Mechanical Engineering
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Item MATHEMATICAL MODELS AND NOVEL BIOMARKERS TOWARD OPTIMIZATION OF BURN INJURY RESUSCITATION(2022) Arabidarrehdor, Ghazal; Hahn, Jin-Oh; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Extensive burn injury is not only devastating but also a significant challenge for healthcare providers. Following a chain of inflammatory responses post-burn, significant amounts of plasma shift from the vascular compartment into the tissues, simultaneously posing the risks of hypovolemic shock and edema. Standard burn resuscitation protocols aim to replace the lost blood volume while not exacerbating the edema through hourly-titrated intravenous fluid infusion. Due to the significant variability in treatment efficacy, there is a substantial ongoing effort to optimize and individualize the burn resuscitation protocols. In this work, we aim to contribute to this effort by (i) developing a platform for the virtual evaluation of burn resuscitation protocols and (ii) identifying biomarkers to guide fluid resuscitation effectively. The first part of this work presents a mathematical model of burn injury and resuscitation, which can be used for the development and non-clinical testing of burn resuscitation protocols and algorithms, as well as to garner knowledge and intuition into this complex pathophysiology. Our mathematical model consists of a multi-compartmental model of blood volume kinetics, a hybrid mechanistic-phenomenological model of kidney function, and novel lumped-parameter models of burn-induced perturbations in volume kinetics and renal function. We examined our mathematical model’s prediction accuracy and reliability using a rich dataset from 16 sheep with extensive burn injuries and clinical data from 233 real-world burn patients. The second part of this work presents the expansion of the mathematical model to incorporate the cardiovascular and renin-angiotensin-aldosterone systems, as well as detailed descriptions of the kidney’s mechanisms, particularly regarding its blood volume and blood pressure regulation roles. This expansion was motivated by the importance of cardiovascular monitoring in the critical care of burn injury patients. We trained and validated the expanded mathematical model for three species: nine sheep subjects and 15 swine subjects with rich cardiovascular and volume kinetics data, and 233 human subjects with demographic and urinary output (UO) data. To the best of our knowledge, our mathematical model may be the first of its kind which is extensively validated for use as a digital twin to replicate realistic burn patients and replace standard large animal pre-clinical testing of burn resuscitation protocols. The third part of this work presents the identification of biomarkers capable of guiding, optimizing, and individualizing burn resuscitation. The UO, the most common endpoint used to titrate burn resuscitation fluid doses, has many limitations as a single variable. Hence, this work aimed to find convenient and reliable biomarkers from arterial blood pressure (ABP) waveform to complement UO in guiding burn resuscitation. Pulse pressure variation (PPV), systolic pressure variation (SPV), and stroke volume variation (SVV) are dynamic indices derived from ABP that have shown promise in hemorrhage resuscitation but are not investigated for different resuscitation paradigms for burn injury. We observed the longitudinal behavior of PPV, SPV, and SVV for 21 porcine subjects with 40% burn injury, which were each either under-resuscitated, adequately resuscitated, or deliberately over-resuscitated. We investigated the features' potential in tracking reference cardiac output (CO) and stroke volume (SV) via linear regression and correlation analysis. PPV, SPV, and SVV showed plausible and statistically different trends for different paradigms. While they performed just as well as UO in tracking CO and SV, their inherent advantage of being available in real-time and their disagreement with UO in determining the subject status suggest that they may potentially complement UO in the hemodynamic assessment of burn patients.Item EFFECT OF MISMATCHED BASE PAIRS ON DNA PLECTONEMES(2022) Desai, Parth Rakesh; Das, Siddhartha; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Base pair mismatches in DNA occur during replication and can result in mutations and certain types of cancer. The exact mechanism by which mismatch repair proteins recognize mismatches is still not well understood. Structures of mismatch recognition proteins bound to a mismatch indicate that the process involves introducing a sharp bend in the DNA and flipping out the mismatched base. Under external torsional stress, an elastic rod with a defect would buckle at the defect, provided the defect reduces the local bending stiffness. In vivo, if the same energetic scenario prevails, it could localize (or pin) the mismatch at the plectoneme end loop (plectoneme refers to a structure formed by the DNA when it buckles, and its helical axis wraps or writhes around itself in the presence of a critical torsional stress) and make the mismatched base pair more accessible to the mismatch repair protein. In genomic DNA, however, the entropic cost associated with plectoneme localization could make pinning unfavorable. Magnetic-tweezers-based studies of DNA supercoiling, performed at high salt concentrations, have shown that in DNA harboring a single mismatch, the plectoneme will always localize at the mismatch. Theoretical studies have predicted that under physiological salt concentrations, plectoneme localization becomes probabilistic, i.e., the plectoneme does not always localize at the mismatch. Plectoneme localization under physiological salt conditions is dependent on the number of mismatches and tension applied to the DNA. However, both experimental and theoretical approaches are currently limited to positively supercoiled DNA. In the current dissertation, we aim to study plectoneme localization, in physiologically relevant conditions, using state-of-the-art molecular dynamics (MD) simulations and single molecule magnetics tweezers-based experiments.In order to simulate plectoneme localization we first develop a framework using the widely available sequence and salt dependent OxDNA2 model. We verify that the OxDNA2 model can quantitively reproduce a reduction in bending rigidity due to the presence of the mismatch(es), similar to all-atom MD simulations. We then verify that the current framework can reproduce the experimentally observed plectoneme pinning (at the location of the mismatches). Next, we simulate plectoneme pinning under physiologically relevant conditions. We find that the plectoneme pinning (at the location of the mismatches) becomes probabilistic and this probability of plectoneme pinning increases with an increase in the number of mismatches. We also simulate a longer 1010 base pair long DNA to study the influence of entropic effects on plectoneme pinning. Next, we extend the simulation framework to simulate a negatively supercoiled, i.e., under-wound, DNA molecule. In vivo, DNA is maintained in a negatively supercoiled state. Negative supercoiling can result in local melting at the mismatched base pairs: this local melting would further reduce the local bending rigidity at the mismatched base pairs and could enhance plectoneme pinning. We find that negative supercoiling significantly enhances plectoneme pinning in comparison with equivalent levels of positive supercoiling. We also find that the mismatched base pairs are locally melted and the plectoneme end loop is bent significantly more as compared to the positive supercoiling case. Additionally, we simulate the 1010 base pair long DNA under two different negative super-helical densities, i.e., two different degrees of unwinding. We find that the super helical density does not affect the plectoneme pinning probabilities. We also conduct simulations of DNA under different stretching forces (0.3 pN, 0.4 pN and 0.6 pN). Negatively supercoiled DNA under relatively high stretching force (~0.6 pN) absorbs tortional stress by locally melting instead of supercoiling. Simulations of DNA under different forces allow us to study the effect of mismatches on the competition between supercoiling and local melting in a negatively supercoiled DNA. We find that higher stretching forces, up to a maximum set by the onset of melting, increase plectoneme pinning at the location of mismatch. Finally, we propose and develop a single molecule assay to validate the simulations results presented in the previous chapters. Previous single-molecule magnetic tweezers measurements of mismatch DNA buckling and pinning were limited to the high force (~2 pN) – high salt (>0.5 M NaCl) regime. We propose to overcome this limitation by attaching a small gold nano-bead via a di-thiol group close to the mismatched base pairs, which permits direct observation of transient DNA buckling at the mismatch. We generate a DNA substrate that can be used to directly observe plectoneme pinning at the mismatch. We perform single-molecule magnetic tweezers measurements to verify that the presence of the di-thiol group does not result in anomalous pinning in an intact DNA molecule.Item PHOSPHOLIPID BEHAVIOR AND DYNAMICS IN CURVED BIOLOGICAL MEMBRANES(2020) JING, HAOYUAN; Das, Siddhartha SD; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Curvature in biological membranes defines the morphology of cells and organelles and serves key roles in maintaining a variety of cellular functions, enabling trafficking, recruiting and localizing shape-responsive proteins. For example, the bacterial protein SpoVM is a small amphipathic alpha-helical protein that localizes to the outer surface of a forespore, the only convex surface in the mother bacteria. Understanding several of these membrane curvature dependent events rely on a thorough understanding of the properties, energetics, and interactions of the constituent lipid molecules in presence of curvatures. In this dissertation, we have used molecular dynamics (MD) simulations to explore how the curvature of the lipid bilayer (LBL), a simplified mimic of the cell membrane, affects the packing fraction and diffusivity of lipid molecules in the LBL, energetics of lipid flip flop in the LBL, and lipid desorption from the LBLs. We have also investigated the interaction between LBLs and a small bacterial protein, SpoVM, which was previously shown to preferentially embed in positively curved membranes. Our work started with simulating convex surface, represented by the nanoparticle supported lipid bilayers (NPSLBLs) in MD. We first quantified the self-assembly, structure, and properties of a NPSLBL with a diameter of 20 nm and showed how the type of the nanoparticle (NP) affects the properties of the NPSLBLs. Second, we studied the energetics of lipid flip flop and desorption from LBLs for the cases of planar substrate supported lipid bilayer (PSSLBL) and NPSLBL. Finally, we investigated the energetics of SpoVM desorption from the PSSLBL and the NPSLBL providing clues to the fundamental driving forces dictating the curvature sensing of SpoVM. In Chapter 1, we discuss the motivation, methods, biological relevance, and the overall structure of this thesis. In Chapter 2, the structure and properties of a pre-assembled NPSLBL were studied. In Chapter 3, we report the MD simulation results on the structure and properties, such as diffusivity, of the lipid molecules within the LBLs of the NPSLBLs formed through the self-assembly route. We compare our findings with that of unsupported lipid bilayer nanovesicles (NVs). Our results show that the structure of the NPSLBLs, although affected by the type of the NPs, is still similar with the free NV consisting of identical number and species of lipid. On the other hand, the properties such as the diffusivity of the lipid molecules within the LBL are significantly different between the cases of NPSLBL and the free vesicle. Results are provided for different combinations of the lipid molecules and the NP materials. The findings described in Chapters 2 and 3 will be eventually useful in long-term for designing new generation of NPSLBLs as drug carrier. In Chapter 4, we focus on the lipid flip-flop and desorption from the LBLs for NPSLBLs and PSSLBLs. We investigated the energetics of a lipid molecule traversing through the lipid bilayer (from inner-to-outer and outer-to-inner leaflet) as a function of the position of the hydrophilic head group of the lipid within the LBL. We obtained the potential of mean force (PMF) by using umbrella sampling. Most importantly, we observed little effect of the curvature in the variation of the lipid flip-flop PMF, establishing that the energetics of lipid migration within the supported bilayer, which implies that energy changes associated with bilayer fluctuations, is independent of the shape of the supported bilayer. The conclusion is supported by the reported experimental results. Next, in Chapter 5, MD simulations are carried out to reveal the energetics of a single SpoVM protein undergoing desorption from LBLs of NPSLBLs and PSSLBLs. The free energy comprises of five different contributions: 1) the free energy change for deforming the protein in the bilayer with respect to the conformation of the protein in the membrane, 2) the free energy change for reorienting the protein in the bilayer about the first Euler angle with the conformation of the protein restrained, 3) the free energy change for reorienting the protein in the bilayer about the second Euler angle with the conformation and the first Euler angle restrained, 4) the free energy change for changing the position of the center of the protein from the membrane to the bulk water with conformation and both Euler angles restrained, and 5) the free energy change for deformation of the protein in the bulk water with respect to the conformation of the protein in the membrane. Through these simulations, we confirmed that SpoVM prefers NPSLBLs rather than PSSLBLs, indicating by a lower free energy change. Additionally, we revealed that the SpoVM membrane sensing is based on the interplay between the packing of the hydrophilic head groups of the lipids and the packing of the acyl chains of the lipids. Our findings reported in Chapter 5 might be helpful in the development of diagnosis and treatment of diseases associated with protein mislocalization.Item BILAYER MEMBRANE ELECTROSTATICS AND CHARGE-REGULATED MEMBRANE-NANOPARTICLE INTERACTIONS(2018) Sinha, Shayandev; Das, Siddhartha; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Nanoparticle (NP) driven targeted drug delivery and NP driven imaging of cells, tumors etc. have been one of the most investigated areas in interfacial and biomedical engineering in recent years involving a massive amount interdisciplinary efforts cutting across disciplines like physics, chemistry, material science, biology, pharmaceutics, and engineering. Drug delivery or imaging with the NPs invariably require the NPs to first adhere to the surface of a cell, which is bound by a cell membrane (also known as plasma membrane or PM). All of these processes occur in an electrolyte medium as the fluids present inside and outside the cell have ions inside them. There have been significant amount of studies on adhesion of nanoparticles but until today, there has been very less number of investigations on the role of the ionic environment on such systems of adhesion. The ions present in the intracellular and the extracellular space produce an electric double layer (EDL) on both sides of the PM. The PM is also a semipermeable membrane i.e it does not let all kinds of ions to pass through it. The moieties that it lets to pass through it is completely dependent on the ion channels present across it and such semi-permeable action dictates the ion distribution around the PM, which in turn would regulate the NP-PM interactions. The main aim of this dissertation is to look into the influence of this ionic environment and the role that it can play on adhesion of NPs. In order to look deeply we first look into the electrostatics of the PMs. We develop a continuum model to investigate the role of the ionic environment or the EDL on the electrostatics present across the membrane. This investigation led us to a very important aspect of membrane electrostatics. We found out charge-inversion (CI) like characteristics on the cytosol side (fluids present inside the cell) of the membrane. There has been no previous reports of such CI like characteristics in either the PM electrostatics or more importantly, in a system consisting of only monovalent electrolyte ions (as is the case we consider). In the next step, we looked into the role of the the surface charge density of the membrane and the concentration of the ions in influencing this PM electrostatics. This led to more interesting results. We found out that for biologically relevant conditions and for standard membrane surface charges, there is a possibility of having the location of CI on the surface of the membrane itself. This is a most remarkable result establishing a positive zeta potential on the surface of the negatively charged PM and we explored the phase-space where such situation of opposite signs of membrane zeta potential and membrane surface charge persists. This electrostatics definitely influences various measurable properties of the membrane. One such very important measurable property of a membrane is the membrane capacitance. It has been widely reported that the ionic environment does not influence the capacitance much. However, with exploration of this phase-space through our continuum simulations we were able to pinpoint a domain where the capacitance can be influenced by as much as 15%. This also stems from the fact that the electrostatics of the system is itself very interesting to study under various conditions. We then move on to explore the effect of this electrostatics on the adhesion of NP on the membranes. Most of these adhesive processes occur through the receptor-ligand (R-L) mechanism. Therefore, until and unless a ligand is able to physically influence a receptor and can get bonded to it, the process of adhesion will never begin. The electrostatics can cause a hindrance to this phenomenon. The main reason is the electrostatic osmotic or disjoining pressure, which causes a repulsion between the ligand-bearing NP and the receptor-bearing cell membrane, and forbids the NP to come to significant proximity of the PM for ensuring that the ligands start to interact with the receptors. Through our analysis, we calculated such repulsion and calculated the distance up to which this repulsion remains strong and can overcome the influence of other attractive effects (e.g., van der Waals forces or thermal forces) that drive the NP closer to the PM. We hypothesize that if the length of the ligand-receptor complex is not larger than this distance up to which the electrostatic repulsion effects remain dominant then the process of adhesion will not even begin. Next, we study what is the role of this ionic environment for the case where the NP adhere to the PMs non-specifically. Such non-specific adhesion (NSA) refers to the adhesion of the NP to the PM by actual physical attachment without involving R-L interactions. Understanding such NSA is vital to gauge the side effects of the NP-based drug delivery -- the dug carrying NP will invariably adhere (non-specifically) to the healthy cells causing damages to the healthy cells. Therefore the current practice necessitates uses of those NPs that demonstrate least cytotoxicity post adhesion and internalization in healthy cells. We show that when metallic NPs non-specifically adhere to the PMs, the resulting destruction of the surface charge effects of PMs would lead to a favorable energy change, which in turn drives the NP NSA to even stiffer membranes (e.g., cell membranes rich in cholesterol). Subsequently, we show that one can use biomimetic NPs (namely NPs encapsulated in PM-derived lipid bilayers) to ensure that electrostatic interactions between the biomimetic NPs and the PM can usher in the most coveted scenario where one can simultaneously ensure the promotion of specific adhesion and prevention of NSA. Finally we address the future directions of this work and how this work can start the discussion about the role of other kinds on nanoparticles in drug delivery and therapy.Item A Proposed Mechanical-Metabolic Model of the Human Red Blood Cell(2014) Oursler, Stephen Mark; Solares, Santiago D; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The theoretical modeling and computational simulation of human red blood cells is of interest to researchers for both academic and practical reasons. The red blood cell is one of the simplest in the body, yet its complex behaviors are not fully understood. The ability to perform accurate simulations of the cell will assist efforts to treat disorders of the cell. In this thesis, a computational model of a human red blood cell that combines preexisting mechanical and metabolic models is proposed. The mechanical model is a coarse-grained molecular dynamics model, while the metabolic model considers the set of chemical reactions as a system of first-order ordinary differential equations. The models are coupled via the connectivity of the cytoskeleton with a novel method. A simulation environment is developed in MATLAB® to evaluate the combined model. The combined model and the simulation environment are described in detail and illustrated in this thesis.Item PLANNING FOR AUTOMATED OPTICAL MICROMANIPULATION OF BIOLOGICAL CELLS(2013) CHOWDHURY, SAGAR; Gupta, Satyandra K.; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Optical tweezers (OT) can be viewed as a robot that uses a highly focused laser beam for precise manipulation of biological objects and dielectric beads at micro-scale. Using holographic optical tweezers (HOT) multiple optical traps can be created to allow several operations in parallel. Moreover, due to the non-contact nature of manipulation OT can be potentially integrated with other manipulation techniques (e.g. microfluidics, acoustics, magnetics etc.) to ensure its high throughput. However, biological manipulation using OT suffers from two serious drawbacks: (1) slow manipulation due to manual operation and (2) severe effects on cell viability due to direct exposure of laser. This dissertation explores the problem of autonomous OT based cell manipulation in the light of addressing the two aforementioned limitations. Microfluidic devices are well suited for the study of biological objects because of their high throughput. Integrating microfluidics with OT provides precise position control as well as high throughput. An automated, physics-aware, planning approach is developed for fast transport of cells in OT assisted microfluidic chambers. The heuristic based planner employs a specific cost function for searching over a novel state-action space representation. The effectiveness of the planning algorithm is demonstrated using both simulation and physical experiments in microfluidic-optical tweezers hybrid manipulation setup. An indirect manipulation approach is developed for preventing cells from high intensity laser. Optically trapped inert microspheres are used for manipulating cells indirectly either by gripping or pushing. A novel planning and control approach is devised to automate the indirect manipulation of cells. The planning algorithm takes the motion constraints of the gripper or pushing formation into account to minimize the manipulation time. Two different types of cells (Saccharomyces cerevisiae and Dictyostelium discoideum) are manipulated to demonstrate the effectiveness of the indirect manipulation approach.Item Microfluidic Planar Phospholipids Membrane System Advancing Dynamics Studies of Ion Channels and Membrane Physics(2012) Shao, Chenren; DeVoe, Donald L; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The interrogation of lipid membrane and biological ion channels supported within bilayer phospholipid membranes has greatly expanded our understanding of the roles membrane and ion channels play in a host of biological functions. Several key drawbacks of traditional electrophysiology systems used in these studies have long limited our effort to study the ion channels. Firstly, the large volume buffer in this system typically only allows single or multiple additions of reagents, while complete removal either is impossible or requires tedious effort to ensure the stability of membrane. Thus, it has been highly desirable to be able to rapidly and dynamically modulate the (bio)chemical conditions at the membrane site. Second, it is difficult to change temperature effectively with large thermal mass in macro device. Third, traditional PPM device host vertical membranes, therefore incompatible with confocal microscopy techniques. The miniaturization of bilayer phospholipid membrane has shown potential solution to the drawbacks stated above. A simple microfluidic design is developed to enable effective and robust dynamic perfusion of reagents directly to an on-chip planar phospholipid membrane (PPM). It allows ion channel conductance to be readily monitored under different dynamic reagent conditions, with perfusion rates up to 20 µL/min feasible without compromising the membrane integrity. It is estimated that the lower limit of time constant of kinetics that can be resolved by our system is 1 minute. Using this platform, the time-dependent responses of membrane-bound ceramide ion channels to treatments with La3+ and a Bcl-xL mutant were studied and the results were interpreted with a novel elastic biconcave distortion model. Another engineering challenge this dissertation takes on is the integration of fluorescence studies to micro-PPM system. The resulting novel microfluidic system enables high resolution, high magnification and real-time confocal microscope imaging with precise top and bottom (bio)chemical boundary conditions defined by perfusion, by integrating in situ PPM formation method, perfusion capability and microscopy compatibility. To demonstrate such electro-optical chip, lipid micro domains were imaged and quantitatively studied for their movements and responses to different physical parameters. As an extension to this platform, a double PPM system has been developed with the aim to study interactions between two membranes. Potential application in biophysics and biochemistry using those two platforms were discussed. Another important advantage of microfluidics is its lower thermal mass and compatibility with various microfabrication methods which enables potential integration of local temperature controller and sensor. A prototype thermal PPM chip is also discussed together with some preliminary results and their implication on ceramide channel assembly and disassembly mechanism.