http://hdl.handle.net/1721.1/7752 http://hdl.handle.net/1721.1/49629 Mitochondrial mutational spectra in human bronchial ephithelial cells of smokers and nonsmokers Coller, Hilary Ann, 1967- Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Toxicology, 1998. Includes bibliographical references (leaves 165-190). http://hdl.handle.net/1721.1/46090 An investigation into a potential relationship between the rise in lanthanide usage and the increased prevalence of childhood asthma Puri, Charu Thesis (M.S.)--Massachusetts Institute of Technology, Division of Toxicology, 1997. Includes bibliographical references (leaves 69-71). http://hdl.handle.net/1721.1/45949 Yeast-based vaccine approaches to cancer immunotherapy Howland, Shanshan W Saccharomyces cerevisiae stimulates dendritic cells and represents a promising candidate for cancer immunotherapy development. Effective cross-presentation of antigen delivered to dendritic cells is necessary for successful induction of cellular immunity. Using a yeast vaccine model, we investigated the phagosome-to-cytosol pathway of cross-presentation. We demonstrate that the rate of antigen release from phagocytosed yeast directly affects cross-presentation efficiency, with an apparent time limit of about 25 min post-phagocytosis for antigen release to be productive. Antigen expressed on the yeast surface is cross-presented much more efficiently than antigen trapped in the yeast cytosol by the cell wall. The cross-presentation efficiency of yeast surface-displayed antigen can be increased by the insertion of linkers susceptible to cleavage in the early phagosome. Antigens indirectly attached to yeast through antibody fragments are less efficiently cross-presented when the antibody dissociation rate is extremely slow. Next, we present a yeast-based cancer vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the dendritic cell phagosome and subsequent translocation for cross-presentation. (cont.) Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast cell wall fragments instead of whole cells, and by conjugating multiple layers of antigen. The novel multi-layer conjugation scheme is site-specific and takes advantage of Sfp phosphopantetheinyl transferase, enabling the antigen dose to grow linearly. We show that whole yeast cells coated with one layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing three layers were able to cross-prime naive CD8+ T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after ten days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied towards the development of other particulate vaccines. Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. Includes bibliographical references. http://hdl.handle.net/1721.1/45948 Development of novel diagnostics and therapeutics for amyotrophic lateral sclerosis Townsend, Seth A. (Seth Alan) Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with diagnostics and treatments that are ineffective at stopping the progression. This thesis examines new ways of both diagnosing and treating ALS, including 1) a gadolinium tetanus toxin C fragment (Gd-TTC) biomarker for axonal retrograde transport, 2) TTC-conjugated biodegradable nanoparticles, and 3) poly(glycerol-co-sebacate) acrylate (PGSA) and 3D scaffolds for human embryonic stem cell (hESC) and neuronal encapsulation.A Gd-TTC conjugate was developed and characterized that was shown to be highly visible under MRI and preserved the functionality of the native TTC protein in vitro. Live animal MRI imaging and immuno fluorescent staining of the spinal cord showed that the conjugate was transported to the central nervous system (CNS) and localized in motor neurons. H&E staining and biodistribution studies showed that GdTTC was well tolerated and bio available. Quantification of MRI and staining images showed that Gd-TTC was retrograde transported and that that this rate decreased during the disease progression of ALS in a transgenic mouse model, suggesting that Gd-TTC could be used as a biomarker for neurodegenerative diseases.TTC-conjugated nanoparticles were developed by synthesizing PLGA-PEG-biotin and using biotin binding proteins (avidin, streptavidin, and neutravidin) to specifically conjugate TTC to the nanoparticle surface. TTC nanoparticles were shown to selectively target neurons and not other cell types in vitro. (cont.) Subsequent in vivo experiments showed that nanoparticles were well tolerated and that TTC was co-localized with neurons unilaterally, suggesting that TTC-conjugated nanoparticles may be a useful drug delivery system. Porous PGSA scaffolds were prepared and characterized by porosity, swelling, mass loss, toxicity and mechanical properties, and subsequently used to encapsulated hESC and neuroblastoma cells in vitro. Neuroblastoma cells proliferated and formed matrix fibrils, and fluorescent staining of undifferentiated hESCs showed the presence of all three germ layers. In vivo experiments showed that porous PGSA scaffolds were well-tolerated and promoted vascular ingrowths. Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. Includes bibliographical references (p. 224-236).