Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 66-75). / (cont.) of transcription factors and subsequently, induction of RNA processing (35% of genes incrementally induced) and kinases involved in protein phosphorylation. In the WT, the response was restricted to a transient repression of fundamental biochemical processes. Interestingly, a gene whose repression is known to mimic apoptosis was found to be repressed in the WT. The overwhelming induction of ribosomal protein synthesis genes in both WT and mgtl in response to MNNG is an unexpected result that could signify a successful recovery following wide-spread cellular damage. / Damage to DNA can occur by means of endogenous biochemical processes or exogenous chemicals such as alkylating agents. If left unrepaired, alkylated bases, most notably, O⁶ Methylguanine (O⁶MeG) can be mutagenic and cytotoxic to the cell. Luckily, DNA methyltransferase (encoded by the gene MGT1 in yeast), repairs this damage. By using transcriptional profiling as a tool, an attempt to elucidate the role of MGT1 has been made. First, the basal expression profile of the mgtl was established. Then, the response of wild-type (WI) yeast and yeast lacking MGT1 (mgt1) to the alkylating agent, MNNG was studied using exponentially growing WT and mgti cultures which were exposed to 30[mu]g/ml of MNNG for 10 to 60 minutes. Basal expression profile of yeast lacking MGT1 showed up-regulation of RETV7, a gene implicated in spontaneous mutagenesis. Response to MNNG was invoked immediately and was dramatic and widespread involving 30% of the genome in both WT and mgt1. Cell-cycle checkpoints, damage signal amplifiers, DNA repair genes (nucleotide excision repair, photoreactive repair, mismatch repair) and chromatin remodeling genes were induced. Genes involved in maintaining mitochondrial structure and mitochondrial genome were also induced. Intriguingly, RPN4, a key regulator of proteasomal system was found to be repressed. Environmental stress response genes were culled out to examine the effects of MNNG on WT and mgtl, more carefully. Temporal gene expression profiles in WT and mgtl were informative in delineating differences in the distinct responses mounted by WT and mgtl. The magnitude of response in mgt1 is more profound than in WT. The differences in the dynamic trends between the two suggest that mgt1 initiates a coordinated response involving repression / by Anoop Rao. / S.M.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 139-141). / The purpose of this thesis was to provide a broad survey of bioprocess options for typical drug production vehicles in the biotechnology industry. This goal was accomplished by comparing the growth and monoclonal antibody production by HPCHO Chinese hamster ovary cells and IB4 hybridoma cells in batch, fed-batch, and three perfusion systems: the stirred ceramic membrane reactor (SCMR), the alternating tangential flow (ATF) hollow fiber membrane system, and the external spin filter (ESF) system. The batch experiments for each cell line were used as base case scenarios for the comparisons of cell growth and monoclonal antibody production. The fed-batch experiments for each cell line did not vastly improve the cell growth and monoclonal antibody production over the batch cases. The SCMR perfusion system greatly improved the cell growth and monoclonal antibody production for each cell line. The maximum viable cell concentration of the IB4 hybridoma cells in the SCMR experiment was over seven times that found in the batch experiment and the monoclonal antibody production in the SCMR experiment was over ten times that found in the batch experiment. / (cont.) The ATF perfusion system greatly increased the cell growth for each cell line over the batch cases, but the shear forces created by the system adversely affected the viability of the cells and the monoclonal antibody production was less than that of the SCMR experiments. The ESF perfusion system damaged the HPCHO Chinese hamster ovary cells. For the production of the IB4 hybridoma cells, the ESF system, as well as a modified ESF system, did not vastly improve the cell growth and monoclonal antibody over the base case due to cell aggregation and filter fouling. The cell growth and productivity data, along with economic considerations, were evaluated for the purpose of recommending the best feeding strategy for each of the two cell lines studied. It was concluded that the most successful operation mode for the two cell lines studied was the SCMR perfusion system. More research is needed regarding the scale-up ramifications of the SCMR perfusion system as well as in the quantification of monoclonal antibodies, the optimization of fed-batch systems, and the use of alternative perfusion systems. / by Kathy Hufford. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 117-127). / One goal underlying synthetic biology is to develop standard biological parts that can be reliably assembled into devices encoding higher-order functions. Here, I developed a framework for engineering post-translational devices, which are devices whose inner workings are modulated by non-covalent protein interactions and covalent protein modifications. To test the framework, I designed a scaffold for engineering post-translational devices in yeast, the Phospholocator, that can be used to assemble peptide parts in order to produce devices that couple upstream kinase activity to regulated nuclear translocation. I used the Phospholocator to design, build, and characterize a Phospholocator device, the Cdc28-Phospholocator, whose location is regulated by the activity of cyclin-dependent kinase Cdc28. I next engineered and tested a Fus3-Phospholocator device, whose location is regulated by the activity of the mitogen-activated protein kinase Fus3, in order to demonstrate that the Phospholocator scaffold supports the engineering of many post-translational devices. I used the Cdc28-Phospholocator to follow Cdc28 activity levels throughout the yeast cell cycle, thereby illustrating the utility of the Cdc28-Phospholocator as a tool for biological inquiry. To implement more complex functions, device engineers will want to connect post-translational devices to build multi-component systems. I thus developed a model for device composition that features a universal signal carrier that is both input into and output from post-translational devices. The universal signal could enable engineers to easily combine devices in any desired order, and thus build many new post-translational systems. / (cont.) I next developed a set of specifications and guidelines for designing prototypical protein parts for engineering post-translational devices that communicate via the universal signal carrier. I used the universal signal model and the corresponding set of device specifications to design and model a proof-of-principle. multi-device post-translational system, a post-translational latch, that functions as designed. Taken together, my initial experiences in engineering post-translational devices, defining universal device signals that enable device interconnectivity, and designing, modeling, and analyzing the model of a functional multi-device system, along with the work of many other groups, are sufficiently encouraging to motivate continued work on post-translational devices. / by Samantha C. Sutton. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 143-160). / Live or attenuated pathogens are the basis of many successful vaccines due in part to the orchestrated response of dendritic cells (DCs) triggered by these immunizations, which includes (1) DC and DC precursor attraction to the immunization site, (2) efficient antigen delivery to class I and class II MHC loading pathways coincident with maturation of DCs, and (3) emigration to draining lymph nodes for T cell activation. We have developed a model immunization system designed to allow these steps in the DC life cycle to be controlled in the context of a subunit vaccine. The system is comprised of microspheres encapsulating chemokines and hydrogel nanoparticles; each nanoparticle contains antigen and DC maturation signals (e.g., TLR ligands). The nanoparticles remain sequestered within the carrier microspheres but the chemokine is released at a controllable rate, creating a local chemoattractant gradient centered on each microsphere. DCs are attracted to individual microspheres where nanoparticles are concentrated; attracted DCs extract nanoparticles from the carrier microspheres, and receive maturation signals coincident with the delivery of antigen into both class I and class II MHC processing pathways. / (cont.) In addition, the nanoparticles may be labeled to allow subsequent tracking of particle-carrying DCs in vivo. These components allow the attraction (or if desired, emigration) of dendritic cells and their precursors to be selectively modulated at an immunization site, and the activation signals received by these cells when they encounter antigen to be tailored. In vitro experiments indicate that chemokine-releasing microspheres effectively attract DCs and monocytes over significant distances, and that the gel nanoparticles efficiently trigger DC maturation and lead to both CD4+ and CD8+ T cell activation in vitro and in vivo. This system provides both a platform for rational immunotherapy as well as a powerful set of tools by which the function of dendritic cells can be manipulated and dissected to improve our understanding of how DC trafficking and functional state impacts immune responses. / by Siddhartha Jain. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / Complex carbohydrates such as the heparin/heparan sulfate glycosaminoglycans (HSGAGs) and the asparagine linked glycans (N-glycans) have emerged as crucial components of many human biological systems. Both classes of molecules have diverse roles in normal and pathological processes including development, cell growth, wound healing, angiogenesis, viral entry, and cancer. Taking advantage of the ability of carbohydrates to affect biological outcomes, several companies have already begun developing sugar-based structures as therapeutics for malaria and breast cancer, among others. However, the structural complexity that allows HSGAGs and N-glycans to exhibit such a wide array of activities also makes them more difficult to study than their protein and nucleotide counterparts. There are currently no effective methods for rapid synthesis of specific HSGAG or N-glycan structures, and until recently, most common analytical techniques were not useful in this field due to sensitivity constraints. Now, with advances in separation techniques and mass spectrometry, the fundamental roles of biological carbohydrates can be elucidated more efficiently. However, there is still a need for improved methodologies for studying these classes of molecules. This thesis describes the development of a novel technique for the direct isolation and sequencing of protein-binding HSGAG oligosaccharides. As a proof-of-concept, we used heparin and HSGAG-binding proteins of known specificity (fibroblast growth factor and antithrombin III), then utilized the technique to identify an oligosaccharide binder for a protein with unknown HSGAG binding specificity (endostatin). Furthermore, this technique can be readily applied to carbohydrate material from the cell surface. We have / (cont.) optimized methods for harvesting and purifying cell surface HSGAGs that are suitable for use in many experimental procedures. Taken together, these methods will allow for the rapid identification of HSGAG sequences from biologically relevant sources that bind to proteins of interest, potentially leading to new pharmaceutical targets. Not only do carbohydrates represent a huge potential drug market, they have an enormous role in the development of protein therapeutics. Since N-glycans can significantly alter the activity and half-life of proteins, it is essential to have the appropriate glycosylation for maximum effect. We therefore optimized a method for analyzing the carbohydrate component of N-glycans from mass-produced proteins, and compared the glycosylation patterns of the same protein produced in different types of bioreactors. Further, we built upon the methods for analyzing N-glycans from single proteins to obtain a complete N-glycan profile from total serum glycoproteins. Searching for patterns in these profiles may yield new information on N-glycan changes in disease states, and lead to more sensitive diagnostic testing. / by Nishla Keiser. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 36-38). / Anti-angiogenic therapy offers many benefits over traditional cytotoxic chemotherapy including fewer toxic side effects and the reduced development of drug resistance. Anti-angiogenics alone have not proven effective in inducing tumor regression in the clinic due to both the cytostatic nature of anti-angiogenic therapy and the potential formation of new regions of hypoxia within the tumor after therapy. The new therapeutic paradigm is for combining both anti-angiogenics and traditional cytotoxic agents for a synergistic effect. The efficacy of cytotoxic agents may be reduced after anti-angiogenic therapy, however, due to limited access to tumor vasculature and hypoxia-induced drug resistance. We propose that loading cytotoxic agents within the tumor prior to blood vessel collapse will enable both greater drug accumulation within the tumor as well as a reduction in the formation of therapy-induced regions of hypoxia. We present here a novel nanodelivery vehicle termed a 'nanocell' for the spatio-temporal recruitment of both anti-angiogenics and cytotoxic agents within the solid tumor to achieve this goal. Nanocells consist of a polymeric nanocore encapsulating the cytostatic agent doxorubicin surrounded by a lipid vesicle containing the anti-angiogenic agent combretastatin A4. / (cont.) Nanocell treatment resulted in an 88% reduction in tumor size in vivo, compared to a 66% reduction in tumor size after delivering combretastatin A4 lipid vesicles and doxorubicin nanocores simultaneously but separately. Nanocell treatment also resulted in a significant reduction in systemic toxicity, fewer metastases to the lung and liver, and a greater degree of tumor apoptosis. / by David A. Eavarone. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Recent studies have developed preliminary wiring diagrams for a number of important biological networks. However, the design principles governing the construction and operation of these networks remain mostly unknown. To discover design principles in these networks, we investigated and developed a set of computational tools described below. First, we looked into the application of optimization techniques to explore network topology, parameterization, or both, and to evaluate relative fitness of networks operational strategies. In particular, we studied the ability of an enzymatic cycle to produce dynamic properties such as responsiveness and transient noise filtering. We discovered that non-linearity of the enzymatic cycle allows more effective filtering of transient noise. Furthermore, we found that networks with multiple activation steps, despite being less responsive, are better in filtering transient noise. Second, we explored a method to construct compact models of signal transduction networks based on a protein-domain network representation. This method generates models whose number of species, in the worst case, scales quadratically to the number of protein-domain sites and modification states, a tremendous saving over the combinatorial scaling in the more standard mass-action model was estimated to consist of more that 10⁷ species and was too large to simulate; however, a simplified model consists of only 132 state variables and produced intuitive behavior. The resulting models were utilized to study the roles of a scaffold protein and of a shared binding domain to pathway functions. / by Bambang Senoaji Adiwijaya. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 80-84). / While many have explored how vascular processes alter gap junction communication and composition few have analyzed the role of specific gap junction connexin proteins in regulating cellular communication and wound healing. Using RNA interference or peptide inhibitors to downregulate specific connexins we examined the role of gap junctions in intercellular diffusion, calcium excitation, and in mediating the expression of vascular regulators transforming growth factor-[Beta][ (TGF-[beta]), prostacyclin, and endothelial nitric oxide synthase (eNOS). siRNA inhibition of connexin 43 in porcine aortic endothelial cells (PAEC) significantly decreased the diffusion distance of Lucifer yellow dye and cytoplasmic calcium levels after mechanical wounding. Wound healing experiments suggested that stimulatory signals travel through gap junctions containing connexin 43, while inhibitory signal travel through gap junctions containing connexin 37. Connexin 43 and connexin 37 inhibition, alone or in combination, reduced the levels of secreted latent TGF-[beta] in confluent PAEC monolayers after 24 hours of incubation. Human umbilical vein endothelial cells (HUVEC) behaved in a similar manner. Inhibition of any one of the three connexins resulted in a marked increase in eNOS concentration. / (cont.) Yet, TGF-P was sensitive to simultaneous inhibition of connexins 37, 40, and 43 and prostacyclin was controlled by connexin 37 and/or connexin 40 but not connexin 43. We have demonstrated how selective inhibition of gap junction connexin expression can reveal the potent gap junction mediation of cellular communication, wound healing, and vascular function. We demonstrate for the first time that connexin proteins play distinct roles in vasoregulation with differential effects on TGF- [beta], eNOS and prostacyclin. This technique in general and findings in specific may help explain density-dependent control of vascular signaling and repair. / by David Morad Elihu. / M.Eng.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007. / Includes bibliographical references. / Background: Investigators have focused on mechano-regulation of upstream signaling and responses at the level of gene transcription, protein translation and post-translational modifications. Intracellular pathways including those involving integrin signaling, mitogen activated protein kinases (MAPKs), and release of intracellular calcium have been confirmed in several laboratories. Studies with IGF-1: Insulin-like growth factor-I (IGF-1) is a potent anabolic factor capable of endocrine and paracrine/autocrine signaling. Previous studies have demonstrated that mechanical compression can regulate the action of IGF-1 on chondrocyte biosynthesis in intact tissue; when applied simultaneously, these stimuli act by distinct cell activation pathways. Our objectives were to elucidate the extent and kinetics of the chondrocyte transcriptional response to combined IGF-1 and static compression in cartilage explants. Discussion: Clustering analysis revealed five distinct groups. TIMP-3 and ADAMTS-5, MMP-l and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed under all conditions tested. In comparing gene expression levels to previously measured aggrecan biosynthesis levels, aggrecan synthesis is shown to be transcriptionally regulated by IGF- 1, whereas inhibition of aggrecan synthesis by compression is not transcriptionally regulated. / (cont.) Conclusion: Many genes measured are responsive the effects of IGF-1 under 0% compression and 50% compression. Clustering analysis revealed strong co-expressed gene pairings. IGF-1 stimulates aggrecan biosynthesis in a transcriptionally regulated manner, whereas compression inhibits aggrecan synthesis in a manner not regulated by transcriptional activity. / by Cameron A. Wheeler. / S.M.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Cancer science is a heavily researched and rapidly changing field. Cutting edge research consistently reveals unique features of tumors that can be exploited for treatment. For example, it is well known that cells of varying tumor types have unique molecular markers and cell-surface receptors - fingerprints of sorts - that set cancerous cells apart from healthy cells. Furthermore, the tumor microenvironment has been explored to the point that its unique fluid mechanical and biochemical properties are well understood in the context of tumor growth and survival. However, very little of this research has penetrated clinical medicine. The purpose of this thesis is to present a recent concept in cancer therapy: an anti-cancer nanocell that is capable of the spatial and temporal targeting of drugs to tumor cells. The combination of targeting mechanisms designed into the nanocell is a product of our current understanding of tumors. The design serves to improve the effectiveness of inexpensive, out-of-patent cytotoxic and anti-angiogenic drugs to standards representative of modern research. Efforts towards improving the efficiency of the nanocell, with regards to both drug loading and tumor cell targeting, are presented and discussed. / (cont.) The synthesis of various polymer-ligand conjugates for use in the improved nanocell is reported, as is the development of a prodrug consisting of a generation three polyamidoamine dendrimer conjugated to methotrexate via an ester bond; cell studies demonstrating the effectiveness of the prodrug are included. The expandability of the nanocell is also explored, because ultimately, the nanocell must be robust enough to accommodate both tumor-type and population variability. / by Samir Awasthi. / M.Eng.