Line Balancing Problem with Multi-Manned Workstations and Resource Constraints: The Case of Electronics Waste Disassembly

The increasing public awareness of environmental protection and the scarcity of rare earth elements have made closed-loop supply chains a necessity in many sectors. In particular, recycling components and parts from end-of-life consumer electronics have drawn the attention of both academics and practitioners. Disassembly line balancing improves the resource efficiency of recycling operations. This study proposes a new mathematical formulation and hybrid metaheuristics for solving the Disassembly Line Balancing Problem (DLBP) considering multi-manned workstations and resource constraints. The transformed AND/OR graph is used for prioritizing disassembly tasks in the modeling process. The method is applied for optimizing a real-world case of laptop disassembly to showcase the usefulness of the approach. The performance of the developed metaheuristics is compared to minimize the number of workstations, operators, and machines involved in the disassembly operations. Further, the results are analyzed through sensitivity analysis. This study concludes by providing practical insights and suggestions for the future development of DLBPs

Functions of Some Capsular Polysaccharide Biosynthetic Genes in Klebsiella pneumoniae NTUH K-2044

The growing number of Klebsiella pneumoniae infections, commonly acquired in hospitals, has drawn great concern. It has been shown that the K1 and K2 capsular serotypes are the most detrimental strains, particularly to those with diabetes. The K1 cps (capsular polysaccharide) locus in the NTUH-2044 strain of the pyogenic liver abscess (PLA) K. pneumoniae has been identified recently, but little is known about the functions of the genes therein. Here we report characterization of a group of cps genes and their roles in the pathogenesis of K1 K. pneumoniae. By sequential gene deletion, the cps gene cluster was first re-delimited between genes galF and ugd, which serve as up- and down-stream ends, respectively. Eight gene products were characterized in vitro and in vivo to be involved in the syntheses of UDP-glucose, UDP-glucuronic acid and GDP-fucose building units. Twelve genes were identified as virulence factors based on the observation that their deletion mutants became avirulent or lost K1 antigenicity. Furthermore, deletion of kp3706, kp3709 or kp3712 (ΔwcaI, ΔwcaG or Δatf, respectively), which are all involved in fucose biosynthesis, led to a broad range of transcriptional suppression for 52 upstream genes. The genes suppressed include those coding for unknown regulatory membrane proteins and six multidrug efflux system proteins, as well as proteins required for the K1 CPS biosynthesis. In support of the suppression of multidrug efflux genes, we showed that these three mutants became more sensitive to antibiotics. Taken together, the results suggest that kp3706, kp3709 or kp3712 genes are strongly related to the pathogenesis of K. pneumoniae K1

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

The Ubiquitin-mediated Regulation of AMPK in Chronological Aging

出芽酵母(Saccharomyces cerevisiae)為分子生物學中重要的模式生物之一。因其 生活史短,基因又具保守性,故適合進行行老老化機制之研究。本實驗以驗證 Ubp8 及 Ubp10 調控 Snf1 的蛋白質降降解並影響老老化性狀狀的路路徑為目標,進行行研究。根據遺 傳資料料庫分析的結果,推測與高等動物具高度度保守性之酵母菌腺苷酸活化蛋白激 酶(AMPK) Snf1 可能為 Ubp8 及 Ubp10 此兩兩個去泛素化酵素 (deubiquitinases, DUBs) 的目標蛋白質。Snf1/AMPK 在細胞中扮演能量量調控的主要角色。正常情況下,Snf1 並不不活化;當環境中葡萄糖糖耗盡,Snf1 被上游激酶活化,透過改變染色質結構、 轉錄錄因子活化、轉錄錄作用調控等方式,進而增加細胞對其他碳源的利利用及對逆境 的耐受性。由西方墨點法的結果,發現 ubp8Δubp10Δ 細胞中的 Snf1 總量量因為 DUBs 的缺乏而有明顯減少的現象,但其 mRNA 表現並無受到阻斷;而在兩兩個 DUBs中, 又以 Ubp8 為主要影響 Snf1 蛋白質穩定性的酵素;在老老化相關路路徑上,此調控主要影響對氧化壓力力以及繼代存活壽命(CLS)的性狀狀,在複製存活壽命(RLS)上則沒有明顯差異異;此外,此突變株的再生能力力異異於目前所知的由基因不不穩定造成的癌化突變。進一步探討 Snf1 的泛素化修飾影響其磷酸化的結果顯示:在 ubp8Δubp10Δ 突變細胞的 Snf1總量量雖然減少,但有高度度磷酸化的現象,而此現象可能導致 Snf1對於壓力力反應序列列(Stress responsive element, STRE)相關之轉錄錄作用的調控,增加 ubp8Δubp10Δ 突變細胞對於逆境的耐受性。透過已知的 Snf1 調控機制,我們歸納出數數個影響 Snf1 高度度磷酸化可能的機制,以及其下游調控的蛋白影響逆境耐受的可能性。Ubiquitin is a small regulatory protein that is expressed ubiquitously in eukaryoticorganisms. Conjugation of ubiquitins to proteins directs them to compartments in the cell, including the proteasome, which destroys and recycles proteins. Based on the genetic interaction analysis, I purposed that Snf1, highly conserved yeast AMPK, may be a target by both of Ubp8 and Ubp10, two highly conserved ubiquitin-specific proteases (DUBs). Snf1 plays an important role in modulating energy status in the cell, which is usually inactive. Only when glucose depleted can Snf1 be activated by its upstream kinases. Via changing chromatin structures, activating transcription factors and modulating transcription, Snf1 enhances the abilities of the cell to use alternative carbon sources and resist stresses. In this study, I aimed to reveal the pathway of the ubiquitin-mediated Snf1 regulation. Western blot analyses demonstrated that the level of Snf1 was dramatically decreased in ubp8∆ubp10∆ cells, but the mRNA level detection did not change significantly. Snf1 has been shown to be involved in aging processes; I first examined the possible roles of Ubp8 and Ubp10 in yeast aging. The deletion of UBP8 and UBP10 affected the cellular resistance to oxidative stresses and chronological life span (CLS) phenotype, possibly due to the decreased Snf1 protein level; while there was no significant change in replicative life span (RLS). In addition, the ubp8∆ubp10∆ cells exhibited regrowth phenotype which is known to be induce accumulated ROS and mutagenesis; however, in those cells I found no evidence of ROS-mediated genome instabilities. Further investigation revealed that despite the protein level was decreased, Snf1 was hyperphosphorylated in ubp8∆ubp10∆ cells. The ubp8∆ubp10∆ cells was able to grow on non-fermentable carbon sources but was not the SNF1 deleted cells. Taken together, my results suggest that Snf1 is likely protected by both Ubp8 and Ubp10 from proteasome-mediated degradation, which diminishes thecellular level of Snf1. Interestingly, the remaining Snf1 in in ubp8∆ubp10∆ cells is hyperphosphorylated via an unknown mechanism. I propose that the hyperphosphorylated Snf1 in ubp8∆ubp10∆ cells are able to activate the stress-responsive (STRE) transcription to maintain the cellular resistance to stresses. The potential pathways that may participate in the hyperphosphorylaiton of Snf1 are discussed.口試委員審定書 ............................................................................................ i 謝誌 ............................................................................................................... ii 摘要 ..............................................................................................................iii Abstract ....................................................................................................... iv Contents....................................................................................................... vi List of Figures ............................................................................................. ix List of Tables ................................................................................................ x Chapter I: Introduction .............................................................................. 1 1.1 Budding yeast as a model organism.......... ..........................................1 1.2 Ubiquitin-mediated regulation .............................................................1 1.2.1 The ubiquitin-proteasome system...................... .............................1 1.2.2 Deubiquitinases: Ubp8 and Ubp10......................................................2 1.3 Structure and regulation of Snf1............................................................3 1.3.1 AMPK..................................................................................................3 1.3.2 Yeast AMPK: Snf1 complex..................................................................4 1.3.3 The regulation of Snf1 complex ....................................... ..................4 1.4 Aging in yeast ........................................................................................5 1.4.1 Replicative life span ............................................................................5 1.4.2 Chronological life span.......................................................................6 1.4.3 Adaptive regrowth .............................................................................7 1.4.4 The UPS in aging................................................................................7 1.4.5 The role of Snf1 complex in RLS.........................................................7 1.4.6 The role of Snf1 complex in CLS........................................................8 1.5 Objective of this study..........................................................................9 Chapter II: Materials and methods.........................................................10 2.1 Materials..........................................................................................10 2.1.1 Yeast strains .................................................................................10 2.1.2 Plasmids.........................................................................................10 2.1.3 Medium..........................................................................................11 2.2 Methods .............................................................................................13 2.2.1 The stability of Snf1-TAP at exponential stage ................................13 2.2.2 The mRNA expression levels of SNF1 in the mutants .......................14 2.2.3 The levels of FLAG-Snf1 protein turn-over .......................................14 2.2.4 The purification of Ub-conjugated proteins .....................................15 2.2.5 Spotting assay for alternative carbon sources and oxidative stresses.........15 2.2.6 Replicative lifespan measuremnt.......................................................15 2.2.7 Chronological lifespan measurement ..............................................16 Chapter III: Results .................................................................................. 17 3.1 Snf1 is the potential target of both Ubp8 and Ubp10...........................17 3.2 Functional interaction between Snf1 and Ubp8/Ubp10 .......................19 3.3 To determine the effect of decreased Snf1 on physiological functions...22 3.3.1 The ability of using alternative carbon sources ................................22 3.3.2 Replicative aging .............................................................................24 3.3.3 Chronological aging ........................................................................24 3.4 The role of Snf1 during aging ..............................................................25 3.4.1 The Snf1 protein levels during CLS ...................................................25 3.4.2 The effect of Snf1 on calorie restriction ............................................27 3.4.3 The stress resistance of aging cells in the absence of Ubp8/Ubp10 ..27 3.4.4 The regrowth phenotype .................... ...........................................28 3.4.5 The phosphorylation of Snf1 in DUB-deficient cells ................ .........31 Chapter IV: Discussion ............................................................................. 33 4.1 The decreased level of Snf1............................. ....................................33 4.2 The decreased Snf1 protein level and aging phenotype .......................34 4.3 AMPK activity in CR pathway.................. ..............................................35 4.4 Hyperphosphorylation of AMPK ...........................................................36 4.5 Furture work........................... ............................................................37 Chapter V: References .............................................................................. 39 Appendix ................................................................................................. 4

Identifying Predictors for Inflammation-Induced Preterm Birth: A Murine Study

Introduction: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. A large proportion of preterm deliveries is affected by intra-amniotic inflammation, which can occur in the presence (intra-amniotic infection) or absence (sterile intra-amniotic inflammation) of microbes. Studies have shown an association between intra-amniotic inflammation, cervical shortening, and changes in the cervicovaginal microbiome. However, their causal relationships are unknown. This study aims to determine the causality of intra-amniotic inflammation, cervical shortening, and cervicovaginal microbiome alterations. Methods: Pregnant C57BL/6 dams received an ultrasound-guided intra-amniotic injection of an endotoxin lipopolysaccharide (LPS) or the alarmin interleukin-1 alpha (IL-1 alpha) on 16.5 days post-coitum (n = 6-8 per group) to model intra-amniotic infection- or sterile intra-amniotic inflammation-associated preterm birth. Control dams were injected with saline (n=6-8). Cervical length was measured by ultrasound at time zero and 6-hours post-injection. In a second cohort of injected dams, cervical and vaginal tissues were collected 6 hours post-injection (n = 6 per group) for cervicovaginal microbiome analyses via 16S rRNA sequencing. Results: Dams that received intra-amniotic injections of LPS and IL-1 showed greater percentage of cervical shortening when compared to controls. Microbiome analyses showed taxonomic differences in the bacterial profiles of the cervical and vaginal tissues. However, there were no differences in bacterial profile richness/heterogeneity, composition/structure, and bacterial taxa abundance between the two contrasting groups using generalized linear models, PERMANOVA, LefSe, and ANCOM-BC analyses, respectively. Conclusion: Alarmin- and endotoxin-induced intra-amniotic inflammation led to cervical shortening, and this was not associated with an acute alteration of the cervicovaginal microbiome