Gene expression, which connects genomic information to functional units in living cells, has received substantial attention since the completion of The Human Genome Project. Quantitative characterization of gene expression will provide valuable information for understanding the behavior of living cells, and possibilities of building synthetic gene circuits to control or modify the behavior of naturally occurring cells. Many aspects of quantitative gene expression have been studied, including gene expression dynamics and noise in E. coli. The gene expression process itself is stochastic, and modelling approaches have been broadly used to study gene expression noise; however, stochastic gene expression models are usually large and time intensive to simulate. To speed up simulations, we have developed a systematic method to simplify gene expression models with fast and slow dynamics, and investigated when we can ignore the gene expression from the background genome when modelling the gene expression from plasmids. When modelling the noise in gene expression, one usually neglected aspect is the slow maturation process of fluorescent proteins, necessary for the protein to give out fluorescence after it is produced. By modelling, we show that the maturation steps can bring large changes to both the mean protein number and the noise in the model. An unstudied aspect of gene expression dynamics is the time dependent gene expression behavior in E. coli batch culture. Contrary to the usual assumption, we have found, in E. coli batch culture gene expression, that there is no steady state in terms of both the mean number of proteins and the noise. Negative feedback is thought to be able to reduce the noise in a system, and experiments have shown that negative feedback indeed suppresses the noise in gene expression, but the modelling shows that negative feedback will increase the noise. We have found that the increase of noise by feedback is due to the exclusion of extrinsic noise from the model, and that negative feedback will suppress the extrinsic noise while increasing the intrinsic noise. Living cells are crowded with macromolecules, which will, predicted by modelling, make the reaction constant time dependent. Our experimental observation has confirmed this prediction.

Elucidating the structure-function relationships of membrane proteins is critical for the design of therapeutic agents to treat disease and for understanding numerous cellular processes such as signal transduction and molecular or ion transport. Recent advances in the application of correlated single molecule imaging techniques have provided new insights into protein-protein and protein-membrane interactions. To demonstrate the potential of these approaches, we have used in situ atomic force microscopy and single molecule fluorescence microscopy to characterize the interactions between membrane receptors and their soluble ligands, examine the monomer-dimer equilibrium in a family of adhesion receptors, and elucidate protein-mediated membrane restructuring of a supported lipid bilayer. Building on these studies, we examined the CorA ion transporter protein. We demonstrated single molecule resolution of reconstituted CorA molecules in supported lipid bilayers using a correlated AFM-TIRF microscopy platform. This approach provided new insights into a purported mechanism of CorA activation that involved ion binding.

Elucidating the structure-function relationships of membrane proteins is critical for the design of therapeutic agents to treat disease and for understanding numerous cellular processes such as signal transduction and molecular or ion transport. Recent advances in the application of correlated single molecule imaging techniques have provided new insights into protein-protein and protein-membrane interactions. To demonstrate the potential of these approaches, we have used in situ atomic force microscopy and single molecule fluorescence microscopy to characterize the interactions between membrane receptors and their soluble ligands, examine the monomer-dimer equilibrium in a family of adhesion receptors, and elucidate protein-mediated membrane restructuring of a supported lipid bilayer. Building on these studies, we examined the CorA ion transporter protein. We demonstrated single molecule resolution of reconstituted CorA molecules in supported lipid bilayers using a correlated AFM-TIRF microscopy platform. This approach provided new insights into a purported mechanism of CorA activation that involved ion binding.

Haematopoiesis requires strict regulation in order to maintain a balanced production of the various blood cell components. Escape from this regulation contributes to the development of cancers such as leukemia. SOCS2 is a member of the Suppressor of cytokine signalling (SOCS) family, and normally functions as a negative regulator of the JAK/STAT pathway. I examined gene expression and promoter methylation in acute myeloid leukemia (AML) cell lines and patient samples. SOCS2 expression was quite variable in AML patients, and very low in acute promyelocytic leukemia (APL) patients. Promoter hyper-methylation was found in these patients, particularly those with high white blood cell count and a FLT3-ITD. I speculate that SOCS2 interacts with an aspect of the signalling complex to inhibit cell growth in these patients, and silencing SOCS2 is necessary for leukemia progression. Treating these patients with a de-methylating agent, such as decitabine, may show promise in the clinic.

The nematode Caenorhabditis elegans possesses a relatively simple nervous system of only 302 neurons, but is able to perform an impressive range of complex behaviors. This dissertation aims to understand the neurobiology of behavior by quantifying, at the systems-level, the sensorimotor response to carefully controlled stimuli. Through neuronal or genetic perturbations to the system, we can begin to uncouple the behavior from the underlying circuitry. The behavior studied here is thermal nociception, an escape response designed to protect an organism from potential tissue damage or harm from noxious heat. Vertebrates and invertebrates alike possess sensory neurons called nociceptors that detect noxious stimuli and relay the stimulus information to elicit an appropriate escape response. C. elegans is known to perform a reversal or forward response when presented with noxious stimuli at the head or tail, respectively. In this work, we develop a novel thermal stimulus assay with precise spatiotemporal control of an infrared pulse that targets small regions along the worm to spatially dissect the noxious response. We comprehensively quantify the nociceptive behavior, and identify key metrics that scale with intensity, such as speed in the escape state and the probability of certain behavioral states after the stimulus. Furthermore, we have mapped the behavioral receptive field of the worm along its body, and show a previously unreported probabilistic midbody behavior distinct from the head and tail responses. Surprisingly, the worm is able to differentiate localized stimuli at the midbody that are as close as 80 microns. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. This suggests PVD could be used as a model to study spatial discrimination at the level of a single nociceptor. This spatial specificity further extends to pharmacological perturbations of the system. In particular, the application of clinically used painkillers to the worm results in a knockdown of this nociceptive response, but does so in a spatially specific manner. These results are promising for future studies building upon the techniques developed here, as they evidentiate the use of C. elegans as a model organism to study pain.

The nematode Caenorhabditis elegans possesses a relatively simple nervous system of only 302 neurons, but is able to perform an impressive range of complex behaviors. This dissertation aims to understand the neurobiology of behavior by quantifying, at the systems-level, the sensorimotor response to carefully controlled stimuli. Through neuronal or genetic perturbations to the system, we can begin to uncouple the behavior from the underlying circuitry. The behavior studied here is thermal nociception, an escape response designed to protect an organism from potential tissue damage or harm from noxious heat. Vertebrates and invertebrates alike possess sensory neurons called nociceptors that detect noxious stimuli and relay the stimulus information to elicit an appropriate escape response. C. elegans is known to perform a reversal or forward response when presented with noxious stimuli at the head or tail, respectively. In this work, we develop a novel thermal stimulus assay with precise spatiotemporal control of an infrared pulse that targets small regions along the worm to spatially dissect the noxious response. We comprehensively quantify the nociceptive behavior, and identify key metrics that scale with intensity, such as speed in the escape state and the probability of certain behavioral states after the stimulus. Furthermore, we have mapped the behavioral receptive field of the worm along its body, and show a previously unreported probabilistic midbody behavior distinct from the head and tail responses. Surprisingly, the worm is able to differentiate localized stimuli at the midbody that are as close as 80 microns. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. This suggests PVD could be used as a model to study spatial discrimination at the level of a single nociceptor. This spatial specificity further extends to pharmacological perturbations of the system. In particular, the application of clinically used painkillers to the worm results in a knockdown of this nociceptive response, but does so in a spatially specific manner. These results are promising for future studies building upon the techniques developed here, as they evidentiate the use of C. elegans as a model organism to study pain.

Master of Science / Department of Physics / Robert Szoszkiewicz / For proteins in living cells, forces are present at all levels. These range from macroscopic to single molecule levels. Single molecule atomic force microscopy (AFM) in force extension (FX) and force clamp (FC) modes can investigate the mechanical properties of proteins, for example, forces at which proteins unfold, or the kinetics of these processes. In the FX-AFM experiments, proteins are pulled at constant velocity, while in FC-AFM experiments, proteins are pulled at constant force. This thesis describes i) how a single molecule FX/FC-AFM was constructed using various components, ii) how it was calibrated and tested using (I27)4 polyprotein, and iii) how it was applied to the studies of a Notch construct. Building up the single molecule FX/FC-AFM system opened a path to investigate the mechanical properties of proteins. Such a system was tested on a known protein construct, hence the usage of the (I27)4 polyprotein. The Notch protein is a signaling protein that plays a role in triggering breast cancer. It is believed that understanding the mechanical properties of Notch can help to understand its oncogenic functions. We have successfully constructed and calibrated the FX/FC-AFM setup. It was found that the AFM worked for the standard calibration protein of (I27)4. The results on a Notch construct revealed our ability to see some conformational transition state in this molecule under force. These results opened a path for further investigations of a Notch construct at various physiologically relevant conditions.

Master of Science / Biochemistry and Molecular Biophysics / John M. Tomich / NC-1007 (CK₄-M2GlyR) (PARVGLGITTVLTMTTQSSGSRAKKKK) is a synthetic peptide modeled after the second transmembrane segment of the spinal cord glycine receptor’s α-subunit, and has demonstrates the capacity to oligomerize to form transmembrane channels with Cl[superscript]- permselectivity. While studies into the effects of truncation on both CK[subcript]4 (C-terminal tetra-lysl adducted) and NK[subscript]4 (N-terminal tetra-lysl adducted) led to more control over solution aggregation in the NK[subscript]4 variant, the work presented explore whether C-terminal sequential substitutions with a tryptophan residue could similarly stabilize the aqueous structure in monomeric form or further define the pore registry in such a way as to promote an increase ion permeability. Tryptophan was substituted for amino acids in the 18[superscript]th, 19[superscript]th, 20[superscript]th, and 21[superscript]st positions of the peptide sequence (SSGS, respectively), and changes in aggregation profiles, secondary structure, and channel ion permeability were observed. Synthesized peptides show circular dichroism spectral profiles indicating that the studied tryptophan substitutions did not result in a reduction of the characteristic helicity of the peptide; however, the tryptophan substitution also did little to decrease solution aggregation as demonstrated by comparative studies by reverse-phase high- performance liquid chromatography. All peptides demonstrated channel activity, directly measured by recordings of transepithelial short-circuit current. with profiles that suggest trends in electrostatic interactions and membrane registry relative to substitution position. One peptide in particular, NC-1007 S21W displayed atypical activity, which could not be effectively described by the standard Hill-based model but may be indicative of an ill-defined registry due to the substituted peptide’s proximity to another strongly pore-defining residue. Further studies in the effects of sequence modification to channel-forming peptides will elucidate how sequences may be altered to optimize synthetic peptide solubility, resistance to in-solution aggregation, and ability to form selective and permeable ion channels. The understanding gained from this study will improve our ability to develop peptides that could serve as a therapeutic treatments for a number of endogenous channelopathies.

Farber disease is a rare lysosomal storage disease (LSD) caused by a deficiency of acid ceramidase (AC). Patients show a classic triad of symptoms including subcutaneous granulomas, laryngeal involvement and painful swollen joints. The most common and severe form has neurological manifestations and patients typically die by the age of two. Current treatment consists of symptomatic supportive care and allogeneic bone marrow transplantation (BMT). However, BMT has shown limited success. Gene therapy has previously been shown to be a promising treatment strategy for monogenetic diseases and has the potential to treat the underlying cause of the disease. Presented here is the first report of in vivo testing of retrovirus-mediated gene therapy strategies for the treatment of Farber disease. Retroviral vectors were engineered to overexpress AC and a cell surface marker, human CD25. Transduction with these viral vectors corrected the enzymatic defect in Farber patient cells and in vivo administration of the lentiviral vector led to long-term expression of the marking transgene as well as increased AC expression in the liver. To determine the effect of over-expression of AC, human CD34+ cells were transduced and transplanted into NOD/SCID animals. It was found that transgene-expressing cells could reconstitute the host. To address the neurological manifestations of Farber disease, vascular endothelial growth factor (VEGF) was investigated as an agent to transiently open the blood brain barrier for entry of lentivirus. It was found that in addition to increasing the amount of therapeutic virus in the brain, VEGF treatment also increased transduction in other organs. Further, to address the concerns of insertional mutagenesis associated with using integrating vectors, an immunotoxin-based strategy was developed as a safety system to clear transduced cells. It was found that a CD25-targeted immunotoxin could eliminate both transduced hematopoietic cells as well as tumor cells over-expressing CD25. This strategy can be employed following gene therapy should an unwanted proliferative event occur. Together, these studies represent considerable advances towards the development of a cure for Farber disease, demonstrating both therapeutic potential and also containing a built-in safety system.

Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.