http://hdl.handle.net/1721.1/7712 2013-06-19T23:47:10Z http://hdl.handle.net/1721.1/79251 Methods for chemical exchange saturation transfer magnetic resonance imaging Scheidegger, Rachel Nora Chemical exchange saturation transfer (CEST) is a relatively new magnetic resonance imaging (MRI) acquisition technique that generates contrast dependent on tissue microenvironment, such as protein concentration and intracellular pH. CEST imaging has the potential to become an important biomarker in a wide range of disorders. As an indicator of tissue pH, CEST imaging may allow the identification of the ischemic penumbra in stroke, and predict chemo- and radiation therapy outcomes in cancer. As a marker of protein concentration, CEST may be able to delineate tumor margins without contrast enhancement, identify disease onset in Alzheimer's disease, and monitor cartilage repair therapies. Despite several promising pilot studies, CEST imaging has had limited clinical application due to two main technical challenges. First, CEST imaging is extremely sensitive to magnetic field inhomogeneity. Images suffer from large susceptibility artifacts unless specialized BO inhomogeneity correction methods are employed that tremendously increase scan time. Second, the CEST contrast cannot be separated from the intrinsic macromolecular magnetization transfer (MT) asymmetry and brain images reflect the MT properties of white and gray matter rather than the desired protein and pH contrast. We have developed a novel CEST imaging acquisition scheme, dubbed saturation with frequency alternating RF irradiation (SAFARI), designed to be insensitive to Bo inhomogeneity and MT asymmetry. Studies in healthy volunteers demonstrate that SAFARI is robust in the presence of BO inhomogeneity and eliminates the need for specialized BO correction, thereby reducing scan time. In addition, results show that SAFARI removes the confounding MT asymmetry. We applied SAFARI imaging towards the study of the saturation transfer contrast in patients with high grade glioma. Results show that the contrast in brain tumors, which was previously attributed to an increase in the CEST signal from amide protons due to an elevated protein concentration, is instead the result of the loss of MT asymmetry found in the normal brain. Therefore, our work has lead to a new understanding of the different sources of signal in saturation transfer images of the brain with important implications for the design and analysis of future CEST studies of brain tumors. Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Division of Health Sciences and Technology, 2013.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 108-126). 2013-01-01T00:00:00Z http://hdl.handle.net/1721.1/79250 Bacteria-targeting nanoparticles for managing infections Radovic-Moreno, Aleksandar Filip Bacterial infections continue to be a significant concern particularly in healthcare settings and in the developing world. Current challenges include the increasing spread of drug resistant (DR) organisms, the side effects of antibiotic therapy, the negative consequences of clearing the commensal bacterial flora, and difficulties in developing prophylactic vaccines. This thesis was an investigation of the potential of a class of polymeric nanoparticles (NP) to contribute to the management of bacterial infections. More specifically, steps were taken towards using these NPs (1) to achieve greater spatiotemporal control over drug therapy by more targeted antibiotic delivery to bacteria, and (2) to develop a prophylactic vaccine formulation against the common bacterial sexually transmitted disease (STD) caused by Chlamydia trachomatis. In the first part, we synthesized polymeric NPs containing poly(lactic-co-glycolic acid)- block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG). We show that these NPs are able to bind to bacteria under model acidic infection conditions and are able to encapsulate and deliver vancomycin to inhibit the growth of Staphylococcus aureus bacteria in vitro. Further work showed that the PLGA-PLH-PEG-based NPs demonstrated the potential for competition for binding bacteria at a site of infection from soluble protein and model phagocytic and tissue-resident cells in a NP composition dependent manner. The NPs demonstrated low toxicity in vitro, were well tolerated by mice in vivo, and circulated in the blood on timescales comparable to control PLGA-PEG NPs. In the second part, we used PLGA-PLH-PEG-based NPs to design a prophylactic vaccine against the obligate intracellular bacterium Chlamydia trachomatis, the most common cause of bacterial STD in the world. Currently, no vaccines against this pathogen are approved for use in humans. We first formulated NPs encapsulating the TLR7 agonist R848 conjugated to poly(lactic acid) (R848-PLA) in PLGA-PLH-PEG-based NPs, then incubated these R848-NPs with UV-inactivated C. trachomatis bacteria in acidity, forming a construct. Mice immunized with this vaccine via genital or intranasal routes demonstrated protection from genital infection post immunization in a primarily CD4⁺ T cell-dependent manner. These results may suggest avenues for future work in designing and developing more targeted drug therapies or vaccine formulations for managing bacterial infections using polymeric nanoparticles. Thesis (Ph. D. in Chemical and Biomedical Engineering)--Harvard-MIT Division of Health Sciences and Technology, 2013.; Cataloged from PDF version of thesis.; Includes bibliographical references. 2013-01-01T00:00:00Z http://hdl.handle.net/1721.1/79249 Neuroimaging investigation of the motor control disorder, dystonia with special emphasis on laryngeal dystonia Makhlouf, Miriam L Laryngeal dystonia (LD) is the focal laryngeal form of the neurological movement disorder called dystonia, a condition that often changes in severity depending on the posture assumed and on voluntary activity of the affected body area. Pathophysiology of dystonia is unknown. This thesis employed a combination of diffusion tensor and functional magnetic resonance imaging (DTI and fMRI) studies to investigate the structure and function of the basal ganglia (BG) in dystonia patients. Fractional anisotropy (FA) and probabilistic diffusion tractography analyses were used to investigate the questions of whether LD patients exhibited altered connectivity between BG and brainstem regions and whether FA and tractography could be used to predict differences in clinical presentations of dystonia. Findings of this study support the hypothesis that connections between the BG and brainstem may play a role in dystonia pathophysiology and may be used to predict differences in clinical presentations of dystonia. An fMRI study was carried out to investigate whether abnormally sustained BG activity observed after performance of a finger tapping task in hand dystonia patients may represent an amplification of a normal motor control mechanism. As dystonia has been hypothesized to result from overactivation of normal postural programs, this study aimed to investigate the question of whether the sustained BG activity was a normal feature observed in motor control tasks requiring more precision. Results suggest that cerebellar cortex is recruited particularly during fine motor control. Thesis (Ph. D. in Speech and Hearing Bioscience and Technology)--Harvard-MIT Division of Health Sciences and Technology, 2013.; Cataloged from PDF version of thesis.; Includes bibliographical references. 2013-01-01T00:00:00Z http://hdl.handle.net/1721.1/79248 Ultra-rapid 2-D and 3-D laser microprinting of proteins Scott, Mark Andrew, Ph. D. Massachusetts Institute of Technology When viewed under the microscope, biological tissues reveal an exquisite microarchitecture. These complex patterns arise during development, as cells interact with a multitude of chemical and mechanical cues in the surrounding extracellular matrix. Tissue engineers have sought for decades to repair or replace damaged tissue, often relying on porous scaffolds as an artificial extracellular matrix to support cell development. However, these grafts are unable to recapitulate the complexity of the in vivo environment, limiting our ability to regenerate functional tissue. Biomedical engineers have developed several methods for printing two- and three-dimensional patterns of proteins for studying and directing cell development. Of these methods, laser microprinting of proteins has shown the most promise for printing sub-cellular resolution gradients of cues, but the photochemistry remains too slow to enable large-scale applications for screening and therapeutics In this work, we demonstrate a novel high-speed photochemistry based on multi-photon photobleaching of fluorescein, and we build the fastest 2-D and 3-D laser microprinter for proteins to date. First, we show that multiphoton photobleaching of a deoxygenated solution of biotin-4-fluorescein onto a PEG monolayer with acrylate end-group can enable print speeds of almost 20 million pixels per second at 600 nanometer resolution. We discovered that the mechanism of fluorescein photobleaching evolves from a 2-photon to 3- and 4-photon regime at higher laser intensities, unlocking faster printing kinetics. Using this 2-D printing system, we develop a novel triangle-ratchet method for directing the polarization of single hippocampal neurons. This ability to determine which neurite becomes an axon, and which neuritis become dendrites is an essential step for developing defined in vitro neural networks. Next, we modify our multiphoton photobleaching system to print in three dimensions. For the first time, we demonstrate 3-D printing of full length proteins in collagen, fibrin and gelatin methacrylate scaffolds, as well as printing in agarose and agarose methacrylate scaffolds. We also present a novel method for 3-D printing collagen scaffolds at unprecedented speeds, up to 14 layers per second, generating complex shapes in seconds with sub-micron resolution. Finally, we demonstrate that 3-D printing of scaffold architecture and protein cues inside the scaffold can be combined, for the first time enabling structures with complex sub-micron architectures and chemical cues for directing development. We believe that the ultra-rapid printing technology presented in this thesis will be a key enabler in the development of complex, artificially engineered tissues and organs. Thesis (Ph. D. in Electrical and Medical Engineering)--Harvard-MIT Division of Health Sciences and Technology, 2013.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 124-135). 2013-01-01T00:00:00Z