Coronary atherosclerotic plaque rupture is a critical event during atherosclerosis disease progression. Clinical consequences of atherosclerotic plaque rupture vary from asymptomatic to acute arterial thrombosis, yet the mechanisms underpinning such divergent biological response remain poorly understood. Novel biological signatures of plaque rupture will confer further insights into the dynamic responses triggered by plaque rupture event(s), and may provide alternative strategies for modulation of this prevalent disease. This thesis aims to investigate the events that accompany coronary plaque rupture and their relations to subsequent, downstream systemic effects. In a prospective clinical study of patients undergoing non-emergency percutaneous coronary intervention (PCI), stenting induced plaque disruption was used as a model of plaque rupture in vivo. Optical coherence tomography (OCT) imaging of the plaque lesion was performed prior to stenting, followed by serial blood sampling from targeted anatomical sites in reference to the plaque disruption event. Coronary plaque debris were also retrieved in a controlled manner. Analysis of candidate markers demonstrated a role of matrix metalloproteinase 9 (MMP9) in the systemic response to plaque disruption. Local and systemic elevations of MMP9 were observed promptly after plaque disruption. The systemic release of MMP9 was independent to the myocardial injury and systemic inflammation as a result of PCI. OCT analysis further suggested that plaque morphology may be a determining factor in the subsequent MMP9 release, as the disruption of lipid rich plaque(s) resulted in more acute elevation 'of ~1'MP9 when compared to disruption of non-lipid lesions. Changes of systemic MMP9 served as an index of response to the stent induced plaque disruption. Subjects with divergent MMP9 responses to the plaque disruption event were put forward for further comprehensive investigation during the discovery phase, using mass spectrometry techniques to investigate the lipidomic, metabolomics, and proteomic signatures of plaque disruption. Coronary atherosclerotic plaque disruption was associated with immediate changes in the plasma lipidomics and metabolomics profiles, which had distinct relations to the subsequent systemic MMP9 release. Coronary plaque debris provided a "catalogue" of proteins which could be acutely liberated into systemic circulation. Several such novel proteins were detected in circulation after plaque disruption, which triggered disparate responses in known canonical pathways. Evidence from this thesis implicates the role of liver X receptor / retinoic acid receptor pathway (LXR/RXR) as a key mediator to the divergent systemic responses after plaque disruption, and pinpoints a direction for future investigations.

Travelling wave ion mobility spectrometry (TWIMS) in combination with ultra-high performance liquid chromatography (UHPLC) and mass spectrometry (MS) has been applied successfully for the untargeted, global metabolic profiling of biofluids such as mouse plasma and saliva. Methods based on UHPLC-MS alone and in combination with ion mobility spectrometry (UHPLC-IM-MS) have been developed and validated for the untargeted metabolite profiling of saliva, obtained non-invasively by passive drool. Three separate metabolic profiling studies have been carried out in conjunction with bioinformatics strategies to identify potential metabolomic biomarker ions that are associated with efficacy of rice bran in colorectal cancer, physiological stress and that have the potential for the diagnosis of asthma. The advantages offered by the utility of ion mobility in UHPLC-MS based metabolic profiling studies, including the increased analytical space, mass spectral clean-up of contaminants such as PEG post-UHPLC-IM-MS analysis, enhancement of the selectivity of targeted metabolites as well as the potential for the identification of metabolites by comparison of ion mobility drift times have been highlighted. Ten potential metabolic biomarker ions of asthma have been identified from the moderate asthmatics from untargeted metabolite profiling of saliva by UHPLC-MS. A predictive model based on partial least squares discriminant analysis (PLS-DA) has been constructed using these ten discriminant ions, which demonstrates good predictive capability for moderate asthmatics and controls. Potential metabolic biomarker ions of physiological stress have been identified through untargeted metabolite profiling analysis of saliva samples collected before and after exercise by UHPLC-IM-MS. Valerolactam has been identified as a potential biomarker of physiological stress from saliva by comparison of retention time, ion mobility drift time and MS/MS spectra with a standard of δ-valerolactam.

Background: Physically injured elder adults present challenges in the emergent injury phase. Oxidative stress contributes to cellular deterioration, resulting in decreases in physiological reserve. Imbalance of oxidative stress pathways lead to damage and drive the aging process and frailty. Goals of this study were to determine if a new plasma biomarker of oxidative stress is related to: 1) oxidation reduction status in patients who have experienced traumatic injury as well as healthy community dwellers, 2) outcomes of patients who have experienced trauma, 3) frailty measured by established frailty scales in healthy community dwellers. Methods: Prospective study included 1) trauma patients ≥65 admitted to Level I trauma center 2) age, gender matched healthy, community-dwelling participants. Plasma samples tested in duplicate for capacity oxidative reductive potential (cORP, μC; antioxidant reserve), and static oxidative reductive potential (sORP, mV; the current state of oxidative stress). Frailty assessments were performed in healthy participants using established frailty scales. ORP measurements were analyzed using correlation analyses. Univariate analysis analyzed cORP and sORP for differences by the variables gender, age, smoking, diabetes, statin use, vitamin use and any alcohol use in both the injured and healthy populations. Results: 186 subjects included in study (N=93 for both groups). Trauma groups's cORP values were significantly lower in patients with diabetes (p<0.05) and patients that smoked (p<0.01). Similarly the healthy group's cORP was significantly lower for those who smoked and those with diabetes (p<0.05). Non-vitamin use in the healthy group was related to lower cORP values (p<0.05). Trauma patients who smoked and those with diabetes exhibited higher sORP values (p<0.05). In the healthy group, sORP did not differ when considering the variables. No12significant differences were found based on gender, statin or alcohol use for either group. Significant correlation was found for both sORP and cORP with CSHA Clinical Frailty Scale in the healthy group. Conclusion: Findings suggest that the variables of smoking and diabetes are contributory to frailty trajectory. Data suggest the capacity to tolerate oxidative stress, measured by cORP, is lower in aged individuals that smoke or are diabetic and contributes to frailty as a result of oxidative damage.

Bone bruise lesions (BBL) are documented on MRIs diagnosing acute knee ligament injury (AKLI). Recent evidence has indicated that a majority of patients that sustain an AKLI, especially anterior cruciate ligament (ACL) knee injury, will develop post-traumatic osteoarthritis (PTOA) 10-20 years following injury. It has been proposed that the initial damage sustained to the articular cartilage overlying BBL causes a cascade of events that may result in PTOA. Researchers have proposed a modification to treatment protocols for more severe BBL, or have stressed the need for the development of protective therapies to protect the articular cartilage. However, there are limited tools available to evaluate the clinical outcome of articular cartilage overlying BBL. Furthermore, damage to the cartilage overlying BBL may be different according to differing BBL severities. Therefore, the use of a cartilage degradation biomarker, serum cartilage oligomeric matrix protein (sCOMP) and the use of a BBL severity classification system may be useful to determine if differences exist between patients with and without BBL, and with differing BBL severities. The purpose of this dissertation was to investigate the utility of sCOMP as a biomarker for acute articular cartilage damage. The purposes of these studies were to determine the inter and intraday reliability of this marker, to document sCOMP longitudinally in collegiate athletes and following AKLI, and to determine if differences in sCOMP and self-reported pain and function exist for patients with and without BBL, and differing BBL following AKLI. The results of these studies indicated sCOMP measures had strong inter and intraday reliability. Additionally, exercise does seem to influence sCOMP levels; however, these elevations may not be clinically meaningful. Furthermore, sCOMP levels were not different between patients with BBL and without, and between differing BBL severities. The results of these studies support the use of a BBL severity classification for future research studies in order to further elucidate the outcomes of these lesions.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly. / text

Brain trauma occurs frequently in children and its consequences cause significant health and financial burden to the patients, their carers and society. This thesis assessed the modulating effects of physiological, genetic, and biochemical factors on the sequelae of childhood brain trauma. Primary brain injury from the mechanical forces of trauma and secondary brain insults consequent on the primary injury are determinants of brain trauma outcome. The most important secondary insults recognised are reduced cerebral perfusion pressure (CPP) and raised intracranial pressure (ICP). CPP is governed by the mean arterial blood pressure and the ICP. During childhood these physiological measures change with age. With continuous physiological recordings, ‘critical’ age-related minimum CPP thresholds for children aged 2-6, 7-10 and 11-15 years were defined as 48, 54 and 58mmHg respectively. Utilising these thresholds and a novel cumulative pressure-time index (PTIc), we have demonstrated that CPP insult still remains a feature in 80% of the severe brain trauma patients and significantly relates to global outcome. Brain trauma and cerebral ischaemia are stimuli to the inflammatory cascade leading to further brain damage. Serum adhesion molecule levels after brain trauma indicate injury severity and predict outcome better than brain specific proteins. Predictability is improved using more than one serum biomarker level. Neuro-inflammatory pathways involving adhesion molecules may have a strong modulating effect on brain trauma outcome but warrants further investigations in relation to CPP insult. Genetic factors such as Apolipoprotein E (APO E) genetic polymorphisms may additionally influence outcome, but it was not known whether genetic factors lessen the quantity of CPP insult or the cellular response to it. We demonstrated that the e4 carriers who had unfavourable outcome had 22 times less CPP insult than the non-e4 carriers, while the e3 homozygous who had good recovery had 26 times more CPP insult than the non-e3 homozygous. This suggests that APO E polymorphisms may affect the patient’s cerebral ischaemic tolerance differently, indicating especially the need to prevent CPP insult among e4 carriers. Cerebral ischaemia may, therefore, be a common pathway through which physiological and genetic factors modulate outcome after brain trauma.

The goal of this dissertation research was to describe the relationships among psychosocial variables, physical activity and physical fitness, and biological measures indicative of cardiovascular health in Mexican American male adolescents using a biobehavioral model. One aim of the research was to describe the predictive relationship of psychosocial variables, perceived benefits, perceived barriers, self-efficacy, and interpersonal influences, on physical activity and physical fitness. A second aim described the predictive relationship among physical activity and physical fitness and the amount of body fat and levels of biological markers indicative of endothelial function in this population. Study participants were 28 Mexican American male adolescents ages 15-19. Psychosocial variables were assessed using instruments developed for adolescents by Pender. Physical activity was measured by a 3-day accelerometer recording of activity counts while physical fitness was measured with cycle ergometry withVO2 max. Biologic measures indicative of cardiovascular health included serum leptin, CRP, adiponectin. Fat mass was assessed using BMI and DEXA scans. Findings demonstrated partial support for the model. Psychosocial variables predictive of physical fitness included perceived benefits of action and interpersonal influences. Perceived benefits of exercise significantly predicted physical fitness, explaining 50% of the variance in physical fitness scores while exercise norms, a measure of interpersonal influence, predicted 17% of the variance. Self-efficacy did not meet criteria as a mediating variable; it directly predicted physical activity. Physical activity predicted 15% of the variance in body fat measured as BMI percentile. Physical fitness predicted Leptin levels accounting for 23% of the variance. Physical fitness also predicted 51% of the variance related to the DEXA-derived body fat measurement and 18% of the variance related to BMI. Additional trends were identified including lack of parental support for exercise. Although the study participants reported high acculturated levels, language spoken at home indicated that the family was less acculturated which may have accounted for the lack of parental support. Higher acculturation levels were also significantly associated with increased perceived benefits of action and higher BMI levels. In conclusion, this study suggests that selected psychosocial variables including interpersonal influences should be considered in designing research with Mexican American adolescent males. In addition results suggest that objectively obtained measures of physical fitness and activity are in part predictive of measures of endothelial function and body fat.

This thesis focuses on how physical and biological factors influence the outcome of exposures to γ/X-rays. That the dose rate changes during real life exposure scenarios is well-known, but radiobiological data from exposures performed at increasing or decreasing dose rates is lacking. In paper I, it was found that an exposure where the dose rate decreases exponentially induces significantly higher levels of micronuclei in TK6 cells than exposures at an increasing or constant dose rate. Paper II describes the construction and validation of novel exposure equipment used to further study this “decreasing dose rate effect”, which is described in paper III. In paper I we also observed a radioprotective effect when cells were exposed on ice. This “temperature effect” (TE) has been known for decades but it is still not fully understood how hypothermia acts in a radioprotective manner. This was investigated in paper IV, where a multiparametric approach was used to investigate the underlying mechanisms. In paper V the aim was to investigate the role of biomarkers and clinical parameters as possible risk factors for late adverse effects to radiotherapy (RT). This was studied in a rare cohort of head-and-neck cancer patients that developed mandibular osteoradionecrosis (ORN) as a severe late adverse effect of RT. Biomarker measurements and clinical factors were then subjected to multivariate analysis in order to identify ORN risk factors. The results suggest that the patient’s oxidative stress response is an important factor in ORN pathogenesis, and support the current view that patient-related factors constitute the largest source of variation seen in the frequency of late adverse effects to RT. In summary, this thesis provides new and important insights into the roles of biological and physical factors in determining the consequences of γ/X-ray exposures. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 5: Manuscript.</p>

Disertacijos tikslas – integruotas teršalų pasklidimo aplinkoje ir jų biologinio poveikio Baltijos jūros Lietuvos zonoje vertinimas. Darbe buvo apibendrinti ilgalaikių aplinkos taršos tyrimų Baltijos jūros Lietuvos zonoje duomenys, aprašyti teršalų pasklidimo ypatumai Klaipėdos uosto, grunto gramzdinimo jūroje, Būtingės naftos terminalo, D-6 naftos platformos ir cheminio ginklo nuskandinimo rajonuose, tirti aplinkos taršos biožymenų jūrų organizmuose iš skirtingų pietrytinės Baltijos jūros rajonų ypatumai bei atliktas eksperimentas sąryšio tarp teršalų ir biožymenų atsako moliuskų audiniuose ypatumams nustatyti. Darbas atitinka HELCOM Baltijos jūros veiksmų plano ir ES Jūrų strategijos pagrindų direktyvos iškeltus tikslus – „Baltijos jūros aplinka nepaveikta pavojingų medžiagų“ ir „teršalų koncentracijos lygis yra toks, kad nesukelia taršos poveikio“. Norint pasiekti šiuos tikslus, būtina atlikti kompleksinį Baltijos jūros aplinkos būklės vertinimą. Šiems tikslams sukurtas HELCOM Cheminės būklės vertinimo įrankis CHASE (angl. Chemical Status Assessment Tool), buvo panaudotas kompleksiškai vertinant Lietuvos Baltijos jūros rajonų aplinkos taršą. Atsiranda vis daugiau moksliniais tyrimais pagristų įrodymų, kad biologinių efektų tyrimai yra svarbūs. Šis darbas yra dar vienas biologinių efektų tyrimų svarbos įrodymas ir tokio pobūdžio tyrimų įtraukimo į valstybinės stebėsenos programą aktualumo patvirtinimas. / The aim of the study is an integrated assessment of the spread of contaminants and their biological effects in the Lithuanian zone of the Baltic Sea. Long-term monitoring data on contaminants in water, sediments and biota were summarized; the peculiarities of distribution of contaminants in the Klaipėda harbour, Būtingė oil terminal, dredged sediments dumping site, adjacent area to the Russian D-6 oil platform and chemical munitions dumpsite were described; biomarker responses in marine organisms from the different areas of the southeastern Baltic Sea and the Curonian Lagoon were evaluated and the impact of contaminants on biomarker responses in mussels was assessed. The thesis reflects the overall goals of the hazardous substances segment of the HELCOM Baltic Sea Action Plan – to achieve a Baltic Sea with life undisturbed by hazardous substances and of the EU Marine Strategy Framework Directive – concentrations of contaminants are at levels not giving rise to pollution effects. The ongoing activities of HELCOM and EU are aimed at the integrated holistic assessments of the environmental state of the Baltic Sea. The combination of contaminant concentrations with the biological effects were used in the HELCOM Chemical Status Assessment Tool CHASE, which also has been applied for Lithuanian waters. Whereas more scientific evidences appear the biological effects become an important issue of the environmental research. This study also reflects the importance of integration of... [to full text]

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma (NHL). FL is clinically designated as an indolent disease with a long median survival of 8-10 years. However, the clinical and biological behavior of FL shows considerable variability, with some patients showing aggressive disease progression and very short survival. Because defects in the regulation of apoptotic cell death are fundamental in FL pathogenesis, we hypothesized that deregulated expression of components of the pRb signaling pathway may promote cell proliferation, thereby complementing antecedent anti-apoptotic mutations and producing more aggressive disease. In the present study we undertook an immunohistochemical (IHC) evaluation of expression of key cell-cycle regulatory proteins in diagnostic biopsies from 127 cases of FL using formalin-fixed, paraffin-embedded tissues (FFPE) in tissue microarray (TMA) sections immunostained for p53, pRb, p16INK4A and cyclin D3. Data analysis revealed that increased abundance of p53 or p16INK4A is associated with reduced overall survival (OS) (p=0.005 and p=0.014 respectively), and with conventional pathological markers of tumour aggressiveness including high histologic grade. Encouraged by this remarkable finding of a counterintuitive association between p16INK4A expression and clinical outcome, we analyzed CDKN2A gene deletion and methylation, as these are the most frequent mechanisms of the CDKN2A gene inactivation in NHL including FL. We determined the deletion and methylation status of CDKN2A in 105 FL cases. Laser-capture microdissection was used to enrich the samples for lymphoma cells. CDKN2A was deleted in 9 cases and methylated in 22 cases. The 29 cases (28%) with CDKN2A deletion or methylation had decreased overall survival (OS) (p=0.046) in all cases and in cases treated with rituximab (p<0.001). Our findings indicate that deleterious alterations of CDKN2A are relatively prevalent in FL at diagnosis and can predict poor clinical outcome. In summary, our data reveal novel insights into the pathogenesis of FL and suggest a relationship between increased p16INK4A expression and CDKN2A deletion or methylation and unfavorable clinical outcome in FL. We hope that the work presented herein will provide a useful prognostic tool for predicting the prognosis and choosing optimal treatment approaches to help patients suffering from FL. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-03-12 23:49:44.541