LLANO PIEDRA, LISSET BARBARA
Infectious diseases represent one of the leading causes of death globally. Prompt diagnosis is essential for the onset of clinical treatment but certain cases of underlying bacterial infection deep in the body can remain undiagnosed for weeks. Hidden bacterial infection is the leading cause of fever of unknown origin (FUO), which is observed in 2 % of all hospital admissions around the world. Molecular imaging of bacterial infections is the ideal non-invasive diagnostic tool, but all available probes also detect inflammation. Two targets were selected for development of bacteria-specific molecular imaging probes, namely iron-uptake pathways and peptidoglycans involved in the synthesis of the cell wall. Both, Gram-positive and Gram-negative bacteria use iron-binding molecules called siderophores to scavenge iron from their surroundings. The structural similarities between Fe3+ and Ga3+ allow siderophores to be radiolabelled with 67/68Ga and visualized by nuclear medicine techniques. The clinically proven siderophore Deferoxamine (Dfo) has a plasma half-life of only 5.5 min that does not favor its direct use as a probe. Dfo derivatives with improved pharmacokinetics properties were designed and tested on Staphylococcus aureus cultures. The ciprofloxacin and the ethyloxycarbonyl derivatives of DFO at the primary amino position were among the most successful conjugates targeting the siderophore active-transport mechanism and reaching high relative uptake rates. Furthermore, the peptidoglycan pathway of Gram-positive bacteria was in vitro targeted with vancomycin conjugated to 67Ga-Dfo which showed even higher labelling capacity than 67Ga-Dfo within a few minutes of exposure. In vitro siderophore studies remain challenging due to the lack of methods for the preparation of rigorously iron-depleted media. We developed an iron chelating method with the goal of creating iron-free growth media. / Thesis / Master of Science (MSc)
31 January 2012
No description available.
Preparation and Evaluation of Molecular Imaging Probes Targeting the Urokinase Plasminogen Activator SystemVito, Alyssa January 2015 (has links)
The aim of this thesis was to develop a molecular imaging probe for the urokinase plasminogen activator (uPA) system, which has been shown to play a critical role in cancer metastasis, tumour aggressiveness and likelihood of progression. Two classes of small molecule inhibitors carrying isotopes of iodine were synthesized and evaluated using in vitro assays and in vivo studies. Lead compounds showed high affinity for the target with Ki values in the low nanomolar range (1b = 1.4 nM, 1e = 6.1 nM, 1g = 2.6 nM and 2a = 2.1 nM). Biodistribution studies of the reversible compounds (1b, 1e, 1g) showed rapid clearance, accumulation in the gall bladder and intestines and little to no tumour uptake (<1 %ID/g). The irreversible inhibitor (2a) showed specificity for the target through SDS-PAGE and biodistribution studies. Analysis of the biodistribution pattern showed retention in the tumour over time reaching a maximum at 24 h post-injection of 1.95 %ID/g with tumour-to-blood ratio being 0.65 at 24 h, 1.13 at 48 h and 1.09 at 96 h post-injection. A parallel strategy reported involved targeting the uPA receptor (uPAR) through the use of antibodies and bioorthogonal chemisty based on radiolabeled tetrazines and transcyclooctene (TCO) functionalized biomolecules. A new tetrazine synthon was developed that can be readily labeled with both 99mTc and 18F where the products were produced in 75 and 31 % radiochemical yields. Stability studies showed the compounds are suitable for use in vivo. Biodistribution studies were carried out in CD1 mice and results showed that both probes had sufficient distribution patterns to warrant use in pre-targeting strategies. Their reactivity with TCO, including functionalized derivatives such as TCO-anti-uPAR, was also demonstrated creating the means to develop PET and SPECT probes for imaging the urokinase system using a single prosthetic group. / Thesis / Master of Science (MSc)
Development and Application of CatalyCEST MRI Contrast Agents for the Study of Enzyme Activities in Tumor ModelsSinharay, Sanhita January 2016 (has links)
The in vivo detection of enzyme activity is a significant biomarker in tumorigenesis. Assessment of enzyme activity relative to enzyme concentration can serve as quite an accurate measurement of several disease states. Chemical Exchange Saturation Transfer (CEST) MRI is a non-invasive imaging technique that can be used to evaluate enzyme activity. Compared to other contrast agents CEST MRI agents have a slower chemical exchange rate and thus have greater specificity for detecting the intended biomarker. Chapter 1 provides an overview of the advances made in the field of molecular imaging for detection of cancer biomarkers. The molecular mechanism of each technique is explained with specific examples and advantages as well as disadvantages of each technique. Chapter 2 investigates the specific example of detection of an enzyme, γ-glutamyl transferase (GGT) in ovarian cancer tumor models using a catalyCEST MRI contrast agent. This chapter discusses the step-by step evaluation of the non-metallic contrast agent, from synthesis to evaluation of its catalytic efficiency with Michaelis Menten kinetics studies and finally in vivo GGT detection in ovarian tumor models of OVCAR-8 and OVCAR-3. Chapter 3 investigates the enzyme, Kallikrein-6 and its detection in HCT116 colon cancer tumor model. In addition to enzyme detection, enzyme inhibition using Antithrombin III inhibitor has also been explored within in vitro media and in vivo HCT116 tumor model. Chapter 4 introduces the catalyCEST agent for detection of sulfatase enzyme. This chapter discusses the synthesis of this agent and its ability to detect sulfatase in bacterial cell suspension and mammalian cell suspension. These examples portray catalyCEST MRI as a platform technology for enzyme activity detection. Finally in Chapter 5 future ideas have been proposed to improve the in vivo detection and broaden the applications of catalyCEST MRI in the field of enzyme studies.
Joshi, Nikhil Vilas
Cardiovascular disease remains the commonest cause of death worldwide. The majority of deaths are caused by atherosclerotic plaque rupture with resultant myocardial infarction or stroke, or rupture of abdominal aortic aneurysms. Conventional imaging modalities have consistently failed to identify atherosclerotic plaques or aneurysms with high-risk pathological features that are at highest risk of rupture or progression. The development of modern molecular imaging techniques targeted at these features could lead to the identification of such high-risk plaques and aneurysms in vivo and guide the development of novel treatment strategies. The aim of this thesis was to evaluate whether novel molecular modalities have a role in providing new insights into biological disease processes, and identify high-risk plaques and aneurysms. Using positron emission tomography-computed tomography (PET-CT), 18F-fluorodeoxyglucose and 18F-fluoride were utilised as markers of metabolic inflammation and active calcification. Cellular inflammation was assessed using ultrasmall superparamagnetic particles of iron oxide (USPIO) enhanced magnetic resonance imaging (MRI). In a prospective trial, 80 patients with myocardial infarction (n=40) and stable angina (n=40) underwent 18F-fluoride and 18F-fluorodeoxyglucose PET-CT, and invasive coronary angiography (Chapter 3). Intense 18F-fluoride uptake localised to recently ruptured plaque in patients with acute myocardial infarction. In patients with stable coronary artery disease, 18F-fluoride uptake identified coronary plaques with high-risk features on intravascular ultrasound. 18F-fluoride PET-CT is the first noninvasive imaging method to identify and localise ruptured and high-risk coronary plaques. Aortic vascular uptake of 18F- fluorodeoxyglucose was studied in patients with myocardial infarction and stable angina (Chapter 4). In a separate outcome of 1,003 patients enrolled in the Global Registry of Acute Coronary Events, we further evaluated whether infarct size predicted recurrent coronary events. Patients with myocardial infarction had higher remote atherosclerotic tracer uptake that correlated with the degree of myocardial necrosis, and exceeded that observed in patients with stable coronary disease. The outcome cohort demonstrated that patients with higher degree of myocardial necrosis had the highest risk of early recurrent myocardial infarction. This supports the hypothesis that acute myocardial infarction exacerbates systemic atherosclerotic inflammation and remote plaque destabilization: myocardial infarction begets myocardial infarction. In a prospective imaging cohort, the role inflammation and calcification was assessed in 63 patients with abdominal aortic aneurysms and 19 age and sex matched patients with atherosclerosis (Chapter 5). Compared to non-aneurysmal segments, enhanced inflammation and calcification was observed within the wall of aortic aneurysmal segments. In comparison to matched controls with atherosclerosis, the entire aorta in those with aortic aneurysm appears more highly inflamed, suggesting presence of a global aortopathy rather than a disease confined only to the abdominal region of the aorta. Aortic aneurysms have greater active inflammation and calcification than atherosclerotic controls suggesting a more intense, destructive and transmural pathological process. A subgroup of fifteen patients with aortic aneurysms underwent imaging with both PET-CT with 18F-fluorodeoxyglucose, and T2*- weighted MRI before and 24 h after administration of USPIO (Chapter 6). Whilst there was a moderate correlation between the two tracers, there were distinct differences in the pattern and distribution of uptake suggesting a differential detection of macrophage glycolytic and phagocytic activity respectively. These studies provide novel insights into vascular biological processes involved in the initiation, progression and rupture of atherosclerotic plaques and aortic aneurysms. Future longitudinal studies are needed to establish whether these techniques have a role in improving the clinical management and treatment of patients with coronary artery disease and aortic aneurysms.
Williamson, Andrew Carl
01 December 2010
Molecular imaging is becoming an important contributor to the development of personalized medicine. Positron Emission Tomography (PET) is a technology that enables molecular imaging by allowing a physician to detect and map the location of various physiological processes. The purpose of this work is to design, fabricate and test a mechanism that would make the production of the PET isotope, copper-64 practical for both researchers and commercial suppliers. In order to have the maximum usefulness, the design needs to fit and operate within several constraints. A one dimensional thermal analysis indicated that operation of the system under existing cooling conditions would be a reasonable solution. Based on the design specifications, a detailed design was completed and fabricated. The design was functionally and operationally tested with the performance meeting expectations. The design was utilized to produce copper-64 isotope with a typical one hour bombardment producing 30 mCi of isotope. The design could be optimized if future isotope demand exceeded current production capacity or if research required the production of other radioisotopes with varying thermal characteristics.
02 July 2009
Kandadai, Nirmala Krishna
12 November 2013
Biomolecular imaging has become one of the most exciting potential applications of the Linear Coherent Light Source (LCLS), which is a source of intense femtosecond X-rays. It has been predicted that a highly intense pulse with pulse lengths on the order of a few femtoseconds should be sufficient to capture the image of a biomolecule before it is destroyed. However, the rate at which a large biomolecule explodes during exposure is a large unknown, and will likely be one of the major factors in determining if such imaging will succeed. Clusters were chosen as a size dependant model system, ideal to study the evolution of complex systems in X-ray fields. From earlier intense near-infrared (IR) experiments, it is known that depending on size and Z constitution, clusters explode by Coulomb or hydrodynamic forces. These two limits have very different cluster explosion times and signatures. Coulomb explosion is too fast to allow imaging, whereas a hydrodynamically expanding cluster is a much slower process. The ionization process leading to cluster explosion is strongly wavelength dependent as one passes from IR through XUV to the X-ray regime because the kinetic energy of the released electrons determines the charge imbalance within the cluster, and therefore, determines the explosion dynamics. Unlike in previous experiments performed with near IR or XUV pulses, irradiation by photons at the LCLS will lead to the ejection of energetic photo- and Auger- electrons which could easily escape from the cluster, leaving behind positive ions. The buildup of this charge during exposure can lead to a Coulomb explosion of the sample. On the other hand, if the charge accumulates, the photoelectrons will be held inside the cluster, where they could contribute to the cluster temperature and form a nanoplasma and expand hydrodynamically. The main goal of the thesis was to study the explosion dynamics of clusters generated due to their interaction with intense X-rays and look at its dependencies on the X-ray energy, photon fluence, absorption cross sections, sample constituency and sample size. This thesis also compares the results from X-rays with the corresponding results obtained using ultrashort XUV and Infrared lasers. / text
Gone Fishing: Synthesis and Design of a Superparamagnetic Nanobait for Trapping Reactive Metabolites In Vivo.Tayyabi, Ehsen January 2018 (has links)
Adverse drug reactions are common causes of medical injuries. Drug-induced hepatotoxicity remains one of the leading causes of emergency room visits, FDA non-approval, and drug withdrawal from the market. We have investigated the ability of endogenous nucleophilic amino acid residues (K, H, and C) to selectively bind to reactive electrophilic drug metabolites, focusing on acetyl-para-aminophenol (APAP, i.e. Tylenol®), for which hepatotoxicity has recently re- emerged as a major health concern for Canadians. Three peptide sequences were synthesized bearing terminal nucleophilic residues, brominated phenylalanine residues, and c-terminal amides. These peptides were coupled to carboxy methyl dextran coated iron oxide nanoparticles (CMX- IONPs) with a hepatocyte targeting group. IONPs are known for their ability to act as T2-weighted MRI contrast agents, giving us the ability to track them in vivo. This study begins to establish a nanotechnology-based method for the in vivo trapping of NAPQI, the reactive metabolite of APAP, using a cysteine bearing IONP.
Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies. Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties. In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules. Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours. The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.
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