In Streptomyces bacteria, the expression of many antibiotic biosynthetic clusters is controlled by both cluster-specific regulators and more globally-acting regulators; however, much remains unknown about the factors that govern antibiotic production. In Streptomyces venezuelae, we have discovered that the broadly-conserved nucleoid-associated protein Lsr2, plays a major role in repressing specialized metabolic cluster gene expression. To understand how Lsr2 exerts its gene silencing effects, we focused our attention on the well-studied, but transcriptionally silent, chloramphenicol cluster in S. venezuelae. We established that Lsr2 represses transcription of the chloramphenicol cluster by binding DNA both within the cluster and at distal positions. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not under Lsr2 control. We discovered that CmlR functions to ‘counter-silence’ Lsr2 activity, alleviating Lsr2 repression and permitting chloramphenicol production, by recruiting RNA polymerase. Lsr2 plays a central role in controlling antibiotic production in Streptomyces; however, beyond this counter-silencing activity, little is known about how Lsr2 is regulated. We identified regulators that could control the expression of lsr2, and found that Lsr2 and LsrL, an Lsr2 homologue that is encoded by all streptomycetes, interact directly with each other, and that their respective DNA-binding activities are altered by the presence of the other protein. These data suggest that LsrL may impact Lsr2 activity in regulating antibiotic production in Streptomyces. Beyond Lsr2, we wanted to develop a comprehensive understanding of the regulatory proteins that impact biosynthetic gene cluster expression. To define the regulatory protein occupancy of antibiotic clusters, we developed ‘in vivo protein occupancy display-high resolution’ (IPOD-HR) technology for use in Streptomyces. This work will lay the foundation for establishing a comprehensive regulatory network map for biosynthetic clusters in Streptomyces, and guide future work aimed at stimulating the expression of metabolic clusters in any Streptomyces species. / Thesis / Doctor of Philosophy (PhD) / Streptomyces bacteria produce the majority of naturally-derived antibiotics, and they have the genetic potential to produce many more antibiotics and antibiotic-like compounds (‘specialized metabolites’). Specialized metabolism is controlled by multiple regulatory systems. In Streptomyces venezuelae, we have discovered that the nucleoid-associated protein, Lsr2, represses the expression of most specialized metabolic clusters, and manipulating Lsr2 activity can stimulate antibiotic production. To better understand how Lsr2 exerts its repressive effect, we explored how Lsr2 controlled the production of a known antibiotic. We ultimately identified multiple regulators that could impact the expression and/or activity of Lsr2. Building on the regulatory foundation provided by Lsr2, we then set out to establish a comprehensive regulatory network that governs biosynthetic gene cluster expression. Collectively, this work improves our understanding of antibiotic gene regulation in Streptomyces bacteria, and has the potential to guide novel strategies aimed at stimulating the production of new antibiotics in Streptomyces.

No description available.

No description available.

No / Despite wide acknowledgment that hand hygiene is the pre-eminent measure in the control of health-care-associated infection, Didier Pittet and colleagues 1 have highlighted that there is still a need for a systematic programme of research that will allow the development of new¿as well as refinement of existing¿approaches to hand cleansing. One of the key priorities Pittet and colleagues identified is the need for investigations into the relative importance of between and within patient cross-transmission

Obesity is tightly linked to the development of liver steatosis which is an important risk factor for the development of metabolic dysfunction associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC). Specifically, obesity increases the risk of developing HCC by over 80% indicating the urgent need to understand and treat the obesity-MASLD-HCC spectrum of diseases. Environmental toxicants including some commonly used pesticides have been linked to the development of obesity, MASLD and HCC however, the mechanisms contributing to these effects are incompletely understood. Mechanistically, defects in fatty acid metabolism including increases in de-novo lipogenesis, mitochondrial dysfunction, and reductions in brown adipose tissue (BAT) metabolic activity are important contributing factors to MASLD and HCC. A key regulator of both liver and BAT metabolism is the AMP-activated protein kinase (AMPK). These data suggest that environmental factors may contribute to obesity, MASLD and HCC by inhibiting AMPK while therapeutic activation of AMPK may exert beneficial effects. In this thesis we find that two commonly used pesticides, chlorpyrifos and deltamethrin, suppressed the metabolic activity of cultured brown adipocytes. Subsequent studies in mice found that chlorpyrifos promoted obesity and liver steatosis and that this was linked to reductions in BAT metabolic activity and AMPK action. In contrast, when deltamethrin was delivered to mice it had no effect on obesity. Lastly, we evaluated the effects of the rheumatoid arthritis drug and AMPK activator Salsalate in mice and found that it reduced MASLD and HCC and enhanced the effectiveness of Lenvatinib, a tyrosine kinase inhibitor used in HCC treatment. This work furthers our understanding of the mechanisms by which commonly used pesticides may promote obesity and MASLD and provides evidence for the potential repurposing of Salsalate as a combinatorial treatment strategy for MASLD driven HCC. / Thesis / Doctor of Philosophy (Medical Science) / Environmental chemicals may affect the body’s metabolic functions and contribute to cancer. In this thesis we studied the effects of two common pesticides chlorpyrifos and deltamethrin, their contribution to obesity and liver disease and tested a new treatment strategy in liver cancer. We found that chlorpyrifos, but not deltamethrin, inhibited brown adipose tissue activity, the body’s metabolic sink which promoted diet induced obesity and fatty liver disease. Next, we studied whether salsalate, a drug with metabolic properties used to treat rheumatoid arthritis, could also be used to combat fat accumulation in the liver and block liver cancer. Salsalate in combination with Lenvatinib, an established cancer therapy for liver cancer, slowed down cancer growth and reduced factors that increase the risk and severity of liver cancer development. This work highlights the need to understand the effects of pesticides on our metabolism and capacity of metabolic strategies in helping combat liver cancer.

Fear and anxiety are fundamental emotional states that are critical for survival. These states are characterized by a variety of coordinated responses, including behavioral and autonomic changes, that need to be properly integrated. For the past decades, most studies have separated the behavioral and autonomic elements, generating a gap in understanding their integrative nature. In this thesis, a framework analysis is presented that allows for the integration of cardiac, behavioral, and neuronal readouts in freely moving mice during different emotional states. Furthermore, a growing body of evidence demonstrates that a vital component of these states is the physiological report of bodily states, or interoception, which allows for quick adaptation to changing situations. A set of distinctive interoceptive pathways has been described from the periphery to the brainstem; however, the circuits that process and integrate cardiac interoceptive signals in higher orders are poorly understood. The midbrain periaqueductal gray (PAG) is a region crucially involved in defensive states through its modulation of both, cardiac and behavioral components. Preliminary studies demonstrate an anatomical connection between the major cardiac interoception brainstem area, the nucleus of the solitary tract, and the PAG; however, the functional characterization and the specific neuronal substrates responsible for interoception in this area have not been described. An interesting particularity of the PAG is that the ventro-lateral subcolumn is the highest order of the neuraxis where inhibitory neurons that express the glycine can be found. In the lower brainstem and spinal cord, glycinergic inhibitory neurons have demonstrated a role in processing sensory and autonomic signals from the periphery, raising the question of whether the PAG glycinergic neurons could be involved in integrating cardiac interoceptive signals as part of a defensive state. In this thesis, using virally mediated trans-synaptic retrograde tracing, I showed that glycinergic PAG neurons receive inputs from cardiac regulatory areas in the brainstem and project massively to forebrain and midbrain regions. By employing advanced techniques such as deep brain calcium imaging with a miniaturized microscope and optogenetics, this study provides compelling evidence for the involvement of glycinergic PAG neurons in controlling heart rate and maintaining cardiac macrostate dynamics within physiological levels. The results of the optogenetic manipulation further revealed that a change in the heart rate macrostate caused by the glycinergic PAG neurons leads to anxiety-like behaviors, providing further evidence for the role of these neurons in regulating defensive states. Overall, by unraveling the neural circuitry underlying interoception in the PAG, our study paves the way to better understand fear and anxiety disorders. / Furcht und Angst sind grundlegende emotionale Zustände, die für das Überleben entscheidend sind. Diese Zustände sind durch eine Vielzahl von koordinierten Reaktionen gekennzeichnet, darunter auch Verhaltensänderungen und autonome Veränderungen, die richtig integriert werden müssen. In den letzten Jahrzehnten haben die meisten Studien die verhaltensbezogenen und autonomen Elemente getrennt, was zu einer Lücke im Verständnis ihrer integrativen Natur führte. In dieser Arbeit wird eine Rahmenanalyse vorgestellt, die die Integration von kardialen, verhaltensbezogenen und neuronalen Messwerten bei sich frei bewegenden Mäusen während verschiedener emotionaler Zustände ermöglicht. Darüber hinaus gibt es immer mehr Belege dafür, dass eine wichtige Komponente dieser Zustände die physiologische Rückmeldung von Körperzuständen an das Gehirn, die sog. Interozeption ist, die eine schnelle Anpassung an sich ändernde Situationen ermöglicht. Es wurde eine Reihe von ausgeprägten interozeptiven Bahnen von der Peripherie bis zum Hirnstamm beschrieben. Jedoch sind die Schaltkreise, die kardiale interozeptive Signale in höherer Ordnung verarbeiten und integrieren, kaum bekannt. Das periaquäduktale Grau des Mittelhirns (PAG) ist eine Region, die sich entscheidend an Verteidigungszuständen beteiligt ist und deren Modulation sowohl kardiale als auch verhaltensbezogene Signale beeinflusst. Vorläufige Studien zeigen eine anatomische Verbindung zwischen dem wichtigsten kardialen Interozeptionsbereich des Hirnstamms, dem Nucleus tractus solitarius, und dem PAG; die funktionelle Charakterisierung und die spezifischen neuronalen Substrate, die für die Interozeption in diesem Bereich verantwortlich sind, sind bislang jedoch nicht beschrieben worden. Eine interessante Besonderheit des PAG ist, dass die ventro-laterale Subkolumne die höchste Ordnung der Hirnachse mit hemmenden glyzinergen Zellkörpern aufweist. Im Hirnstamm und im Rückenmark haben diese hemmenden Neuronen eine Rolle bei der Verarbeitung sensorischer und autonomer Signale aus der Peripherie gezeigt, was die Frage aufwirft, ob die glyzinergen Neuronen des PAG an der Integration kardialer interozeptiver Signale als Teil eines Verteidigungszustandes beteiligt sein könnten. In dieser Arbeit habe ich mit Hilfe von viral vermitteltem trans-synaptischem retrograden Tracing gezeigt, dass glyzinerge PAG-Neuronen Inputs von kardialen Regulationsbereichen im Hirnstamm erhalten und massiv in Vorder- und Mittelhirnregionen projizieren. Durch den Einsatz fortschrittlicher Techniken, wie z. B. Tiefenhirn- Kalziumbildgebung mit einem miniaturisierten Mikroskop und Optogenetik, liefert diese Studie überzeugende Beweise für die Beteiligung glyzinerger PAG-Neuronen an der Kontrolle der Herzfrequenz und der Aufrechterhaltung der kardialen Makrostate-Dynamik innerhalb physiologischer Grenzen. Die Ergebnisse der optogenetischen Manipulationen zeigten außerdem, dass eine durch die glyzinergen PAG-Neuronen verursachte Veränderung des Makrostates der Herzfrequenz zu angstähnlichem Verhalten führt, was einen weiteren Beweis für die Rolle dieser Neuronen bei Furcht und Angst darstellt. Insgesamt ebnet unsere Studie durch die Entschlüsselung der neuronalen Schaltkreise, die der Interozeption im PAG zugrunde liegen, den Weg zu einem besseren Verständnis von Angst und Angststörungen.

Nosocomial infections are a cause of concern for hospital patients and the incidence rates of these infections are greater in intensive care units (ICUs) due to the invasive nature of treatments, additional risk factors and comorbidities, and therapies used. Invasive devices, such as vascular central lines, Foley catheters, and mechanical ventilators pose a risk for critically ill patients in the ICUs to develop device-related, healthcare-associated infections (HAI). The purpose of this study was to describe the epidemiological characteristics of patients who developed device-related HAIs within 3 ICU units (medical-surgical, cardiovascular, and neurosurgical) of an academic medical facility. The ecosocial theory of disease distribution provided the theoretical framework for the study to describe how ecological and social determinants interact and affect health variances. Secondary data were analyzed using analysis of variance (ANOVA), Pearson correlations, and chi-square statistical tests. A total of 4,213 patients admitted to the 3 ICUs from 2010-2014 were identified. According to the chi-square analysis, there was significant association between race/ethnicity and type of device-associated infection; between gender and types of infection; and between risk factors (diabetes, obesity, smoking habits) and kinds of infection, all of which the statistical significance had varied for each individual ICU. Bacterial differences were noted between device-associated infections. The potential positive social change from this study could be insight on possible new processes and interventions to reduce nosocomial infections and improve adult ICU patient outcomes such as decreased HAIs, decreased length of stay, comorbidities, and cost for both the patient and the hospital.

Background Clostridium difficile is a Gram-positive bacteria found in the large bowel or colon that causes mild to severe intestinal conditions and sometimes death. The primary risk factors for development of Clostridium difficile infection (CDI) include healthcare exposure and recent antimicrobial use. The purpose of this study is to compare risk factors associated with CDI occurring in the Community to those associated with Healthcare Facility Associated CDI in the metro Atlanta population from September 1, 2009 – April 30, 2011. Methods Patients were identified through C. difficile surveillance program of the Georgia Emerging Infections Program (EIP). Prospective, population based, laboratory based surveillance for all positive C. difficile cases in the Georgia Health District 3 (HD3). Due to the sampling scheme, for this analysis CO-HCFA and HCFO cases were combined to make a Healthcare Facility Associated (HCFA) classification. Using SAS, a logistic regression analysis was performed to compare the associated risks between CA and HCFA classifications. Results The rate of CDI in the HD3 counties in Georgia is 84 per 100,000. The median age of infection is 63 and the age range in this study is 1 to 102 years old. CA cases represented 38% of the sampled population. CDI cases 65 and older were more likely to have a Healthcare association compared to CA-CDI cases (p Conclusion This study supports literature about CDI and antimicrobial use and looks further in to the role underlying conditions play as a risk factor for HCFA-CDI cases.

Background: The enhancement of quality of life (QOL) is a principal health care objective. Surprisingly, few studies have investigated this outcome in ANCA associated vasculitis (AAV), a complex chronic disease. Existing studies have, however, identified fatigue as a specific problem amongst this population. Although its aetiology is unknown, there is evidence, from other populations, to support a neural basis for this symptom. Aims: This study aimed to characterise QOL and its determinants amongst patients with AAV. A secondary study examined the association of AAV related fatigue with alterations of the brain. Methods: An AAV case-control study was conducted, incorporating a comparison and within-case analysis, using two groups of population and chronic disease controls. All participants completed a questionnaire comprising measures of QOL and putative determinants of QOL impairment. Concurrently, putative clinical determinants were collected from cases. The secondary study recruited AAV cases based on fatigue status. A further group with idiopathic fatigue was recruited from the general population. All subjects underwent magnetic resonance (MR) brain scanning incorporating structural and physiological imaging. Results: Compared to population controls, cases were substantially more likely to report low QOL and levels were equable to disease controls. Potentially modifiable biological and psycho-social factors were independently associated with poor QOL, of which fatigue was found to be of principal importance. In the secondary study, structural and physiological differences were observed between AAV patients with and without fatigue, as well as fatigued population subjects. Conclusions: AAV patients experienced significant QOL impairment. A bio-psychosocial approach to AAV health care is likely to improve QOL outcomes, although a better understanding of specific mechanisms is necessary to fully manage these problems. MR techniques have suggested a neural basis for AAV related fatigue. In the future they may help delineate the mechanisms of fatigue and consequently improve QOL in AAV.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.116-124