Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 105-112). / Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of complex diseases like cancer. Development of new assay formats based on microfluidic, label-free platforms enable radical reductions in assay complexity and reagent requirements with the potential for such applications. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can measure mass with femtogram precision. Biomolecules such as proteins and nucleic acids are denser than water, and their presence can thus be quantified by their buoyant mass, or increase in mass relative to the solution they displace. This thesis presents two approaches to conducting label-free, buoyant-mass immunoassays with SMRs with potential for clinical applications. The sensor surface can be functionalized to bind targets directly, or individually weighed polystyrene beads can be used as mobile supports. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from non-specific binding. We demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR SiO2 surfaces act as ultra-low fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL. Decoupling the complexity of surface modifications from the sensor precludes the need for specialized reagents. Monodisperse, micron-scale polystyrene beads are widely available and can be used as mobile supports, with the mean mass of a bead population quantifying target binding onto bead surfaces. Inherent mass variability in the bead population is masked by matching solution density to bead density. We demonstrate that by weighing hundreds of beads in 30 min, mean mass can be estimated with a resolution of 100 attograms. A proof-of-principle assay is demonstrated that quantifies IgG binding onto functionalized beads at 5.20 femtograms per bead. / by Marcio Goldani von Muhlen. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 221-242). / Macaques are the most commonly used non-human primate in cognitive neuroscience research due to similarities between the macaque and human brain. Cephalic recording chambers (CRCs) are often surgically implanted to obtain neuronal recordings. CRCs represent a persistent source of microbial contamination, which can occasionally progress to clinical sequelae of meningitis and brain abscesses. In this thesis, we first examined aerobic and anaerobic bacterial species colonizing CRCs using both traditional culture-dependent methods and 16S microbiota culture-independent methods. We evaluated the most prevalent species, and compared CRC bacterial communities to skin, oral and fecal bacterial communities. Our results indicated that CRC bacterial communities are predominantly composed of anaerobic flora and are relatively unique between individual macaques. Additionally CRC bacterial communities are more similar to skin and oral bacterial communities than fecal bacterial communities, indicating that fecal contamination of CRCs is a less likely source of contamination. Aerobic culture and sensitivity data from samples collected in 2011 identified Staphylococcus aureus, Enterococcus faecalis and Proteus spp. as the most prevalent species isolated, and that E.faecalis isolates displayed marked resistance to multiple antimicrobial classes. Routine CRC sanitization procedures were revised in September 2014 to prohibit antimicrobial use within CRCs, and we evaluated how E.faecalis lineages persisted and evolved between 2011 and 2017. We identified a shift in sequence type (ST) from ST4 and ST55, predominating in 2011, to ST48 predominating in macaques implanted after 2013. ST48 lineages were less resistant to antimicrobials and stronger biofilm producers as compared to ST4 and ST55 lineages. We concluded that loss of selective pressure from antimicrobial use within CRCs permitted ST48 to emerge as the predominant lineage due to its strong biofilm-forming abilities. Finally, we evaluated alternative E.faecalis biofilm treatment strategies. We isolated lytic bacteriophages with activity against ST55 E.faecalis and evaluated the use of phages and antimicrobial peptides LL-37 and PR-39 against E. faecalis biofilm, alone, and in combination with antimicrobials. Our results identified that bacteriophages successfully decreased biofilm produced by ST55 and ST4 E. faecalis isolates and should be evaluated further for treatment of animal and human enterococcal-associated biofilm infections. / by Mia Tova Rock Lieberman. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / 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. / Malaria remains a major burden to global public health. Antimalarial drugs are a mainstay in efforts to control and eventually eradicate this disease. However, increasing drug resistance threatens to reverse recent gains in malaria control, making the discovery of new antimalarials critical. Antimalarial discovery is especially challenging due to the unique biology of malaria parasites, the scarcity of tools for identifying new drug targets, and the poorly understood mechanisms of action of existing antimalarials. Therefore, this work describes the development of two chemical biology tools to address unmet needs in antimalarial drug discovery. A particular challenge in antimalarial development is a shortage of validated parasite drug targets. Potent antimalarials with demonstrated clinical efficacy, like the aminoquinolines and artemisinins, represent a promising basis for rational drug development. Unfortunately, the molecular targets of these drugs have not been identified. While both are thought to interact with parasite heme, linking in vitro heme binding with drug potency remains challenging because labile heme is difficult to quantify in live cells. This work presents a novel genetically-encoded heme biosensor and describes its application to quantify labile heme in live malaria parasites and test mechanisms of antimalarial action. Another challenge is posed by the widespread malaria parasite Plasmodium vivax, which, unlike P. falciparum, cannot be propagated in vitro, hindering research into parasite biology and drug target identification. P. vivax preferentially invades reticulocytes, which are impractical to obtain in continuous supply. The basis for this invasion tropism remains incompletely understood, mainly because current tools cannot directly link molecular binding events to invasion outcomes. This work presents novel methods for immobilizing synthetic receptors on the red blood cell surface. These receptors are used in proof-of-concept experiments to investigate requirements for efficient invasion via a well-characterized P. falciparum invasion pathway, suggesting this method can be used to elucidate molecular mechanisms underlying parasite invasion tropisms. Future receptor designs could promote the invasion of P. vivax into mature red blood cells and potentially facilitate practical in vitro culture. Taken together, these tools present new opportunities for drug discovery to aid efforts in malaria control and eventual eradication. / by James R. Abshire. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 119-128). / Angiogenesis is crucial during many physiological processes, and is influenced by various biochemical and biomechanical factors. Models have proven useful in understanding the mechanisms of angiogenesis and the characteristics of the capillaries formed as part of the process. We have developed a 3D hybrid, agent-field model where individual cells are modeled as sprout-forming agents in a matrix field. Cell independence, cell-cell communication and stochastic cell response are integral parts of the model. The model simulations incorporate probabilities of an individual cell to transition into one of four states - quiescence, proliferation, migration and apoptosis. We demonstrate that several features such as continuous sprouts, cell clustering and branching that are observed in microfluidic experiments conducted under controlled conditions using few angiogenic factors can be reproduced by this model. We also identify the transition probabilities that result in specific sprout characteristics such as the length and number of continuous sprouts. We have used microfluidics to study cell migration and capillary morphogenesis. The experiments were conducted under different concentrations of VEGF and Ang I. We demonstrated that capillaries with distinct characteristics can be grown under different media conditions and that characteristics can be altered by changing these conditions. A two-channel microfluidic device fabricated in PDMS was used for all experiments. The rationale underlying the design of the experiments was twofold: the first goal was to generate reproducible and physiologically relevant results in a microfluidic device, and the second goal was to quantify the capillary characteristics and use them to estimate the transition parameters of the model. We developed stable, well-maintained sprouts by using human microvascular endothelial cells in 2.5 mg/ml dense collagen I gel and by using media supplemented with 40 ng/ml VEGF and 500 ng/ml Ang 1 for two days. It has been shown in many studies that VEGF acts as an angiogenic factor and Ang 1 acts as stabilizing factor. Here we showed that their roles are maintained in the 3D microenvironment, and the sprout characteristics obtained by using this baseline condition could be altered by changing the concentrations of these two growth factors in a systematic way. Sprout and cell characteristics obtained in the experiments and simulations were analyzed by adapting Decision Tree Analysis. This methodology provides us with a useful tool for discerning the impact of different growth factors on the process of cell migration or proliferation as they alter general sprout morphology. The imprints obtained via experiments and simulations were compared; by choosing appropriate values of the transition probabilities, the model generates capillary characteristics similar to those seen in experiments (R2 ~ 0.82- 0.99). Thus, this model can be used to cluster sprout morphology as a function of various influencing factors and, within bounds, predict if a certain growth factor will affect migration or proliferation as it impacts sprout morphology. This was demonstrated in the case of anti-angiogenic agent, PF4. We showed that at high concentration of PF4 (- 1000 ng/ ml), the transition to migration is more profoundly affected while at low concentrations of - 10 ng/ ml, PF4 does not have much of an effect on either migration or proliferation. / by Anusuya Das. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / "December 2012." Cataloged from PDF version of thesis. / Includes bibliographical references. / Multiple signaling pathways are employed by endothelial cells to differentially respond to distinct hemodynamic environments and acquire functional phenotypes, including regulation of inflammation, angiogenesis, blood coagulation, and the vascular tone. In order to understand how these pathways interact, this thesis applies a systems biology approach through a two-step process. First, we constructed an integrated mathematical model for shear-stress-induced nitric oxide (NO) production to assemble the current understanding of this signaling system. Second, we conducted experiments to define how shear stress dynamically modulates the expression of components of the endothelial glycocalyx, a mechanosensor that regulates shear-stressdependent NO production. Nitric oxide produced by vascular endothelial cells is an anti-inflammatory mediator and a potent vasodilator. In order to understand the rich diversity of responses observed experimentally in endothelial cells exposed to shear stress, we assembled four quantitative molecular pathways previously defined for shear-stress-induced NO production. In these pathways, endothelial nitric oxide synthase (eNOS) is activated (a) via calcium release, (b) via phosphorylation reactions, and (c) via enhanced protein expression. To these pathways we added (d) an additional pathway describing the actual NO production from the interactions of eNOS with its various protein partners. These pathways were then combined and simulated. The integrated model is able to describe the experimentally observed change in NO production with time following the application of fluid shear stress, and to predict the specific effects to the system following interventional pharmacological or genetic changes. Importantly, this model reflects the up-to-date understanding of the NO system and provides a platform to aggregate information in an additive way. The endothelial glycocalyx is a glycosaminoglycan layer located on the apical surface of vascular endothelial cells. Previous studies have documented a strong correlation between the glycocalyx expression, local hemodynamic environment, and atheroprotection. Based on these observations, we hypothesized that the expression of components of the endothelial glycocalyx is differentially regulated by distinct hemodynamic environments. In order to test this hypothesis, human endothelial cells were exposed to shear stress waveforms characteristic of atherosclerosis-resistant or atherosclerosis-susceptible regions of the human carotid, and the expression of several components of the glycocalyx was then assessed. Interestingly, we found that heparan sulfate expression is higher and evenly distributed on the apical surface of endothelial cells exposed to the atheroprotective waveform, and is irregularly present in cells exposed to the atheroprone waveform. Furthermore, the expression of a heparan sulfate proteoglycan, syndecan-1, is also differentially regulated by the two waveforms, and its suppression mutes the atheroprotective-flow-induced cell surface expression of heparan sulfate. Collectively, these data links distinct hemodynamic environments to the differential expression of critical components of the endothelial glycocalyx. Taken together, these projects present in this doctoral thesis increase our understanding of endothelial mechano-activated pathways, and have demonstrated how we could use systems biology approach to unravel complex biological problems. / by Andrew Jia-An Koo. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / 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. / In this thesis, we developed two synthetic biology frameworks to facilitate the construction of useful genetic circuits, with a focus on circuits that sense miRNAs. miRNAs are an attractive biomarker for sensing since they regulate virtually all biological pathways in plants and animals, and because miRNA sensors can be easily designed by incorporating sequences complementary to the miRNA into a genetic circuit. Therefore, circuits that sense endogenous miRNAs can dynamically respond to cellular signaling or classify between cell types. However, the development of genetic circuits, and especially multi-input miRNA sensors, has traditionally been iterative, costly, and time-consuming. To this end, we have developed a framework to measure miRNA activity and generate accurate predictions for sensors with multiple miRNA inputs. We started by building the largest library of miRNA sensors to date (620 sensors) and used the library to measure miRNA activity in several cell lines. We then constructed multi-input sensors and determined design rules for predicting their function, namely that miRNAs repress targets synergistically in opposite UTRs and antagonistically within the same UTR. In our second framework, we developed a 'one-pot' method for high-information transfection and analysis that allows researchers to quickly determine performance of many tuned circuit variants in a single well. We used our one-pot method to quickly characterize a variety of genetic elements and to optimize the design of a miRNA sensor with inverted logic, a circuit topology we found difficult to design using traditional methods. / by Jeremy Jonathan Gam. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Joint injuries are common and often result in damage to cartilage, which has a limited ability to repair itself. Tissue engineering is a promising approach for improving cartilage healing in which biomaterials and chemical factors are supplied to direct cells to create a new tissue. The objective of this thesis was to optimize cartilage-like extracellular matrix production by investigating the effects of Dexamethasone (Dex) and HB-IGF- I (heparin-binding insulin-like growth factor-1) on cells encapsulated in the self-assembling peptide RAD and agarose hydrogels. Dex is a synthetic corticosteroid that has been shown to improve cartilage-like tissue production by bone marrow stromal cells (BMSCs), but the mechanisms underlying BMSC response to Dex are not understood. The hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine BMSCs in RAD and agarose hydrogels was tested. The effects of Dex were dependent on the hydrogel material and the species/age of the BMSCs. Importantly, Dex reduced aggrecanase-mediated degradation of matrix in both agarose and RAD hydrogels and for both young bovine and adult human BMSCs. HB-IGF-1, a fusion protein of the heparin binding domain of HB-EGF and IGF-1, can be retained in cartilage matrix and stimulate proteoglycan synthesis with a single dose, whereas unmodified IGF-1 easily diffuses out of cartilage tissue. The RAD peptide was used as a scaffold for retaining growth factor to stimulate encapsulated chondrocytes and adjacent cartilage tissue. RAD was modified by adsorption of HB-IGF-1 before and after RAD assembly, as well as adsorption of heparan sulfate (HS) and IGF-1. The RAD material retained HS adsorbed pre-assembly and HB-IGF-1 delivered in both adsorption methods. Adsorbed HB-IGF-1 and IGF-1 led to increased aggrecan content regardless of the method of adsorption. A trend was found for increased proteoglycan synthesis in adjacent explants as well. RAD self-assembling hydrogels are a promising material for culturing BMSCs undergoing chondrogenesis, retaining, and delivering HB-IGF-1. Dex decreases aggrecanase activity of differentiating BMSCs and adsorbed HB-IGF-1 appears to enhance aggrecan production by encapsulating chondrocytes and adjacent tissue. These findings show potential for improving cartilage repair in vivo. / by Emily Marie Florine. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2008. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 126-138). / Technologies that provide information about the concentrations or activities of specific molecules in living subjects have the potential to greatly advance science and medicine. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task because of its ability to image deep inside tissues at relatively high spatial and temporal resolution. However, the range of molecular phenomena currently accessible to MRI is limited by a lack of suitable molecular sensors. Most efforts to create such sensors have focused on synthetic contrast agents, whose complicated structures make them difficult to engineer, synthesize and deliver to target tissues. If MRI sensors could instead be made of proteins, a number of these difficulties could be mitigated. Here, we describe two platforms for the development of protein-based molecular sensors for MRI. The first is based on the heme domain of the bacterial cytochrome P450-BM3, which produces changes in TI contrast in MRI in response to small molecule binding. We developed a high-throughput assay that allowed us to evolve this protein into a sensor of the neurotransmitter dopamine (DA). We then used it to image DA release from cultured cells and cocaine-induced changes in DA transport in the brains of living rats. The second platform is based on the human iron storage protein ferritin (Ft), which enhances T2 contrast in MRI upon self-aggregation. We developed a system to express self-assembled Ft nanoparticles incorporating multiple surface functionalities, and used it to create a sensor for protein kinase A activity. Our results provide a proof of concept for two novel platforms for protein-based MRI sensor development, and highlight some key advantages of this approach over the synthetic methods used previously. / by Mikhail G. Shapiro. / Ph.D.

Thesis (M. Eng. in Biomedical Engineering)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 37-40). / Spinal Cord injury (SCI) leads to paralysis, decrease in quality of life and high lifetime medical costs. Direct nerve Functional Electrical Stimulation (FES) induces muscles to contract by electrically stimulating nerves, which shows promise for clinical applications in restoring muscle function in SC. However, Functional Electrical Stimulation is limited by the lack of graded response in muscle contraction and by high fatigability due to the reversal of recruitment order of motor units. Previous work showed that ion-selective membranes can be used to modulate Ca 2 ions in situ, decreasing the current threshold for nerve stimulation and eliciting a more graded muscle contraction response. This work developed polyimide-based cuff ion-selective electrodes to enable the future application of this technique in vivo. The developed electrodes were flexible, elastic and conductive. In vitro tests of the electrodes by stimulation of frog sciatic nerve reproduced the decrease in stimulation current threshold, which had been observed in planar glass-based electrodes, in the flexible polyimide-based electrodes. Additionally, cuffing the stimulated nerves with ion-selective electrodes was more effective at decreasing current threshold than planar stimulation. This work also analyzed data on twitch width, contraction time and relaxation time to infer effects of ion-selective electrodes on recruitment order. Stimulation with the ion-selective electrodes had higher twitch width, contraction time and relaxation time than traditional electrical stimulation at all force levels. The difference was particularly high at low force levels, indicating an effect of Calcium ion depletion on recruitment order. / by Ragheb Mohamad Fawaz El Khaja. / M.Eng.in Biomedical Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 189-211). / While a link between chronic inflammation and cancer has been established, the mechanisms of genotoxicity in inflammatory environments remain poorly understood. We hypothesized that inflammation may provide cues that allow cells to survive in the face of significant DNA damage as well as cues that foster cell proliferation. This study sought to elucidate changes that influence hepatocyte decision processes under conditions of chronic inflammation by using a set of extracellular signals, both soluble and matrix-related, which can be varied systematically to create a diverse range of intracellular signaling states and phenotypic outcomes. We developed an easily translatable model system that can maintain primary mouse hepatocytes in a differentiated state and study the independent extracellular cues which regulate hepatocyte behavior during chronic inflammation. This model system allowed systematic variation of oxygen concentration as well as matrix composition and stiffness. Collagen, polyacrylamide, and RADA gels were used to create extracellular environments resembling the various stages of normal and fibrotic liver. Through careful control of medium depth and incubator oxygen levels, we determined that oxygen tension and extracellular matrix affected hepatocyte differentiation independently, and that both high oxygen and a compliant matrix environment are necessary for prolonged maintenance of primary mouse hepatocytes in vitro. Optimization of a quantitative imaging solution enabled the capture of rare events occurring in individual cells within a larger cell population. Using TNF-alpha, Fas ligand and, IL-6 cytokines in conjunction with oxygen and matrix cues, we investigated how extracellular stimuli influence cell fate in a pseudo-inflammatory environment and demonstrated that partial execution of apoptosis is a possible mechanism for genotoxicity in hepatocytes during chronic inflammation. The results of this study improve our understanding of how cues in the extracellular environment combine to influence the behavior primary mouse hepatocytes. The system we developed can be used as a platform for a multitude of in vitro applications including studies regarding drug toxicity and inflammation. / by Lorenna Dianne Buck. / Ph. D.