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Ultrasound contrast agents (CAs) are typically used in clinic for perfusion studies (blood flow through a specific region) and border delineating (differentiate borders between tissue structures) during cardiac imaging. The CAs used during ultrasound imaging usually consist of gas filled microbubbles (MBs) (diameter 1-5 μm) that are injected intravenously into the circulatory system. This thesis partially involves a novel polymer-shelled ultrasound CA that consists of air filled MBs stabilized by a polyvinyl alcohol (PVA) shell. These MBs could be coupled with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as a combined CA for ultrasound and magnetic resonance imaging. The first three papers (Paper A-C) in this thesis investigate the structural characteristic and the elimination process of the CA. In Paper A, two types (PVA Type A and PVA Type B) of the novel CA were analyzed using transmission electron microscopy (TEM) images of thin sectioned MBs. The images demonstrated that the SPIONs were either attached to the PVA shell surface (PVA Type A) or embedded in the shell (PVA Type B). The average shell thickness of the MBs was determined in Paper B by introducing a model that calculated the shell thickness from TEM images of cross-sectioned MBs. The shell thickness of PVA Type A was determined to 651 nm, whereas the shell thickness of PVA Type B was calculated to 637 nm. In Paper C, a prolonged blood elimination time was obtained for PVA-shelled MBs compared to the lipid-shelled CA SonoVue used in clinic. In addition, TEM analyzed tissue sections showed that the PVA-shelled MBs were recognized by the macrophage system. However, structurally intact MBs were still found in the circulation 24 h post injection. These studies illustrate that the PVA-shelled MBs are stable and offer large chemical variability, which make them suitable as CA for multimodal imaging. This thesis also involves studies (Paper D-E) of the molecular chaperones (Hsp21 and DNAJB6). The small heat shock protein Hsp21 effectively protects other proteins from unfolding and aggregation during stress. This chaperone ability requires oligomerization of the protein. In Paper D, cryo-electron microscopy together with complementary structural methods, obtained a structure model which showed that the Hsp21 dodecamer (12-mer) is kept together by paired C-terminal interactions.The human protein DNAJB6 functions as a very efficient suppressor of polyglutamine (polyQ) and amyloid-β42 (Aβ42) aggregation. Aggregation of these peptides are associated with development of Huntington’s (polyQ) and Alzheimer’s (Aβ42) disease. In Paper E, a reconstructed map of this highly dynamic protein is presented, showing an oligomer with two-fold symmetry, indicating that the oligomers are assembled by two subunits. / <p>QC 20160527</p>

Magnetic nanoparticles are of great interest for a wide range of applications. This work has focused on three primary forms of iron based nanoparticles and combinations thereof: α-iron, iron oxide, and iron carbide or cementite. The synthesis of several core-shell particles including cementite-iron oxide, α-iron-cementite, and α-iron-iron oxide was accomplished through reverse micelle routes and high temperature decomposition of iron pentacarbonyl in various media. Structural analysis to confirm the structures was performed using extended x-ray absorption fine structure (EXAFS) techniques. A rapid characterization technique was developed utilizing a correlation between Fourier transform infrared spectroscopy and EXAFS to determine the full metal cation distribution between the octahedral and tetrahedral sites in manganese zinc ferrite (MZFO). This method was then used to show that the initial Fe3+ to Fe2+ ratio in MZFO synthesis could be used to design a desired cation distribution and affected the zinc incorporation levels into the resultant ferrite. Functionalization of nanoparticles for aqueous dispersions and ferrofluids has varying degrees of importance, depending on the application. In applications such as magnetic resonance imaging (MRI) where the targets are biological systems, it was important to produce solutions that will not aggregate in the high magnetic field of the MRI. It was also vital to characterize decomposition mechanisms and products that would be presented to the body after use as a contrast agent. This work has provided insight into both the preparation of magnetic samples for MRI applications and implications of the biocompatibility of reactive and decomposition products. Three successful methods of forming dispersions that would not aggregate in the high magnetic field of the MRI were comprised of cysteine/polyethylene glycol (PEG), PEG based ferrofluids, and dopamine/PEG. The dopamine functionalization however showed reactivity with the iron/iron oxide nanoparticles and led to the formation of the cytotoxic dopamine quinone and resulted in the destruction of the nanoparticles. Using all three types of dispersions to compare the iron based nanomaterials, the MRI measurements concluded with the iron oxide ferrofluid yielding the highest R2 enhancement.

The topic of my Master degree thesis is the development of a conceptually new class of contrast agents for the 31P magnetic resonance imaging (31P MRI). These agents are based on nanoparticles of calcium(II) phytate. Phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate) is largely present in plants, seeds and grains. It is non-biodegradable but nontoxic for animals and human beings and most importantly around 22% of its mass is phosphorus, so it is easily detectable by 31P NMR/MRI. These nanoparticles of Ca(II) phytate were doped with paramagnetic Fe3+ ions which broaden the 31P signal, making the nanoparticles invisible in healthy tissues. In the presence of bacteria producing siderophores (for example in Helicobacter pylori in gastric ulcers), Fe3+ is released from the gel and 31P MRI signal becomes detectable. In vitro simulation of this release was performed with deferroxamine, a compound possessing high affinity to Fe3+ ions forming coloured complex with it exploitable for the UV-VIS evaluation. The Ca(II) phytate can be synthesized in two possible ways. The first way is by direct precipitation of the Ca2+ salt with sodium phytate. The second way is ion exchange of phosphate in the nanoparticles of hydroxyapatite which creates electrostatically stabilized calcium phytate nanoparticles. Both...

O ferro é essencial para a vida do ser humano, desempenhando um papel fundamental no metabolismo. Contudo, quando não armazenado em compartimentos biológicos adequados, o metal apresenta um potencial tóxico ao organismo, uma vez que contribui para a formação de espécies reativas de oxigênio. A sobrecarga de ferro é uma condição desfavorável para portadores de algumas disfunções genéticas, como a hemocromatose, ou de anemias crônicas que requeiram transfusões de sangue periódicas, como é o caso da talassemia. Os fármacos atuais que controlam a patologia, como a desferrioxamina (DFO), requerem infusão subcutânea lenta, causando desconforto em pacientes e podendo trazer um série de complicações, como insuficiência hepática e renal. A modificação dessas moléculas com biopolímeros é uma proposta para minimizar efeitos colaterais e aumentar a biodisponibilidade do fármaco no organismo. Dentre esses biopolímeros, destacam-se as albuminas proveniente do soro bovino (BSA) e do ovo (OVA), que têm baixa toxicidade, baixo custo e abundância de sítios reativos, que quando modificados, favorecem reação com a desferrioxamina. Como resultado, houve a reação dos biopolímeros com a desferrioxamina, com mudanças em suas estruturas secundárias e possível dimerização, resultando na formação de conjugados possuem afinidade com íon ferro e capacidade antioxidante semelhante ao fármaco original, características que tornam os compostos bons candidatos a uma alternativa à terapia de quelação. Os conjugados BSA-DFO e OVA-DFO podem reagir, além do ferro, com gadolínio, fazendo com o que os complexos tenham uma potencial aplicação como agentes de contraste em ressonância magnética de imagem (MRI). Neste trabalho, vimos que o complexo entre Gd(III) e BSA-DFO apresentou uma relaxatividade de 52,92 s-1 mM-1 para T2 e 45,37 s-1 mM-1 para T1 , um valor bem superior aos fármacos disponíveis no mercado, que apre-sentam relaxatividade entre 4 e 5 s-1 mM-1, o que foi explicado por sua elevada massa molecular, indicando que poderia ter bons efeitos na qualidade de MRI, com menores doses. / Iron is essential for human life, playing a fundamental role in metabolism. However, when not stored in appropriate biological compartments, the metal presents a toxic potential to the body, contributing to the formation of reactive oxygen species (ROS). Iron overload is an unfavorable condition for people with certain genetic disorders, such as hemochromatosis, or chronic anemias that require periodic blood transfusions, as thalassemia. Current drugs that control the pathology, as desferrioxamine, require slow subcutaneous infusion, causing discomfort in patients and may lead to a number of complications, such as hepatic and renal failures. As a result, the biopolymers were reacted with desferrioxamine, with changes in their secondary structures and possible dimerization, resulting in the formation of conjugates with iron ion affinity and antioxidant capacity similar to the original drug, characteristics that make the compounds good candidates for an alternative chelation therapy As a result, the reaction of the biopolymers with desferrioxamine caused a change in the secondary structure, with possible formation of dimers and showing different mobility when exposed to an electric potential difference. Not all polymer chains have reacted with DFO, however BSA-DFO complex has antioxidant capacity similar to the original drug. The BSA-DFO and OVA-DFO conjugates can react, in addition to iron, with gadolinium, making the complexes potential contrast agents for magnetic resonance imaging (MRI). In this work, the complex between Gd(III) and BSA-DFO presented a relaxativity of 52,92 s-1 mM-1 for T2 and 45,37 s-1 mM-1 for T1, values higher than the available drugs in the market (4 - 5 s-1 mM-1) which was explained by the high molecular weight, indicating a good effects on the quality of MRI, with lower doses.

Magnetic Resonance Imaging (MRI) is the most prominent non-invasive technique used in clinical diagnosis and biomedical research. Its development as an imaging technique has been aided by contrast agents (CAs) which enhance contrast to noise ratio in the images. This dissertation deals with paramagnetic lanthanide- and superparamagnetic iron-based nanoparticles (NPs) which are potential CAs at clinical field of 3 T and a high field of 9.4 T. Chapter 1 provides a brief overview of colloidal nanoparticles, with an emphasis on their surface chemistry and magnetic properties for bio-applications. Chapter 2 employs europium as an optical probe to illustrate the contribution of inner, second and outer sphere relaxation towards longitudinal and transverse relaxivities of paramagnetic NP-based CAs. Chapter 3 investigates the positive and the negative contrast enhancement abilities and magnetization of paramagnetic NPs comprising a core of sodium dysprosium fluoride with a sodium gadolinium fluoride shell. Their surface chemistry is tuned to target prostate cancer specifically. The application of these NPs is further extended in Chapter 4 to track an intraneuronal protein called tau following mild traumatic brain injury. Chapter 5 deals with facile synthesis and long-term stability of superparamagnetic iron NPs for their potential application as CAs. Chapter 6 illustrates the concept of MRI correlation using ‘T1-only’ and ‘T2-only’ NPs. Chapter 7 investigates on the dynamics involved in the phospholipids coating the surface of NPs. Chapter 8 concludes on the work detailed in the previous chapters and outlines the future outlook. / Graduate / 2020-01-15

"Magnetic resonance imaging (MRI) has increasingly become the preferred imaging modality in modern day research to study disease. MRI presents an imaging technique that is practically non-invasive and without any ionizing radiation. This dissertation presents the use of contrast agents in MRI studies to differentiate compartments, to study dose dependence of relaxation times, and to characterize tumors using signal amplifying enzymes in the brain. Differentiating compartments in the brain can be useful in diffusion studies to detect stroke at an early stage. Diffusion-weighted NMR techniques have established that the apparent diffusion coefficient (ADC) of cerebral tissue water decreases during ischemia. However, it is unclear whether the ADC change occurs due to changes in the intracellular (IC) space, extracellular (EC) space, or both. To better understand the mechanism of water ADC changes in response to ischemic injury, making IC and EC compartment specific measurements of water diffusion is essential. The first study was done where manganese (Mn2+) was used as an IC contrast agent. Mn2+ uptake by cells causes shortening of the T1 relaxation time of IC water. The relative difference in T1 relaxation times between the IC and EC compartments can be used to discriminate between the MR signals arising from water in the respective compartments. Mn2+ is also widely used in manganese-enhanced MRI (MEMRI) studies to visualize functional neural tracts and anatomy in the brain in vivo. In animal studies, the goal is to use a dose of Mn2+ that will maximize the contrast while minimizing its toxic effects. The goal of dose study was to investigate the MRI dose response of Mn2+ in rat brain following SC administration of Mn2+. The dose dependence and temporal dynamics of Mn2+ after SC injection can prove useful for longitudinal in vivo studies that require brain enhancement to persist for a long period of time to visualize neuroarchitecture like in neurodegenerative disease studies. Contrast agents, in addition to their use in compartmental differentiation and dose studies, can be used for imaging tumors. The last study in this dissertation focuses on imaging EGF receptors in brain tumors. We tested a novel pretargeting imaging approach that includes the administration of humanized monoclonal antibody (anti-EGFR mAb, EMD72000) linked to enzymes with complementing activities that use a low-molecular weight paramagnetic molecule (diTyr-GdDTPA) as a reducing substrate administered following the mAb conjugates. We analyzed the differential MR tumor signal decay in vivo using orthotopic models of human glioma. The patterns of MR signal change following substrate administration revealed differences in elimination patterns that allowed distinguishing between non-specific and specific modes of MR signal decay. "

O uso de agentes de contraste em técnicas de diagnóstico por imagem é uma prática médi-ca rotineira. Certos compostos, presentes em agentes de contraste, possuem propriedades paramagnéticas que podem afetar os sinais da tomografia por ressonância magnética, \"Mag-netic Resonance Imaging\"(MRI). Em estudos aplicados ao trato gastrintestinal (GI), os meios de contraste são amplamente utilizados por via endovenosa, mas também podem ser admi-nistrados oralmente. Porém, a adoção do uso oral é limitada, principalmente, porque os agentes convencionais são caros e causam, geralmente, efeitos colaterais. Desta forma, a-presentamos a caracterização e os resultados preliminares da implementação da polpa do fruto da Euterpe olerácea para um possível uso clínico como agente de contraste oral em MRI do trato GI. A polpa da Euterpe olerácea, conhecida como Açaí, de origem amazônica, apre-senta um aumento de sinal de MRI ponderado em T1 equivalente ao do Gd-DTPA e, tam-bém, um decréscimo de sinal em imagens ponderadas em T2. Investigamos propriedades intrínsecas que possam estar correlacionadas com o aumento de sinal em T1 e à opacidade em T2. O espectro de absorção atômica revelou a presença de íons Fe, Mn e Cu no Açaí, o que contribui para o valor susceptométrico encontrado de -4,83 . 10-6. Essa medida fomen-ta a hipótese de que as mudanças de contraste nas imagens são devido à presença de mate-rial paramagnético, revelando um contraste clinicamente satisfatório nas porções superiores do trato GI. Estudos preliminares indicaram que a homogeneidade e a intensidade do sinal da polpa do Açaí (Euterpe olerácea), no estômago, e duodeno, são próximos daquele encon-trado em agentes convencionais. Além disso, ele não apresentou efeito colateral algum. Devido ao aumento de contraste associado ao Açaí, podemos observar a parede gástrica de forma singular. Ainda, este agente contribuiu para o diagnóstico das vias pancreática e biliar em exames de colangiopancreatografia por ressonância magnética, Magnetic Resonance Colan-giopancreatography (MRCP), com seqüências ponderadas em T2, por reduzir o sinal das alças intestinais. / The use of contrast agents is a common practice in medical imaging protocols. Paramagnetic properties of certain compounds present in contrast agents can affect Magnetic Resonance Imaging (MRI) signals. For abdominal applications, they are usually injected, but may also be administered orally. However, their use as a routine technique is limited, mainly due to the lack of appropriate oral contrast agent. Standard agents are expensive and cause, generally, some kind of side effect. We herein present the preliminary characterization and results for implementation of Euterpe olerácea (popularly named Açaí) as a possible clinical oral contrast agent for MRI of the gastrointestinal (GI) tract. The pulp of Açaí, a fruit from the Amazon area, presented an increase in T1-weighted MRI signal, equivalent to that of Gd-DTPA, and a signal decrease in T2-weighted images. We looked for intrinsic properties that could be responsible for the T1 signal enhancement and T2 opacification. Atomic Absorption spectra revealed the presence of Fe, Mn and Cu ions in Açai. The presence of such ions contribute to the susceptometric value found of -4.83 x 10-6 . This finding assents with the hypothesis that image contrast changes were due to the presence of paramagnetic material. The first measurements in vivo demonstrate a clear increase of contrast due to signal intensity and homogeneity in stomach and bowel walls with the pulp of Açaí, which look like the effects related to standard agents. Consistently, the increase in T1-weighted and the opacification in a T2-weighted acquisition was evident, revealing a biphasic contrast on gastric tissues. Besides, the pulp does not present any side effect. It still has contributed to the diagnostic of pancreatobiliary system at Magnetic Resonance Cholangiopancreatography (MRCP), by reducing overlap of the surround tissues and those structures.

<p>Current challenges in neuronal tract tracing include sacrificing the animal, detailed sectioning of the brain, and cumbersome reconstruction of slices to gather information, which are very tedious, time consuming, and have low-throughput. In this regard, Manganese-enhanced Magnetic Resonance Imaging (MEMRI) has been an emerging methodology for fiber tract tracing <i>in vivo</i>. <i></i>The manganese ion (Mn²⁺) is paramagnetic and is analogous to calcium ions (Ca²⁺), which allows it to enter excitable cells through voltage-gated calcium channels, thereby reporting cellular activity in T₁-weighted MR images<i>. </i>Moreover, once the Mn²⁺enters the cell, it will move along the axon by microtubules, release at the synapse, and then uptake by post-synaptic neurons, hence revealing the pathway of Mn²⁺transportation. While most MEMRI neuronal tracing studies have focused on mapping circuitries within the brain, MEMRI has rarely been applied to trace peripheral nerve projections into the brain. </p><p>In this thesis, I will propose the use of MEMRI to trace vagal nerve projections into the central nervous system by showing enhancement of neuronal pathways with an optimized protocol. This protocol demonstrates <i>in vivo </i>monitoring of manganese transport into the brain from the nodose ganglion and shows how the enhancement in MR images can be promoted with vagus nerve stimulation (VNS). Additionally, I will present preliminary findings, for the very first time, that show the downstream projection of the sympathetic pathway from the brainstem. In sum, the technique presented in this thesis will shed light on the use of MEMRI to study the functional results of using clinically-based VNS settings</p>

<p>The purpose of this thesis was to investigate the utility of the Ultrasmall Superparamagnetic Iron Oxide Particle (USPIO), NC100150 Injection for assessment of macro- and microvascular morphology and function using magnetic resonance imaging. The feasibility of NC100150 Injection was tested for the following applications: Coronary angiography, Thrombus detection, Cardiac function, Myocardial perfusion, Assessment of myocardial blood volume and water exchange and finally assessment of endothelial integrity of the myocardium.</p><p>The test method included computer simulations, in vitro, animal and clinical experiments. The computer simulations included propagation of longitudinal magnetization in non-steady state acquisitions. Animal models used were coronary artery stenosis in pigs, thrombus formation in the jugular vein in pigs, normal pig myocardium and transplanted hearts in rats. A human study of patients with suspected coronary artery disease was also performed.</p><p>The results showed that angiography using an USPIO is less efficient in the coronary arteries than in the peripheral arteries. Direct targeting to thrombus using an USPIO is possible but will be limited by the spatial resolution. An USPIO will enhance gradient echo imaging of cardiac function. T2-weighted myocardial perfusion imaging is feasible as well as assessment of myocardial blood volume and endothelial integrity.</p><p>Due to physiological limitations and technical development the utility of NC100150 Injection for assessment of the macrovascular morphology is limited.However for the assessment of microvascular function, especially perfusion and permeability, NC100150 Injection may contribute to a successful implementation of these methods.</p>