Cardiovascular complications are the leading cause of death in patients with chronic kidney disease (CKD). Uraemic cardiomyopathy (UCM) is characterised by structural and cellular remodelling including left ventricular hypertrophy (LVH), metabolic remodelling and mitochondrial dysfunction. Although ex vivo studies have highlighted evidence of enhanced glucose utilisation in the hypertrophied heart, cardiac glucose metabolism in uraemia has yet to be established in vivo. In addition, little is known about mitochondrial morphology or the impact of iron therapy on cardiac mitochondrial function in CKD. The aims of this study were to (I) investigate cardiac glucose metabolism in vivo using 18F-flurodeoxyglucose positron emission tomography (18F-FDG PET) during the development of UCM and (II) characterise mitochondrial morphology and the impact of iron therapy on cardiac mitochondrial function in uraemia. Experimental uraemia was induced surgically in male Sprague-Dawley rats via a subtotal nephrectomy. Dynamic PET/CT scans were acquired at 5, 9 and 13 weeks post-surgery using 18F-FDG PET. The rate and distribution of 18F-FDG uptake were determined using Patlak and polar map analysis. In a separate series of experiments the iron complex, ferumoxytol, was administered 6 weeks post-surgery and mitochondrial respiratory rates and enzyme activities determined following sacrifice 6 weeks later. Cardiac mitochondrial morphology was characterised by probing the expression of key mitochondrial fusion and fission proteins and evaluating mitochondrial size and structure in left ventricular tissue and isolated mitochondria. Renal dysfunction was prominent in uraemic animals by 12 weeks as evidenced by elevated serum creatinine, urea and the presence of anaemia. LVH was associated with moderately increased 18F-FDG uptake in the uraemic heart at 5, 9 and 13 weeks. This was paralleled at the cellular level by altered mitochondrial morphology, characterised by a more sparsely packed cristae, and increased mitochondrial state 4 respiration, indicative of reduced efficiency. However, ferumoxytol treatment did not impact on cardiac mitochondrial function at this stage of uraemia. Collectively these data suggest there is evidence of enhanced glucose utilisation in the uraemic heart in vivo and these changes are associated with altered mitochondrial structure and bioenergetics.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. Page 104 blank. / Includes bibliographical references. / Tissue engineering can be applied to develop therapeutic techniques for osteoarthritis, a degenerative disease caused by the progressive deterioration of cartilage in joints. An inherent goal in developing cartilage-replacement treatments is ensuring that tissue-engineered constructs possess the same properties as native cartilage tissue. Biochemical assays and imaging techniques can be used to study some of the main components of cartilage and assess the value of potential therapies. Agarose and self-assembling peptides have been used to make hydrogels for in vitro culture of bovine bone marrow stromal cells (BMSCs) which can differentiate into chondrocytes, undergo chondrogenesis, and produce cartilage tissue. So far, differences in cell morphology that characterize chondrogenesis had been observed in peptide hydrogels like KLD and RAD but not in the 2.0% agarose hydrogels typically used for culture. A tissue engineering study was conducted to determine if a suitable environment for cell proliferation and differentiation could be obtained using different agarose compositions. BMSCs were cultured in 0.5%, 1.0%, and 2.0% agarose hydrogels for 21 days following TGF-p1 supplementation. Results indicate that the 0.5% agarose hydrogels are clearly inferior scaffolds when compared to the 1.0% and 2.0% agarose hydrogels, which are generally comparable. Since agarose gels appear to be suboptimal in promoting chondrogenesis, self-assembling peptides should be used in future studies. In addition to the biochemical assays traditionally used in cartilage tissue engineering studies, atomic force microscopy (AFM) can be used to image aggrecan, one of the main components of cartilage. Imaging studies were carried out using fetal bovine epiphyseal aggrecan to optimize previous extraction and sample preparation procedures, as well as an AFM imaging protocol, for samples containing aggrecan. Experiments were conducted with 10, 25, and 50 ptg/mL aggrecan solutions to find the minimum concentration needed to create aggrecan monolayers on APTES-mica that would yield acceptable AFM images (25 [mu]g/mL). AFM instrument and software parameters were optimized to find the working range of the integral and proportional gains (0.2 - 0.4 and 0.6 - 0.8, respectively) and to increase the resolution, showing fields at the 800 nm level. Finally, an image processing protocol relevant to these molecules was established. / by Nicole Casasnovas Ortega. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / DNA damage contributes to cancer, aging, and heritable diseases. Ironically, DNA damaging agents are also commonly used in current cancer treatment. We therefore need robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. The single cell gel electrophoresis (comet) assay has become a standard method for DNA damage analysis, however, it is not well suited for use in clinical and epidemiological settings due to issues of low throughput, poor reproducibility, and a laborious image analysis requirement. To overcome these limitations, we applied microfabrication techniques to engineer an arrayed cell comet platform that maximizes the number of analyzable cells and provides spatial encoding for automated imaging and analysis. Additionally, we developed complementary software that eliminates the inherent bias of manual analysis by automatically selecting comets from the defined array. In its 96-well format, the so-called CometChip integrates with high throughput screening technologies, further increasing throughput and removing user error. This improved approach enables multiple cell types, chemical conditions, and repair time points to be assayed in a single gel with improved reproducibility and processing speed, while maintaining the simple protocol and versatility of the comet assay to assess a wide range of DNA damage. Using the CometChip, we evaluated a variety of DNA damaging agents, revealing repair profiles that can be used to gain insight into biological mechanisms of damage sensitivities. We confirmed the ability of the CometChip to identify deficiencies in four major DNA repair pathways, supporting the use of the assay in determining pathway sensitivities that may be useful in guiding treatment strategies that more selectively target cancerous cells and reduce side-effects. We also used the platform to evaluate potential inhibitors of DNA repair, which are emerging as promising adjuvants in cancer management. Taken together, the CometChip enables high throughput genotoxic evaluation of chemical exposures, discovery of novel chemotherapeutic strategies, and measurement of DNA repair kinetics for identification of susceptible populations and disease prevention. The CometChip is a significant advancement in DNA damage and repair technology, providing high throughput, objective, and quantitative measurements that have the potential to become a new standard in DNA damage analysis. / by David McGregor Weingeist. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 116-129). / The emergent field of synthetic biology is different from many other biological engineering efforts, in that its roots, design principles, and forward engineering perspective have been adopted from electrical engineering and computer science. Synthetic biology is uniquely poised to make great contributions to numerous fields such as bio-fuel, energy production, agriculture and eco-remediation, national defense, and biomedical and tissue engineering. Considerable progress has been made in engineering novel genetic circuits in many different organisms. However, not much progress has been made toward developing a formal methodology to engineer complex genetic systems in mammalian cells. One of the most promising areas of research is the study of embryonic and adult stem cells. Synthetic biology has the potential to greatly impact the progression and development of research in this area of study. A critical impediment to the development of stem cell engineering is the innate complexity, little to no characterization of parts, and limited compositional predictive capabilities. In this thesis, I discuss the strategies used for constructing and optimizing the performance of signaling pathways, the development of a large mammalian genetic part and circuit library, and the characterization and implementation of novel genetic parts and components aimed at developing a foundation for mammalian synthetic biology. I have designed and tested several orthogonal strategies aimed at cell-cell communication in mammalian cells. I have designed a characterization framework for the complete and proper characterization of genetic parts that allows for modular predictive composition of genetic circuits. With this characterization framework I have generated a small library of characterized parts and composite circuits that have well defined input-output relationships that can be used in novel genetic architectures. I also aided in the development of novel analysis and computational tools necessary for accurate predictive composition of these novel circuits. This work collectively provides a foundation for engineering complex intracellular transcriptional networks and intercellular signaling systems in mammalian cells. / by Noah Davidsohn. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student submitted PDF version of thesis. / Includes bibliographical references. / Gastric cancer is the 2nd leading cause of cancer death worldwide and the 4th most commonly diagnosed cancer worldwide. Helicobacter pylori infection is the major risk factor of gastric cancer, and as such, this bacterium has been classified as a type 1, or definite, carcinogen by the International Agency for Research on Cancer. H. pylori infects the gastric mucosa of more than half of the world's population and promotes gastric carcinogenesis by inducing chronic inflammation. Over decades of persistent H. pylori infection and chronic inflammation, the stomach goes through a well characterized pathological progression involving chronic gastritis, atrophy, intestinal metaplasia, dysplasia, and ultimately cancer. Interestingly, there are strong gender differences in the development of gastric cancer, as men are twice as likely to develop the disease than women. Given the importance of H. pylori and chronic inflammation in gastric carcinogenesis, this thesis investigated the role of gender in modulating host immune responses to H. pylori. The aims of this thesis explored 1) the effect of gender on H. pylori's ability to induce mutations and 2) the effect of estrogen and the anti-estrogen, Tamoxifen, on H. pyloriinduced gastric cancer. For the first aim, the gpt delta mouse model, a murine mutational analysis model, was used to study chronic infection with H. pylori. Increased frequency of point mutations was observed in infected female mice at 12 months post infection. These mutations were not observed in infected male mice. Further analysis revealed that H. pylori induced a greater immune response in female mice in this model, as measured by increased severity of gastric lesions, decreased bacterial counts and the higher levels of Th1 antibodies for H. pylori. The spectra of mutations pointed towards oxidative damage as the underlying cause of induction. This study revealed that gender differences in mutagenesis were mediated by the severity and duration of the immune response. In the second aim, 17[beta]-estradiol prevented the formation of gastric cancer in the INSGAS mouse model, which develops gastric cancer in a male-predominant manner. Unexpectedly, this study led to the discovery that Tamoxifen may act as an agonist in this model of gastric cancer, as it was able to prevent gastric cancer using mechanisms similar to 17[beta]- estradiol. Both compounds downregulated pathways associated with cellular movement and cancer. CXCL1, a murine homolog of IL-8, was downregulated by treatment at both local and systemic levels, which led to a decreased neutrophilic infiltrate. 17[beta]-estradiol and Tamoxifen mediated the disruption of a positive feedback loop coupling CXCL1 secretion with neutrophil recruitment, which dampened the activation of proinflammatory and oncogenic pathways, leading to protection against gastric cancer. In conclusion, these studies provide further insight into the role of gender modulation of host immune response in H. pylori-induced mutagenesis and carcinogenesis. / by Alexander Sheh. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 191-207). / Articular cartilage is avascular, and defects have limited capacity for spontaneous healing. Angiogenesis may interfere with maturation of naturally avascular tissues. Our rationale is that the use of endostatin, a potent angiogenesis inhibitor, will facilitate the formation of hyaline cartilage during regeneration. The objective of this thesis was to develop a system with a novel approach for treating cartilage defects, namely endostatin-producing cartilaginous constructs. The constructs were engineered using nonviral gene therapy, through evaluation of select variables, including regulators (culture media, endostatin plasmid load, method of pEndo lipoplex incorporation, and oxygen tension), scaffold formulation, and cell type. We also investigated select aspects of the in vivo cartilage defect model in which the construct can be implanted, including the post-surgical rehabilitation protocol and the use of osteogenic protein (OP)- 1. The principal achievement was the engineering of endostatin-expressing cartilaginous constructs in vitro using chondrocytes and mesenchymal stem cells, collagen sponge-like scaffolds and hydrogels, and chondrogenic medium. Peaks in endostatin protein were observed during the first few days of culture, followed by decreases. The endostatin levels were comparable to therapeutic levels in vitro and physiological levels in vivo. Most of the endostatin protein was released into the expended medium; little retention was observed, including in scaffolds supplemented with heparan sulfate, chondroitin sulfate, and heparin. In vivo work examining chondral defects in the goat knee demonstrated that long-term post-operative immobilization, even with periodic passive motion exercise, resulted in significant joint degeneration. Cell-seeded scaffolds were observed in the defect 2 months following implantation and short-term immobilization, and yielded results at least as good as historical data obtained using other treatment techniques, including autologous chondrocyte implantation and microfracture, suggesting that a cell-seeded scaffold is a viable option for cartilage repair. There was no significant benefit of multiple treatments of OP-I on chondral defects. Neovascularization was observed in the largely fibrous reparative tissue filling the chondral defects, providing further rationale for the use of endostatin. A notable finding was the observation of laminin and type IV collagen, 2 common basement membrane molecules, in both in vitro engineered cartilaginous constructs and in vivo cartilage repair samples. / by Lily Jeng. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Recent advances in chemotherapeutic development have targeted vital mechanisms that ensure survival of cancer cells; these include the ability to evade immune surveillance, undergo metabolic adaptations and form a defense mechanism against oxidative stress. Cancer cells often possess higher endogenous levels of reactive oxidative species (ROS) compared to normal cells due to the cumulative effects from genomic instability, inflammation and oncogene activation, and they become increasingly reliant on the cell antioxidant network to prevent this elevated oxidative stress from becoming toxic. Thus, chemotherapeutics that inhibit the antioxidant network and thereby elevating ROS are thought to be promising candidates that can selectively eliminate tumor cells. Despite the promises of these molecules, chemotherapeutics modulating ROS levels have mostly fallen short of their projected impact. We believe that a thorough understanding of the quantity, location and duration of ROS generation needed to cause tumor cell toxicity, will be important for understanding the mechanism of current successful chemotherapeutics and designing future ROS-based drug candidates. In this thesis, we explored the use of genetically encoded sensors and generators of ROS, H₂O₂ in particular, to answer these important redox biology questions. We began by developing a deeper understanding of how to use these protein-based peroxide sensors in a quantitative manner. We created a technique quantifying intracellular peroxide levels by converting the fluorescence signal outputs from these sensors into more meaningful intracellular concentrations. This was accomplished via a combination of kinetic modeling, biochemical measurements and image analysis techniques. We also explored the cell to cell heterogeneity in sensor response to H₂O₂ stimulation, and found that the intracellular expression level of the sensor is correlated with the ratio-metric response of the probe. Further kinetic modeling analysis showed that the slow recycling step of activated sensor was responsible for the correlation. In the second part of the thesis, we used these sensors in combination with enzymatic generators that can produce H202 endogenously in a kinetically controlled manner. These tools allowed us to quantitatively determine that there are two toxicity thresholds, a total accumulation of H₂O₂ and intracellular concentration, that are needed for H₂O₂ -mediated cell death. We also applied these tools to investigate the mechanism of two ROS-based chemotherapeutics, phenethyl isocyanate (PEITC) and piperlongumine. We found that depletion of the glutathione antioxidant by these drugs was unimportant to the toxicity mechanism, and the amount of oxidative stress generated by these compounds was not enough to induce significant toxicity by itself. The final part of the thesis involves technology development for a next generation enzymatic ROS generator. We explored the use of P450-BM3, an enzyme that can generate superoxide and hydrogen peroxide through a reaction that requires only NADPH and oxygen. While this reaction in the wild type protein is slow, it can be engineered to have much higher catalytic rates. We demonstrated through various protein engineering approaches that we could create P450-BM3 proteins with enhanced generation of H₂O₂. We also were able to express correctly folded, active enzymes inside mammalian cells that utilize intracellular NADPH and oxygen to produce H₂O₂. / by Beijing Kara Huang. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student submitted PDF version of thesis. / Includes bibliographical references (p. 162-176). / It is well established that extracellular pH (pHe) becomes acidic in several important physiological and pathological contexts, including the tumor and wound microenvironments. Although it is known that acidic pHe can have profound effects on cell adhesion and migration processes integral to tumor progression and wound healing, the molecular mechanisms underlying the cellular responses to acidic pHe are largely unknown. Transmembrane integrin receptors form a physical linkage between cells and the extracellular matrix, and are thus capable of modulating cell adhesion and migration in response to extracellular conditions. In this thesis, computational and experimental approaches are used to investigate the role of acidic extracellular pH in regulating activation and binding of integrin [alpha]v[beta]3, and to characterize the consequences for downstream subcellular- and cellular-scale processes. Molecular dynamics simulations demonstrate that opening of the integrin [alpha]v[beta]3 headpiece occurs more frequently in acidic pHe than in normal pHe, and that this increased headpiece opening can be partially attributed to protonation of ASP[beta]127 in acidic pHe. These computational data indicate that acidic pHe can promote activation of integrin [alpha]v[beta]3. This is consistent with flow cytometry and atomic force microscope-enabled molecular force spectroscopy experiments, which demonstrate that there are more activated [alpha]v[beta]3 receptors on live [alpha]v[beta]3 CHO-B2 cell surfaces at acidic pHe than at normal pHe 7.4. Put together, these atomistic- and molecular-level data suggest a novel mechanism of outside-in integrin activation regulation by acidic extracellular pH. Next, the consequences of acid-induced integrin activation for subcellular- and cellular-scale processes are investigated. Kymography experiments show that [alpha]v[beta]3 CHO-B2 cell membrane protrusion lifetime is increased and protrusion velocity is decreased for cells in pHe 6.5, compared to cells in pHe 7.4. Furthermore, [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 form more actin-integrin adhesion complexes than cells in pHe 7.4, and acidic extracellular pH results in increased cell area and decreased cell circularity. Cell migration measurements demonstrate that [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 migrate slower than cells in pHe 7.4, and that the fibronectin ligand density required for peak migration speed is lower for cells in pHe 6.5. Together, these data show that acidic pHe affects subcellular- and cellular-scale processes in a manner that is consistent with increased integrin activation in this condition. Finally, the migration behavior of [alpha]v[beta]3 CHO-B2 cells, bovine retinal microvascular endothelial cells, and NIH-3T3 fibroblasts in an extracellular pH gradient is investigated. Results demonstrate that NIH-3T3 fibroblasts do not exhibit directional preferences in the pHe gradient, but that [alpha]v[beta]3 CHO-B2 cells and bovine retinal microvascular endothelial cells migrate preferentially toward the acidic end of the gradient. These data suggest that acidic extracellular pH may serve as a cue that directs migration of angiogenic endothelial cells to poorly vascularized regions of tumors and wounds. Overall, this thesis research results in multiscale, in-depth understanding of extracellular pH as a critical regulator of cell function, with associated implications for tumor growth, wound healing, and the role of proton pumps in cell migration. / by Ranjani Krishnan Paradise. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 125-133). / Using molecular imaging in vivo, biomolecular and cellular phenomena can be investigated within their relevant physiological context, addressing a central challenge for 21st century biomedicine and basic research. To advance neuroscience in particular, molecular-level measurements across the brain inside the intact organism are required. However, existing imaging strategies and available probes have been limited by serious constraints. Magnetic resonance imaging (MRI) provides deeper tissue penetration depth than optical imaging and better spatial resolution and greater versatility in sensor design than radioactive probes. The most important drawback for MRI probes has been the need for high concentrations in the micromolar to millimolar range, leading to analyte sequestration, complications for noninvasive brain delivery, and toxicity. Efforts to address the sensitivity problem, such as nuclear hyperpolarization, introduce their own technical constraints and so far lack generality. Here, we introduce a conceptually novel molecular imaging technique based on artificially induced physiological perturbations, enabling molecular MRI with nanomolar sensitivity. In this imaging strategy, we take advantage of blood as an abundant endogenous source of contrast compatible with multiple imaging modalities including MRI and optical imaging to decouple the concentration requirement for molecular sensing from the concentration requirement for imaging contrast. Highly potent vasoactive peptides are engineered to respond to specific biomolecular phenomena of interest at nanomolar concentrations by inducing dilation of the microvasculature, increased local bloodflow, and consequently, large changes in T₂*-weighted MRI contrast. This principle is exploited to design activatable probes for protease activity based on the calcitonin gene-related peptide (CGRP) and validate them for brain imaging in live rats; to use CGRP as a genetic reporter for cell tracking; and to create fusions of a vasoactive peptide from flies to previously characterized antibodies capable of crossing the blood-brain barrier (BBB), suggesting the possibility of minimally invasive brain delivery of such probes. We demonstrate the feasibility of highly sensitive molecular MRI with vasoactive probes at concentrations compatible with in situ expression of probes and delivery across the BBB, and show that vasoactive peptides are a versatile platform for MRI probe design which promises unprecedented in vivo molecular insights for biomedicine and neuroscience. / by Adrian L. Slusarczyk. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Animal noses have evolved the ability to rapidly detect a seemingly infinite array of odors at minute concentrations. The basis of this sensitivity are the olfactory (smell) receptors - a large, highly related class of sensory G-protein coupled receptors that function together combinatorially to allow discrimination between a wide range of volatile and soluble molecules. However, the structural and functional mechanisms of these amazing receptors are not currently known. In order to begin to investigate the molecular mechanism(s) of olfaction, I have developed a mammalian expression system for the large-scale production and purification of functional olfactory receptor (OR) proteins in milligram quantities. Expressed OR genes were fabricated from scratch using PCR-based gene synthesis, which facilitated codon optimization and attachment of different affinity tags for purification. Established methods for the production and purification of rhodopsin were adapted to olfactory receptors through extensive optimization (including a full-spectrum screening of over 45 detergents). Key to the efficient extraction and solubilization of olfactory receptors tested is the use of novel zwitter-ionic fos-choline detergents. Following initial experiments on the inducible expression of a human olfactory receptor (hOR17-4) in adherent HEK293S cell cultures, the system was successfully scaled up using a suspension bioreactor. Large-scale culture allowed the purification of >10 milligrams of hOR17-4 monomer at >90%, which was suitable for subsequent X-ray crystallization screening trials. The purified protein was also characterized using several spectroscopic methods and shown to possess the correct secondary structure and several predicted post-translational modifications. To assay the functionality of purified (nonmembrane- bound) hOR17-4, we successfully developed an in vitro assay method using surface plasmon resonance (SPR) to demonstrate that the receptor retains functional selectivity in binding specific odorant ligands in a concentration-dependent manner. The application of these techniques to other olfactory receptors already shows promise and could lead to a generalized method for obtaining large quantities of any olfactory receptor in a rapid and simple manner. Such methods could prove extremely useful in elucidating the structural and functional mechanism(s) of olfactory receptors and in their integration into OR-based biosensor devices. / by Brian Lee Cook. / Ph.D.