Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 35-40). / The study of stem cells is one of the most fascinating topics in biology. Adult stem cells (ASC), which play the prime role in the maintenance and restoration of tissues, are thought to hold great potential for the advancement of medicine. It has been postulated that adult stem cells are able to retain "immortal" DNA template strands over successive generations by non-random chromosome co-segregation, and in so doing, to protect the long-term genomic fidelity of whole tissue compartments. The investigation of this theory may yield insights into areas such as the development of cancer and the process of aging. In addition, it may lead to the discovery of an effective method for the unique identification of adult stem cells, the study of which has thus far suffered from the lack of unique identifiers. Thus, the goal of this research was to develop an assay for the detection of immortal DNA strand co-segregation that could be applied to the detection and analysis of adult stem cells. It is proposed that such an assay may in itself serve as a unique identification method for adult stem cells. In this thesis, the development of such an assay is described. This assay, referred to as the label release assay, has provided further evidence for the existence of immortal strand co-segregation in model cell lines, and will potentially be useful in the study of adult stem cells in tissues. / by Jennifer J. Cheng. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 111-127). / To shed light on the cell's response to its mechanical environment, we examined cell rheology at the single cell level and quantified it with nanometer spatial and microsecond temporal resolutions over a five-decade frequency range (- 0.5 Hz to 50 kHz). To this end, we developed and optimized an instrument for fast fluorescence laser tracking microrheology (FLTM). This novel method aims at experimentally deriving cellular viscoelastic properties from the passive monitoring of fluorescent microspheres undergoing Brownian motion inside the tested sample. Further instrument enhancement even broadens the FLTM frequency span up to seven decades by modulating data acquisition speed or complementing FLTM with a two-particle microrheology modality. In living cells, FLTM accurately characterizes the solid-like vs. liquid-like cytoskeletal behavior from measurements based on endocytosed micron-sized beads, independently of probe size or surface chemistry. FLTM also demonstrates the existence of two distinct rheological regimes on the cell surface and in the cell interior: While the former surface investigations show power-law frequency variations of the complex shear modulus G*(co), the latter intracellular experiments identify multiple time and length scales affecting cell rheological features. Finally, FLTM evaluates frequency-specific stretch-induced cell mechanics and thus promises to broaden and diversify the scientific knowledge on mechanotransduction, from a molecular and cellular standpoint. / (cont.) FLTM also demonstrates the existence of two distinct rheological regimes on the cell surface and in the cell interior: While the former surface investigations show power-law frequency variations of the complex shear modulus G*(co), the latter intracellular experiments identify multiple time and length scales affecting cell rheological features. Finally, FLTM evaluates frequency-specific stretch-induced cell mechanics and thus promises to broaden and diversify the scientific knowledge on mechanotransduction, from a molecular and cellular standpoint. / by Maxine Jonas. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2006. / "February 2006." / Includes bibliographical references (leaves 56-59). / Following exposure to DNA damage, cells initiate a stress response involving multiple protein kinase signaling cascades. The DNA damage response results in one of several possible cell-fate decisions, or cellular responses: induction of cell-cycle arrest, initiation of DNA repair, activation of transcriptional programs, and either apoptosis, necrosis or cell senescence. The mechanisms by which cells make these decisions, and how cell fate depends upon variables such as DNA damage type and dose, and other environmental factors, is unknown. The process by which cells select among alternate fates following such stimuli, or "cues" is likely to involve a dynamic, multi-variate integration of signals from each of the kinase signaling components. A major goal of signal transduction research is to understand how information flows through signal transduction pathways downstream of a given cue, such as DNA damage, and how signals are integrated, in order to mediate cellular responses. Mathematical modeling approaches are necessary to advance our understanding of these processes. / (cont.) Indeed, statistical mining and modeling of large datasets, consisting of quantitative, dynamic signaling and response measurements, is capable of yielding models that identify key signaling components in a given cue-response relationship, as well as models that are highly predictive of cellular response following novel cues that perturb the same network. We have validated a novel assay system that allows for the high throughput collection of quantitative and dynamic signaling data for 7 protein kinases or phospho-proteins known to be "hubs" in the DNA damage response and/or general stress response networks, including ATM, Chk2, H2AX, JNK, p38, ERK and p53. This novel high-throughput In-cell Western assay is based on immuno-fluorescent staining and detection of target proteins in a "whole cell" environment, performed and visualized in a 96-well plate format. This assay allows for the detection and measurement of up to 7 target proteins in triplicate, over up to 3 treatment regimes, or up to 21 signals for a single treatment, simultaneously. Pre-processing steps, and steps involved in the protocol itself are significantly fewer (and require smaller amount of most reagents and biological material), / (cont.) as compared to traditional signal measurement methods, such as quantitative Western analysis and kinase assays. We have used this novel high-throughput In-cell Western assay to investigate the DNA damage response after the specific induction of DNA double strand breaks (DSB). We have measured the dynamics of seven "hub" proteins modified with activating phosphorylations (as a surrogate measure of protein activity) that span major branches of the DNA damage, stress, and death signaling networks, following the specific induction of DNA double strand breaks. Signaling proteins measured include ATM, Chk2, H2AX, JNK, p38, ERK and p53. In parallel with these signaling measurements, we have quantitatively measured corresponding phenotypic responses, such as cell cycle profile and apoptosis. In future work, we will use a Partial Least Squares (PLS) regression analysis approach to construct a statistical model using this data, which is predictive of the cellular responses included in our measurements, following perturbation of this branch of the DNA damage response network. This analysis should reveal key signaling components involved in the decision-making process (possible molecular targets for the improvement of cancer therapy regimens that rely upon the induction of DSB, e.g. the topoisomerase inhibitor, cisplatin), and provide a basis for constructing new, and improving existing, physics-chemical models of this branch of the DNA damage response network. / by Andrea R. Tentner. / S.M.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaf 39). / Dynamic material properties of the isolated mouse tectorial membrane (TM) were quantified by applying sinusoidal shearing forces to the TM with a microfabricated probe (contact area 30 x 30 [micro]m²). Forces in the range 30-300 nN with frequency in the range 10-9000 Hz were applied tangentially to the surfaces of four TM specimens. We found that TM displacement was linear with respect to force. The TM exhibited both elastic and viscous characteristics, with the elastic behavior being more prominent. The elastic and viscous components of TM impedance remained proportional through two decades of frequency. The radial impedance was approximately three times larger than the longitudinal impedance. The point stiffness of the TM increased with frequency when radial forces were applied but showed no trend with frequency for longitudinal forces. Displacement of surrounding tissue decreased as distance from the probe increased. Space constants were on the order of tens of micrometers. These results represent the most detailed shear measurements to date of the isolated TM and are consistent with those obtained using the magnetic bead method [AF00]. / by Jianwen Wendy Gu. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / A multi-faceted approach was applied to the molecular design of substrates for the selective adhesion, proliferation and differentiation of connective tissue progenitors (CTPs) from human bone marrow aspirates. The basic premise of the thesis is that integrin-specific adhesion peptides, when presented in a biophysically appropriate spatial arrangement against an inert background, allow enrichment of CTPs in vitro. Comb copolymer comprising a methyl methacrylate backbone with 10-mer poly(oxyethylene) sidechains was selected as the vehicle to present small adhesion peptides at the surface. This polymer shows excellent performance in cell resistance studies and offers sufficient functionalizable sites to create high local densities of ligands. Methods for preparing comb copolymer substrates with peptides in [approx.] 300 nm² clusters with inter-ligand spacings closer than integrins were developed. This nanometer-scale clustered presentation was favorable to integrin binding. Cells were more spread on RGD peptide substrates with a higher degree of nanoscale clustering but of the same overall peptide surface density as comparable substrates with lower degree of peptide clustering. We evaluated adhesion peptides for their ability to support CFU formation of marrow-derived CTPs using colony forming unit (CFU) assay. / (cont.) The results, analyzed with a statistical model implemented to capture characteristics of CFU assay, showed that while RGD substrates supported a moderate amount of alkaline phosphatase -positive CFU (CFU-AP) formation, the bone sialoprotein peptide FHRRII(A, and two [alpha]4 [beta]1 peptides demonstrated the best performance in promoting CFU-AP formation. Patient variability in CFU data could be partially explained by the variations in marrow aspirate cell integrin expression, particularly [alpha]5 and [alpha]v [beta]3. The high level of ECM protein association seen with aspirate cells, as revealed by immunoblotting, may inhibit cell adhesion and account for the fairly low CFU counts observed. Treatments of marrow aspirate with phosphate saline buffer (PBS) and RGD solution reduced a significant amount of protein association. A comprehensive study showed that patients' marrow aspirates were naturally partitioned into two groups of very different colony formation behavior and integrin and AP expressions but consistency was observed within each group. Upon treatment of marrow cells with divalent ion-free PBS, CFU-AP formation on RGD substrates drastically increased in one group of patients. The designs and assays developed in this thesis could be applied for the further understanding of marrow aspirates, such as their interaction with a high-affinity [alpha]5 [beta]1 peptide, and with that knowledge, further optimize the surface design of bone marrow grafts. / by Ada Au. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2008. / "May 2008." / Includes bibliographical references. / Despite increased awareness of the obesity epidemic and a higher incidence of the metabolic syndrome in humans, the incidence of obesity and its comorbidities-most notably, type II diabetes (T2D) and cardiovascular disease (CVD)-continues to increase. Although numerous animal models are available to study the molecular pathways, genetics and therapeutic/prevention strategies for T2D, no model completely recapitulates T2D or it's comorbidities in humans. Therefore, novel animal models represent valuable research tools in understanding T2D. To develop a novel mouse model of T2D, I characterized an outbred mouse discovered at MIT that displayed clinical signs of diabetes. Prevalence of glucosuria in the Swiss Webster colony reached 60% (n=70) in males 8 weeks to 6 months of age. Despite severe obesity in some females, no females were diabetic. Pathologic findings in affected males included cachexia, dilated gastrointestinal tracts with poor muscular tone, pancreatic islet degeneration and atrophy with compensatory metaplasia and/or neogenesis, bacterial pyelonephritis, membranous glomerulopathy, and late-onset hepatic tumors with macrosteatosis, microsteatosis and hydropic change in aged males. Serum insulin correlated with blood glucose in a nonlinear pattern suggestive of islet exhaustion. Circulating leptin levels showed a weak inverse correlation with glucose. Diabetic males were bred with obese colony females to produce 20 male and 20 female offspring. Prevalence of diabetes in male offspring was 80% (16/20) with a median age of onset of 18 weeks. In contrast, no diabetic females were identified, despite being significantly more obese than males. Male predominance is likewise a feature of T2D in humans. / (cont.) To our knowledge, this is the first documentation of hepatocellular carcinoma and islet metaplasia and/or neogenesis in a spontaneous outbred mouse model of T2D. The SW availability and histolopathologic features represent a promising new model for the study of T2D. Further studies are required for complete molecular and genetic characterization of the diabetic SW mouse. These studies are outlined in this thesis. / by Laura B. Lemke. / S.M.

Thesis (Ph. D. in Bioengineering)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Leaf 117 blank. / Includes bibliographical references. / A goal for gene delivery research is to design vectors capable of (a) delivering transgenes to target cells, (b) yielding efficient gene expression, and (c) minimizing any immune, inflammatory, or cytotoxic response. Current research has focused on developing such vehicles using end gene expression as the benchmark. While transgene protein production is the overall objective of successful gene delivery, such qualitative treatment of gene delivery, especially for non-viral vectors, may result in unoptimized vectors and potential rate limiting steps unidentified. Quantitative analysis of the gene delivery pathway is essential for the characterization, comparison, and design of vectors. The complex nature of the mechanisms for gene delivery, particularly at the cellular level, contains multiple potentially rate limiting steps to successful gene expression. Through quantitative methodologies, vector efficacy can be related to molecular characteristics and specific processes within the gene delivery pathway. These potentially rate limiting steps include, but are not limited to, cell surface association, subcellular trafficking, endosomal escape, nuclear translocation, vector unpackaging, and gene expression. Design of synthetic gene delivery vectors seeks to develop molecular systems mimicking virus-like infection behavior, including cell membrane attachment and rapid internalization followed by endosomal escape, nuclear localization, and finally gene expression. To explore such opportunities for vector optimization and design, a model human hepatocellular carcinoma cell line by sets of transfection agents complexed with a plasmid. Time courses of plasmid numbers were determined both from whole cells and from isolated nuclei by real-time quantitative PCR. / (cont.) This enabled determination of values for parameters characterizing the key intracellular trafficking processes for a validated mass-action kinetic model of cellular gene delivery, in concert with model parameter values obtained from literature data. Quantitative parameter sensitivity analyses were performed for the individual gene delivery vectors, permitting elucidation of the particular rate-limiting processes specific to each vector. The resulting model predictions were then extended to test effect of increased delivery by polyethylenimine based gene delivery vectors and thus model utility. Additionally, viral vector performance was measured, providing insight into the extreme efficiency of such vectors. As no single process was found to be rate-limiting for all vectors, nor was the rate-limiting process necessarily the kinetically-slowest process. Thus, a single design factor will not likely improve all types of vectors, but rather each vector must be improved with respect to its own specific rate-limiting process(es) and improvements in vehicle design may best arise from quantitative analysis of the contributions of each to the integrated system operation. / by Csanad M. Varga. / Ph.D.in Bioengineering

Thesis (Ph. D. in Applied Biosciences and Biotechnology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references (p. 113-123). / Glycosaminoglycans (GAGs) are complex polysaccharides that reside in the extracellular matrix and on the surfaces of all cells. The same complexity that contributes to the diversity of GAG function has also hindered their chemical characterization. Recent progress in coupling bacterial GAG-degrading enzymes with sensitive analytical techniques has led to a revolution in understanding the structure-function relationship for an important subset of GAGs, namely heparin/heparan sulfate-like glycosaminoglycans (HSGAGs). The study of chondroitin sulfate and dermatan sulfate (CS/DS), an equally important subset of GAGs, has lagged behind partially due to a lack of enzymatic and analytical tools akin to those developed for HSGAGs. The Flavobacterial heparinases have proven indispensable in characterizing the fine structure of HSGAGs responsible for their different biological functions. As a continuation of ongoing research, a combination of chemical modification, peptide mapping, and site-directed mutagenesis was employed to explore the role of histidine in the activity of heparinase III. Of the thirteen histidines in the enzyme, His295 and His510 were found to be critical for the degradation of heparan sulfate by heparinase III. As a first step to developing the chondroitinases as enzymatic tools for the characterization of CS/DS oligosaccharides, recombinant expression and purification schemes were developed for chondroitinase AC and B from Flactobacterium heparinum. The recombinant enzymes were characterized using biochemical techniques and kinetic parameters were determined for their respective CS/DS substrates. / (cont.) By combining the modeling a tetrasaccharide substrate into the active site of chondroitinase B with site-directed mutagenesis studies, a variety of residues were identified as critical for substrate binding and catalysis. A subsequent co-crystal structure of chondroitinase B with DS-derived hexasaccharide revealed a catalytic role for a calcium ion and provided further clarity into the role of individual active site amino acids. Additionally, using a variety of defined DS-derived oligosaccharides coupled with sensitive analytical techniques, chondroitinase B was identified as an endolytic, non-random, non-processive enzyme that preferentially cleaves longer oligosaccharides compared to shorter ones. Taken together, these studies represent a critical step in developing the chondroitinases as enzymatic tools for the characterization of CS/DS oligosaccharides in a fashion akin to the use of the heparinases to characterize HSGAGs. / by Kevin R. Pojasek. / Ph.D.in Applied Biosciences and Biotechnology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (leaves 148-156). / Mechanical stimulation of 3-D chondrocyte cultures increases extracellular matrix (ECM) production and mechanical stiffness in regenerating cartilage. The goal of this study was to examine the progression of chondrocyte signaling responses to mechanical stimulation in 3-D culture during tissue regeneration. To investigate the role of integrins in chondrocyte mechanotransduction, function-blocking antibodies and small-molecule antagonists were used to disrupt integrin-matrix interactions during dynamic compression of chondrocytes in 3-D agarose culture. At early days in culture, blocking [alpha]v[beta]3 integrin abolished dynamic compression stimulation of proteoglycan synthesis, independent of effects in free-swell culture, while blocking [alpha]5[beta]1 integrins abolished the effect of compression only when blocking in free-swell increased proteoglycan synthesis. This suggests that disrupting [alpha]v[beta]3 and [alpha]5[beta]1 interactions with the ECM influences proteoglycan synthesis in distinct pathways and that [alpha]v[beta]3 more directly influences the mechanical response. To further distinguish individual mechanotransduction pathways, we investigated the temporal gene transcription response of chondrocytes to ramp-and-hold compression on Days 1, 10, and 28 in 3-D agarose culture. Clustered and individual gene expression profiles changed temporally and in magnitude over time in culture. Day 1 cultures differed from Days 10 and 28, reflecting changes in cell microenvironment with development of pericellular and extracellular matrices. Comparisons with the response of intact tissue to compression suggested similar regulatory mechanisms. We further investigated MAPkinase (ERK1/2, p38, JNK) and Akt activation on Days 1 and 28 in agarose culture through phosphorylation state-specific Western blotting. / (cont.) Compression induced transient ERK1/2 phosphorylation on both days, with Day 28 levels similar to intact tissue. Unique from tissue behavior, only slight transient p38 phosphorylation was observed on Day 28, and SEK phosphorylation was undetected. Akt was uniquely regulated in intact cartilage compared to MAPks, with decreased total Akt levels over time under static compression. In contrast, compression transiently decreased pAkt levels in agarose cultures, with no changes in total Akt. Changes in the chondrocyte responses to compression with time in agarose culture suggest that cells sense different forces and respond differently with time; further studies may help optimize mechanical loading for tissue-engineering purposes. These studies provide a basis for further examination of mechanotransduction in cartilage. / by Diana H. Chai. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / "June 2006." / Includes bibliographical references (leaves 175-187). / Molecular markers for adult stem cells (ASCs) are highly demanded for research and clinical applications. The development of specific molecular markers for ASCs has been difficult mainly due to the technical barriers in the identification and isolation of rare ASCs. Previously, reported transcriptional profiling studies for defining molecular features of ASCs were compromised by the use of impure ASC preparations. Thesis for this research was that the study of asymmetric self-renewal, a defining property of ASCs, might provide key clues to understanding ASC function and lead to discovery of novel molecular markers for ASCs. Fifty two self-renewal pattern associated (SRPA) genes were identified by cDNA microarray analysis with cell culture models whose self-renewal pattern could be reversibly regulated, instead of using heterogeneous ASC-enriched populations. From evaluation of whole genome transcript levels to expand the SRPA gene pool, 543 SRPA genes were discovered. Both microarray studies showed that asymmetric self-renewal associated (ASRA) genes were highly represented in ASC-enriched populations but not in embryonic stem cells. The SRPA gene expression signature successfully distinguished isolated ASC-enriched populations from non-stem cell populations by principal component analysis (PCA). / (cont.) The SRPA gene signature clustered and classified putative epidermal stem cell-enriched populations better than reported stemness gene signatures in PCA. Therefore, gene microarray analyses for studying self-renewal pattern per se confirmed for the first time that asymmetric self-renewal is an essential molecular feature of ASCs in vivo. Chromosome mapping of the SRPA genes identified two SRPA chromosome gene cluster regions. One chromosome cluster contained primarily ASRA genes, whereas the other contained primarily symmetric self-renewal associated (SSRA) genes. These two SRPA chromosome cluster regions are frequently rearranged or deleted in particular human cancers. Functional and expression analysis of several selected ASRA and SSRA gene-encoded proteins implicated them in control of asymmetric self-renewal and non-random chromosome co-segregation, respectively. Moreover, one plasma membrane bound ASRA protein, CXCR6, had properties of one of the most specific molecular markers for ASCs described to date. In conclusion, this research strongly supported the precept that asymmetric self-renewal is a unique molecular feature for understanding ASCs, their relation to cancer, their unique function, and for their eventual exclusive identification. / by Minsoo Noh. / Ph.D.