Thesis (S.M.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999. / Includes bibliographical references (leaves 57-62). / by Jose Angel Márquez, Jr. / S.M.

Acidithiobacillus ferrooxidans is an important industrial organism used in the mining industry where it participates in passive bioleaching processes used to produce about 20% of the world’s copper supply. The bacterium thrives in strong mineral acids at ambient temperatures and derives metabolic energy from the oxidation of ferrous iron, sulfur, and reduced inorganic sulfide compounds to fix CO2 and N2. This unique metabolism provides new opportunities to engineer this organism for the production of fuels and chemicals from CO2. While A. ferrooxidans has been studied extensively for 60 years, the tools and methods necessary for a robust genetic system to manipulate and further study this bacterium are not well developed and published techniques are generally difficult to reproduce. This research focuses on developing the means to genetically modify this species to experimentally study its physiology and engineer the organism for the production of chemicals from CO2. This includes developing a robust and reproducible system to generate and select mutant strains, heterologous expression of exogenous genes, characterizing endogenous inducible promoters, and developing new plasmids to expand the repertoire of tools available for this organism.

<p> Spherical Mean technique (SMT) is a novel method of quantifying the diffusion properties of the nerve fibers bundles in the central nervous system. It does this by calculating the spherical mean of the diffusion signal and fitting it to a parametric equation to obtain per voxel diffusion coefficients. We used Expectation&ndash;Maximization to obtain Gaussian Mixture Models (GMM) to find distinct clusters in per voxel coefficient space. We found that the diffusion properties of all the white matter fibers were clustered into a single Gaussian distribution in 867 brain volume samples. This implies that the diffusion properties of the white matter fibers are relatively homogeneous. Then, we checked this result by comparing the clusters obtained using GMM with tissue classification outputs obtained by clustering Fractional Anisotropy (obtained using Diffusion Tensor modeling), T1 weighted image intensity and B0 image intensity for 867 brain volume samples; we observed that the specific clusters of per voxel diffusion coefficients obtained using GMM represent specific tissue types (grey matter fibers, white matter fibers, cerebrospinal fluid). Since the parameters derived from SMT represent the physical diffusion properties that are independent of microscopic fiber orientation and the distribution of diffusion coefficients of white matter can be modeled by a single Gaussian distribution, we can conclude that the diffusion properties of all white matter fiber are homogeneous.</p><p>

Age-related macular degeneration (AMD) is a devastating ocular disease affecting one third of the elderly population in the western world. Some cases of AMD develop neovascular membranes, which are characterized by the pathologic growth of new blood vessels into the retina. This pathology may be initiated by proteins capable of activating endothelial cells to become angiogenic or inflammatory, later causing them to grow abnormally. This investigation aimed to determine the causes of pathologic blood vessel growth in AMD. Human eyes with AMD have been shown by us and others to have abnormal activities of angiogenin, complement component C5 anaphylatoxin (C5a), and/or elastin fragments. We therefore employed methods including PCR, immunoblotting, immunohistochemistry, morphometrics, tissue culture, ultrastructural observations, and functional assays to determine the effects angiogenin, C5a, and elastin fragments on the angiogenic and inflammatory changes of choroidal endothelial cells in vitro and in vivo. It was shown that choroidal endothelial cells express the surface receptor for C5a. Also, these cells increase their expression of ICAM-1, a surface protein that mediates leukocyte trafficking, in response to elevated levels of C5a in organ culture. This indicates that increased levels of C5a associated with AMD increase the inflammatory behavior of choroidal endothelial cells. It was demonstrated that choroidal endothelial cells are able to internalize angiogenin, a potent inducer of angiogenesis. Although cells from the choroid did not increase their angiogenic responses to this protein, their ability to internalize it indicates that they may respond to it by a different mechanism. Elevated levels of elastin fragments, however, did increase the migratory response of choroidal endothelial cells in culture, which is a key event in angiogenesis. Elevated levels of elastin fragents also increased the amount of collagen IV deposition within Bruch's membrane in a mouse model. This is relevant to AMD pathology as deposits within Bruch's membrane are common manifestations associated with AMD. This body of work has provided new insights into the roles of angiogenin, C5a, and elastin fragments in activating choroidal endothelial cells to becoming inflammatory or angiogenic. These endothelial cell behaviors are common characteristics found in neovascular AMD. These new findings will help aid in the further understanding of the pathobiology of this disease in hopes to provide improved treatments in the future.

Glaucoma is a leading cause of blindness throughout the world and is estimated to affect 80 million by 2020. This disease causes progressive loss of vision and, left untreated, can lead to complete blindness. With treatment, however, disease progression can be slowed dramatically. This makes early detection and intervention crucial in preserving the vision of affected individuals. Onset and progression of glaucoma are associated with structural changes to an anatomical feature known as the optic nerve head (ONH). The ONH is the site of attachment between the retina and the optic nerve that carries all visual information to the brain. As glaucoma progresses, characteristic changes related to cell death and loss of vision can be observed in the three-dimensional structure of the ONH. A common modality used to observe these changes is stereo fundus imaging. This modality captures three-dimensional information via stereo imaging and is commonly used in clinical settings to diagnose and monitor glaucoma. A limitation of using stereo fundus images is the need for review by glaucoma specialists to identify disease related features of ONH structure. Further, even when expert evaluation is possible, the subjective nature of the process can lead due large discrepancies in the evaluations and resultant clinical decisions. The work presented here seeks address these concerns by providing automated, computational tools that can be used to characterize ONH structure. Specifically, this thesis outlines the development of computational methods for inferring three-dimensional information from stereo fundus images and identifying objective, quantitative measurements of ONH structure. The resulting computational tools were applied to image and clinical data collected from a large cohort of individuals to identify hidden relationships between ONH structure, clinical measurements, and glaucoma. These tools were then applied to develop methods for estimating the impact of individual genetic factors on the ONH. Finally, using a longitudinal dataset collected over more than a decade, computational analysis was used to investigate how ONH structure changes over time in response to aging, other disease-related factors, and glaucoma progression.

Most intracranial aneurysms (IA) that are unruptured during clinical screening remain stable over time with no measurable change or symptoms if left untreated. However, a few do grow larger and may rupture. The ability to preemptively identify aneurysms that will become unstable over time (i.e., those that will grow and/or rupture) can result in timely intervention for these few patients while avoiding unnecessary treatment for countless others. So far, reports have primarily assessed potential rupture factors in IAs between ruptured lesions and unruptured lesions. These factors (that discriminate rupture status) do not necessarily distinguish unruptured aneurysms that tend towards growth and/or rupture over a period of time from those that remain stable. The hypothesis motivating this study is that aneurysm shape morphology provides critical information that will indicate instability in unruptured aneurysms. We tested this hypothesis in a large population multi-center prospective longitudinal cohort study of unruptured IAs. A total of 198 study subjects were recruited at four clinical centers - Penn State Hershey Medical Center, Massachusetts General Hospital, Thomas Jefferson University Hospital and University of Iowa Hospitals and Clinics. All had at least one unruptured IA and were placed on follow-up without immediate intervention. Three-dimensional volumetric scans were obtained from the clinics. Three dimensional models were created from the source data using levelset segmentation techniques implemented in the Vascular Modeling ToolKit (VMTK). Five size and six shape indices were calculated on the isolated aneurysm geometry: Size indices - Height (H), max. diameter (Dmax), neck diameter (Dn), volume (V), and surface area (SA) and Shape Indices - Undulation index (UI), Aspect ratio (AR), Ellipticity index (EI), Non-sphericity index (NSI), Bulge Location (BL), and Bottleneck factor (BF). Pressure-induced wall tension was calculated using Finite element analysis. An isotropic, polynomial model was used after a comparative study of different modeling choices was performed and the 95th percentile max. principal stress (Peak Wall Stress - PWS) was chosen to be the index of maximum wall tension. Independent studies were performed to document validity of methods used, to quantify sensitivity of these indices (in turn, of the segmented geometries) to imaging modality and to quantify variability in segmented geometries between investigators. Researchers were blinded from the follow-up status of IAs to avoid any user bias in the analysis. During follow-up, aneurysms were labeled as "grown" or "stable" by radiologists. Study subjects were assigned to one of two groups based on clinical outcome: Unstable group (grown or ruptured) or Stable group (remained same or lesser size). Statistical analysis was then performed on the computed indices. One hundred ninety eight aneurysms were followed for an average of 607 days. Over the follow-up period, 26 were lost to follow up, 150 were found to be stable, 20 had grown, 1 deceased, 1 could not be located on the angiogram and none had ruptured. Also, of the total 198, 53 were electively treated during follow-up. Non-parametric Mann-Whitney U-tests were performed for index to test for statistically significant difference between the stable (n1 = 150) and the unstable group (n2 = 21). None of the indices differentiated the two groups in a statistically significant manner. Receiver Operating Characteristics (ROC) curve analysis was consistent with these findings. Is aneurysm morphology (size and shape) a risk factor for IA growth/rupture? We sought to test this hypothesis in a study design that is unique in many ways - it is the first large population study of longitudinal cohorts; the study population was prospectively recruited at multiple clinical sites; the study population was predominated by small aneurysms (88% of IAs in the study population were less than 7 mm). The results of this large population cohort study suggest that previously reported metrics of aneurysm cannot serve as prognostic indicators of longitudinal outcome.

Automated localization of the optic disc and fovea are important in the field of analysis of fundus images. We introduce a simultaneous detection method for optic disc and fovea with an enhancement and correction step. In the first step, a set of features are extracted from the color fundus image, and the relationship between the feature set and a distance variable d is established during training phase. For a test image, the same set of features is measured and the distance to the optic disc and fovea can be estimated using k-nearest-neighbor regression. A probability image is generated during this step. In the second, a second k-nearest-neighbor regression is applied on the probability image. Detected high likelihood regions from the first step can be enhanced only if they satisfy the trained relationship. The detected regions that do not get support from the other detected structure will be suppressed. 150 color fundus images were used to train the system. 50 color fundus images were used to test the system. The distance error for the optic disc is 9.8±8.3 pixels. The distance error for the fovea is 13.7±6.6 pixels.

All-terrain vehicles (ATVs) are used widely throughout the United States both for recreational activities, such as hunting and riding, and in occupational settings, such as agriculture. In both such activities, ATVs can pose a risk of injury and death resulting from a number of factors, including operator error, operating in an altered state, and misuse. In a study by Milosavlijevic et al. (2011), a survey of agricultural workers in New Zealand displayed that roughly 60% of workers who operated an ATV experienced a loss of control event. The same study showed that nearly 50% of those occurred while riding on a steep slope. Despite these high levels of incidence, little research has been performed to understand the mechanisms that lead to loss of control or instability. In order to study loss of control and instability, a four-degree-of-freedom human-in-the-loop ATV simulator was developed and integrated with a shaking table in a real-time interaction. The simulator allowed for operators to approach conditions by their own means and to study mechanisms of each individual’s approach to traversing a terrain. Inertial measurement units were used to measure the acceleration and angular velocity of each subject at the S1, T10, C7, and head. Inertial measurement units were also mounted on the ATV and table. The ATV was instrumented with 32 force sensors to sense reaction forces applied to the machine at the feet, hands, saddle, and seat. A five-camera motion capture system was used to capture posture throughout the time series of each terrain. Data were reviewed and analyzed at a finer resolution of roughly three seconds in select subjects at moments when there were peaks in both force and acceleration. Analysis showed that a cycle of instability and stability existed as moments of sudden input accelerations caused reactions in the spine and head. Further, component accelerations were analyzed to identify the acceleration of the subject relative to the ATV. The cycle and the time from input acceleration to bracing on the ATV to regain stability serve as a baseline for the subject’s reaction to input acceleration and the time between input acceleration and regaining stability.

Computation predictions of organic crystal structure and thermodynamics are essential for material design, crystal engineering and drug development. However, accurate computational tools for organic crystal thermodynamics calculations are lacking, and experimental data set for validation of computational methods is limited. Most crystal structure predictions and stability calculations depend solely on potential energy, which is an insufficient representation of thermodynamics. This thesis proposes and validates both absolute and relative free energy calculation of small organic compounds, thus presenting an accurate computational tool that overcomes the shortcomings of potential-energy-based models. The solubility of organic molecules can be computed from a thermodynamic cycle that decomposes standard state solubility into the sum of solid-vapor sublimation, i.e. thermodynamic stability of the crystal, and vapor-liquid solvation free energies ΔG°solubility=ΔG°sub+ΔG°solv. Crystal polymorphs have different ΔG°sub thus different solubility, which of critical importance to the pharmaceutical industry, however, robust computational methods to predict this quantity from first principles are lacking. Over the past few decades, alchemical simulation methods to compute solvation free energy using classical force fields have become widely used. However, analogous methods for determining the free energy of the sublimation/deposition phase transition are currently limited. This thesis describes an absolute thermodynamics approach based on growth of the asymmetric unit into a crystal via alchemy (GAUCHE). GAUCHE computes deposition free energy ΔG°dep=-ΔG°sub=ΔG°Vol+ΔG°Au+ΔG°Au→UG as the sum of an entropic term to account for compressing a 1 M vapor into the molar volume of the crystal asymmetric unit (VAU) plus two simulation steps. In the first simulation step, the deposition free energy ΔG°AU for a system composed of only NAU asymmetric unit (AU) molecule(s) is computed beginning from an arbitrary conformation in vacuum. In the second simulation step, the change in free energy ΔG°AU�UG to expand the asymmetric unit degrees of freedom into a unit cell (UC) composed of NUC independent molecules is computed. This latter step accounts for the favorable free energy of removing the constraint that every symmetry mate of the asymmetric unit has an identical conformation and intermolecular interactions. The current work is based on NVT simulations, which requires knowledge of the crystal space group and unit cell parameters from experiment, but not a priori knowledge of crystalline atomic coordinates. GAUCHE was applied to 5 organic molecules whose sublimation free energy has been measured experimentally, based on the polarizable AMOEBA force field and more than a microsecond of sampling per compound. The mean unsigned and root-mean-square errors were only 1.6 and 1.7 kcal/mol, respectively, which indicates that GAUCHE is capable of accurately predicting sublimation thermodynamics. For polymorphic systems, we propose a relative thermodynamics approach, that is similar to the second simulation step of GAUCHE, where ΔG°P1→P2 is calculated instead of ΔG°AU→UG. A relative approach reduces statistical uncertainty upon convergence compared to an absolute calculation; thus, it is more appropriate due to the thermodynamic stability difference between polymorphs are often fairly small. For our paracetamol test system, the experimental free energy difference was only 0.93 kcal/mol. Although both quantum and AMOEBA potential calculations predict the form II of paracetamol as more stable crystal form than form I, our relative free energy calculation predict the opposite stability ranking, which agrees with the experiment. Decomposition of free energy into entropy and enthalpy indicates that the favorable entropy change contributes to the greater thermodynamic stability of form I over form II. Although the exact magnitude of entropy and enthalpy changes differs across literature data as well as our data, the favorable entropic contribution is consistent. Further calculations over the temperature range from 100 to 308 K show the temperature dependence of free energy, which follows the parabolic trend observed in experiments. Our results show that our relative polymorph stability methods can accurately capture temperature dependence of free-energy-based stability ranking and overcome the limitations of potential-energy-based ranking. Thus, the importance of crystal structure predictions based on free energy can be further emphasized.

Obstacles to the realization of polymer scaffolds to be used for cell replacement therapies often include the challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization (TPP) of acrylated poly(caprolactone) (PCL) offers a means of achieving precise microstructural control of a material in a biocompatible and biodegradable platform. Two obstacles inhibit the efficient development and research of this method: there is not a commercially available acrylated PCL and the fabrication time using TPP can be lengthy. Initially in this work, TPP parameters and molecular structures were varied to find the ideal relationship for our prototype. Increasing the concentration of reactive groups, either by increasing number of acrylate groups per molecule or decreasing molecular weight, was found to increase the similarity of the scaffold to the computer generated model. Sub-retinal implantation of TPP PCL scaffolds in a porcine model of retinitis pigmentosa was well tolerated, and did not cause inflammation, infection, or local or systemic toxicity after one month. However, this acrylated PCL was not as pure as originally thought; there were remaining reactants present that prevented accurate quantification of reaction success. Without the ability to measure the number of acrylates that were bonded to the molecule we could not ensure that batches were consistent, preventing a standard to be developed for quality and control. A method to purify the products of our PCL synthesis was delineated; a series of washes and separations with concentrated sodium bicarbonate successfully purified the PCL. The more thoroughly the phases were combined during these washes, the more effectively the reactant was removed. To address the time scale issues of TPP, the implementation of a two-dimensional photo-masked (PM) polymerized PCL film was adopted. Since films are faster and simpler to make, they can be developed and tested first and used as a predicate device for FDA approval of TPP PCL scaffolds. Therefore, the PM scaffolds were formulated similarly to the TPP materials and then the minimum intensity of light and time necessary to polymerize a film was determined. Time and light intensity displayed an inverse relationship, and the films at each intensity and exposure require more research into their quality and elastic modulus . A representative film was selected, seeded with dissociated retinal organoids, and, after one week, the cells that successfully adhered to the scaffold maintained their neuronal lineage. The density of cells seeded using 16 retinal organoids was estimated and will provide insight into how many organoids to use in the future. These results represent an important step towards understanding how photopolymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications.