Structural and Functional Characterization of SPOA Domains in Salmonella Typhimurium Type III Secretion Systems

Once optimistically believed to be a relic of the pre-antibiotic era, bacterial pathogens remain a substantial threat to human health, and the growing epidemic of antibiotic resistance has raised concerns for the long term prospects of antimicrobial therapy. By understanding the mechanisms used by bacteria to manipulate their host and cause disease, it is hypothesized that we might more rationally approach anti-infective therapeutic design. Type III secretion systems (T3SS) are employed by some gram-negative human pathogens to manipulate the host environment. One T3SS subtype, known as the “injectisome,” delivers virulence factors directly into host cells. The other T3SS subtype secretes the the polymeric flagellar filament used for motility. While both systems share related elements of a cytoplasmic “sorting platform” that facilitates the hierarchical secretion of protein substrates, the structural mechanism of its assembly remains unclear. The work described in this thesis makes strides towards the mechanistic understanding of T3SS sorting platform assembly by applying structural, biochemical, and genetic techniques to the characterization of the SctQ/FliM/FliN protein family and their interactions with other sorting platform components. These proteins uniquely possess Surface Presentation Of Antigens (SPOA) folds, and I will present the molecular structures of distinct homotypic and heterotypic SPOA-SPOA interactions in the Salmonella typhimurium SPI-1 sorting platform protein SpaO (Chapter 2). In Chapter 3, I structurally characterize the interaction of the heterotypic SPOA complex with a regulator of the SPI-1 ATPase and demonstrate the necessity of the interaction for T3SS secretory function. Then, I will present the homologous structures from the S. typhimurium flagellar apparatus and compare and contrast them with their SPI-1 homologues, providing an explanation for the observed subtype specificity in sorting platform assembly (Chapter 4). In Chapter 5, biochemical evidence for an interaction of the SpaO amino-terminal domain(s) with the homotypic SPOA complex is presented. These results provide a model for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design (Chapter 6)

A gradient-based parameter identification method for time-delay chaotic systems

In this paper, the parameter identification problem for a general class of time-delay chaotic systems is considered. The objective of the problem is to determine optimal values for an unknown time-delay and unknown system parameters such that the dynamic model of the system best fits given experimental data. We propose a gradient-based optimization algorithm to solve this problem, where accurate values for the partial derivatives of the error function are obtained by solving a set of auxiliary time-delay systems. Simulation results for two example problems show that the proposed algorithm is robust and efficient

By force or by fraud: optimizing U.S. information strategy with deception

Military deception (MILDEC) operations have a long and illustrious place in America's battlefield history. To great effect, MILDEC has enabled countless victories in every U.S. conflict since the Revolutionary War. However, the United States has allowed its deception capability to atrophy. Possible explanations for our MILDEC divestiture range from structural insufficiencies to an ethical framework that emphasizes truth and transparency. Simultaneously, the onset of the Information Age has leveled the playing field between state and non-state actors (NSA) and proved that lasting victory cannot be achieved by force alone. Yet, due in part to the difficulty involved in quantifiably measuring information strategy, the contemporary military's acceptance and understanding of information warfare has been limited. This necessitates the re-examination of U.S. information strategy formulation to address more effectively the challenges and complexities encountered in the human domain. To overcome this impediment, this thesis examines the intangible aspects of information warfare and proposes a structured decision-making tool capable of generating precise computations of optimal information strategies. By Force or by Fraud is a quantitative assessment of MILDEC's utility on the modern battlefield that is qualitatively tested against historic cases of information warfare.http://archive.org/details/byforceorbyfraud1094549457Major, United States ArmyMajor, United States ArmyApproved for public release; distribution is unlimited

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

Bis-(3 ',5 ') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K-d similar to 2 mu M). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence

Retrospective Study of Midazolam Protocol for Prehospital Behavioral Emergencies

Introduction: Agitated patients in the prehospital setting pose challenges for both patient care and emergency medical services (EMS) provider safety. Midazolam is frequently used to control agitation in the emergency department setting; however, limited data exist in the prehospital setting. We describe our experience treating patients with midazolam for behavioral emergencies in a large urban EMS system. We hypothesized that using midazolam for acute agitation leads to improved clinical conditions without causing significant clinical deterioration.Methods: We performed a retrospective review of EMS patient care reports following implementation of a behavioral emergencies protocol in a large urban EMS system from February 2014–June 2016. For acute agitation, paramedics administered midazolam 1 milligram (mg) intravenous (IV), 5 mg intramuscular (IM), or 5 mg intranasal (IN). Results were analyzed using descriptive statistics, Levene’s test for assessing variance among study groups, and t-test to evaluate effectiveness based on route.Results: In total, midazolam was administered 294 times to 257 patients. Median age was 30 (interquartile range 24–42) years, and 66.5% were male. Doses administered were 1 mg (7.1%) and 5 mg (92.9%). Routes were IM (52.0%), IN (40.8%), and IV (7.1%). A second dose was administered to 37 patients. In the majority of administrations, midazolam improved the patient’s condition (73.5%) with infrequent adverse events (3.4%). There was no significant difference between the effectiveness of IM and IN midazolam (71.0% vs 75.4%; p = 0.24).Conclusion: A midazolam protocol for prehospital agitation was associated with reduced agitation and a low rate of adverse events

Time-delay estimation for nonlinear systems with piecewise-constant input

We consider a general nonlinear time-delay system in which the input signal is piecewise-constant. Such systems arise in a wide range of industrial applications, including evaporation and purification processes and chromatography. We assume that the time-delays—one involving the state variables and the other involving the input variables—are unknown and need to be estimated using experimental data. We formulate the problem of estimating the unknown delays as a nonlinear optimization problem in which the cost function measures the least-squares error between predicted and measured system output. The main difficulty with this problem is that the delays are decision variables to be optimized, rather than fixed values. Thus, conventional optimization techniques are not directly applicable. We propose a new computational approach based on a novel algorithm for computing the cost function’s gradient. We then apply this approach to estimate the time-delays in two industrial chemical processes: a zinc sulphate purification process and a sodium aluminate evaporation process

Preliminary results using a P300 brain-computer interface speller: a possible interaction effect between presentation paradigm and set of stimuli

Fernández-Rodríguez Á., Medina-Juliá M.T., Velasco-Álvarez F., Ron-Angevin R. (2019) Preliminary Results Using a P300 Brain-Computer Interface Speller: A Possible Interaction Effect Between Presentation Paradigm and Set of Stimuli. In: Rojas I., Joya G., Catala A. (eds) Advances in Computational Intelligence. IWANN 2019. Lecture Notes in Computer Science, vol 11506. Springer, ChamSeveral proposals to improve the performance controlling a P300-based BCI speller have been studied using the standard row-column presentation (RCP) par-adigm. However, this paradigm could not be suitable for those patients with lack of gaze control. To solve that, the rapid serial visual presentation (RSVP) para-digm, which presents the stimuli located in the same position, has been proposed in previous studies. Thus, the aim of the present work is to assess if a stimuli set of pictures that improves the performance in RCP, could also improve the per-formance in a RSVP paradigm. Six participants have controlled four conditions in a calibration task: letters in RCP, pictures in RCP, letters in RSVP and pictures in RSVP. The results showed that pictures in RCP obtained the best accuracy and information transfer rate. The improvement effect given by pictures was greater in the RCP paradigm than in RSVP. Therefore, the improvements reached under RCP may not be directly transferred to the RSVP.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech