Malaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for the design of rational intervention strategies. To achieve this goal, we performed an isobaric tag-based approach for a system-wide quantitative analysis of protein expression and site-specific phosphorylation events of the Plasmodium asexual developmental cycle in the red blood cells. This study identified 2,767 proteins, 1,337 phosphoproteins, and 6,293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Because the links between Plasmodium protein kinases as key cell cycle regulators to cellular events are largely unknown, it is of importance to define their cognate physiological substrates. To test the hypothesis that genetic screening would be a useful approach for discovery of candidate substrates of a protein kinase, we used the orphan kinase PfPK7 as a model. Our comparison of the phosphoproteome profiles between the wild-type 3D7 and PfPK7- parasites identified 146 proteins with 239 phosphorylation sites exhibiting decreased phosphorylation in the absence of PfPK7 at the developmental stages where nuclear division and merozoite formation occur. Further analysis of the decreased phosphorylated events revealed three motifs that are enriched among phosphorylated sites in proteins that are down regulated. In vitro kinase assays were done to validate the potential substrates of PfPK7 and to elucidate the signaling events that are regulated by PfPK7. In parallel to our experimental analysis, we used a computational approach for substrate prediction from our phosphoproteome dataset. This analysis identified 43 distinct phosphorylation motifs and a range of proline-directed potential MAPK/CDK substrates. To identify substrates/ interactors of Plasmodium CDK-like kinases, we also used HA-tagged CDK-like kinases, PfPK6 and Pfmrk lines. Co-immunoprecipitation of the HA-tagged PfPK6 and Pfmrk baits, followed by mass spectrometric analyses, identified the components of the protein interaction complexes of these kinases. Our analyses of HA-PfPK6 and HA-Pfmrk immunoprecipitates identified 15 and 21 proteins in the interaction complex, respectively. The ability of recombinant PfPK6 and Pfmrk to interact and/or utilize any of the proteins identified in the interaction complex as substrates was verified through in vitro kinase assays and pull-down analysis. This study is the most comprehensive definition of the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, and offered an insight into the dynamics of phosphorylation during the asexual cycle progression [1]. In summary, this study has 1) defined the constitutive and regulated expression of the Plasmodium proteome during its asexual life cycle, 2) demonstrated that fluctuation and reversible phosphorylation is important for the regulation of P. falciparum*s unique cell cycle, 3) provided the foundation for quantitative phosphoproteomic analysis of kinase negative mutants to understand their function, 4) provided a major step towards defining kinase-substrate pairs operative within parasite*s signaling networks, and 5) generated a preliminary interactome for PfPK6.

Developing diagnostic modalities that utilize nanomaterials and miniaturized detectors can have an impact in point-of-care diagnostics. Diagnostic systems that (i) are sensitive, robust, and portable, (ii) allow detection in clinical samples, (iii) require minimal sample preparation yielding results quickly, and (iv) can simultaneously quantify multiple targets, would have a great potential in biomedical research and public healthcare. Bacterial infections still cause pathogenesis throughout the world (Chapter I). The emergence of multi-drug resistant strains, the potential appearance of bacterial pandemics, the increased occurrence of bacterial nosocomial infections, the wide-scale food poisoning incidents and the use of bacteria in biowarfare highlight the need for designing novel bacterial-sensing modalities. Among the most prominent disease-causing bacteria are strains of Escherichia coli, like the E. coli O157:H7 that produces the Shiga-like toxin (Stx). Apart from diarrheagenic E. coli strains, others cause disease varying from hemolytic uremic syndrome and urinary tract infections to septicemia and meningitis. Therefore, the detection of E. coli needs to be performed fast and reliably in diverse environmental and clinical samples. Similarly, Mycobacterium avium spp. paratuberculosis (MAP), a fastidious microorganism that causes Johne's disease in cattle and has been implicated in Crohn's disease (CD) etiology, is found in products from infected animals and clinical samples from CD patients, making MAP an excellent proof-of-principle model. Recently, magnetic relaxation nanosensors (MRnS) provided the first applications of improved diagnostics with high sensitivity and specificity. Nucleic acids, proteins, viruses and enzymatic activity were probed, yet neither large targets (for instance iv bacterial and mammalian cells) nor multiple bacterial disease parameters have been simultaneously monitored, in order to provide thorough information for clinical decision making. Therefore, the goal of this study was to utilize MRnS for the sensitive identification of multiple targets associated with bacterial pathogenesis, while monitoring virulence factors at the microorganism, nucleic acid and virulence factor levels, to facilitate improved diagnosis and optimal treatment regimes. To demonstrate the versatility of MRnS, we used MAP as our model system, as well as several other pathogens and eukaryotic cell lines. In initial studies, we developed MRnS suitable for biomedical applications (Chapter II). The resulting MRnS were composed of an iron oxide core, which was caged within a biodegradable polymeric coating that could be further functionalized for the attachment of molecular probes. We demonstrated that depending on the polymer used the physical and chemical properties of the MRnS can be tailored. Furthermore, we investigated the role of polymer in the enzyme-mimicking activity of MRnS, which may lead to the development of optimized colorimetric in vitro diagnostic systems such as immunoassays and small-molecule-based screening platforms. Additionally, via facile conjugation chemistries, we prepared bacterium-specific MRnS for the detection of nucleic acid signatures (Chapter III). Considering that MAP DNA can be detected in clinical samples and isolates from CD patients via laborious isolation and amplification procedures requiring several days, MRnS detected MAP's IS900 nucleic acid marker up to a single MAP genome copy detection within 30 minutes. Furthermore, these MRnS achieved equally fast IS900 detection even in crude DNA extracts, outperforming the gold standard diagnostic method of nested Polymerase Chain v Reaction (nPCR). Likewise, the MRnS detected IS900 with unprecedented sensitivity and specificity in clinical isolates obtained from blood and biopsies of CD patients, indicating the clinical utility of these nanosensors. Subsequently, we designed MRnS for the detection of MAP via surface-marker recognition in complex matrices (Chapter III). Milk and blood samples containing various concentrations of MAP were screened and quantified without any processing via MRnS, obtaining dynamic concentration-dependent curves within an hour. The MAP MRnS were able not only to identify their target in the presence of interferences from other Gram positive and Gram negative bacteria, but could differentiate MAP among other mycobacteria including Mycobacterium tuberculosis. In addition, detection of MAP was performed in clinical isolates from CD patients and homogenized tissues from Johne's disease cattle, demonstrating for the first time the rapid identification of bacteria in produce, as well as clinical and environmental samples. However, comparing the unique MAP quantification patterns with literatureavailable trends of other targets, we were prompted to elucidate the underlying mechanism of this novel behavior (Chapter IV). We hypothesized that the nanoparticle valency – the amount of probe on the surface of the MRnS – may have modulated the changes in the relaxation times (ΔΤ2) upon MRnS – target association. To address this, we prepared MAP MRnS with high and low anti-MAP antibody levels using the same nanoparticle formulation. Results corroborated our hypothesis, but to further bolster it we investigated if this behavior is target-size-independent. Hence utilizing small-moleculeand antibody-carrying MRnS, we detected cancer cells in blood, observing similar detection patterns that resembled those of the bacterial studies. Notably, a single cancer vi cell was identified via high-valency small-molecule MRnS, having grave importance in cancer diagnostics because a single cancer cell progenitor in circulation can effectively initiate the metastatic process. Apart from cells, we also detected the Cholera Toxin B subunit with valencly-engineered MRnS, observing similar to the cellular targets' diagnostic profiling behavior. Finally, as bacterial drug resistance is of grave healthcare importance, we utilized MRnS for the assessment of bacterial metabolism and drug susceptibility (Chapter V). Contrary to spectophotometric and visual nanosensors, their magnetic counterparts were able to quickly assess bacterial carbohydrate uptake and sensitivity to antibiotics even in blood. Two MRnS-based assay formats were devised relying on either the Concanavalin A (Con A)-induced clustering of polysaccharide-coated nanoparticles or the association between free carbohydrates and Con A-carrying MRnS. Overall, taking together these results, as well as those on pathogen detection and the recent instrumentation advancements, the use of MRnS in the clinic, the lab and the field should be anticipated.

Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative seleniumdependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.

The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are essential for cardiovascular development as embryos lacking NE/EPI begin to die abruptly between embryonic days 10.5 and 11.5 due to apparent cardiac failure. The objective of this research aims to elucidate the mechanism of the embryonic fatality observed in the NE/EPI deficient mouse model. Here we utilized the dopamine β- hydroxylase knockout (Dbh-/-) mouse model, which lacks the gene and subsequent enzyme necessary for the conversion of dopamine to norepinephrine. Embryonic mouse hearts were extracted from Dbh+/+ (control) and Dbh-/- (experimental model) mice for mRNA transcript expression profiling. These studies were performed using the Affymetrix Mouse Genome 430A 2.0 Arrays and quantitative real-time RT-PCR. Gene expression data suggest a novel connection between the ability of the heart to synthesize adrenergic hormones and the gene expression of enzymes involved in the synthesis of retinoic acid. Specifically, we found a statistically significant change in transcriptional expression of the retinol binding protein-1 (Rbp-1), retinol dehydrogenase 12 (Rdh-12) and beta carotene monooxygenase-1 (Bcmo-1) genes in the E10.5 Dbh-/- mouse heart. The gene expression of Rbp-1 and Rdh-12 were increased 1.4 fold and 2.1 fold on the microarray, respectively. The proteins translated from these genes play central roles in the transport and enzymatic conversion of precursor molecules in the pathway of retinoic acid biosynthesis. Additionally, we found that the expression of Bcmo-1, an enzyme responsible for the breakdown of beta carotene to the retinoic acid iii precursor retinal, was down regulated 2.7 fold in the Dbh-/- heart based on microarray assessment. Bcmo-1 is a well known retinoic acid responsive gene, suggesting that the loss of adrenergic hormones in the Dbh-/- mouse heart may result in a deregulation of retinoic acid synthesis and further an alteration in the concentration of retinoic acid present in the embryonic tissue of adrenergic hormone deficient embryos. In addition, we utilized a genetic mouse model that expresses β-galactosidase (β-Gal) in cells capable of synthesizing epinephrine in order to identify the spatial and temporal distribution of adrenergic-derived cells in the developing heart. The model was designed so that cells capable of expressing the gene phenylethanolamine Nmethyltransferase (Pnmt), which is responsible for the synthesis of epinephrine, also produce the enzyme β-Gal as a reporter. The resulting presence of the β-Gal enzyme can be visualized using a β-Gal substrate called XGAL, which is converted into a blue precipitate when cleaved by the β-Gal enzyme. Evaluation of the location of these cells in the embryonic heart showed a preferential distribution at the atrioventricular sulcus at E10.5, and later at E18.5 a more widely distributed ventricular pattern was observed. In addition, the right atrium showed a cluster of XGAL positive cells (blue cells) near the region of the sinoatrial node, while the distribution of XGAL positive cells in the left atrium was quite diffuse. Interestingly, when the adult heart was examined, it was discovered that cells capable of synthesizing epinephrine (adrenergic-derived) are found predominately on the left side of the heart. This left-sided distribution appears to be non-random and non-uniform, since specific regions are consistently XGAL positive, but not every cell in each region. Whole mount and 3-dimensional reconstruction of the iv β-Gal staining showed that these cells traverse the depth of the heart at the midventricular and apical regions. This finding is quite interesting and may provide new knowledge about the functional and structural characteristics of the adult heart. One observation is that these cells may contribute to the cardiomyopathy known as TakoTsubo or "Broken Heart" syndrome. The syndrome is characterized by left ventricular dysfunction during bouts of stress. Also, of particular intrigue is the anatomical correlation of the adrenergic derived cells and the helical ventricular myocardial band (HVMB). Careful examination of the spatial and directional pattern of these cells within the myocardium suggests they contribute primarily to a specific section of the HVMB. The significance of this finding is yet to be uncovered. Taken collectively, this study has shown a novel connection between two crucial developmental signaling pathways. Adrenergic hormone and retinoic acid signaling can now be viewed as cooperative partners in the development of the embryonic heart. In addition, this study has also shown that adrenergic derived cells in the adult heart have a distinctive left-sided distribution, which is non-random, non-uniform, and shows interesting features suggesting an anatomical connection to the HVMB and a clinical association to Tako-Tsubo syndrome. These findings will appreciably contribute to the knowledge base of the scientific community

Summary Autonomous vehicles are a growing trend in society and are anticipated to change the wholetransportation system as we know it today. Many vehicle manufacturers are focusing ondevelopment of different kinds of self-driving vehicles. The variety of automation in vehiclescan be divided into six levels of automation, according to the Society for Automotive Engineers(SAE) International’s division. As the level of automation varies between vehicles, the level ofneeded driver action also varies. As the automation level gets higher the drivers role changesmore and more towards being a supervisory controller for the automation. This leads to adifferent need of information presented to the driver in an autonomous vehicle compared towhat is provided in a manual vehicle. The driver must be able to understand what the automationis doing and predict how it will behave in different situations. Hence, the design of the Human-Machine Interface (HMI) is utterly important for the safety of autonomous driving. The main objective for the thesis work was to evaluate the driving compartment, from acognitive ergonomics point of view, for a special type of professional autonomous road vehicle. A literature study focusing on evaluation and testing of HMIs in autonomous vehicles wereperformed and the evaluation was done through user tests in a simulator with 22 professionaldrivers. The tests were followed by questionnaires and interviews about the test participants’experience of the designed HMI. In the literature study, only a few publications, were found addressing the validation of thewhole human machine interaction in autonomous vehicles. Most of the references had focus ontesting of limited subparts of the HMI, such as trust in automation, situational awareness andtransitions between manual and automatic driving mode. The designed HMI worked as intended, since the test participants noticed and acted upon allHMI signals and were able to takeover and handover the control from/to the automation. Somerecommendations were, however, provided for the future development work of the evaluatedautonomous vehicle. For example, since there were some limitations in the simulator used andin the test design, a next iteration of the test was recommended for future development. This evaluation was done as a first exploratory test and the aim of the thesis work has beenfulfilled. The design of the HMI, the manoeuvring devices for the autonomous vehicle and theconcept of two driver roles are, at this stage, considered adequate for continuing the project. It was also concluded that the driving behaviour of the automation is a crucial aspect forfacilitation of user-trust in the autonomous vehicle and that clear procedures for communicationare needed to ensure traffic safety. Further, it was concluded that standardization of frameworks and methods for evaluatingautonomous vehicles is beneficial for developers, authorities as well as end-users.

No description available.

Allergic asthma is a chronic inflammatory disease of the airways. Importantly, the chronic nature of this disease imparts specific additional consequences that would not otherwise be observed in a strictly acute setting. The development ofvarious structural alterations to the airway wall, collectively tem1ed airway remodeling, represents one such example. Decades of research have provided a great deal of insight into the acute allergic asthmatic response and the processes that govern it. However, less is known about the impact of protracted allergen exposure and chronic immune-inflammatory responses. To this end, the research presented in this thesis explores the consequences of chronic allergen exposure and persistent airway inflammation on asthma pathogenesis, using a mouse model of allergic airway disease induced by respiratory exposure to house dust mite (HDM) allergens. Specifically examined are: i) the impact of continuous allergen exposure and the resulting immune-inflammatory response on the development of de nova sensitization to newly encountered allergens (Chapter 2) and, ii) the roles oftransforming growth factor (TGF)-~ and eosinophils, two putatively critical components of the allergic inflammatory response, in the generation of airway remodeling (Chapters 3 and 4). Our data show that chronic exposure to HDM facilitates the development of the full 'asthmatic phenotype' towards an innocuous antigen. Moreover, they demonstrate that, unlike what has been previously observed in ovalbumin-based models, neither TGF-~ nor eosinophils are critically required for remodeling to develop in the context of HDM exposure. These findings highlight the importance of the lung microenvironment in influencing the type of immune response that develops upon initial antigen encounter and, furthermore, underscore the notion that the role of a particular cell type or molecule in the asthmatic response is contextual and not necessarily broadly applicable. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

The cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter, GLUT-1, and two glycolytic enzymes, Hexokinase II (HXKII) and phosphofructokinase-1 (PFK1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Re-addition of IL-7 to cytokine deprived lymphocytes restored the transcription of the HXKII gene within 2 hours, but not that of GLUT-1 or PFK1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-Bromopyruvate or specific siRNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, while over-expression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. This IL-7 mediated HXKII gene expression was abrogated with inhibition of JNK pathway. IL-7 also increased activation of AP-1 complex and DNA binding of JunD, a transcriptional complex thought to be negative regulator of proliferation. We found that over expression of HXKII caused cell cycle arrest and cell death, indicating that a potent IL-7 signal could produce negative growth signals. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII through activation of JNK-JunD pathway, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes.