Introduction: Civilian trauma remains a significant health care problem in North American society. Hemorrhagic shock and resuscitation (S/R) have been shown to prime the immune system for an exaggerated response to subsequent otherwise innocuous inflammatory stimuli such as lipopolysaccharide (LPS), resulting in multiple organ failure or death. Using a rodent model of lung injury, we previously demonstrated that antecedent S/R leads to augmented LPS-induced lung injury by way of heightened NF-κB nuclear translocation, resulting in increased elaboration of pro-inflammatory cytokines in alveolar macrophages. Further studies revealed that oxidative stress generated during S/R is responsible for this priming phenomenon. Our group recently identified two significant alterations to LPS signaling under oxidative stress conditions in macrophages: 1) the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, and 2) the reprogramming of LPS signaling to a Src-dependent pathway involving phosphatidylinositol 3-kinase (PI3K). Major Objective and Hypothesis: The objective of this thesis is to elucidate the molecular mechanisms underlying the augmented cellular responsiveness observed in macrophages following oxidative stress. The central hypothesis is that oxidative stress regulates LPS signaling by altering the activation and assembly of TLR4 receptor signaling components through generation of the lipid ceramide. Summary of Findings: In the first paper, we demonstrate that the antioxidant stilbazulenyl nitrone (STAZN), a novel second-generation azulenyl nitrone, is protective in a rodent two-hit model of lung injury involving hemorrhagic S/R and subsequent intra-tracheal LPS injection. Resultant oxidative stress and lung injury in vivo were significantly reduced by STAZN following S/R and LPS. In the second paper, we explore the mechanism underlying oxidant-induced surface up-regulation of TLR4 in macrophages. Using immunofluorescence microscopy and flow cytometry techniques, hydrogen peroxide in vitro and hemorrhagic S/R in vivo are shown to induce TLR4 translocation in macrophages in a ceramide and Src-dependent manner, and the enzyme acid sphingomyelinase (ASM) is shown to mediate ceramide generation. In the third paper, the role of ceramide in oxidant-induced macrophage priming for LPS signaling is investigated. Ceramide generation via ASM is shown to have a prominent upstream role in oxidant activation of the PI3K/Akt pathway via Src kinases in macrophages. Furthermore, oxidative stress is shown to reprogram LPS signaling to a ceramide dependent pathway. Conclusion: Together, these findings highlight the role of oxidative stress in mediating augmented cellular responsiveness following S/R, and describe the role of ceramide as a central upstream mediator of oxidant priming in macrophages. The hierarchy of signaling molecules and interactions described herein represent novel targets for modulating oxidative stress in the treatment of critical illness and organ injury.

Age-dependent changes in the kidney are often debilitating, can be life-threatening and are a significant cause of increasing health costs worldwide. Excessive fibrosis, a general lack of regenerative ability and an increase in apoptosis in cells that determine healthy renal function work together to cause chronic kidney disease (CKD). This thesis reviewed the literature and then tested hypotheses developed from this review, to provide information on the molecules and mechanisms that determine the age-dependent changes of CKD. Results in this thesis provide a comprehensive analysis of the molecular, structural and functional changes of age-related CKD, with particular attention paid to the longevity gene p66Shc. The present studies were able to make use of established ageing rodent colonies of various phenotypes. In the first of the research Chapters, rat models of age-related CKD linked with obesity and hypertension were used. The research tested the hypothesis that each cause of age-related renal change (ageing, obesity or hypertension) would have differing underlying genetic modifications that could explain any differences in renal structure and function. In particular, alterations in oxidant handling and energy metabolism were investigated to identify markers for age-related CKD. Young (3 months) and old (20-24 months) spontaneously-hypertensive rats (SHR), normotensive Wistar- Kyoto (WKY) and Wistar rats (normotensive, with excess visceral and peri-renal fat in ageing) (N = 4 per group) were compared for renal functional and physiological parameters, fibrosis, inflammation and oxidative stress. All of the analyses indicated the old obese Wistars had the greatest renal injury, inflammation and markers of oxidative stress. In particular, % phosphop66/ p66Shc, considered an oxidant stress marker, was significantly increased in these animals (p<0.05). These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD, and that obesity alone may be a key causative mechanism of age-related CKD. x Oxidative stress is thought to be a major cause of age-related CKD. In chapter 5, the following hypotheses were tested: (1) that the added stress of ischemia-reperfusion (IR) injury on the ageing kidney would create an environment for increased injury; and (2) that this injury could be modulated by using a short-term anti-ageing strategies. Old (20-24 month) and young (3 month) WKY rats (females, N = 4 per group) were used to compare the effects of bilateral, 45-minute, IR injury with and without calorie restriction (a 40% reduction in food from baseline) or vitamin E (daily gavage of 1000IU) for 10 days prior to IR surgery and then for the length of recovery from IR (4 days). Histological, functional and molecular analyses were used. Old rats had significantly worsened renal injury compared with young rats with IR. Proteins involved in oxidative stress (HO-1, p66Shc and phospho-p66Shc), survival (PKB and phospho-PKB), apoptosis (Bax, Bcl-2), inflammation and fibrosis (NF-κB, tumour necrosis factor-α/TNFα, transforming growth factor-β/TGFβ) were differentially expressed according to age and development of IR injury. Vitamin E-supplemented animals showed minimal improvement and calorie restriction generally worsened the outcome in both young and old animals. Changes in protein expression support the notion that these short-term calorie-restricted animals were in a catabolic state, perhaps similar to protein energy wasting seen in some of the human dialysis population. In chapter 6 in vitro experimental models using primary human renal proximal tubular epithelial cells (PTECs) were utilised. Successive passaging in culture of the PTECs showed increasing markers of senescence and oxidative stress. The degree of senescence correlated with expression of the oxidative stress marker phospho-p66Shc and alterations in other key signalling molecules. Hydrogen peroxide (5mM for 1 hour) was used to simulate a burst of oxidative stress and the effects of leptin and resveratrol was examined. Histological and molecular analyses demonstrated some links with the previous in vivo results, for example the involvement of phospho-p66Shc in the development of cell senescence but generally the in vitro experiments did not replicate in vivo xi findings. The lack of complex, heterogeneous, cellular and growth factor/cytokine interactions of the in vivo environment are thought to be a factor in this disappointing result. The tendency for development of CKD differs in males and females in ageing humans. In chapter 7 characteristics of age-related CKD in old male and female rats were compared, summarising data on the WKY rats from Chapters 3 to 5. Minor differences between males and females in histology, function and protein expression are described, but these do not adequately reflect the findings of gender dimorphisms in development of CKD, reported from human and experimental in vivo studies. These experiments demonstrate some of the pathogenetic mechanisms of age-related CKD. The results indicate pathways or molecules that may be targeted in future therapies or may be used as biomarkers of early development of age-related CKD. In particular, the modification of p66Shc may one day be used to minimize renal damage and promote health in the elderly.

Thesis (Ph.D.) - University of Glasgow, 2008. / Includes bibliographical references.

Thesis (M.Sc. Biology)--University of Waikato, 2007. / Title from PDF cover (viewed May 2, 2008) Includes bibliographical references (p. 131-144)

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 182 - 209. Also available online via the Internet.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 121-129). Also available on the World Wide Web.

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (M.S.)--University of North Carolina at Greensboro, 2007. / Title from PDF title page screen. Advisor: Allan H.Goldfarb; submitted to the School of Health and Human Performance. Includes bibliographical references (p. 49-57).

Recently, oxidative stress has been highlighted as a potential mediator underlying life-history trade-offs in animals. However, despite growing interest in the role of oxidative stress as a mechanistic explanation of trade-offs, the importance of oxidative stress in wild populations remain poorly understood. In this thesis, I use four commonly applied markers of oxidative stress. I apply two markers of oxidative damage, protein carbonyls and malondialdehyde, and two markers of antioxidant protection, superoxide dismutase activity and total antioxidant capacity. These were applied to samples collected in 2010-2013 and 2015 from the wild population of Soay sheep (Ovis aries), St Kilda, Scotland. I investigate correlations among different markers of oxidative stress, and their within individual repeatability over time. In addition, I examine the role played by oxidative stress in mediating life-history traits at different life-stages; specifically, I test for associations between oxidative stress, growth and survival in Soay sheep lambs. I also investigate the oxidative costs of reproduction in adult Soay sheep females and I test whether any such costs vary with age. My findings reveal, firstly, that plasma markers of oxidative stress vary dramatically among years, and that the different markers of oxidative damage and antioxidant protection applied throughout this thesis, are uncorrelated with each other (Chapter 2). This indicates that oxidative stress is a multifaceted process, where each oxidative stress marker may reflect different and potentially uncoupled biochemical processes (Chapter 2). Second, faster lamb growth shows a weak, positive association with malondialdehyde. However, growth is not associated with variation in the other three markers (Chapter 3). In addition, lamb survival also shows marker dependent associations; lambs with higher superoxide dismutase activity are more likely to survive their first winter, as are male but not female lambs with lower protein carbonyl content. Survival does not vary with malondialdehyde or antioxidant capacity. Thus, different markers of oxidative stress capture different aspects of the complex relationships between individual oxidative state, physiology and fitness (Chapter 3). Third, protein carbonyl content and superoxide dismutase activity measured at birth and at four month old in the lambs, show no within individual repeatability, although there is a significant difference in mean marker values over time (Chapter 4). This indicates that these markers of oxidative stress might reflect transient, rather than general, physiological states. Finally, I find some evidence for an oxidative cost of reproduction and for age-related variation in oxidative stress (Chapter 5). However, once again, effect are highly marker and year dependent, and I find no consistent patterns of variation across the two oxidative damage markers or across the two antioxidant protection markers, as they all show different responses to both breeding and age (Chapter 5). Together, my results provide some support for the association between oxidative stress and life-histories, but the effects are both marker and year dependent. Furthermore, the lack of correlation among different markers and the lack of within individual repeatability of oxidative stress markers highlight the need for careful selection and interpretation of plasma oxidative stress markers in the wild.