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- Tang, Ka-Wei, 1983
(författare)
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Herpes Simplex Virus 1 DNA replication and its role in recombination and transcription
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Herpes simplex virus 1 (HSV-1) is one of nine different herpesvirus infecting man. They are all capable of establishing a life-long latent state following the primary infection. HSV-1 as well as other herpesviruses may reactivate from the latent state and give rise to a productive infection with clinical symptoms or asymptomatic shedding. HSV-1 infections are primarily treated by targeting the viral DNA replication carried out by a molecular machinery, a replisome, encoded by the virus. Here we examine the mechanism of initiation of viral DNA replication and also how viral DNA replication interacts with DNA recombination and gene expression. In our first study we examined the initiation-step of HSV-1 replication. The origin binding protein (OBP) initiates replication by binding to the origin of replication (oriS and/or oriL). We showed, using phylogenetics and biochemical experiments, that there was a step-wise evolutionary development of herpesvirus DNA replication initiation. The initial divergence was seen in herpesviruses acquiring an amino-acid motif RVKNL in OBP which binds the sequence TTCGCAC in the oriS. The next step was the development of an ICP8 binding motif at the C-terminus of OBP and finally the arrangement of the binding-sites for OBP in the oriS-sequence and the ability to form a stable hairpin. We presented molecular in vitro data to support the phylogenetic analysis and thereby defining essential motifs in OBP for protein-protein and protein-DNA interactions. The next study was focused on genetic recombination between different HSV-1 strains. We followed the propagation of HSV-1 in cells infected with one to three genotypes of HSV-1 and calculated the number of recombination events. We found evidence for Rad51 and Rad52 involvement in recombination of the unique long and unique short gene segments of HSV-1. We also observed an increased recombination rate in cells with retarded ligation of Okazaki-fragments. The fidelity of recombination in virus propagated through mixed infections appears to be high since expansion or shortening of repeated sequences in the US7 gene was not detected. In the third study we examined the replication-coupled transcription of HSV-1 late genes, which are known to depend on DNA replication for efficient expression. Using chromatin immuno-precipitation we could determine that recruitment of RNA polymerase II to late gene promoters occur even in the absence of replication. Recruitment was dependent on ICP4, but delayed in comparison with early gene promoters. These observations suggested the involvement of transcription elongation and/or maturation in the expression of gamma genes. By using the drug DRB, which inhibits the kinase CDK9, a component of the positive transcription elongation factor B, and siRNA against Spt5, a transcription processivity factor, we could show a specific impairment of late gene expression, with only minimal effect on early gene expression and DNA synthesis. We suggest that CDK9 and Spt5 are specifically recruited to replicated late genes and mediate a maturation of transcribed mRNA for nuclear exit. In summary, we have studied essential molecular processes in the HSV-1 life cycle and identified molecular interactions as well as mechanistic pathways, which may serve as future drug targets.
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| 2. |
- Wei, Shuoguo, 1986-
(författare)
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Core Acquisition Management in Remanufacturing : Current Status and Modeling Techniques
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Remanufacturing is an important product recovery option that benefits our sustainable development. Cores, i.e. the used products/parts, are essential resources for remanufacturing. Without cores, there will not be any remanufactured products. Challenges in the core acquisition process are mainly caused by the uncertainties of: return volume, timing and core quality. Core Acquisition Management actively attempts to reduce these uncertainties and achieve a better balance of demand and return for the remanufacturers. The aim of this dissertation is to extend the knowledge of Core Acquisition Management in remanufacturing, by investigating the current status of research and industrial practice, and developing quantitative models that assist the decision making in the core acquisition process.In the dissertation, a literature review is firstly conducted to provide an overview about the current research in Core Acquisition Management. Possible further research interests, for example, more studies based on non-hybrid remanufacturing systems and imperfect substitution assumption are suggested. Through an industrial survey carried out in a fast developing remanufacturing market - China, environmental responsibility and ethical concerns, customer orientation and strategic advantage are identified as the most important motives for the remanufacturers, while customer recognition is their most serious barrier at present. Suggestions for further improving the Chinese remanufacturing industry from the policy-makers’ perspective are provided. After the above investigation, mathematical models are then developed to assist the acquisition decisions in two aspects: to deal with the uncertainties of return volume and timing, and to deal with the uncertainties of core quality.Acquisition decision about volume and timing is firstly studied from a product life cycle perspective, where the demands for remanufactured products and the core availability change over time. According to industrial observations, the remanufacturing cost decreases with respect to its core inventory. Using optimal control theory, core acquisition and remanufacturing decisions are derived to maximize the remanufacturer's profit. It is found that besides a simple bang-bang type control policy (either collecting as much as possible, or nothing), a special form of synchronizing policy (adjusting the core collection rate with demand rate) also exists. Furthermore, the acquisition decision depends greatly on the valuation of cores, and Real Option Valuation approaches are later used to capture the value of flexibility provided by owning cores when different aspects of remanufacturing environment are random. More specifically, the value of disposing a core earlier is investigated when the price of remanufactured product is uncertain, and the impact of the correlation between stochastic demand and return is also studied.To deal with the uncertainties of core quality, refund policies with different numbers of quality classes are studied. Under the assumption of uniformly distributed quality, analytical solutions for these refund policies are derived. Numerical examples indicate that the customers’ valuation of cores is an important factor influencing the return rates and the remanufacturer’s profit. Refund policies with a small number of quality classes could already bring major advantages. Credit refund policies (without deposits) are included for comparisons. In addition, within a game theory framework, the trade-off of two types of errors of the quality inspection in a deposit-refund policy is studied. The salvage values of different cores show great influences on the remanufacturer’s policy choices. The value of information transparency about the inspection errors are studied under different conditions. Interestingly, the customer may actually return more low quality cores when the inspection accuracy is improved.
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