Ebola viruses cause sever disease in humans and non-human primates. The resulting disease, Ebola virus disease (EVD), can have hemorrhagic manifestations and has mortality rates ranging from 20-90%. There is a strong need for better understanding of the disease as well as improved diagnostics and prognostics. One approach to improving diagnostic and prognostics for severe viral diseases such as EVD is to define how the host response to infection develops and produces indicators of disease and outcome. To create a better means to understand if a patient is likely to survive or succumb to Ebola (EBOV) infection, I have sought to develop an understanding of the host response to EBOV infection in humans from the recent outbreak. I analyzed RNA-Seq samples from the 2013-2016 West Africa outbreak. I identified that the innate immune pathways are in general over activated in EVD and is stronger in patients who succumbed to disease. Furthermore, I developed a set of 10 genes that can perform as a prognostic indicator of disease independent of the viral load. This is the first demonstration that the circulating transcriptional immune response to EBOV infection can be used to predict infection outcome. To work towards a diagnostic platform of disease, I analyzed multiple studies of time-resolved datasets in animal models of disease. I analyzed RNA-Seq and NanoString data coupled with telemetry data in EBOV-challenged macaques. The earliest and strongest changes seen in the pre-symptomatic stage of disease is the up-regulation of many innate immune genes. I used this information to develop a NanoString codeset that can act as a pre-symptomatic indicator of disease that was tested in further animal studies as a diagnostic in pre-symptomatic stages of disease. Together, this work has identified a sets of genes that can work as a diagnostic for pre-symptomatic patients of EBOV and act as a prognostic indicators of disease. In future outbreaks, this type of information will be important to help track primary contacts of infected individuals and first responders, as well as better inform clinical management of patients. This lays the groundwork for similar analysis to be performed on other severe viral diseases such as Lassa Fever and Marburg Fever. / 2019-11-27T00:00:00Z

The communities of microorganisms associated with humans and other animals are characterized by a large degree of diversity and unexplained variation across individual hosts. While efforts to explain this variation in host-associated systems have focused heavily on the effects of host selection, community assembly theory emphasizes the role of dispersal and stochastic demographic processes, otherwise known as ecological drift. In this dissertation, I characterize the communities of microorganisms associated with the zebrafish, Danio rerio, intestine, and assess the importance of microbial dispersal and drift to their assembly. First, I describe changes in the composition and diversity of the zebrafish intestinal microbiome over zebrafish development and show that while host development is a major driver of community composition over time, there remains a large amount of unexplained variation among similar hosts of the same age. I go on to show that random dispersal and ecological drift alone in the absence of host selection are sufficient to explain a substantial amount of this variation, but the ability of these processes to predict the distribution of microorganisms across hosts decreases over host development. Finally, I present an experimental test of dispersal in host-associated systems, and show that not only does dispersal among individual zebrafish hosts have a large impact on the composition and diversity of associated microbial communities, but it can also overwhelm the effects of important host factors, such as the innate immune system. As a whole, this work demonstrates that the composition and diversity of microbial communities associated with animal hosts are not solely the result of selection by the host environment, but rather dispersal and stochastic processes have important and often overwhelming effects on their assembly. To fully understand the assembly of host-microbe systems, we must broaden our focus to include scales beyond that of an individual host and their associated microorganisms. / 10000-01-01

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No description available.

Network operators need detailed understanding of their networks in order to ensure functionality and to mitigate security risks. Unfortunately, legacy networks are poorly suited to providing this understanding. While the software-defined networking paradigm has the potential to, existing switch-based implementations are unable to scale sufficiently to provide information in a fine-grained. Furthermore, as switches are inherently blind to the inner workings of hosts, significantly hindering an operator's ability to understand the true context behind network traffic. In this work, we explore a host-based software-defined networking implementation. We evaluation our implementation, showing that it is able to scale beyond the capabilities of a switch-based implementation. Furthermore, we discuss various detailed network policies that network operators can write and enforce which are impossible in a switch-based implementation. We also implement and discuss an anti-reconnaissance system that can be deployed without any additional components.

Ions play pivotal roles both biologically and environmentally. The effects felt from the impact of ions, much like their relative charge, can be positive or negative. Ions are responsible for catalyzing and executing precise control over many of the essential processes that occur in our bodies. Ions can also be major contributors as environmental pollutants having catastrophic effects. There is a great deal of interest in better understanding the role and effect of ions in the surrounding environments. The ability to study ions of interest relies on efficient recognition and sensing of these targets. The field of supramolecular chemistry is particularly well suited for this task as it utilizes non-covalent molecular interactions in much the same way natural system involving ions operate. Broadly, this body of research seeks to explore the subtle interactions of various targeted ions with supramolecular receptors. This research is an effort to further understand the nature of these interactions for potential recognition and sensing applications as well as better understanding the highly complex systems found in biology. Chapter I provides a brief overview of various mechanisms of ion coordination in supramolecular chemistry and emphasizes some key examples demonstrating the importance of the various types of coordination as it pertains to the research presented herein. Chapter II highlights a unique class of phosphine oxide-based tripodal ditopic receptors and presents studies on their interactions with alkali metals and halides. Chapter III covers the synthesis and study of anionic binding trends for a series of heteroaromatic-containing urea-based receptors and discusses the influence of anion binding on receptor conformation. Chapter IV provides preliminary results on the application of the bipyridal bisurea-based anion receptor, presented in Chapter III, as ligand for metal salts. Chapter V focuses on a series of pyridal bisurea-based receptors with regard to their ability to serve as chloride sensitive probes in cellular environments. This dissertation contains both previously published and unpublished co-authored material. / 10000-01-01

Citrobacter rodentium is a gut pathogen, which infects the distal colon of mice. It has many similarities to human Enteropathogenic and Enterohemorrhagic E.coli in terms of mechanisms of pathogenicity and methods of transmission. Like many other gram negative bacteria, C. rodentium has developed a complex and highly specialised protein secretion system, known as type three (T3SS), to deliver bacterial proteins into eukaryotic cells. By injecting effector proteins into host cell cytoplasm, the pathogens are able to modulate host cellular functions to facilitate their own survival and replication. There is growing evidence that Attaching Effacing (AE) pathogens can inject effector proteins into gut epithelial cells, which dampen pro-inflammatory responses. There is also evidence that EPEC, Yersinia and Shigella can inject effectors into immune cells and also modulate their function. The objective of this work was to visualise and identify the host cells targeted for type III secretion by C. rodentium, and consequently determine the effect on host immune responses. The method chosen to detect cells targeted for effector protein delivery was the β-lactamase reporter system, where cells loaded with the fluorogenic substrate CCF2-AM emit a green FRET signal upon excitation by UV light, but emit a blue signal when cleaved by β-lactamase. By creating reporter strain of C.rodentium expressing fusion proteins between NleD effector and β-lactamase, I was able to show that C.rodentium is capable of injecting NleD in a wide variety of murine cell lines including Swiss 3T3 fibroblasts, J774 macrophages, CMT93 epithelial cells and BW715 T cells in a dose and time dependent manner in vitro. In addition, I found that C.rodentium has the ability to inject proteins into the cytoplasm of immune cells isolated from mouse lymphoid tissues including the spleen, mesenteric lymph nodes and Peyer's patches. Detailed analysis of the types of cells injected with effectors in vitro showed that NleD- injected cells represented B cells, dendritic cells and T cells. After inoculation of mice with the reporter strain of CitropACYCnleD, the plasmid encoded reporter fusion remained stable throughout infection and was able to inject cells in vitro after passage through the mouse gut. Unfortunately under the conditions described in this study, we were unable to visualise any gut cells targeted for protein delivery by C. rodentium in vivo, thus highlighting the complex nature of the host pathogen relationships in the gut. Although there is a need to develop better strategies to visualise effector translocation in vivo, our study has demonstrated, for the first time, the ability of C. rodentium to target immune cells for effector injection in vitro.

Supramolecular chemistry allows the rapid formation of complex systems through self-assembly. These systems often possess unique properties not observed for conventional covalent constructs and have potential applications in areas such as sensing, drug delivery and catalysis. Metallosupramolecular container compounds have been shown to catalyse reactions with both regio- and stereo-selectivity in methods analogous to enzyme type catalysis. Separately, visible-light photoredox catalysis has recently gained considerable interest as an efficient, green and mild method for the rapid synthesis of many chemical compounds. In order to combine the favourable properties of both supramolecular catalysis and visible-light photoredox catalysis, a number of photoredox active metallosupramolecular assemblies were designed, synthesised and analysed. Initial steps were taken to stabilise a known iridium-based M6L4 luminescent cage compound to allow guest encapsulation to take place. The incorporation of isocyanide donors as strong ligands improved the stability of model compounds but synthesis of an analogous three-dimensional assembly was unsuccessful. Instead, a “complex-as-ligand” approach was taken that allowed the straight-forward formation of Pd2L4 systems from a range of photoactive iridium complexes. Importantly, unlike many other photoactive systems, the complexation to palladium did not drastically affect the photoredox properties of the constituent iridium complexes. Multiple approaches were then taken to improve both the stability and guest binding properties of the photoactive assembly in an effort to achieve supramolecular photoredox catalysis. Utilising a model system, a general method was thus developed for enhancing the association constants of neutral guests in organic solvents by switching to large, non-coordinating counter ions that provided reduced competition for the internal binding site. In combination with this increased binding affinity, a range of guest properties were adjusted by association with the hydrogen bond donor environment of the internal cavity. The encapsulation of quinone based oxidants led to unexpected and novel reaction pathways not observed in the bulk phase. As such, this work represents a significant advancement in development of metallosupramolecular systems capable of regio- and stereo-selective photoredox catalysis.

Immune responses are presumed to contribute to host fitness, either by fighting off infections or via immunopathology. Research in this thesis sought to relate the magnitude of a putative immune response to infection and host and parasite fitness. The experiments and field studies presented here all focus on the interactions between the freshwater crustacean, Daphnia magna and its sterilizing bacterial endoparasite, Pasteuria ramosa, using the number of circulating haemocytes as a measure of host immune activity. I found substantial genetic variation in Daphnia’s cellular response to P. ramosa, and that Daphnia genotypes that mount the strongest cellular responses are the most likely to get infected and suffer sterilization. Thus, a strong cellular response is associated with low, as opposed to high host fitness potential. There were also some host genotypes that mounted a weaker cellular response and did not go on to suffer infection, and some that lacked a cellular response and also never suffered infection with P. ramosa. These findings led to a heuristic two-stage model for infection, where the parasite has to (1) pass from the host gut to haemolymph and then (2) successfully overcome haemolymph-based immune effectors to reproduce and achieve fitness. I also demonstrate that both the magnitude of host cellular response and likelihood of infection increases with initial parasite dose in susceptible host genotypes, and that host cellular response is associated with likely infection under both host and parasite genetic variation. Parasitised Daphnia also have substantially more circulating haemocytes than their healthy counterparts in both the laboratory and in the wild, where there is substantial genetic and environmental variation. This is one of the very few examples of how an immune response designates low host and high parasite fitness potential in a wild system. Finally, using a mixture of field study and common garden experiment, I demonstrate evolution in parasite infection traits over the course of an epidemic in a wild population, and that this evolution is associated with a decline in host abundance.

Homing- und Chemokin-Rezeptoren können wichtige Informationen liefern über Aktivierungsstand und Organspezifität der einzelnen Zelle, aber auch über Homöostase und Gleichgewicht von T-Zellen untereinander. Mittels FACS-Analyse konnte in murinen Transplantationsmodellen durch die Hochregulation einzelner Homing-Rezeptoren auf T-Zellen die Entwicklung einer aGvHD vorhergesagt werden. Ob sich ein solches diagnostisches Fenster auch beim Menschen darstellen könnte, sollte in einer klinischen Pilotstudie untersucht werden. Zu diesem Zweck wurde der Expressionsverlauf von 19 verschiedenen Aktivierungsmarkern, Chemokin- und Homingrezeptoren auf T-Zell-Subpopulationen bei allogen transplantierten Patienten charakterisiert. Anschließend wurde versucht, eine Assoziation zwischen der Höhe der jeweiligen Rezeptorexpression und der späteren Entwicklung einer akuten Graft-versus-Host-Disease herzustellen. Die Expression der meisten untersuchten Rezeptoren nahm im Zeitverlauf ab und war insgesamt nur schwach ausgeprägt. Dabei zeigte sich insbesondere im frühen Verlauf nach SZT eine hohe relative Expression dieser Marker bei Patienten ohne aGvHD verglichen mit Patienten mit aGvHD im Verlauf. Insofern scheint eine hohe relative Expression dieser Rezeptoren kein Hinweis auf eine gesteigerte Alloreaktivität der jeweiligen T-Zellen zu sein. Gleichzeitig schien die absolute Anzahl an rezeptorpositiven Zellen eher positiv mit einer aGvHD korreliert zu sein. Die erhöhte Anzahl rezeptorpositiver Zellen errechnete sich allerdings aus der höheren absoluten Anzahl aller T-Zellen bei Patienten mit aGvHD. Damit war diese nicht rezeptorspezifisch, so dass sich Rezeptoren vom Expressionstyp 1 nicht eignen, um in der klinischen Routine eine aGvHD vorherzusagen. Einige Rezeptoren waren auf T-Helfer-Zellen auf mittlerem Niveau konstant exprimiert. Für manche von diesen zeigten sich unterschiedlich hohe relative Expressionsniveaus vor GvHD-Beginn bei Grad 2-4 verglichen mit Grad 1. Die absolute Anzahl rezeptorpositiver Zellen war bei allen Rezeptoren bei aGvHD Grad 2-4 höher als bei Grad 1. Die Expression einiger Marker war auf T-Zellen ausgeprägt, aber im Zeitverlauf fluktuierend. Für die Expression von beta 7 integrin allein und auch für dessen Koexpression zusammen mit CD 49d alpha 4 deuteten sich bei Patienten mit aGvHD Grad 2-4 höhere relative und absolute Werte an als für Patienten mit aGvHD Grad 1. Die Expression der übrigen hoch exprimierten Rezeptoren schien nicht vom Ausmaß der später einsetzenden aGvHD beeinflusst zu werden. Ob durch FACS-Analyse der Rezeptoren CLA, CCR 4, CD 45RA und/oder alpha 4 beta 7 allein oder in Kombination tatsächlich die alloreaktiven T-Zellen der aGvHD charakterisiert und quantifiziert werden können, muss an einem größeren Patientenkollektiv untersucht werden. Da zum jetzigen Zeitpunkt der prädiktive Wert dieser Marker weder postuliert noch ausgeschlossen werden kann, erscheint eine weitergehende Untersuchung sinnvoll. / Homing and chemokine receptors can provide important information about the activation and organ specifity of the cell itself as well as about homeostasis and equilibrium among t cells in general. In murine transplantion models, FACS analysis of t cells could detect the upregulation of homing receptors on t cells prior to the clinical onset of aGvHD. We tried to invesigate if such a diagnostic window could also be found in a clinical setting. Therefore we characterized the expression of 19 homing and chemokine receptors on t cells subsets in allogeneicly transplantated patients. We then looked for any association between the amount of the very receptor expression and the later development of aGvHD. The expression of most studied receptors was decreasing over the course of time and overall low. Especially in the first weeks after alloHCT, a high percentage of receptor expressing cells could be seen in patients without aGvHD compared to patients who developed aGvHD though. Therefore a relatively high expression of these receptors during lymphopenia did not seem to be associated with alloreactivity of the t cells. At the same time, an increased amount of receptor positive cells seemed to correlate positively with aGvHD. Yet, this was because patients with aGvHD showed overall higher rates of leukocytes and lymphocytes than the controls. The effect did not seem to be receptor specific so that receptors of the expression type 1 do not qualify for predicting aGvHD in clinical routine. Some receptors were constantely expressed on a medium level on helper t cells. A part of those showed higher expression levels when patients developed aGvHD grade 2-4 compared to patients with milder grade 1. The absolute amount of receptor positive cells was higher in severe aGvHD in all markers classified as expression type 2. Type 3 is characterized by an overall high but fluctuating homing receptor expression on t-cells. The receptors beta 7 integrin and CD 49 d alpha 4 were upregulated before patients developed severe aGvHD grade 2-4. The expression of the other type 3 receptors didn't seen to be influenced by later aGvHD. Further investigation in a larger cohort is needed to test the value of CLA, CCR4, CD 45 RA, beta 7 integrin and CD 49 d alpha 4 as predictive aGvHD biomarkers.