Toward an Understanding of How Hypercapnia Affects Apoptosis in Human Promyeloblasts in 3D Suspension Culture Systems

Stem cells have more recently attracted interest in scientific research and their use is increasingly developing in many therapeutic applications. A great deal of cell-based work is conducted at 5% CO2 concentration and is predominantly cultured on two-dimensional surfaces. These methods are straightforward and of low economic costs but do not necessarily replicate the microenvironment; also, evidence to provide justification for selecting this specific concentration of CO2 in hematopoietic progenitor cell (KG-1a) proliferation studies is lacking. Our work herein pursues to prove the following hypotheses: (a) Hypercapnia decelerates proliferation rate but increases overall expansion of KG-1a cells without disturbing cell surface integrity, (b) Apoptosis of KG-1a cells is delayed under the effect of hypercapnia in both 2D and 3D culture systems, (c) KG-1a cells can successfully proliferate within a 3D bioreactor system to yield higher cell concentrations than 2D systems. This work was successful in proving that in both 2D and 3D culture systems, hypercapnia decelerates proliferation rate but increases overall expansion of KG-1a without disturbing cell surface integrity in comparison to hypocapnic and control populations. Also, we established that the apoptotic capacity of these KG-1a cells is delayed under the effect of hypercapnia in both 2D and 3D culture systems. And finally, this work has demonstrated that KG-1a cells can successfully proliferate within a 3D bioreactor system to yield comparable cell concentrations to traditional 2D systems. This study has succeeded in adding to the current body of knowledge that exists on hematopoietic stem cell expansion and it may potentially provide the foundation to enhancing proliferation systems of hematopoietic stem cells and therefore contribute to resolving numerous diseases

Co-registered photoacoustic and ultrasound tomographic imaging of human colorectal and ovarian cancer: light delivery, system development, and clinical study

Ovarian cancer remains the deadliest of all the gynecological malignancies. Conventional screening tests, including pelvic examination, transvaginal ultrasound (TVUS), and blood testing for cancer antigen 125 (CA-125), lack sufficient specificity for early ovarian cancer diagnosis. Imaging modalities such as computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI) have been used for surgical guidance. However, all of these modalities have limitations in detecting small lesions. Globally, colorectal cancer is the second most commonly diagnosed malignancy and the fourth most common cause of cancer mortality. Accurate staging and post-treatment surveillance of this prevalent disease are critical because treatment strategies are predicated upon the stage at presentation and the patient’s response to pre-surgical therapy – in some instances, detailed imaging allows certain patients to avoid surgery altogether. While colonoscopy and biopsy are the gold-standard diagnostic tests for colorectal cancers, multiple imaging modalities are also utilized, including optical imaging, endoscopic ultrasound (EUS), pelvic magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). Unfortunately, each of these modalities has critical weakness in evaluating colorectal tumors. In staging colorectal tumors and evaluating their therapeutic response, more precise imaging modalities could transform the standard of care. This dissertation explores co-registered photoacoustic and ultrasound tomographic imaging of two fatal cancers: ovarian cancer and colorectal cancer. It is composed of three main parts: light delivery optimization/fiber optics, system development and optimization, and pilot patient and sample study. To begin, we designed, optimized, and evaluated an hand-held photoacoustic and ultrasound probe suitable for endo-cavity subsurface tumor imaging. Compared to previous designs, the prototype probe, consisting of four 1 mm multi-mode optical fibers attached to 1.5 mm diameter ball-shaped fiber tips sandwiched between a transvaginal ultrasound transducer and a custom-made sheath, demonstrated a higher light output and better beam homogeneity on the tissue subsurface. Second, we developed a novel fiber diffuser tip using microspheres dispersed within an ultraviolet (UV) adhesive to scatter light. This diffuser keeps the skin surface fluence under the maximum permissible exposure (MPE), while enabling higher laser energy injection to enhance the photoacoustic (PA) signal generated from the tissue. Third, we proposed an improved beamformer, named lag-based delay multiply and sum combined with coherence factor (DMAS-LAG-CF). Simulations and phantom experiments demonstrate that compared with conventional delay and sum (DAS), the proposed algorithm can provide 1.39 times better resolution and 10.77 dB higher contrast. For patient data, similar improvements in contrast ratios have been observed. However, since diagnostic accuracy in distinguishing between cancer and benign/normal groups is the significant measure, we have extracted the photoacoustic histogram features of mean, kurtosis, and skewness. When mean and skewness are used as features, DMAS-LAG-CF can improve cancer diagnosis, with an AUC of 0.91 in differentiating malignant from benign ovarian lesions. Fourth, to investigate the ability of co-registered photoacoustic and ultrasound tomographic imaging to assess human colorectal cancer, we conducted a pilot study on 23 ex-vivo human colorectal tissue samples that were collected immediately after surgical resection. Co-registered photoacoustic images of malignancies showed significantly increased PAT signals compared to normal regions of the same sample. We found statistically significant differences between untreated colorectal tumors and normal tissues, based on the quantitative relative total hemoglobin concentration (rHbT) computed from four optical wavelengths, spectral features such as the mean spectral slope and 0.5 MHz intercept extracted from PAT and US spectral data, and image features such as the first and second order statistics along with the standard deviation of the mean radon transform of PAT images. Using either a logistic regression model or a support vector machine, the best set of parameters of rHbT and PAT intercept achieved AUC values of 0.97 and 0.95 for the training and testing data sets, respectively, in predicting histologically confirmed invasive carcinoma. One limitation of the current system is its poor image resolution (~ 250 μm axial resolution) limited by the commercial endo-cavity ultrasound transducer array (6 MHz central frequency, 80% bandwidth). For better image resolution in high frequency PAT/US imaging, we first decoded the pin configuration of a high-frequency transducer array (15 MHz central frequency, 9-18 MHz bandwidth) and adapted it to our home-made 128 channel ultrasound pulsing and receiving system (sampling rate: 40 MHz). To further improve the performance, we are building a 64-channel FPGA-based high frequency photoacoustic imaging system with a sampling rate of 80 MHz and signal-to-noise ratio (SNR) of 40 dB. For in-vivo patient study, this system will be integrated with an endo-rectal probe with a side-firing fiber tip. Fifth, we investigated the laser safety of photoacoustic imaging, in preparation for its use in clinical reproductive medicine. Using preimplantation mouse blastocyst stage embryos, we assessed potential DNA damage from photoacoustic laser exposure. Different embryo groups were exposed to either 5- or 10- minute 15-Hz laser doses (typical clinical doses), or a 1-minute 1-kHz laser dose (a significantly higher dose). We demonstrated that typical lasers and exposure times used for photoacoustic imaging do not induce increased cell death in mouse blastocysts. Sixth, we demonstrated a novel fiber endface photoacoustic generator using IR 144 laser dye dispersed within an ultraviolet (UV) adhesive. The generator provides wide acoustic bandwidth in the transducer frequency range of 2-7 MHz, high thermal conversion efficiency (\u3e 90%), good PA intensity controllability (via the easily controlled IR 144 concentration), and high feasibility (simple procedures). Through a series of experimental validations, we show this fiber-based endface photoacoustic generator can be a useful tool for a broad range of biomedical applications, such as calibrating the local absorption coefficient of biological tissue for quantitative photoacoustic tomography

Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer

Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer

Methods and Standards for the Analysis and Imaging of Latent Fingerprints and Trace Contraband using Ambient Ionization Mass Spectrometry and Secondary Ion Mass Spectrometry

A recent report from the National Academy of Sciences (NAS), evaluating the state of forensic science, identified the need to more rapidly, accurately, and reproducibly provide scientifically validated forensic analyses of evidence to support the criminal justice system. The NAS report also highlighted the need for the forensic science community to collaborate with universities and national laboratories, as well as to forge relationships with the National Institute of Standards and Technology (NIST) to address method development, validation, and evaluation of new analytical techniques of relevance to forensic science. This thesis was completed as part of a unique collaboration between the University of Maryland (UM), the National Institutes of Standards and Technology (NIST) and the Defense Forensic Science Center (DFSC) to address several of the existing research needs in current forensic science practice while meeting the requirements for a Department of Defense (DOD) Science Mathematics and Research for Transformation (SMART) fellowship and supporting NIST efforts in trace contraband detection. Two distinct areas of research were pursued. First, studies were conducted on method development and validation for the detection of explosives using four different ambient ionization mass spectrometry (AI-MS) techniques relevant to routine casework at DFSC and to other federal labs that screen for trace contraband. Additional method validation studies for the detection of adulterants in beverages and the analysis of bank dye are also presented. All methods were developed in accordance with the requirements specified by the International Organization of Standardization (ISO) 17025 guidelines, which is the accreditation standard for practicing forensic laboratories. The second track of this thesis involved exploration of emerging analytical methods, and several novel applications, for mass spectrometry based chemical imaging of both endogenous and exogenous components in latent fingerprints. This work was driven by a recent National Science Foundation (NSF) report that identified mass spectral imaging (MSI) as a key goal for the future development of forensic science. Both AI-MS and secondary ion mass spectrometry (SIMS) techniques were utilized and evaluated for their ability to chemically image fingerprints. To support these studies, a novel standard fingerprint test material was also developed during this research. As a result of this work there are now validated methods for the screening of trace explosives, as well as other types of forensic evidence such as adulterants and bank dye, that can be reliably employed into the casework scheme. Also, there are new applications and capabilities for MSI of fingerprints and an artificial fingerprint material that allows for the reproducible deposition of test fingerprints

Inferring structures, free energy differences, and kinetic rates of biological macromolecular assemblies by integrative modeling

Biological macromolecular assemblies play crucial roles in most cellular processes. The determination of their structures, thermodynamics, and kinetics is essential to understand their function, evolution, modulation, and design. Determining such models, however, remains challenging. One particularly powerful approach to constructing models in general is integrative modeling. Integrative modeling aims to maximize the accuracy, precision, and completeness of models, by simultaneously utilizing all available information, including experimental data, physical principles, statistical analyses, and other prior models. The goal of this thesis is to expand the scope of integrative modeling to the inference of spatial, thermodynamic, and kinetic aspects of macromolecular assemblies. In Chapter I, I introduce the integrative modeling framework for spatiotemporal modeling of biological macromolecular assemblies. In Chapter II, I demonstrate how the synergy between multi-chemistry cross-linking mass spectrometry and integrative modeling can map the structural dynamics of macromolecular assemblies, by application to the human Cop9 signalosome complex. In Chapter III, I present a method for determining structures, free energy differences, and kinetic rates of macromolecular assemblies along their functional cycle, mainly from negative stain electron microscopy (EM). We apply the method to the yeast Hsp90 to estimate the free energy differences and kinetic parameters along its nucleotide hydrolysis cycle, which includes open and closed states of Hsp90. In Chapter IV, I describe a validation of stochastic sampling in integrative modeling. The remaining chapters describe applications of integrative modeling to assemblies of various sizes and scales, using various sources of information, thus illustrating the flexibility of the integrative modeling approach. Specifically, I apply integrative modeling to the human ECM29-Proteasome assembly under oxidative stress (Chapter V), the yeast nuclear pore complex (NPC) cytoplasmic mRNA export platform (Chapter VI), the major membrane ring component of the yeast NPC (Chapter VII), the entire yeast NPC (Chapter VIII), and the reconstruction of 3D structures of MET antibodies (Chapter IX)

Armchair geography : speculation, synthesis, and the culture of British exploration, c.1830-c.1870

This thesis recovers the practice of ‘armchair geography’ as an overlooked, yet significant aspect of the mid-nineteenth-century culture of exploration. These histories are popularly associated with such famed explorers as Dr David Livingstone and John Hanning Speke, who travelled across Africa. Yet, far from the field, there were other geographers, like William Desborough Cooley and James MacQueen, who spoke, wrote, theorised, and produced maps about the world based not on their own observations, but on the collation, interpretation, speculation, and synthesis of existing geographical sources. The dominant historical trope of geography through the nineteenth century is one of transition, shifting from an early modern textual practice of the ‘armchair’ to a modern science in the ‘field’. This thesis challenges such a limited view by demonstrating how critical practices continued to be a pervasive presence in the period 1830–1870, and how these two modes of geography co-existed and overlapped, and were combined and contested. It seeks to dismantle the static binarism that positions the critical geographer as both separate and in opposition to the field explorer. The chapters move to survey explorers that sit; explorers that read; critical geographers that move; books that travel; and libraries that lay out the world. In so doing, it identifies and attends to the unsettled physical and spatial boundaries between modes and methods of geography. It examines the role of the ‘armchair geographer’ in developing geographical thought and practice, and in negotiations concerning credible knowledge at the newly founded Royal Geographical Society. Crucially, this thesis expands the history of ‘armchair’ practices in geography beyond an entertaining tale of ‘conflict’ in exploration, and presents a critical examination of the many spatial manifestations of the ‘field’ and ‘fieldwork’ in geography’s disciplinary identity. This thesis contributes a spatially sensitive account of geographical knowledge making that interrupts and challenges current histories of the development of geography as a field of knowledge and set of practices in the nineteenth century