Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 73-79). / Neutrophils in the pulmonary microcirculation are subjected to mechanical deformation while traveling through capillaries of sizes much smaller than the mean neutrophil diameter. This deformation has been shown to result in significant reductions in both the shear storage and shear loss moduli of the cell, with subsequent recovery towards their initial values. Also, deformation above a threshold stimulus results in neutrophil activation, evidenced by pseudopod projection from the cell. These two events are thought to occur via independent pathways, yet little is known about the mechanosensing signaling involved. Other work has demonstrated that physiological deformation of neutrophils induces a marked increase in the levels of cytosolic calcium, suggesting that this occurrence may trigger the biomechanical response observed in the cell. The aim of this thesis was to elucidate the role of calcium in the neutrophil response to the mechanical deformation experienced during transit through the pulmonary capillaries. / (cont.) Chelating intracellular calcium in neutrophils resulted in (i) decreased deformability of the cells into a microchannel, (ii) attenuation of the drop in shear storage modulus (G') observed in untreated cells upon deformation, and (iii) shorter activation times. These findings suggest that cytosolic calcium holds an important function in the neutrophil transit through the capillaries, and inhibition of normal calcium release within the cell can lead to leukostasis-like conditions. / by Jeffrey J Hsu. / M.Eng.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (p. 33-37). / Label-free detection is the detection of biomolecules and their interactions, without the use of a molecule external to the interaction, used as a reporter to indicate presence and/or location. The suspended microchannel resonator offers the opportunity to perform such label-free measurements. The goal of this work is to open new avenues of possible applications for the suspended channel. I introduce the concept of detecting mass subtraction as a new approach, rather than the conventional detection of mass addition. In a model implementation scenario of this approach, a mass-intensifying tag bound to a small ligand molecule will be equilibrated with surface-immobilized receptors, and later displaced by an identical, but label-free, ligand molecule. This approach offers opportunities to extend the sensitivity range of the device, as well as introduces new functionality for it. It enables researchers to follow, label-free, real-time enzymatic reactions, relative affinities of different ligands to a receptor, and presence of small molecules in a solution. / by Shelly Levy-Tzedek. / S.M.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 59-61). / One of the major challenges in liver research today lay in the understanding of the complex relationship between liver structure and function. The highly orchestrated events that take place in the liver to maintain homeostasis require the presence of all liver cell types. In vivo experiments offer only a snapshot of the liver, and usually involve perturbation of normal function through injury or experimental disease. The role of cell-cell interactions in maintaining normal liver function is far less understood than in pathological conditions. This may be because of the lack of methods in monitoring normal function in vivo. Culturing systems may capture pieces of the puzzle, but often capture only two cell types, and involve mediators presented to the cells in concentrations much higher than physiological values. In addition, the liver lobule contains a 3-dimensional metabolic zonation, and liver cell types comprise a heterogeneous population from the portal triad where the blood flows into the sinusoids to the central vein area where the blood flows out of the sinusoids. Liver cell types are dynamic responders to environmental cues from soluble factors to heterotypic cell interactions, to extracellular matrix proteins. Therefore, a system that serves to promote the health of all liver cell types through a 3-dimensional, perfused scaffold, and allows for self-organization of the liver cells in response to the engineered environment, would serve as a useful tool in understanding some of these complex, orchestrated events. In the research presented here, methods were developed to detect and characterize the heterogeneous population that makes up the hepatic stellate cell population inside the liver bioreactor (Griffith et. al). / (cont.) This cell type, comprising a small percentage of total liver cells (approximately 5-10%), rapidly change their phenotype in response to liver injury, and, similarly, upon being taken out of the liver and cultured in 2-D on tissue culture plastic. This cell type plays a major role in relaying signals to and from both parenchymal and other nonparenchymal cells; stellate cells are also in charge of maintaining the components of the Space of Disse and are the key players in the pathology of liver fibrosis. They are found to be tightly complexed with sinusoidal endothelial cells and at the same time found to be tightly interacting with hepatocytes, sometimes even penetrating the hepatic plate. Stellate cell function, is therefore, highly dependent upon its interaction with other liver cells in maintaining the tightly knit structure-function relationship. For this reason, the liver bioreactor serves as a highly useful tool, in order to better understand the hepatic stellate cell's role in these complex situations. In this dissertation, detection and characterization methods are developed with the goal of capturing the heterogeneous stellate cell population as a whole with a toolbox of characterization markers, as well as to learn more about their functionality and location within tissue structures. These tools can be used to detect and characterize the population at various timepoints during tissue formation inside the bioreactor, as well as after exposure to physiologically-relevant concentrations of toxins, viruses, pharmaceuticals, etc. ... / author: Kathryn E. Wack. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / MIT Science Library copy: printed in leaves. / Also issued printed in leaves. / Includes bibliographical references (p. 127-141). / Utilizing molecular recognition and self-assembly, material-specific biomolecules have shown great promise for engineering and ordering materials at the nanoscale. These molecules, inspired from natural biomineralization systems, are now commonly selected against non-natural inorganic materials through biopanning random combinatorial peptide libraries. Unfortunately, the challenge of studying the biological inorganic interface has slowed the understanding of interactions principles, and hence limited the number of downstream applications. This work focuses on the facile study of the peptide-inorganic interface using Yeast Surface Display. The general approach is to use combinatorial selection to suggest interaction principles followed by rational design to refine understanding. In this pursuit, two material groups-II-VI semiconductors and synthetic sapphire (metal oxides)-are chosen as inorganic targets due to their technological relevance and ease of study. First, yeast surface display (YSD) was established as a broadly applicable method for studying peptide-material interactions by screening a human scFv YSD library against cadmium sulfide (CdS), a II-VI semiconductor. The presence of multiple histidine residues was found to be necessary for mediating cell binding to CdS. As a follow-up, a systematic screen with yeast displayed rationally designed peptides was performed on a panel of II-VI semiconductors and gold. Cell binding results indicated that peptide interaction was mediated by a limited number of amino acids that were influenced by locally situated residues. Interpretation of the results facilitated design of new peptides with desired material specificities. Next, the nature of peptide/metal oxide binding interface was interrogated using sapphire crystalline faces as model surfaces. / (cont.) Biopanning a random peptide YSD library and subsequent characterization of the identified binding partners revealed the importance of multiple basic amino acids in the binding event. Study of rationally designed basic peptides revealed a preference for those amino acids to be spaced in such a manner that maximized simultaneous interaction with the surface. Fusing peptides to maltose binding protein (MBP) allowed for quantitative affinity measurement with the best peptides having low nanomolar equilibrium dissociation constants. Finally, peptides were demonstrated as facile affinity tags for protein immobilization in micro-patterning and biosensor assays. / by Eric Mark Krauland. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Leaf 69 blank. / Includes bibliographical references (leaves 54-58). / Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Highly oxygen-demanding hepatocytes are commonly maintained under oxygen-deficient culture conditions, limited by culture medium thickness as well scaffold thickness. Thus, the cells are forced into anaerobic metabolic states that degenerate liver specific functions. Furthermore, cells separated from their extracellular matrix and disconnected from the synergistic interactions between other hepatic cells types further exacerbate hepatocellular function. This study aims to improve hepatospecific activity, especially CYP3A2 - a biomarker that is notoriously known to quickly lose expression in primary cultures, by creating a platform based on collagen sandwich cultures. The modified sandwich cultures are substituted with self-assembling peptide, RAD16-I, combined with integrin-binding sequence RGD or laminin receptor binding sequence YIGSR functional peptide motifs to create a cell-instructive peptide scaffold. To facilitate oxygen and nutrient diffusion and exchange, plasma modification technology is employed to control peptide layer dimension. We have successfully shown that plasma engineering can be used to optimize peptide thickness. / (cont.) Likewise, we have shown that the incorporation of the functional motifs enhanced hepatospecific activity. CYP3A2 expression from cultures on our platform improved over 256 times the levels found in collagen sandwich cultures, the current standard for hepatocyte cultures. This study demonstrates the capability of sandwich cultures with modified instructive self-assembling peptides and the importance of thinner cultures scaffolds to promote better oxygen and nutrient exchange. We believe that our novel bioengineered platform has the potential to greatly improve existing hepatocyte culture methods and be invaluable to future in vitro hepatocyte studies as well as toxicity tests. / Jonathan Wu. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 86-96). / Filamentous bacteriophage (M13) are excellent biological build block due to their multiple peptide display system including type 8 (complete peptide display at pVIII) and type 83 (complete peptide display at both pVIII and pIII) display systems. Unlike the phagemid systems, the advantage of these systems is that we can get homogenous peptide display on pVIII resulting in uniform placement of selected molecules as well as defined length and width. In this thesis, type 8 and type 83 phage were constructed and used as biological scaffolds to meet the following four specific aims. First, the self-assembly of engineered M13 bacteriophage as a template for Co-Pt crystals was demonstrated. An phage library with an octapeptide library on the major coat protein (pVIII) was used for selection of binders to cobalt ions. Fibrous structures with directionally ordered phage were obtained by interaction with cobalt ions. Co-Pt alloys were synthesized on the fibrous scaffold, and their magnetic properties were characterized. The mineralization showed organized nanoparticles on fibrous bundles with superparamagnetic properties. Second, an in vitro molecular selection method in non-biological conditions for inorganic synthesis was introduced. / (cont.) A phage display peptide library which is resistant to ethanol was constructed and used for selection against titania in 90% ethanol. The selected peptide, with a conserved basic amino acid sequence, promotes nanoparticle formation (- 60 nm) during titania synthesis by the traditional sol-gel method. Third, storage of proteins in smectically aligned phage film was demonstrated. [Beta]-galactosidase and a green fluorescent protein variant were stored in the phage film with increased stability. In addition, streptavidin conjugated phycoerythrin were aligned in a SI phage film, in which streptavidin binding peptides are displayed at the end of the phage particles. The alignment showed increased fluorescent intensity of phycoerythrin molecules. Finally, the potential of type 8 and type 83 phage as a nano-structural scaffold were studied 'or device application. An Au binding peptide was selected using type 8 phage library. Self-assembly of gold particles on phage was observed. In addition, type 83 phage which display both streptavidin binding peptide at pIII and Au binding peptide at pVIII were constructed for complex assembly of both Au nanoparticles and streptavidin conjugated nanoparticles. Genetically engineered bacteriophage show promise for application including biologically compatible materials and functional bio-inorganic hybrid materials. / by Soo-Kwan Lee. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Osteoarthritis is characterized by synovial joint degeneration, and its cardinal pathological feature is degeneration and loss of the articular cartilage joint surface. While the aetiology of osteoarthritis is unknown, risk factors include gender, age, obesity, and prior joint injury. Joint injuries, including tears of the anterior cruciate ligament (ACL) and meniscus, increase the risk for the development of OA and involve both mechanical damage to cartilage, meniscus and synovial tissues, and tissue degradation associated with cytokine-induced inflammation. While the role of inflammatory cytokines in OA is still controversial, their role in rheumatoid arthritis is evidenced by the successful use of anti-TNF-ca and anti-IL-1 therapies to abrogate disease symptoms and progression. In vitro, both IL-13 and TNF-ca promote chondrocyte-mediated matrix degradation and inhibit cartilage matrix synthesis, while mechanical damage causes cell death, matrix damage, and decreased cell biosynthesis. Understanding the similarities and differences in cartilage responses to inflammatory cytokines and mechanical injury is important in understanding the catabolic-anabolic shifts that typify OA progression. Therefore, the objectives of this thesis were (1) to identify the role of TNF-a and IL-1 induced nitric oxide (NO) as a mediator of cartilage tissue damage; / (cont.) (2) to characterize and compare the regulation by IL- 1 P, TNF-cc, and mechanical injury of secreted factors, matrix degradation, and mechanisms of chondrocyte cell death using an SDS-PAGE-LC/MS/MS protein profiling approach; and (3) to further quantify the effects of IL-1 3, TNF-ca and injury using an isobaric isotope labeling (iTRAQ) based 2D-LC/MS/MS approach. Together these studies were designed to provide better understanding of matrix degradation, cell death, immune response, and evidence of cell-mediated repair processes. NO is produced by chondrocytes in response to inflammatory cytokines TNF-a, IL-1 3 and IL-17, and can mediate cellular and extracellular events through cGMP signaling, protein modifications (e.g., S-nitrosation or tyrosine nitration), altered transcript stability, and altered sugar and lipid chemistry. Cartilage was treated with IL-13 or TNF-ca left untreated in the presence or absence of the NO synthase inhibitor, L-N-methylarginine (L-NMA), and changes in gene expression and matrix breakdown were measured. We found that L-NMA treatment partially inhibited TNF-a-induced, aggrecanase-mediated aggrecan degradation as indicated by a decrease in sGAG loss to the medium and by an increase in the generation of aggrecanase-specific aggrecan fragments. / (cont.) No change was observed upon addition of L-NMA to IL- 1P treated explants, but addition of L-NMA to combined IL-1 3 and TNF-a treated explants increased sGAG loss, suggesting that the effects of NO may be contextual. We hypothesized that this might be due to differences in aggrecanase expression (ADAMTS4 vs. ADAMTS5) or post-translational modification, but no aggrecanase was consistently identified in the samples. No difference in MMP expression or activation was noted following addition of L-NMA, and no change in NO chemistry between IL-1 3 and TNF-a treatment was evident by nitrate and nitrite production. Gene expression analysis was conducted on a battery of 32 genes, including matrix proteins, inflammatory mediators, proteases, cytokines and growth factors, and housekeeping proteins. While IL-113 and TNF-a both increased the expression of proteases and inflammatory mediators, addition of L-NMA did not significantly affect expression of the genes tested. We concluded that the effects of TNF-a and IL-1 p-induced NO production may depend on differences in cellular responses to each of these cytokines and possibly to differences in signaling or aggrecanase expression. / (cont.) In the second study, newborn bovine calf cartilage explants were treated with 10 ng/ml IL-1 p, 100 ng/ml TNF-a, radially-unconfined injurious compression (strain: 50%; strain rate 1000/o/sec), or no treatment, and cultured for five days. Pooled medium was subjected to SDS-PAGE-LC/MS/MS, and data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, differences between treatments and matrix protein proteolysis. Over 250 proteins were identified among the four protein groups including CD 109, platelet derived growth factor like protein, and scrapie responsive protein, which have not been previously identified in cartilage. IL-13 and TNF-a caused an increase in YKL39, YKL40, complement factor B, MMP-3, ECM-1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins including GRP58, GRP78, alpha 4 actinin, pyruvate kinase, and vimentin, suggesting a loss of membrane integrity in a population of chondrocytes. Data on actin release within the first 24 hours suggested that this loss of membrane integrity occurred by mechanical cell disruption. Injurious compression also caused proteolysis of collagen type VI subunits, collagen type II, and COMP. Thrombospondin 1 fragments were seen in all treatment groups, and aggrecan proteolysis was predominant with cytokine treatment. / (cont.) Cartilage explants subjected to injurious compression released intracellular proteins and showed enhanced degradation of matrix proteins, while explants subjected to IL- 13 or TNF-ct released proteins involved in innate immunity and stress response. In the third study, cartilage explants were subjected to injurious compression, TNF-a (100 ng/ml) or IL-13 (10 ng/ml), or no treatment, cultured in equal volumes of medium, and the medium was collected, pooled and the proteins deglycosylated by treatment with chondroitinase ABC. The proteins were subjected to trypsinization, and the peptides were labeled with one of four iTRAQ labels each containing a unique signature ion. The labeled peptides were subjected to nano-2D-LC/MS/MS on a QStar, quadrupole time of flight instrument. The study was done in analytical replicate on a pooled sample of greater than 70 explants from a total of 6-12 different animals. Data were analyzed by ProQuant to obtain a ProGroup peptide report containing identified spectra, which were combined to achieve a peptide, and then a protein level output of mean ratios, standard deviations of those ratios, and significance based on either Wilcoxan sign rank or Student's t-test both corrected for multiple comparisons. / (cont.) Because of our interest in catabolic and anabolic shifts, a targeted data analysis approach was taken in addition to a systems level PCA and K-means clustering approach. By focusing on particular protein domains, we identified a decrease in the synthesis of most fibrillar collagen subunits (p<0.05), and an increase in the release of the aggrecan G2 and G3 domains with IL-13 and TNF-ta treatment (p<0.05). We also noted a significant increase in MMP-1, MMP-3, MMP-9, and MMP-13 in at least one condition and, in most cases, all conditions compared to the untreated sample. Increases in proteins involved in innate immunity and immune cell recruitment were noted with IL-1 [3 and TNF-a treatment, while an increase in intracellular protein release was seen most dramatically with mechanical compression injury. Since anabolic effects are often driven by the insulin-like growth factor family and the TGF-[3 superfamily, we specifically identified members of these pathways to understand which factors may mediate early repair processes. / (cont.) At the systems level, 2 principal components were sufficient to describe 97% of the covariance in the data. IL- 1 and TNF-a caused a similar response in proteins identified; in contrast, a 'Y'-shaped distribution was observed upon projection of proteins based on their response injury vs. cytokine treatment. K-means clustering revealed six main clusters to further characterize the biology of mechanical injury versus cytokine effects on cartilage. / by Anna L. Stevens. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / "June 2004." / Includes bibliographical references (p. 75-79). / There is reason to believe that embryonic stem cells would grow favorably in a hypoxic environment. These cells come from the pre-implantation embryo, whose native environment is the hypoxic mammalian reproductive tract. Growing stem cells in an environment with higher oxygen levels (i.e. the atmospheric oxygen levels that are normally used to cultivate stem cells) could be bad for the cells, as oxygen is toxic due to its powerful oxidative capacity. In this experiment, J1 murine embryonic stem cells were grown at 0%, 2%, 5%, 10%, and 20% oxygen by volume to assess the effects that lower oxygen levels have on stem cell growth, plating efficiency, and metabolism. Two reaction vessels were built so that the cells could be grown in a controlled environment. Premixed gas cylinders were used to flush the vessels and an optical probe was used to measure the headspace oxygen concentration. Cells were found to grow faster than atmospheric conditions when in a moderately hypoxic environment where the oxygen concentration was between 2% and 10%, and considerably slower than atmospheric conditions as the headspace oxygen concentration approached zero. Maximum cell density decreased, glucose-lactate yield increased, glucose-cell yield decreased, and glutamine-cell yield decreased as headspace oxygen decreased. There were no strong correlations between oxygen concentration and glutamine-ammonia yield or plating efficiency. From the results of this study, it appears that it may be preferable to grow murine embryonic stem cells in a moderately hypoxic environment around 10% oxygen to increase the growth rate while minimizing the maximum cell density and metabolic inefficiency compromises. / by Daryl St. Laurent. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Mitotic homologous recombination is a critical pathway for the repair of DNA double-strand breaks and broken replication forks. Although homologous recombination is generally error-free, recombination between misaligned sequences can lead to deleterious sequence rearrangements, and conditions that stimulate homologous recombination are associated with an increased risk of cancer. To study homologous recombination in vivo, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which a homologous recombination event at a transgene yields a fluorescent cell. To study homologous recombination using FYDR mice, we developed one- and two-photon in situ imaging techniques that reveal both the frequency and the sizes of isolated recombinant cell clusters within intact pancreatic tissue. We then applied these tools to analyze the effects of cancer risk factors such as exposure, genetic predisposition and age on homologous recombination in vivo. To determine the effect of exposure to exogenous carcinogens on homologous recombination, FYDR mice were treated with two different chemotherapeutic agents, cisplatin and mitomycin-C. / (cont.) Results show that exposure to these DNA damaging agents causes an induction of recombinant pancreatic cells in vivo, indicating that homologous recombination is an active repair pathway in adult pancreatic cells and that exposure to certain carcinogens stimulates recombinational repair. As a first step towards exploring the effect of genetic predisposition to genomic instability on homologous recombination in vivo, FYDR mice were crossed with mice carrying a defect in p53, a critical tumor suppressor that is mutated in almost 50% of all human tumors. Although loss of p53 is known to promote genomic instability, results show that p53 status does not significantly affect the spontaneous recombinant cell frequency in the pancreas in vivo or the rate of homologous recombination in cultured fibroblasts in vitro. Age is a risk factor for many types of cancers. Here we examined the effect of age on homologous recombination in two tissues of FYDR mice, pancreas and skin. In the pancreas, a dramatic accumulation of recombinant cells is seen with age, resulting from both de novo recombination events and clonal expansion of recombinant cells. In contrast, the skin shows no increase in recombinant cell frequency with age. / (cont.) In vitro studies using primary fibroblasts indicate that the ability to undergo homologous recombination in response to endogenous and exogenous DNA damage does not significantly change with age, suggesting that these skin cells are able to undergo de novo homologous recombination events in aged mice. Thus, we propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of these cells to persist and clonally expand within the tissue. To further characterize the FYDR mice as a tool for studying homologous recombination, we exploited positive control FYDR-Recombined mice in which all cells carry the full-length coding sequence for enhanced yellow fluorescent protein. Studies show that expression of the FYDR transgene varies among mice, among tissues, and even among cells within a tissue. However, the variation in FYDR expression does not significantly change with age or exposure to exogenous carcinogens. Furthermore, positive control mice reveal that several tissues, in addition to the pancreas and skin, may be amenable for studying homologous recombination in the FYDR mice. / (cont.) Thus, our studies demonstrate that FYDR mice combined with in situ imaging technology provide powerful tools to study the effects of cancer risk factors on homologous recombination in vivo. Ultimately, by applying these techniques to study additional cancer risk factors, we may better understand the relationship between DNA damage, homologous recombination and cancer. / by Dominika M. Wiktor-Brown. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / Adult tissue differentiation involves the generation of distinct cell types from adult stem cells (ASCs). Current understanding of tissue differentiation mechanisms is based on studies of protein and RNAs that asymmetrically segregate between daughter cells during embryogenesis. Whether or not other types of biomolecules segregate asymmetrically has not been widely studied. In 1975, John Cairns proposed that ASCs preferentially segregate the oldest parental template DNA strands to themselves and pass on newly replicated DNA strands to their differentiating progeny in order to protect the stem cell from inheriting DNA replication mutations. This laboratory has shown non-random chromosome segregation in murine fetal fibroblasts that model asymmetric self-renewal like ASCs. In these cells, chromosomes that contain the oldest DNA strands co-segregate to the cycling daughter stem-like cells, while chromosomes with more recently replicated DNA segregate to the non-stem cell daughters. Previously, cytological methods were reported to elucidate non-random segregation in these cells. This dissertation research provides additional confirmation of the mechanism using physicochemical methods. Specifically, buoyant density-shift experiments in equilibrium CsCl density gradients were used to detect co-segregated "immortal DNA strands" based on incorporation of the thymidine base analogue bromodeoxyuridine. In addition, DNA from cells undergoing non-random mitotic chromosome segregation was analyzed for unique DNA base modifications and global structural modifications (by HPLC and melting temperature analyses). To date, these studies show no significant differences compared to control randomly segregated DNA. Components of the mitotic chromosome separation / (cont.) apparatus that might play a role in the co-segregation mechanism were also evaluated. Two homologous proteins, essential for proper chromosome segregation and cytokinesis, Aurora A kinase and Aurora B kinase, were highly reduced in expression in cells retaining immortal DNA strands and may indicate a role for them in the immortal strand mechanism. These studies independently confirm the immortal strand mechanism and provide methods for its detection in other cell lines. In addition, observed changes in chromosome segregation proteins that are potential candidates for involvement in the mechanism have revealed a new area of investigation in the laboratory. These findings are relevant to understanding normal tissue development, cancer, and aging. / y Janice A. Lansita. / Ph.D.