Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm – with or without anchoring them in the plasma membrane –, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised neither by adding a C-terminal stabilisation signal nor by anchoring the protein at the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

System design and resource management in the next-generation integrated WLAN and 3G cellular networks

Next-generation integrated wireless local area network (WLAN) and 3G cellular networks aim to take advantage of the roaming ability in a cellular network and the high data rate services of a WLAN. To ensure successful implementation of an integrated network, many issues must be carefully addressed, including network architecture design, resource management, quality-of-service (QoS), call admission control (CAC) and mobility management. ^ This dissertation focuses on QoS provisioning, CAC, and the network architecture design in the integration of WLANs and cellular networks. First, a new scheduling algorithm and a call admission control mechanism in IEEE 802.11 WLAN are presented to support multimedia services with QoS provisioning. The proposed scheduling algorithms make use of the idle system time to reduce the average packet loss of realtime (RT) services. The admission control mechanism provides long-term transmission quality for both RT and NRT services by ensuring the packet loss ratio for RT services and the throughput for non-real-time (NRT) services. ^ A joint CAC scheme is proposed to efficiently balance traffic load in the integrated environment. A channel searching and replacement algorithm (CSR) is developed to relieve traffic congestion in the cellular network by using idle channels in the WLAN. The CSR is optimized to minimize the system cost in terms of the blocking probability in the interworking environment. Specifically, it is proved that there exists an optimal admission probability for passive handoffs that minimizes the total system cost. Also, a method of searching the probability is designed based on linear-programming techniques. ^ Finally, a new integration architecture, Hybrid Coupling with Radio Access System (HCRAS), is proposed for lowering the average cost of intersystem communication (IC) and the vertical handoff latency. An analytical model is presented to evaluate the system performance of the HCRAS in terms of the intersystem communication cost function and the handoff cost function. Based on this model, an algorithm is designed to determine the optimal route for each intersystem communication. Additionally, a fast handoff algorithm is developed to reduce the vertical handoff latency.^

Foundational forensic techniques for cellular and ad hoc multi-hop networks

The Internet has become an integral part of our nation’s critical socio-economic infrastructure. With its heightened use and growing complexity however, organizations are at greater risk of cyber crimes. To aid in the investigation of crimes committed on or via the Internet, a network forensics analysis tool pulls together needed digital evidence. It provides a platform for performing deep network analysis by capturing, recording and analyzing network events to find out the source of a security attack or other information security incidents. Existing network forensics work has been mostly focused on the Internet and fixed networks. But the exponential growth and use of wireless technologies, coupled with their unprecedented characteristics, necessitates the development of new network forensic analysis tools. This dissertation fostered the emergence of a new research field in cellular and ad-hoc network forensics. It was one of the first works to identify this problem and offer fundamental techniques and tools that laid the groundwork for future research. In particular, it introduced novel methods to record network incidents and report logged incidents. For recording incidents, location is considered essential to documenting network incidents. However, in network topology spaces, location cannot be measured due to absence of a ‘distance metric’. Therefore, a novel solution was proposed to label locations of nodes within network topology spaces, and then to authenticate the identity of nodes in ad hoc environments. For reporting logged incidents, a novel technique based on Distributed Hash Tables (DHT) was adopted. Although the direct use of DHTs for reporting logged incidents would result in an uncontrollably recursive traffic, a new mechanism was introduced that overcome this recursive process. These logging and reporting techniques aided forensics over cellular and ad-hoc networks, which in turn increased their ability to track and trace attacks to their source. These techniques were a starting point for further research and development that would result in equipping future ad hoc networks with forensic components to complement existing security mechanisms.

Cancer therapy combining the modalities of hyperthermia and chemotherapy: In vitro cellular response after rapid heat accumulation in the cancer cell

Hyperthermia is usually used at a sub-lethal level in cancer treatment to potentiate the effects of chemotherapy. The purpose of this study is to investigate the role of heating rate in achieving synergistic cell killing by chemotherapy and hyperthermia. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. The cytotoxicity, mode of cell death, induction of thermal tolerance and P-gp mediated MDR following the two different modes of heating were studied. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. A slow rate hyperthermia was provided by a cell culture incubator. The results show that the potentiating effect of hyperthermia to chemotherapy can be maximized by increasing the rate of heating as evident by the results from the cytotoxicity assay. When delivered at the same thermal dose, a rapid increase in temperature from 37°C to 43°C caused more cell membrane damage than gradually heating the cells from 37°C to 43°C and thus allowed for more intracellular accumulation of the chemotherapeutic agents. Different modes of cell death are observed by the two hyperthermia delivery methods. The rapid rate laser-ICG hyperthermia @ 43°C caused cell necrosis whereas the slow rate incubator hyperthermia @ 43°C induced very mild apoptosis. At 43°C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified. This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

Mitigating congestion by integrating time forecasting and realtime information aggregation in cellular networks

An iterative travel time forecasting scheme, named the Advanced Multilane Prediction based Real-time Fastest Path (AMPRFP) algorithm, is presented in this dissertation. This scheme is derived from the conventional kernel estimator based prediction model by the association of real-time nonlinear impacts that caused by neighboring arcs’ traffic patterns with the historical traffic behaviors. The AMPRFP algorithm is evaluated by prediction of the travel time of congested arcs in the urban area of Jacksonville City. Experiment results illustrate that the proposed scheme is able to significantly reduce both the relative mean error (RME) and the root-mean-squared error (RMSE) of the predicted travel time. To obtain high quality real-time traffic information, which is essential to the performance of the AMPRFP algorithm, a data clean scheme enhanced empirical learning (DCSEEL) algorithm is also introduced. This novel method investigates the correlation between distance and direction in the geometrical map, which is not considered in existing fingerprint localization methods. Specifically, empirical learning methods are applied to minimize the error that exists in the estimated distance. A direction filter is developed to clean joints that have negative influence to the localization accuracy. Synthetic experiments in urban, suburban and rural environments are designed to evaluate the performance of DCSEEL algorithm in determining the cellular probe’s position. The results show that the cellular probe’s localization accuracy can be notably improved by the DCSEEL algorithm. Additionally, a new fast correlation technique for overcoming the time efficiency problem of the existing correlation algorithm based floating car data (FCD) technique is developed. The matching process is transformed into a 1-dimensional (1-D) curve matching problem and the Fast Normalized Cross-Correlation (FNCC) algorithm is introduced to supersede the Pearson product Moment Correlation Co-efficient (PMCC) algorithm in order to achieve the real-time requirement of the FCD method. The fast correlation technique shows a significant improvement in reducing the computational cost without affecting the accuracy of the matching process.

Forecasting enrollment in differential assessment programs using cellular automata

Urban growth models have been used for decades to forecast urban development in metropolitan areas. Since the 1990s cellular automata, with simple computational rules and an explicitly spatial architecture, have been heavily utilized in this endeavor. One such cellular-automata-based model, SLEUTH, has been successfully applied around the world to better understand and forecast not only urban growth but also other forms of land-use and land-cover change, but like other models must be fed important information about which particular lands in the modeled area are available for development. Some of these lands are in categories for the purpose of excluding urban growth that are difficult to quantify since their function is dictated by policy. One such category includes voluntary differential assessment programs, whereby farmers agree not to develop their lands in exchange for significant tax breaks. Since they are voluntary, today’s excluded lands may be available for development at some point in the future. Mapping the shifting mosaic of parcels that are enrolled in such programs allows this information to be used in modeling and forecasting. In this study, we added information about California’s Williamson Act into SLEUTH’s excluded layer for Tulare County. Assumptions about the voluntary differential assessments were used to create a sophisticated excluded layer that was fed into SLEUTH’s urban growth forecasting routine. The results demonstrate not only a successful execution of this method but also yielded high goodness-of-fit metrics for both the calibration of enrollment termination as well as the urban growth modeling itself.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

Two adaptation mechanisms regulate cellular migration in Dictyostelium discoideum

Dictyostelium discoideum is a simple model widely used to study many cellular functions, including differentiation, gene regulation, cellular trafficking and directional migration. Adaptation mechanisms are essential in the regulation of these cellular processes. The misregulation of adaptation components often results in persistent activation of signaling pathways and aberrant cellular responses. Studying adaptation mechanisms regulating cellular migration will be crucial in the treatment of many pathological conditions in which motility plays a central role, such as tumor metastasis and acute inflammation. I will describe two adaptation mechanisms regulating directional migration in Dictyostelium cells. The Extracellular signal Regulated Kinase 2 (ERK2) plays an essential role in Dictyostelium cellular migration. ERK2 stimulates intracellular cAMP accumulation in chemotaxing cells. Aberrant ERK2 regulation results in aberrant cAMP levels and defective directional migration. The MAP Phosphatase with Leucine-rich repeats (MPL1) is crucial for ERK2 adaptation. Cells lacking, MPL1 (mpl1- cells) displayed higher pre-stimulus and persistent post-stimulus ERK2 phosphorylation, defective cAMP production and reduced cellular migration. Reintroduction of a full length Mpl1 into mpl1- cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild type cells (Wt). These results suggest Mpl1 is essential for proper regulation of ERK2 phosphorylation and optimal motility in Dictyostelium cells. Cellular polarization in Dictyostelium cells in part is regulated by the activation of the AGC-related kinase Protein Kinase Related B1 (PKBR1). The PP2A regulatory subunit, B56, and the Glycogen Synthase Kinase 3 (GSK3) are necessary for PKBR1 adaptation in Dictyostelium cells. Cells lacking B56, psrA-cells, exhibited high basal and post-stimulus persistent phosphorylation of PKBR1, increased phosphorylation of PKBR1 substrates, and aberrant motility. PKBR1 adaptation is also regulated by the GSK3. When the levels of active GSK3 are reduced in Wt and psrA- cells, high basal levels of phosphorylated PKBR1 were observed, in a Ras dependent, but B56 independent mechanism. Altogether, PKBR1 adaptation is regulated by at least two independent mechanisms: one by GSK3 and another by PP2A/B56.

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

Foundational Forensic Techniques for Cellular and Ad Hoc Multi-hop Networks

The Internet has become an integral part of our nation's critical socio-economic infrastructure. With its heightened use and growing complexity however, organizations are at greater risk of cyber crimes. To aid in the investigation of crimes committed on or via the Internet, a network forensics analysis tool pulls together needed digital evidence. It provides a platform for performing deep network analysis by capturing, recording and analyzing network events to find out the source of a security attack or other information security incidents. Existing network forensics work has been mostly focused on the Internet and fixed networks. But the exponential growth and use of wireless technologies, coupled with their unprecedented characteristics, necessitates the development of new network forensic analysis tools. This dissertation fostered the emergence of a new research field in cellular and ad-hoc network forensics. It was one of the first works to identify this problem and offer fundamental techniques and tools that laid the groundwork for future research. In particular, it introduced novel methods to record network incidents and report logged incidents. For recording incidents, location is considered essential to documenting network incidents. However, in network topology spaces, location cannot be measured due to absence of a 'distance metric'. Therefore, a novel solution was proposed to label locations of nodes within network topology spaces, and then to authenticate the identity of nodes in ad hoc environments. For reporting logged incidents, a novel technique based on Distributed Hash Tables (DHT) was adopted. Although the direct use of DHTs for reporting logged incidents would result in an uncontrollably recursive traffic, a new mechanism was introduced that overcome this recursive process. These logging and reporting techniques aided forensics over cellular and ad-hoc networks, which in turn increased their ability to track and trace attacks to their source. These techniques were a starting point for further research and development that would result in equipping future ad hoc networks with forensic components to complement existing security mechanisms.

Analysis of the lateral diffuse metal-oxide semiconductor (LDMOS) power amplifier for cellular base stations

The primary purpose of this thesis was to present a theoretical large-signal analysis to study the power gain and efficiency of a microwave power amplifier for LS-band communications using software simulation. Power gain, efficiency, reliability, and stability are important characteristics in the power amplifier design process. These characteristics affect advance wireless systems, which require low-cost device amplification without sacrificing system performance. Large-signal modeling and input and output matching components are used for this thesis. Motorola's Electro Thermal LDMOS model is a new transistor model that includes self-heating affects and is capable of small-large signal simulations. It allows for most of the design considerations to be on stability, power gain, bandwidth, and DC requirements. The matching technique allows for the gain to be maximized at a specific target frequency. Calculations and simulations for the microwave power amplifier design were performed using Matlab and Microwave Office respectively. Microwave Office is the simulation software used in this thesis. The study demonstrated that Motorola's Electro Thermal LDMOS transistor in microwave power amplifier design process is a viable solution for common-source amplifier applications in high power base stations. The MET-LDMOS met the stability requirements for the specified frequency range without a stability-improvement model. The power gain of the amplifier circuit was improved through proper microwave matching design using input/output-matching techniques. The gain and efficiency of the amplifier improve approximately 4dB and 7.27% respectively. The gain value is roughly .89 dB higher than the maximum gain specified by the MRF21010 data sheet specifications. This work can lead to efficient modeling and development of high power LDMOS transistor implementations in commercial and industry applications.

Community control in cellular protein production : consequences for amino acid starvation

The authors thank M. C. Romano, I. Stansfield, L. Ciandrini, A. Kort, and M. Rehberg for helpful discussions. This work was funded by BBSRC grants BB/F00513/X1 and BB/G010722, and the Scottish Universities Life Science Alliance (SULSA).

Community control in cellular protein production : consequences for amino acid starvation

The authors thank M. C. Romano, I. Stansfield, L. Ciandrini, A. Kort, and M. Rehberg for helpful discussions. This work was funded by BBSRC grants BB/F00513/X1 and BB/G010722, and the Scottish Universities Life Science Alliance (SULSA).