Functional imaging of the lungs using magnetic resonance imaging of inert fluorinated gases
SUPERVISOR: Dr. Mitchell Albert (Chemistry)
ABSTRACT: Fluorine-19 (19F) magnetic resonance imaging (MRI) of the lungs using inhaled inert fluorinated gases can potentially provide high quality anatomical and functional images of the lungs. This technique is able to visualize the distribution of the inhaled gas, similar to hyperpolarized (HP) helium-3 (3He) and xenon-129 (129Xe) MRI. Inert fluorinated gases have the advantages of being nontoxic, abundant, and inexpensive compared to HP gases. Due to the high gyromagnetic ratio of 19F, there is sufficient thermally polarized signal for imaging, and averaging within a single breath-hold is possible due to short longitudinal relaxation times. Since inert fluorinated gases do not need to be hyperpolarized prior to their use in MRI, this eliminates the need for an expensive polarizer and expensive isotopes. Inert fluorinated gas MRI of the lungs has been studied extensively in animals since the 1980s, and more recently in healthy volunteers and patients with lung diseases. This thesis focused on the development of static breath-hold inert fluorinated gas MR imaging techniques, as well as the development functional imaging biomarkers in humans and animal models of pulmonary disease. Optimized ultrashort echo time (UTE) 19F MR imaging was performed in healthy volunteers, and images from different gas breathing techniques were quantitatively compared. 19F UTE MR imaging was then quantitatively compared to 19F gradient echo imaging in both healthy volunteers and in a resolution phantom. A preliminary comparison to HP 3He MR imaging is also presented, along with preliminary 19F measurements of the apparent diffusion coefficient (ADC) and iv gravitational gradients of ventilation in healthy volunteers. The potential of inert fluorinated gas MRI in detecting pulmonary diseases was further explored by performing ventilation mapping in animal models of inflammation and fibrosis. Overall, interest in pulmonary 19F MRI of inert fluorinated gases is increasing, and numerous sites around the world are now interested in developing this technique. This work may help to demonstrate that inert fluorinated gas MRI has the potential to be a viable clinical imaging modality that can provide useful information for the diagnosis and management of chronic respiratory diseases.
Bioremediation of contaminated soils from mine sites using native plants in Northwestern Ontario
SUPERVISOR: Dr. Peter Lee (Biology)
ABSTRACT: Practical and scientific importance can be found in this research topic since the results directly apply to remediation of industrial and mined lands in the boreal forest region. Plants suitable for phytostabilization of As, Mo and Sb are identified as well as two hyperaccumulators of Zn. Using phytostabilization practices, metals are immobilized by the below ground components of the plants therefore restricting the flow into the ecosystem and lessening the impacts of metal pollution to the surrounding area. As long as there is little disturbance of the soil physically or chemically, the plants will continue to stabilize the metal in the organic portion of the deceased plants. The ease of replanting a site could incorporate successional ecosystem in the region by focusing on trees and shrubs that are earlier in the revegetation process after a disturbance. The addition of woodbark to the reestablishment of the top soil increases potential nutrients, organic matter, water holding potential as well as diluting potential harmful metal content of the soil and providing a mulching effect. Some concerns exist by using agronomic plant species as the sole part of revegetation as they have the potential to impact the wildlife in the region through excess Mo. Results from this thesis could be helpful for future mine closure plans and in the rehabilitation of other industrial sites.
Hydrogen sulfide modulates gluconeogenesis and mitochanodrial biogenesis in mouse primary hepatocytes
SUPERVISOR: Dr. Lingyun Wu (formerly with Biology)
ABSTRACT: Among many endogenous substances that regulate hepatic energy production is the gasotransmitter hydrogen sulfide (H2S). In the liver, H2S production is largely catalyzed by cystathionine γ-lyase (CSE) and, to a lesser degree, by cystathionine β-synthase. We previously showed that H2S stimulates glucose production in an immortalized carcinoma liver cell line (HepG2 cells) as well as induce ATP generation in isolated vascular smooth muscle cells (VSMCs). Furthermore, we found that H2S upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in rat VSMCs. PGC-1α is a crucial regulator of hepatic gluconeogenesis and mitochondrial biogenesis. Both of these PGC-1α-mediated energy processes are pivotal to maintain whole-body energy homeostasis, whereby their sustained disturbance may lead to the development of type 2 diabetes and metabolic syndrome. Therefore, we investigated the regulation of gluconeogenesis and mitochondrial biogenesis by CSE-generated H2S under physiological conditions in isolated mouse hepatocytes. We found that CSE-knockout (KO) mice had a reduced rate of gluconeogenesis, which was reversed by administration of NaHS (an H2S donor) (i.p.). Interestingly, isolated CSE-KO hepatocytes exhibited a reduced glycemic response to chemical-induced activation of the cAMP/PKA and glucocorticoid pathways compared to wild-type (WT) hepatocytes. Treatment with the inhibitors for PKA (KT5720) or glucocorticoid receptor (RU-486) significantly reduced H2S-stimulated glucose production from both WT and CSE-KO mouse hepatocytes. NaHS treatment upregulated the protein levels of key gluconeogenic transcription factors, such as PGC-1α and CCAAT-enhancer-binding proteins-β (C/EBP-β). Moreover, exogenous H2S augmented the S-sulfhydration of the rate-limiting gluconeogenic enzymes and PGC-1α and increased their activities, which were lower in untreated CSE-KO hepatocytes. Finally, knockdown of PGC-1α, but not C/EBP-β, significantly decreased NaHS-induced glucose production from the primary hepatocytes. After determining that H2S stimulates hepatic glucose production through the PGC-1α signaling pathway, we focused on whether or not H2S induces hepatic mitochondrial biogenesis. We found that CSE-KO hepatocytes produced less mtDNA compared to WT hepatocytes. Mitochondrial content was decreased in CSE-KO hepatocytes compared to normal hepatocytes, which was restored with NaHS treatment. CSE-KO hepatocytes exhibited lower levels of mitochondrial transcription factors and the mitochondrial transcription coactivator, peroxisome proliferator-activated receptor-γ coactivator-related protein (PPRC) compared to WT hepatocytes. Interestingly, NaHS administration upregulated PPRC, yet downregulated PGC-1β protein level in mouse hepatocytes. Moreover, exogenous H2S induced the S-sulfhydration of PPRC, which was lower in untreated CSE-KO hepatocytes, but not that of PGC-β. Finally, knockdown of either PGC-1α or PPRC significantly decreased NaHS-stimulated mitochondrial biogenesis in hepatocytes, where knockdown of both genes were required to completely abolish NaHS-induced mitochondrial biogenesis. Overall this thesis demonstrates the stimulatory effect of endogenous H2S on liver glucose production and reveals four underlying mechanisms. 1) H2S upregulates the expression levels of PGC-1α and PEPCK via glucocorticoid receptor pathway. 2) H2S upregulates the expression level of PGC-1α through the activation of the cAMP/PKA pathway, as well as PGC-1α activity via S-sulfhydration. 3) H2S upregulates the expression and the activities (by S-sulfhydration) of G6Pase and FBPase. 4) H2S augments the protein expression level and activity (via S-sulfhydration) of PPRC. By stimulating the combined activities of PPRC and PGC-1α, H2S induces mitochondrial biogenesis, thereby supplying energy to support its induction of hepatic glucose production. This study may offer clues to the regulation of hepatic energy homeostasis under physiological conditions and its dysregulation in insulin-resistance diseases.
Modeling the molecular mechanisms of biocompatibility of artificial materials
SUPERVISOR: Dr. Oleg Rubel (formerly with Chemistry)
ABSTRACT: One of the most common reasons for implant failure is immune rejection. Implant rejection leads to additional surgical intervention and, ultimately, increases health cost as well as recovery time. Within a few hours after implantation, the implant surface is covered with host proteins. Adsorption of fibrinogen, a soluble plasma glycoprotein, is responsible in triggering the immune response to a given material and, subsequently, in determining its biocompatibility. The work presented here is focused on modeling the interaction between artificial surfaces and plasma proteins at the microscopic level by taking into account the physico-chemical properties of the surfaces. Carbon-based nanomaterials are chosen as a model system due to their unique bioadhesive and contradictory biocompatible properties as well as the possibility of functionalization for specific applications. Graphene and its derivatives, such as graphene oxide and reduced graphene oxide, demonstrate controversial toxicity properties in vitro as well as in vivo. In this study, by covalently adding chemical groups, the wettability of graphene surfaces and the subsequent changes in its biocompatibility are being examined. An empirical force field potential (AMBER03) molecular dynamic simulation code implemented in the YASARA software package was utilized to model graphene/biomolecule interactions. The accuracy of the force field choice was verified by modeling the adsorption of individual amino acids to graphene surface in a vacuum. The obtained results are in excellent agreement with previously published ab initio findings. In order to mimic the natural protein environment, the interaction of several amino acids with graphene in an explicit solvent was modeled. The results show that the behaviour of amino acids in aqueous conditions is drastically different from that in vacuum. This finding highlights the importance of the host environment when biomaterial-biomolecule interfaces are modeled. The surface of Graphene Oxide (GO) has been shown to exhibit properties that are useful in applications such as biomedical imaging, biological sensors and drug delivery. An assessment of the intrinsic affinity of amino acids to GO by simulating their adsorption onto a GO surface was performed. The emphasis was placed on developing an atomic charge model for GO that was not defined before. Next, the simulation of a fibrinogen fragment (D-domain) at the graphene surface in an explicit solvent with physiological conditions was performed. This D-domain contains the hidden (not expressed to the solvent) motifs (PI 7190-202 and P2 7377-395, and specifically P2-C portion 7383-395) that were experimentally found to be responsible for attracting inflammatory cells through CDllb/CD18 (Mac-1) leukocyte integrin and, consequently, promoting the cascade of immune reactions. It was hypothesized that the hydrophobic nature of graphene would cause critical changes in the fibrinogen D-domain structure, thus exposing the sequences and result in the foreign body reaction. To further study this issue, molecular mechanics was used to stimulate the interactions between fibrinogen and a graphene surface. The atomistic details of the interactions that determine plasma protein affinity modes on surfaces with high hydrophobicity were studied. The results of this work suggest that graphene is potentially pro-inflammatory surface, and cannot be used directly (without alterations) for biomedical purposes. A better understanding of the molecular mechanisms underlying the interaction between synthetic materials and biological systems will further the ultimate goal of understanding the biocompatibility of existing materials as well as design of new materials with improved biocompatibility.
Cardiac oxidative stress and antioxidant status in response to radiation and monocrotaline-induced cardiac dysfunction
SUPERVISOR: Dr. Neelam Khaper (NOSM, Biology adjunct)
ABSTRACT: Cardiovascular disease is the leading cause of death and disability worldwide. Oxidative stress has been implicated in many types of cardiovascular disease. Chronic cardiac stress conditions have been shown to be associated with an increase in myocardial oxidative stress following myocardial infarction, which in turn may lead to depressed contractile function, myocardial remodeling and heart failure. Antioxidants play a protective role against oxidative stress damage through the removal of free radical intermediates and inhibition of oxidation reactions. An imbalance of free radicals and antioxidants results in cellular and sub-cellular damage. Therefore, treatment with non-enzymatic antioxidants may provide protection against high levels of free radicals. We investigated, in three different studies, if treatment with antioxidants can protect the heart under conditions of oxidative stress. In the first study, a complex dietary supplement composed of numerous antioxidants and anti-inflammatory components was given to C57BL/6 mice that received a whole body radiation dose of 5 Gy to investigate potential cardioprotective effects by measuring cardiac antioxidant status and apoptosis. In the second study, the same complex dietary supplement was given to Thy1-GFP mice that received a radiation dose of 10 Gy to the head to investigate the abscopal effect on the heart by measuring cardiac inflammation and fibrosis. In the final study, the potential cardioprotective effects of secoisolariciresinol diglucoside, a compound found in flaxseed, was investigated in a Wistar rat model of pulmonary arterial hypertension by correlating cardiac functions with oxidative stress. We have shown that treatment with antioxidants may offer some protection to the heart in these models of oxidative stress though it is important to consider the extent of oxidative stress and when developing a antioxidant treatment protocol.