Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the eukaryotic replication complex, has been shown to be extensively regulated by post translational modifications, during its assembly. Therefore, it is not inconceivable that the process to unload the replication complex would also be a conserved and regulated process. In 2014, our lab discovered that the CMG complex undergoes post-translational modification in the form of ubiquitylation on one of the subunits of CMG, leading to its disassembly from the chromatin. Though the main players in the disassembly of CMG were known, viz the E3 ligase SCFDia2 and segregase Cdc48, very little was known about the mechanism of CMG disassembly. In the process of learning more about the disassembly of the replicative helicase from chromatin, I reconstituted the ubiquitylation of CMG and thereafter the disassembly of CMG helicase in vitro. My work resulting in the reconstitution of CMG disassembly in vitro is the first example of the disassembly of a multi-subunit physiological substrate of Cdc48. Though CMG is ubiquitylated in yeast extracts in vitro, it does not lead to its disassembly and therefore led me to find conditions necessary for the efficient ubiquitylation of CMG. I have further shown that purifying the E3 ligase associated CMG can be efficiently ubiquitylated in a semi-reconstituted system consisting of purified factors, necessary for the ubiquitylation of substrate. I investigated whether this efficiently ubiquitylated CMG can be disassembled by purified Cdc48 and associated co-factor Ufd1/Npl4 in vitro and found that disassembly is dependent on K48 linked poly-ubiquitylation of CMG. I have found that the reconstituted poly-ubiquitylation of CMG is restricted to the Mcm7 subunit of CMG, recapitulating the ubiquitylation of CMG in vivo, and my data points out that there are multiple sites of ubiquitylation on Mcm7. Through this work, I have also found that ubiquitylated Mcm7 no longer associates with the rest of the CMG components after disassembly of CMG. My assays and findings, open the door towards dissecting the molecular mechanism of the disassembly of CMG in greater detail.

Majority of the products get discarded at end-of-life (EoL), causing environmental pollution, and resulting in a complete loss of all materials and embodied energy. Adopting a closed-loop material flow approach can aid preventing such losses and enable EoL value recovery from these products. Design and engineering decisions made and how products are used impact the capability to implement EOL strategies such as disassembly and remanufacturing. Some underlying factors affecting the capability to implement these EOL strategies have been discussed in previous studies. However, relevant metrics and attributes are not well defined and comprehensive methods to quantitatively evaluate them are lacking. This study will first identify key lifecycle oriented metrics affecting disassemblability and remanufacturability. Then a methodology is proposed for the quantitative evaluation of these strategies considering the quality of returns, product-design characteristics and process technology requirements. Finally, an industrial case-study is presented to demonstrate the application of the proposed method.

Le cytosquelette est un assemblage de protéines intracellulaires qui assure le maintien de la forme des cellules et la production de force. Ce cytosquelette est formé de trois types de polymères, dont les filaments d'actine qui sont impliqués dans des fonctions essentielles telles que la motilité cellulaire, la division cellulaire ou encore la morphogénèse. Les filaments d'actine s'agencent en structures organisées dont la dynamique est assurée par la polymérisation et le désassemblage des filaments, contrôlés spatio-temporellement. La plupart des structures d'actine sont dans un état stationnaire dynamique où l'assemblage est compensé par le désassemblage, ce qui permet de maintenir une concentration de monomères intracellulaire élevée. En effet, le réservoir d'actine in vivo est limité et la formation de nouvelles structures de filaments d'actine est dépendante d'un désassemblage efficace des structures les plus âgées. Le but de ma thèse a été d'étudier comment l'organisation architecturale des structures d'actine influence le désassemblage par la machinerie protéique composée de l'ADF/cofiline et d'un de ses cofacteurs Aip1.J'ai d'abord pu montrer que l'efficacité du désassemblage dépendait de l'agencement des filaments d'actine. Quand les réseaux branchés ne requièrent que l'action de l'ADF/cofiline pour être désassemblés efficacement, les faisceaux de filaments d'actine ont besoin de la présence simultanée de l'ADF/cofiline et de l'Aip1. Une étude à l'échelle moléculaire a ensuite été menée pour comprendre le mécanisme du désassemblage des filaments d'actine par ces deux protéines au niveau du filament individuel.Dans un second temps, j'ai développé un système expérimental composé de micropuits de taille comparable à la cellule. Cette technologie nous a permis de réaliser des expériences en milieu confiné, dans lequel le réservoir d'actine était limité de la même manière que le réservoir d'actine cellulaire. J'ai mis ce système a profit pour reconstituer le turnover d'une comète d'actine, un réseau branché formé à la surface d'une bille recouverte de nucléateurs de l'actine.Ce travail de thèse a permis d’établir des lois fondamentales contrôlant la dynamique de l’actine et plus particulièrement comment l’architecture de l’actine et l’environnement peuvent influencer le désassemblage de structures complexes. / The actin cytoskeleton is a major component of the internal architecture of eukaryotic cells. Actin filaments are organized into different structures, the dynamics of which is spatially and temporally controlled by the polymerization and disassembly of filaments. Most actin structures are in a dynamic steady state regime where the assembly is balanced by the disassembly, which maintains a high concentration of intracellular actin monomers. In vivo the pool of actin monomers is limited and the formation of new actin filament structures is dependent on an effective disassembly of the older structures. The goal of my thesis was to study the influence of different architectures of actin by the disassembly machinery made of ADF/cofilin and its cofactor Aip1.Firstly, I showed that the efficiency of the disassembly was dependent on the architecture of actin filaments organizations. Although the branched networks need only ADF/cofilin to be efficiently disassembled, the actin cables require the simultaneous action of ADF/cofilin and Aip1. Further investigations at the molecular scale indicate that the cooperation between ADF/cofilin and Aip1 is optimal above a certain threshold of molecules of ADF/cofilin bound to actin filaments. During my PhD I demonstrated that although ADF/cofilin is able to dismantle selectively branched networks through severing and debranching, the stochastic disassembly of actin filaments by ADF/cofilin and Aip1 represents an efficient alternative pathway for the full disassembly of all actin networks. We propose a model in which the binding of ADF/cofilin is required to trigger a structural change of the actin filaments, as a prerequisite for their disassembly by Aip1.Secondly, I developed an experimental system made of cell-sized microwells. This technology allowed us to develop experiments in a closed environment in which the actin pool is limited in the same way as the cellular environment. I used this experimental system to study how a limited pool of components limits both the assembly and the disassembly of a branched network.This thesis highlights the importance of developing new tools to obtain more “physiological” reconstituted systems in vitro to establish some of the general principles governing actin dynamics.

One of the largest product categories within all the electronic products is portable ­computers, also known as notebooks, is slowly replacing stationary computers and is ­predicted to be the mainstream choice in the near future. But the current state of ­notebook shows these products are not accommodated for an effective large scale ­recycling process. As the notebooks are becoming more compact, its inner ­structures have become more complicated. Permanently fastening methods, multi-material ­compositions and many other factors contribute to an ineffective, expensive and tedious recycling process. The goal of the project treated in this report was to investigate the possibilities of a simplified structure adapted for an effective disassembly and recycling process. And to develop a conceptual notebook adapted for the stated purpose. The project resulted in a conceptual notebook named Sixten with an enhanced and ­simplified inner structure. All permanently fastening methods was replaced by ­detachable locking and securing devices. The number of tools required for dismantling was reduced, as the usage of screws. The variation of materials used in the notebook, with ­electrical components apart, is low. These materials, ABS plastics and aluminum were chosen based on their recyclable and processing possibilities. But also because of the durability and desired mechanical properties the structure required. The limited number of materials used in Sixten makes it environmentally beneficial since every material ­requires its own specific recycling process, and by reducing the number of materials energy can be saved on using only a few recycling methods. Sixten fulfills the principals of design for disassembly as a notebook that is easy to ­disassemble in comparison with notebooks on the market today. The disassembly time has been reduced, from the 45 minutes which may be required to fully disassemble a conventional notebook, down to an estimated disassembly time of 6 minutes, which is the time Sixten requires. It is in this short disassembly time where the economic benefits of Sixten lie.

In this thesis, we consider a Disassembly Line Balancing Problem (DLBP) with fixed number of workstations. We aim to maximize the total value of the recovered parts. We assume that there is a limited supply for the products to be disassembled. Different components can be obtained by disassembling different units of the product. Our aim is to assign the tasks to the workstations of the disassembly line so as to maximize the total value of the recovered parts. We present several upper and one lower bounding procedure. The results of our computational study have revealed the satisfactory behavior of our bounding mechanisms.

In this thesis, we work on adisassembly lot-sizing problem for multiple products with parts commonality,i.e., general product structure. We assume that supply of discarded products is infinite. When a product (or a subassembly) is disassembled, all its immediate child items are obtained,i.e., complete disassembly case.Intermediate and leaf items obtained are demandedbyexternal suppliers or remanufacturers. The maximum possible salesfor each intermediate and leaf item are known.Sales of the intermediate and leaf items are the revenue sources. The discarded products are purchased ata unit purchasing cost. The disassembly operation incurs a fixed and a variable disassembly cost. Due to this cost structure, intermediate and leaf items can be stocked incurring an inventory holding cost. We develop an integer programming formulation to determine the time and quantity of the discarded products to be purchased / thetime and quantity of the discarded products and the intermediateitemsto be disassembled / and the time and quantity of intermediate and leaf items to be soldin order tomaximizethe total profit over a finite planning horizon. We state that ourproblem is NP-hard by refering the study of Kim et. al. (2009). We propose a heuristic solution approach that solves the problem in a reasonable computational time and generates near optimal solutions. The solution approach is based on the idea of sequentially solving a relaxed version of the problem and one-period integer programming models.In a computational study, the performance of the heuristic approach is assessed for a number ofrandomly generated problem instances.The results of the computational study show that the solutions of the heuristic approacharevery close to the optimal and the best feasible solutions obtained within the time limit.

In this study, we consider the disassembly problem of end-of-life (EOL) products for recovering valuable parts or assemblies. All parts obtained by disassembly processes of an EOL product may not be profitable due to their high recovery costs. Our problem is to select the parts to be released and determine the associated disassembly tasks so as to maximize the total profit. We first tackle the simple part selection problem, and then introduce a time constraint for the tasks to be performed for selected parts and search for incomplete time constrained sequences. We formulate our first problem as a Mixed Integer Problem and show that the constraint set of this formulation is totally unimodular. We also provide the dual formulation of our problem and its interpretation. For time-constrained part selection problem we propose a branch-and-bound algorithm. We first develop some reduction mechanism to reduce the size of the problem. Our solution procedure is capable of solving problems with up to 94 parts and tasks.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references (p. 73-93).

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 63-71, 126-140.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.