endosomal sorting for lysosomal degradation. Epsin1 is an endocytic adaptor protein with three tandem UIMs (Ubiquitin Interacting Motifs) which are responsible for the highly specific interaction between epsin and ubiquitylated receptors. Epsin1 is also an oncogenic protein and its expression is upregulated in some types of cancer. Recently it has been shown that novel K11 and K63 mixed-linkage polyubiquitin chains serve as internalization signal for MHC I (Major Histocompatibility Complex I) molecule through their association with the tUIMs of epsin1. However the molecular mode of action and structural details of the interaction between polyubiquitin chains on receptors and tUIMs of epsin1 is yet to be determined. This information is crucial for the development of anticancer therapeutics targeting epsin1. The molecular basis for the linkage-specific recognition of K11 and K63 mixed-linkage polyubiquitin chains by the tandem UIMs of the endocytic adaptor protein epsin1 is investigated using a combination of NMR methods.

Design and Pilot Study for an Efficient High-Throughput Automated Computer-Vision Guided Intelligent De-Calyxing Machine for Post-Harvest Strawberry Processing

Strawberries harvested for processing as frozen fruits are currently de-calyxed manually in the field. This process requires the removal of the stem cap with green leaves (i.e. the calyx) and incurs many disadvantages when performed by hand. Not only does it necessitate the need to maintain cutting tool sanitation, but it also increases labor time and exposure of the de-capped strawberries before in-plant processing. This leads to labor inefficiency and decreased harvest yield. By moving the calyx removal process from the fields to the processing plants, this new practice would reduce field labor and improve management and logistics, while increasing annual yield. As labor prices continue to increase, the strawberry industry has shown great interest in the development and implementation of an automated calyx removal system. In response, this dissertation describes the design, operation, and performance of a full-scale automatic vision-guided intelligent de-calyxing (AVID) prototype machine. The AVID machine utilizes commercially available equipment to produce a relatively low cost automated de-calyxing system that can be retrofitted into existing food processing facilities. This dissertation is broken up into five sections. The first two sections include a machine overview and a 12-week processing plant pilot study. Results of the pilot study indicate the AVID machine is able to de-calyx grade-1-with-cap conical strawberries at roughly 66 percent output weight yield at a throughput of 10,000 pounds per hour. The remaining three sections describe in detail the three main components of the machine: a strawberry loading and orientation conveyor, a machine vision system for calyx identification, and a synchronized multi-waterjet knife calyx removal system. In short, the loading system utilizes rotational energy to orient conical strawberries. The machine vision system determines cut locations through RGB real-time feature extraction. The high-speed multi-waterjet knife system uses direct drive actuation to locate 30,000 psi cutting streams to precise coordinates for calyx removal. Based on the observations and studies performed within this dissertation, the AVID machine is seen to be a viable option for automated high-throughput strawberry calyx removal. A summary of future tasks and further improvements is discussed at the end.