Interaction of hepatitis C virus polymerase with host cell proteins

Hepatitis C virus (HCV) interacts with host cell proteins to modify cellular pathways creating a favourable environment that facilitates its replication and persistence. The purpose of the work presented in this thesis was to identify cellular proteins that can interact with NS5B, the virus's RNA-dependent RNA polymerase, that may contribute to the virus's biology. A number of cellular proteins were found to interact with NS5B using the yeast two-hybrid system. These proteins were involved in cellular pathways such as interferon signalling, lipid transport and metabolism, protein trafficking, cell proliferation and apoptosis. Of these, phospholipid scramblase 1 (PLSCR1) and zinc finger protein 143 (ZNF143) were selected for further investigation. The interactions were confirmed in vitro, and, for PLSCR1, the region that interacted with NS5B was determined to be within the amino-terminal region of the protein (61-137 a.a.). NS5B interacted with PLSCR1 and ZNF143 via a single interacting region localized in its N-terminus (1-153 a.a.).Expression of PLSCR1 or ZNF143 enhanced the ability of interferon to stimulate transcription from an interferon-stimulated response element (ISRE) reporter construct. Co-expression with NS5B was found to down-regulate this activity. Expression of a number of interferon-stimulated genes was investigated in the presence of NS5B, PLSCR1 or ZNF143 but no significant effect was observed. Overexpression of PLSCR1 had no effect on HCV sub-genomic replicon replication, while reduction of its expression by short hairpin RNA (shRNA) enhanced replication. Overexpression of ZNF143 was found to have a suppressive effect on replication but downregulating its expression did not enhance replication. In addition to using the yeast two-hybrid system to identify NS5B- interacting proteins, an in vitro pulldown assay coupled with mass spectrometry identified α- and β -tubulin associated with NS5B in vitro and in vivo. Subsequently this association was demonstrated to be an indirect interaction but the intermediatory partner was not identified. The domain that mediated the association with α- and β-tubulin was determined to be within the N-terminus of NS5B (1-153 a.a.). Nocodazole, an inhibitor of tubulin polymerization, had a marked effect on the association of α -tubulin with NS5B displacing it from the complex but had no effect on β -tubulin's association. Utilizing an HCV sub- genomic replicon, nocodazole was shown to have a significant inhibitory effect on replication. Taken together the data presented in this thesis showed that NS5B had a multitude of potential interactions with a variety of cellular proteins. The biological significance of some of these interactions on the cellular response to IFN and replicon replication was investigated. This work has generated a number of novel observations on the interaction between the virus and the cell that warrant future investigatio

Modeling Cotton and Winter Wheat Growth and Yield Responses to Irrigation Management in the Texas High Plains and Rolling Plains

A significant portion of the intensively cultivated agricultural areas in the U.S. is located in the Texas High Plains and Rolling Plains. In recent years, decreasing ground water supplies and precipitation variability are presenting challenges for profitable cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) production in these regions. A field study was conducted in 2012 and 2013 at Chillicothe, TX, to investigate the growth, yield, and water use efficiency (WUE) responses of cotton cultivars under different irrigation and tillage treatments. Results revealed that deficit irrigation of 45% of cotton evapotranspiration (ET) increased the dryland yield and WUE by 260% and 39%, respectively. The irrigation-by-variety interaction showed that the 90% ET replacement treatment involving PHY375 produced the greatest lint yield and WUE. Tillage did not significantly affect lint yield, WUE, and fiber quality. Increasing irrigation resulted in a linear increase in fiber length and strength, and a linear decrease in fiber micronaire. Two vegetation indices, Normalized Difference Vegetation Index (NDVI) and Normalized Difference Water Index (NDWI) were calculated using spectral reflectance dat. During the peak growing season, NDWI performed better compared to NDVI as no saturation problems were observed. The Crop Water Stress Index (CWSI) calculated using canopy and air temperature measurements showed significant differences among irrigation treatments. It was also observed that the CWSI and NDWI were negatively correlated. A modeling study was performed using the cotton growth simulation model, Cotton2K, to investigate the lint yield, WUE, and economic return responses using 31 years weather records (1980 – 2010) from the Texas Rolling Plains. Results revealed that replacing 112% ET maximized the yield while economic return was maximized at 108% ET. When water resources are limited, deficit irrigation at 80% ET replacement can be used to improve cotton WUE without significant yield and economic reductions in the semi-arid Texas Rolling Plains. A third study was performed on winter wheat using the DSSAT-CERES-Wheat model to investigate winter wheat growth and yield responses to irrigation management in the Texas High Plains using long-term weather datasets available for the Texas High Plains region (1980-2012). Results of winter wheat response to irrigation indicated that deficit irrigation between jointing and anthesis could significantly increase winter wheat grain yield and WUE. Application of 100 mm of irrigation at jointing and 120 mm at anthesis was found to produce a grain and biomass yields and WUE similar to full irrigation with significant amount of water saving

P-Wave Dispersion and Its Relationship with the Severity of the Coronary Artery Disease in Patients with Stable Angina

Background: In Electrocardiography (ECG), the term "P-wave dispersion" (PWD) refers to variation between maximum and minimum P-waves recorded from various Electrocardiography leads. P-wave analysis in ECG may become more widely used marker in many clinical contexts as a result of improved methods for capturing and analyzing this ECG. The link between (PD) and severity of coronary artery disease (CAD) was established among personnel who had stable form of CAD. Objective: We wanted to determine if there was an association between P wave dispersion and CAD severity in patients who had stable CAD.Patients and methods: At Al-Hussein University Hospital Cath Lab, 51 consecutive patients were referred for diagnostic coronary angiography in a cross-sectional trial. They were classified into group A (control group, n=8), who were found to have normal coronary angiography and group B (n = 43), who were found to have coronary artery disease. Results: P wave dispersion was significantly different across cases, with 95.3 percent of patients having abnormal P wave dispersion compared to 100 percent of control cases. P wave dispersion and Gensini score were statistically significantly different between cases and controls where cases had significantly higher scores than controls. Dispersion of P waves was statistically linked to number of vessels affected as well as Gensini scores. The P wave dispersion and the Gensini score showed a strong positive association.Conclusion: Stable coronary artery disease (CAD) patients had aberrant PWD, according to the findings of this study, in patients with stable coronary artery disease, severity of the CAD is correlated with P wave dispersion

Fatigue cycles and performance evaluation of accelerating aging heat treated aluminum semi solid materials designed for automotive dynamic components

The A357-type (Al-Si-Mg) aluminum semi solid casting materials are known for their excellent strength and good ductility, which make them materials of choice, preferable in the manufacturing of automotive dynamic mechanical components. Semi-solid casting is considered as an effective technique for the manufacturing of automotive mechanical dynamic components of superior quality performance and efficiency. The lower control arm in an automotive suspension system is the significant mechanical dynamic component responsible for linking the wheels of the vehicle to the chassis. A new trend is to manufacture this part from A357 aluminum alloy due to its lightweight, high specific strength, and better corrosion resistance than steel. This study proposes different designs of a suspension control arm developed, concerning its strength to weight ratio. Furthermore, this study aims to investigate the effect of accelerating thermal aging treatments on the fatigue life of bending fatigue specimens manufactured from alloy A357 using the Rheocasting semi-solid technology. The results revealed that the multiple aging cycles, of WC3, indicated superior fatigue life compared to standard thermal aging cycles. On the other hand, the proposed designs of automotive suspension control components showed higher strength-to-weight ratios, better stress distribution, and lower Von-Mises stresses compared to conventional designs

Digital enhancement techniques for fractional-N frequency synthesizers

Meeting the demand for unprecedented connectivity in the era of internet-of-things (IoT) requires extremely energy efficient operation of IoT nodes to extend battery life. Managing the data traffic generated by trillions of such nodes also puts severe energy constraints on the data centers. Clock generators that are essential elements in these systems consume significant power and therefore must be optimized for low power and high performance. The focus of this thesis is on improving the energy efficiency of frequency synthesizers and clocking modules by exploring design techniques at both the architectural and circuit levels. In the first part of this work, a digital fractional-N phase locked loop (FNPLL) that employs a high resolution time-to-digital converter (TDC) and a truly ΔΣ fractional divider to achieve low in-band noise with a wide bandwidth is presented. The fractional divider employs a digital-to-time converter (DTC) to cancel out ΔΣ quantization noise in time domain, thus alleviating TDC dynamic range requirements. The proposed digital architecture adopts a narrow range low-power time-amplifier based TDC (TA-TDC) to achieve sub 1ps resolution. Fabricated in 65nm CMOS process, the prototype PLL achieves better than -106dBc/Hz in-band noise and 3MHz PLL bandwidth at 4.5GHz output frequency using 50MHz reference. The PLL achieves excellent jitter performance of 490fsrms, while consumes only 3.7mW. This translates to the best reported jitter-power figure-of-merit (FoM) of -240.5dB among previously reported FNPLLs. Phase noise performance of ring oscillator based digital FNPLLs is severely compromised by conflicting bandwidth requirements to simultaneously suppress oscillator phase and quantization noise introduced by the TDC, ΔΣ fractional divider, and digital-to-analog converter (DAC). As a consequence, their FoM that quantifies the power-jitter tradeoff is at least 25dB worse than their LC-oscillator based FNPLL counterparts. In the second part of this thesis, we seek to close this performance gap by extending PLL bandwidth using quantization noise cancellation techniques and by employing a dual-path digital loop filter to suppress the detrimental impact of DAC quantization noise. A prototype was implemented in a 65nm CMOS process operating over a wide frequency range of 2.0GHz-5.5GHz using a modified extended range multi-modulus divider with seamless switching. The proposed digital FNPLL achieves 1.9psrms integrated jitter while consuming only 4mW at 5GHz output. The measured in-band phase noise is better than -96 dBc/Hz at 1MHz offset. The proposed FNPLL achieves wide bandwidth up to 6MHz using a 50 MHz reference and its FoM is -228.5dB, which is at about 20dB better than previously reported ring-based digital FNPLLs. In the third part, we propose a new multi-output clock generator architecture using open loop fractional dividers for system-on-chip (SoC) platforms. Modern multi-core processors use per core clocking, where each core runs at its own speed. The core frequency can be changed dynamically to optimize for performance or power dissipation using a dynamic frequency scaling (DFS) technique. Fast frequency switching is highly desirable as long as it does not interrupt code execution; therefore it requires smooth frequency transitions with no undershoots. The second main requirement in processor clocking is the capability of spread spectrum frequency modulation. By spreading the clock energy across a wide bandwidth, the electromagnetic interference (EMI) is dramatically reduced. A conventional PLL clock generation approach suffers from a slow frequency settling and limited spread spectrum modulation capabilities. The proposed open loop fractional divider architecture overcomes the bandwidth limitation in fractional-N PLLs. The fractional divider switches the output frequency instantaneously and provides an excellent spread spectrum performance, where precise and programmable modulation depth and frequency can be applied to satisfy different EMI requirements. The fractional divider has unlimited modulation bandwidth resulting in spread spectrum modulation with no filtering, unlike fractional-N PLL; consequently it achieves higher EMI reduction. A prototype fractional divider was implemented in a 65nm CMOS process, where the measured peak-to-peak jitter is less than 27ps over a wide frequency range from 20MHz to 1GHz. The total power consumption is about 3.2mW for 1GHz output frequency. The all-digital implementation of the divider occupies the smallest area of 0.017mm2 compared to state-of-the-art designs. As the data rate of serial links goes higher, the jitter requirements of the clock generator become more stringent. Improving the jitter performance of conventional PLLs to less than (200fsrms) always comes with a large power penalty (tens of mWs). This is due to the PLL coupled noise bandwidth trade-off, which imposes stringent noise requirements on the oscillator and/or loop components. Alternatively, an injection-locked clock multiplier (ILCM) provides many advantages in terms of phase noise, power, and area compared to classical PLLs, but they suffer from a narrow lock-in range and a high sensitivity to PVT variations especially at a large multiplication factor (N). In the fourth part of this thesis, a low-jitter, low-power LC-based ILCM with a digital frequency-tracking loop (FTL) is presented. The proposed FTL relies on a new pulse gating technique to continuously tune the oscillator's free-running frequency. The FTL ensures robust operation across PVT variations and resolves the race condition existing in injection locked PLLs by decoupling frequency tuning from the injection path. As a result, the phase locking condition is only determined by the injection path. This work also introduces an accurate theoretical large-signal analysis for phase domain response (PDR) of injection locked oscillators (ILOs). The proposed PDR analysis captures the asymmetric nature of ILO's lock-in range, and the impact of frequency error on injection strength and phase noise performance. The proposed architecture and analysis are demonstrated by a prototype fabricated in 65 nm CMOS process with active area of 0.25mm2. The prototype ILCM multiplies the reference frequency by 64 to generate an output clock in the range of 6.75GHz-8.25GHz. A superior jitter performance of 190fsrms is achieved, while consuming only 2.25mW power. This translates to a best FoM of -251dB. Unlike conventional PLLs, ILCMs have been fundamentally limited to only integer-N operation and cannot synthesize fractional-N frequencies. In the last part of this thesis, we extend the merits of ILCMs to fractional-N and overcome this fundamental limitation. We employ DTC-based QNC techniques in order to align injected pulses to the oscillator's zero crossings, which enables it to pull the oscillator toward phase lock, thus realizing a fractional-N ILCM. Fabricated in 65nm CMOS process, a prototype 20-bit fractional-N ILCM with an output range of 6.75GHz-8.25GHz consumes only 3.25mW. It achieves excellent jitter performance of 110fsrms and 175fsrms in integer- and fractional-N modes respectively, which translates to the best-reported FoM in both integer- (-255dB) and fractional-N (-252dB) modes. The proposed fractional-N ILCM also features the first-reported rapid on/off capability, where the transient absolute jitter performance at wake-up is bounded below 4ps after less than 4ns. This demonstrates almost instantaneous phase settling. This unique capability enables tremendous energy saving by turning on the clock multiplier only when needed. This energy proportional operation leverages idle times to save power at the system-level of wireline and wireless transceivers

Conceptual framework for off-site roof stacking construction

A great deal of interest in off-site construction has been remarkable over the last decade. However, building on the rooftops of existing building has not been given a significant importance as a subject of research, despite its dependence on off-site construction and prefabrication. Thus, this paper develops a novel conceptual framework to support a multidisciplinary decision making for selecting off-site prefabricated constructional system for roof stacking. The multidisciplinary approach includes each of safety, logistics, cost, time, environmental impact, and quality of construction as major criteria in the decision making process. This paper is the outcome of an exhaustive investigation of more than 136 roof stacking projects built during the last 20 years. The development of framework is supported by a feedback validation loop based on semi-structured interviews with experts in the field of roof stacking and off-site construction.DenCity Prototype: Concepts of Zero Energy 28 Lightweight Construction Households for Urban Densificatio

Detection of A2142G, A2142C and A2143G clarithromycin mutations in Helicobacter pylori in Alexandria University Pediatric Hospital

Background: Helicobacter pylori (H. pylori)colonizes the stomach and affect almost 50% of the world’s population. Clarithromycin is considered a cornerstone for H. pylori treatment. Emergence of clarithromycin resistance (CLR-R) has played a major role in failure of H. pylori eradication both in adults and children.  Clarithromycin resistance is mostly due to mutations in 23S rRNA gene: A2142G, A2142C, and A2143G. The aim of the current study is to determine the prevalence of CLR-R among H. pylori infected children with prior clarithromycin treatment. Materials and Methods: Multiple endoscopic gastric biopsies were collected from 50 H. pylori infected children after cessation of clarithromycin-based treatment. Samples were subjected to histopathological examinations, rapid urease test (RUT) and simultaneous molecular detection of H. pylori infection as well as CLR-R by multiplex Real-Time polymerase chain reaction (PCR). Results: Histopathological examinations and RUT revealed H. pylori in 74% and 92% of samples respectively. Molecular detection of CLR-R showed that 62.5% positive H. pylori cases were not harboring any of the tested mutations, while 25% harbored 2143A-G single mutation. Double mutations (2142A-C and 2143A-G) were detected in only 4 cases. Statistical significant correlation existed between both RUT and PCR results as well as between histopathological findings and PCR test results. Conclusions: A combination of histopathogy, RUT and multiplex PCR procedures offers a real benefit in the simultaneous diagnosis of H. pylori infection along with clarithromycin resistance status. Other mechanisms of clarithromycin resistance need to be investigated to explain treatment failure in absence of the previously detected mutations

Phenotypic and Genotypic Identification of Vancomycin Resistant Enterococci from Different Sources

Enterococci are reservoirs for transmission of the most clinically important antimicrobial resistances such as vancomycin resistance. Therefore, this work aimed to determine the occurrence of enterococci and their respective vancomycine resistance genes (vanA and vanB) from different sources. Two hundred and twenty-four samples from chickens, turkey, fish and human urine, as well as, two types of human food including milk (raw and milk from mastitic animals) and sausage were tested for isolation of Enterococcus species. The isolates were identified morphologically and biochemically using catalase test, sodium chloride tolerance and growth at pH 9.6 and 10- 45˚C. The vancomycin resistance profile of the isolates was verified by both disc diffusion and agar dilution methods. The genotypic enterococcal identification at both genus and species levels and their vancomycine resistance genes were also ascertained using PCR amplification of the respective genes for 28 isolates. Enterococci isolation rate was 70% of the examined samples with a higher percentage of vancomycine resistance (53.5%) and the minimum inhibitory concentrations (MICs) ranged from 16 to 512 µg/mL. Molecular identification of 28 enterococcal isolates revealed the dominance of E. faecalis (42.8%) and clarified a higher proportion of vanA (78.5%) and vanB (67.8%) genes. In conclusion, administration of the antimicrobials mainly vancomycin may be considered as a pronounced stress factor in the veterinary and human practices. In addition, VRE can act as a reservoir for vancomycin resistance

Methodology for design decision support of cost-optimal zero-energy lightweight construction

peer reviewedThe interest to find cost-optimal zero-energy solutions for building, using multi-objective optimization, has risen dramatically over the last decade. Accordingly, several studies have been carried out, proposing new methods and tools. None, however, has introduced a simplified approach that is viable by a broader range of users. This study addresses this lack, offering a methodology that supports the decision making process on cost-optimal zero energy building, using a novel approach, namely Multi-Objective Parametric Analysis (MOPA), rather than optimization algorithms. This study adds to the domain of roof stacking construction by setting the weight of construction as a third objective. The current methodology is applied to a newly developed theoretical Reference Building (RB) for a Belgian passive roof stacking house. Different options of the building’s superstructure components (walls, roof, and windows) have been examined. MOPA follows three consecutive steps: modeling setup, parametric simulation, and ends up with evaluation and selection. The results show cost-optimal zero-energy and lightweight packages of design variables for the building envelope.DenCity Prototype: Concepts of Zero Energy 28 Lightweight Construction Households for Urban Densificatio