Oesophageal cancer is the eight most common cancer and the sixth leading cause of deaths worldwide. There are two major histological subtypes of oesophageal cancer, with the most predominant subtype in Europe and the USA being oesophageal adenocarcinoma (OAC). The standard of care for OAC patients with locally advanced disease is neoadjuvant therapy and surgical resection. Unfortunately, ~70% of patients do not respond to neoadjuvant therapy and non-responders gain no benefit from the aggressive treatment regimen whilst compromising their quality of life. There is an unmet clinical need for biomarkers predicative of patient's response to neoadjuvant chemoradiation therapy (neo-CRT). In a pre-treatment setting, predictive biomarkers indicative of patient response could enable the stratification of patients and would ensure individual patients receive the most effective treatment. However, the greater clinical benefit for patients may come from the development of new or combination therapeutics for OAC. Novel chemosensitising and radiosensitising therapeutics could be administered with neo-CRT to enhance tumour sensitivity, improve CRT efficacy and increase survival rates for OAC patients. MicroRNAs (miRNA/miRs) are short non-coding RNA that function to regulate gene expression at the post-transcriptional level. A single miRNA can potentially target hundreds or thousands of mRNA and subsequently alter the expression of multiple genes and proteins. As essential regulators of gene expression, miRNA are involved in all cellular processes and are dysregulated in cancer and other diseases. Furthermore, miRNA have been identified as predictors and modifiers of tumour sensitivity to chemotherapy and radiotherapy in numerous cancer types. Playing a causal role in disease development and progression, miRNA are promising biomarkers and therapeutic targets. In this study miRNA were investigated as biomarkers of OAC patient response to neo-CRT and as potential therapeutic targets through which to enhance tumour sensitivity to neo-CRT. In pre-treatment OAC biopsies, 67 miRNA that were differentially expressed between responders and non-responders to neo-CRT were identified. MiR-330-5p and miR-187 were downregulated in the pre-treatment biopsy samples of the neo-CRT non-responders. The functional roles of miR-330-5p and miR-187 were investigated as modulators of tumour sensitivity to CRT. In vitro the silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to radiation. Furthermore, silencing miR-330-5p altered the expression of extracellular proteases and protease inhibitors, including MMP1. In vivo a pilot study indicated miR-330-5p silencing enhanced tumour growth and may alter tumour sensitivity to cisplatin. In vitro miR-187 overexpression enhanced cellular sensitivity to radiotherapy and cisplatin, implying that the downregulated miR-187 expression in the non-responders may confer resistance to CRT. Furthermore, miR-187 induced apoptosis in vitro and induction of apoptosis is a potential mechanism by which miR-187 enhances radiosensitivity. MiR-187 altered the expression of genes encoding extracellular proteins, including C3 and other immune related genes. Both miR-330-5p and miR-187 are potential regulators of the secretome, thus emphasising the role of miRNA as modulators of the tumour microenvironment. This study has identified miR-330-5p and miR-187 as potential therapeutic targets that could augment OAC tumour sensitivity to neo-CRT.

Cardiovascular diseases remain a leading cause of morbidity and mortality in the world, despite advances in drug and therapeutic developments. The complex nature of cardiac diseases such as myocardial infarction and heart failure require innovative approaches to elucidate disease mechanisms, identify molecular targets and develop novel therapies. The advent of human pluripotent stem cell (hPSC) technologies allowed for robust and reliable generations of contracting human cardiomyocytes (CMs) in vitro. hPSC-CMs hold great promise for a broad range of research and clinical applications including studying myocardial physiology, modeling cardiac diseases, and transplanting healthy cells to repair the damaged heart. However, one major limitation of hPSC-CMs differentiated in vitro is that they are relatively immature and resemble embryonic CMs. These cells lack well defined cellular edges and mature sarcomeres, which makes it difficult to quantitatively assess contractile functions using traditional edge detection technologies. In addition, hPSC-CMs cultured in traditional glucose rich media lack metabolic and functional maturity, utilizing mainly glycolysis for energy production, similar to the embryonic heart. To address these limitations, we first devised a novel technology to simultaneously quantify hPSC-CMs’ contractile kinetics, force generation and electrical activities at the single cell resolution. This methodology allowed us to examine the impact of energy substrates and metabolic pathway utilization on CM physiology and function. We identified that Hypoxia Inducible Factor 1 alpha (HIF1α) and its transcriptional target Lactate Dehydrogenase A (LDHA) are aberrantly upregulated in hPSC-CMs cultured in traditional glucose rich media. By using small molecules and siRNA, we demonstrated that inhibition of HIF1α/LDHA shifts hPSC-CMs’ metabolism from glycolysis to oxidative phosphorylation, which resulted in improved CM structural and functional maturation. Furthermore, we investigated the energy substrate dependency of hPSC-CMs in response to in vitro hypoxic and ischemia-reperfusion injuries. We observed that hPSC-CMs cultured in glucose rich media lack physiological responses to hypoxic insults. On the other hand, in vitro coverslip ischemia-reperfusion resulted in CM death and apoptosis, independent of glucose cultures. These findings highlighted the importance of bioenergetics in modeling cardiac diseases in vitro and provided us with the basis for a potential drug screening platform using hPSC-CMs. / 2020-07-02T00:00:00Z

The completion of the Human Genome Project has increased the need for investigation of genetic sequences and their biological functions, which will significantly contribute to the advances in biomedical sciences, human genetics and personalized medicine. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an attractive option for DNA analysis due to its high accuracy, sensitivity and speed. In the first part of the thesis, we report the design, synthesis and evaluation of a novel set of mass tagged, cleavable biotinylated dideoxynucleotides (ddNTP-N3-biotins) for DNA polymerase extension reaction and its application in DNA sequencing and single nucleotide polymorphism (SNP) genotyping by mass spectrometry. These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute large mass differences among the nucleotides to increase resolution in MS analysis. It has been demonstrated that these modified nucleotides can be efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides can be captured by streptavidin coated beads and efficiently released using tris(2-carboxyethyl) phosphine in aqueous solution which is compatible with DNA and downstream procedures. Reversible solid phase capture (SPC) mass spectrometry sequencing using ddNTP-N3-biotins was performed, and various DNA templates, including biological samples, were accurately sequenced achieving a read-length of 37 bases. In mass spectrometric SNP genotyping, we have successfully exploited our reversible solid phase capture (SPC)-single base extension (SBE) assay and been able to detect as low as 2.5% heteroplasmy in mitochondrial DNA samples, with interrogation of human mitochondrial genome position 8344 which is associated with an important mitochondrial disease (myoclonic epilepsy with ragged red fibers, MERRF); we have also quantified the heteroplasmy level of a real MERRF patient and determined several mitochondrial MERRF mutations in a multiplex approach. These results demonstrated that our improved mass spectrometry genotyping technologies have great potential in DNA analysis, with particular applications in sequencing short-length targets or detecting SNPs with high accuracy and sensitivity requirements, such as DNA fragments with small indels, or SNPs in pooled samples. To truly implement this mass spectrometry-based genotyping method, we further explored the use of a lab-on-a-chip microfluidic device with the potential for high throughput, miniaturization, and automation. The microdevice primarily consists of a micro-reaction chamber for single base extension and cleavage reactions with an integrated micro heater and temperature sensor for on-chip temperature control, a microchannel loaded with streptavidin magnetic beads for solid phase capture, and a microchannel packed with C18-modified reversed-phase silica particles as a stationary phase for desalting before MALDI-TOF analysis. By performing each functional step, we have demonstrated 100% on-chip single base incorporation, sufficient capture and release of the biotin-ddNTP terminated single base extension products, and high sample recovery from the C18 reverse-phase microchannel with as little as 0.5 pmol DNA molecules. The feasibility of the microdevice has shown its promise to improve mass spectrometric DNA sequencing and SNP genotyping to a new paradigm. DNA sequencing by synthesis (SBS) appears to be a very promising molecular tool for genome analysis with the potential to achieve the $1000 Genome goal. However, the current short read-length is still a challenge. Therefore, the second part of the thesis focuses on strategies to overcome the short read-length of SBS. We developed a novel primer walking strategy to increase the read-length of SBS with cleavable fluorescent nucleotide reversible terminators (CF-NRTs) and nucleotide reversible terminators (NRTs) or hybrid-SBS with cleavable fluorescent nucleotide permanent terminators and NRTs. The idea of the walking strategy is to recover the initial template after one round of sequencing and re-initiate a second round of sequencing at a downstream base to cover more bases overall. The combination of three natural nucleotides and one NRT effectively regulated the primer walking: the primer extension temporarily paused when the NRT was incorporated, and resumed after removing the 3' capping group to restore the 3'-OH group. We have successfully demonstrated the integration of this primer walking strategy into the sequencing by synthesis approach, and were able to obtain a total read-length of 53 bases, nearly doubling the read-length of the previous sequencing. On the other hand, we explored the sequencing bead-on-chip approach to increase the throughput of SBS and hence the total genome coverage per run. The various prerequisite conditions have been optimized, allowing the accurate sequencing of several bases on the bead surface, which demonstrated the feasibility of this approach. Both of these approaches could be integrated into current SBS platforms, allowing increased overall coverage and lowering overall costs. As a step beyond genotyping, the in vivo visualization of biomolecules, like DNA and its encoded RNA and proteins, provides further information about their biological functions and mechanisms. The third part of the thesis focuses on the development of a novel quantum dot (QD)-based binary molecular probe, which takes advantage of fluorescent resonance energy transfer (FRET), for detection of nucleic acids, aiming at their eventual use for detection of mRNAs involved in long term memory studies in the model organism Aplysia californica. We reported the design, synthesis, and characterization of a binary probe (BP) that consists of carboxylic quantum dot (CdSe/ZnS core shell)-DNA (QD-DNA) conjugated donor and a cyanine-5 (Cy5)-DNA acceptor for the detection of a sensorin mRNA-based synthetic DNA molecule. We have demonstrated that in the absence of target DNA, the QD fluorescence is the main signal observed (605 nm); in the presence of the complementary target DNA sequence, a decrease of QD emission and an increase of Cy5 emission at 667 nm was observed. We have demonstrated the distance dependence of FRET, with the finding that the target with 16 base separation between the QD and Cy5 after probe hybridization gave the most efficient FRET. Further studies are in progress to evaluate the effectiveness of this QD-based probe inside a cell extract and in living cells.

The predominant approach for cartilage tissue engineering involves cell transplantation with or without cytokine delivery, biomaterial scaffolds, bioreactors, applied mechanical stimulation and altered oxygen tension. Despite its scientific merit, cell delivery faces drawbacks including scarce cell availability, donor site trauma, possible immune rejection and potential tumorigenesis. Tissue regeneration by cell homing is a novel concept and may offer the advantage of accelerated clinical translation. Promising results have been shown using a cell homing approach to engineer a number of tissue types including dental pulp, vascular tissue and bone. Various stem/progenitor cell populations are present in tissues adjacent to an articular cartilage defect including subsets of cells that have the ability to differentiate into cartilage-like tissue. Furthermore, several factors have been elucidated that stimulate stem/progenitor cell homing and selected cytokines have been discovered to be potent at inducing chondrogenic differentiation of stem/progenitor cells. Cell homing is an exciting area of regenerative medicine but many critical questions remain such as cell origin, homing distance, and effective chemotactic cues. In addition to currently studied cell homing cues, other cytokines present during inflammation that are not typically known for their homing abilities might be helpful in recruiting additional cells to the scaffold and improving the quality of cartilage tissue formation. The effect of concurrently exposing a cell population to multiple cytokine signals, similar to conditions that cells experience in vivo, has not been fully investigated. Determining which cytokine or groups of cytokines that induce high levels of chemotaxis would be critical for designing effective bioactive scaffolds for cell recruitment and chondrogenesis. This thesis develops novel systems to characterize stem/progenitor cell migration and uses the knowledge gained from these systems to develop new methods for inducing chondrogenesis by cytotactic homing. First, the concept of stem/progenitor cell homing for cartilage tissue regeneration is reviewed (Chapter 1). Next, a system was developed for the in vitro recruitment and chondrogenesis of Adipose Stem Cells (ASCs), Mesenchymal Stem Cells (MSCs) and Synovium Stem Cells (SSCs), all of which are natively located adjacent to a full-thickness articular cartilage defect (Chapter 2). Using microfluidic principles, novel assay systems were designed and built to characterize the process of stem cell migration in the presence of single and competing cytokine signals (Chapter 3). An in-depth study was conducted investigating the process of stem/progenitor cell migration in the presence of competing cytokine signals (Chapter 4). Lastly, the knowledge gained through extensive chemokine testing using these novel assay systems was used to develop a bioactive scaffold to induce cell homing and chondrogenesis for rhinoplasty augmentation in a rat model (Chapter 5). The novel migration devices developed herein offer a rare opportunity for screening of cell homing efficacy, potentially applicable to any stem cell population including embryonic, iPS, skeletal, muscular, neural, cardiac and adipose. A number of basic biological concepts have been examined by studies using these devices such as cell motility behavior and optimal migratory distances. The competitive cytotactic assay system provided new insight into stem cell behavior in response to gradients of multiple cytotactic cues, thus mimicking native in vivo conditions. By determining combinations of cytokines effective at maximizing cell homing, novel approaches for cartilage tissue engineering without the need for cell delivery, were developed for rhinoplasty augmentation. These systems for inducing chondrogenesis by chemotactic homing were shown to be an effective alternative to cell transplantation for cartilage tissue regeneration therapies.

The development of a multicellular organism from an embryo is one of the nature's most remarkable phenomena. Deciphering how this transformation occurs is a fundamental challenge in biology with profound biomedical implications. Insights into the molecular signals guiding developmental patterning may provide design strategies to promote multicellular structure formation in applications such as tissue engineering and regenerative medicine. In this thesis, we explored the applications of controllable gene expression techniques in combination with engineering strategies in regulation and patterning of cell differentiation and pluripotency, by pursuing three related research projects: 1. Reversible immortalization of cardiomyocytes to enable their proliferation necessary for obtaining large cell numbers 2. Patterning of the delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector, to mesenchymal stem cells cultured in a microfluidic 3. Patterning of the Nanog gene expression in embryonic stem cells, using a microfluidic device, to establish differentiation - pluripotency boundaries that mimic the developmental processes in vivo. In the first project, we developed a novel strategy for controlled expansion of non-proliferating primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then the reversal of the phenotype of expanded cells back to the cardiomyocytes state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (α-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state, as evidenced by molecular and functional properties inherent to terminally differentiated cardiomyocytes. This strategy for controlled expansion of primary cardiomyocytes by reversible gene transfer could provide large amounts of a patient's own cardiomyocytes for cell therapy, and enable controlled in vitro study of cardiogenesis. In the second project, we developed a novel patterning strategy by using inducible gene expression systems in conjunction with simple multi-laminar fluidic techniques, which can directly pattern the expression of particular gene at transcriptional level. Using osteogenic differentiation of human mesenchymal stem cells as a model, we describe a novel approach to spatially regulate the expression and secretion of bone morphogenetic protein (BMP-2) in a two-dimensional field of cultured cells, by flow patterning the modulators of inducible BMP-2 gene expression. We first demonstrated a control of gene expression, and control of osteogenic differentiation of the cell line with inducible expression of BMP-2. Then we designed laminar flow systems, with patterned delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector. The patterned concentration profiles were verified by computational simulation and dye separation experiments. Experiments conducted in the flow systems for a period of three weeks showed the Dox concentration dependent osteogenic differentiation, as evidenced by mineral deposition. This strategy combining inducible gene expression with laminar flow technologies provides an innovative way to engineer tissue interfaces. In the third project, we further developed the patterning strategy for gene expression to form boundaries of different gene expression domains in cultures of mouse embryonic stem cells. Using Nanog safeguarded embryonic pluripotency as a model; we demonstrated controlled Nanog expression, which lead to controlled early differentiation under the exposure or withdrawal of varied small molecules, as evidenced by alkaline phosphatase (AP) staining, immunofluorescent staining, and gene expression analysis. By patterning Nanog gene expression, as well as soluble factors in the laminar fluidic system, we successfully developed varied differentiation - pluripotency boundaries between Nanog expressing pluripotency zones and Nanog suppressed early differentiation zones from the same population of cells, which mimic the development process in vivo. Mechanistic insights can be gained on dissecting the signaling pathways that drive multicellular patterning during the natural processes of embryonic and adult development. In summary, we demonstrated that controlled expansion of non-proliferating primary cells can achieved by reversible genetic manipulation, and that varied continuous, graded pluripotency - differentiation boundaries can established by patterning the expression of target genes via a simple laminar fluidic system. Taken together, these approaches provide innovative models to modulate cell function at the transcriptional level. Additional cooperative research was conducted during my graduate training. The manuscript of this study "Micropatterned Mammalian Cells Exhibit Phenotype-Specific Left-Right Asymmetry" was submitted to Proc Natl Acad Sci U S A., and it is currently under review. We attached this manuscript in appendix.

The key to effective treatment of neurological diseases resides in the safe opening of the blood-brain barrier (BBB), a specialized structure that impedes the delivery of therapeutic agents to the parenchyma. Despite the fact that several approaches have been successful in overcoming the BBB impermeability, none of them can induce localized BBB opening noninvasively except for focused ultrasound (FUS) in conjunction with microbubbles. The physical mechanism behind the opening, however, has not been identified. Insight into the mechanism can be critical for delineating the safety profile for in both small and large animals alike. Therefore the purpose of this dissertation is to first determine the physical mechanism of FUS-induced BBB opening in mice and then translate this approach to non-human primates. To accomplish this goal, an in vivo transcranial cavitation detection system was developed and tested, built in phantoms and in vivo, to monitor the behavior of the microbubbles in the FUS bean, and to determine the type of cavitation, i.e., the activation of bubbles in an acoustic field, during BBB opening. We showed that the inertial cavitation (IC), a collapse of a bubble, which can vary from a fragmentation of the bubble to shock wave and liquid jets depending on the pressure, thereby damaging the endothelial cells of the brain capillaries, was not required to induce BBB opening in mice. With this system, the role of microbubble properties, including the diameter and shell components, in the BBB opening were determined. When the BBB opens with stable cavitation (SC), i.e., relatively moderate amplitude changes in the bubble size, the bubble diameter is similar to the capillary diameter (i.e., at 4-5, 6-8 µm) while with inertial cavitation it is not (i.e., at 1-2 µm). The bubble may thus have to be in closer proximity to the capillary wall to induce BBB opening without IC. The BBB opening properties, such as volume and permeability, however, were not affected by the shell component of the microbubbles in mice. The connection between the physical and physiological mechanism was then investigated to identify the lowest peak rarefactional pressure BBB opening threshold at 1.5 MHz (0.18 MPa). A sufficiently long pulse (pulse length = 0.5 ms) was required for the SC to induce BBB opening at the lowest pressure. However, the tight junctions, the main formation of the BBB, were found not to be disrupted after sonication at both low (0.18 MPa) and high (0.45 MPa) pressures. Therefore, the transcellular pathway may be the main route of the FUS-induced BBB opening. Finally, the cavitation-guided BBB opening system was used to induce reversible BBB opening in non-human primates. This is a major step towards clinical feasibility. In conclusion, a transcranial cavitation detection system was developed, in order to characterize the physical mechanism, the role of the microbubbles, and the corresponding physiological response of the FUS-induced BBB opening.

Cardiovascular disease is a rising epidemic worldwide, and curative therapies remain elusive. Heart and vascular disease remain some of the hardest to cure due to the limited capacity of the heart to repair itself, necessitating a cell or organ based therapy to cure the inevitable descent into heart failure. Tissue engineering is uniquely poised to significantly alter this disease burden though the fabrication of cardiac and vascular tissues in vitro. However, the challenges for achieving these aims are significant - for cardiac tissues, the therapy must adhere to strict requirements of adequate perfusion and functional integration with the damaged heart. Vascular tissues are required to be amenable to surgical anastomosis while at the same time provide nutrient transport on the cellular level. Recently, a new set of technologies based from the semiconductor industry, have enabled micron level control over the cellular environment and cells themselves and may enable novel approaches to fulfill these tissue engineering requirements. In this dissertation, these microtechnologies will be leveraged to address some of the current obstacles that limit the use of tissue engineering approaches for functional therapy. Specifically, microtechnologies were used to screen the effect of electrical stimulation on the function and maturation of human embryonic stem cell derived cardiomyocytes, which resulted in the ability to program specific individual beating frequencies of the cells while improving contractile function and led to the identification of a channel specific effect for frequency modulation. These technologies were also used to distinguish the vasculogenic potential of different mesenchymal stem cell sources for nascent vessel stabilization, and enabled the development of a powerful hydrogel docking platform with the novel capability to spatially pattern any number of cells, cytokines or drugs on the microscale, while permitting scale up for larger tissue generation without the loss of precision. Finally, these technologies were used to create vascular networks with hierarchical branching patterns that could be implanted and used in vivo fulfilling a major criterion of vascular tissue function - surgical compatibility with microscale architecture for tissue perfusion. Therefore, these microtechnologies support novel interrogation of cell function and enable new methods to engineer cardiovascular tissues.

The importance of atherosclerotic disease in coronary artery has been a subject of study for many researchers in the past decade. In brief, the aim is to understand progression of such a disease, detect plaques at risks (vulnerable plaques), and treat them selectively to prevent mortality and immobility. Consequently, several imaging modalities have been developed and among them intravascular ultrasound (IVUS) has been of particular interest since it provides useful information about tissues microstructures and images with sufficient penetration as well as resolution. In general, the ultimate goal is to provide interventional cardiologists with reliable clinical tools so they can identify vulnerable plaques, make decisions confidently, choose the most appropriate drugs or implant devices (i.e. stent), and stabilize them during catheterization procedures with minimal risk. In this work, we review existing atherosclerotic tissue characterization algorithms including the state-of-the-art virtual histology (VH) framework, which has been implemented in the Volcano (Rancho Cordova, CA) IVUS clinical scanners using 64-elements 20 MHz phased-array transducer. Initially, we intended to extend this technique for data acquired with 40 MHz single-element transducers. For this reason, we started acquiring in vitro IVUS data and studied involved challenges from specimen preparation toward classification. We observed inconsistency among extracted features along with transducer's spectral parameters (i.e. bandwidth, center frequency). This, in addition to infeasibility of construction of reliable training dataset due to heterogeneity of atherosclerotic tissues motivated us to develop an unsupervised texture-based atherosclerotic tissue characterization algorithm. We proposed a two-dimensional multiscale wavelet-based algorithm to expand IVUS backscattered signals and/or grayscale images onto orthogonal symmetric quadrature mirror filters (QMF) such as Lemarie-Battle. At the bottom of decomposition tree, we employed ISODATA to cluster enveloped detected features in an unsupervised fashion and classify atherosclerotic plaque constitutes into fibrotic, lipidic, calcified, and no tissues. For the first time, we studied numbers of factors that were necessary for extension of in vitro derived classifier for in vivo applications such as reliability of classified tissues behind arc of calcified plaques and effects of pressure changes as well as flowing blood on constructed tissue color maps, called prognosis histology (PH) images. The second half of this dissertation is devoted to automatic detection of lumen borders in IVUS grayscale images acquired with high frequency (40 MHz up) transducers where more scattering exhibited within lumen area that makes the problem of interest more challenging. We established our framework on three-dimensional expansion of IVUS sub-volumes onto orthonormal brushlet basis function. The rational behind our framework was presence of incoherent (i.e. blood) versus coherent (i.e. plaque, surrounding fat) textural patterns along pullback direction, which was motivated by what an interventional cardiologist does to locate the lumen border visually by going back and forth among IVUS frames. We studied the feasibility of brushlet analysis through filtering blood speckles and supervised classification of blood versus non-blood regions. Our preliminary study confirmed that the most informative features reside in the innermost cubes, representing low-frequency components in transformed domain. Finally, we explored that tissue responses to IVUS signals are proportionally preserved in brushlet coefficients and it enabled us to classify blood regions in complex brushlet space. Subsequently, we employed surface function actives (SFA) to estimate the lumen borders after regularization. In a comparison study, we quantified our results with two of existing algorithms, employing IVUS grayscale images acquired with 40 MHz and 45 MHz single-element transducers. Overall, our proposed algorithm outperformed and the resulting automated detected borders showed good correlation with manually traced borders by an expert.

580,350 (35%) of 1,660,290 cancer patients are estimated to die in the US in 2013. Routine monitoring by X-Rays and CT scans are hazardous and evaluating this disease is time consuming. Magnetic Resonance Spectroscopy (MRS) has changed this mal-routine significantly in the past few years. MRS can help with better understanding of tumor pathology, study of tumor vascularization and progress, and having a predicting value for the treatment response and disease-free survival of the patients even before they start their treatment. Unfortunately, MRS is still not a common practice among the medical community because of three main reasons: First and far most is the fact that MRS acquisition is usually very time consuming. For a classic 1H 3D MRS with a spatial matrix of 20x18x10 with TR = 1000 ms, the scan time is about 1 hour which is "practically" impossible to acquire on a patient. Second, MR time is extremely expensive. Depending on the site, specific procedure, and strength of the magnet a simple MR study can cost somewhere between 1000 to 3500 US dollars. Finally, non-standardized MRS acquisitions and analysis protocols could create havoc in interpretation and usefulness of the technique. MRS scan parameters such as spatial resolution and echo times have been used non-uniformly in variety of different combinations in research and clinical studies. These parameters must be chosen with utmost care as they have direct impact on signal to noise ratio, quantification of the metabolites, and an overall interpretation of the results. For the reasons said, having a method that could shorten the length of an MRS scan, reduce the cost, and potentially become a sensible routine in clinical practice is of a huge value. 3D Zero J-modulation Echo Planar Chemical Shift Imaging (3D ZJ-EPSI) is a fast MRS technique that can not only achieve all that was mentioned above, it can also provide additional detailed anatomical/pathological information due to its 3D nature. 3D ZJ-EPSI technique acquires proton magnetic resonance spectroscopy with time to acquisition (TE') of less than 1.7 ms and zero J-modulation effects. 3D ZJ-EPSI consisted of a slab excitation, followed by two phase encoding gradients and an echo planar switching readout gradient. The Free induction decay (FID) acquisition occurred during the plateaus of the switching gradient. The lipid suppression was achieved via ten Regional Saturation Technique (REST) pulses placed prior to the main slab excitation RF. The water suppression technique was a chemical shift selective (CHESS) pulse with RF-80º-80º-160º that was placed prior to lipid suppression pulses. The sequence was tested on a brain metabolite phantom with spatial resolution of 15×15×6 mm3 in 4:04 min, yielding spectra with comparable quality to the spectra obtained using conventional chemical shift imaging (CSI) technique taking 56:34 min. The sequence was also tested on human subjects with spatial resolution of 15×15×6 mm3 and 7.5×7.5×6 mm3 and the metabolic ratios were calculated and compared to literature values. Signals of coupled resonances were improved due to near zero TE' and zero J-modulation effects, while the macromolecules were more pronounced in the spectra. With non-water suppressed sequence, variations of waterline shape of different tissues in three spatial dimensions could be studied. The 3D ZJ-EPSI technique addresses the need for a fast MRS method that allows for a better quantification capability by acquiring proton spectra with zero J-modulation. The short acquisition time and near zero TE' make this methodology suitable for uniform quantification of metabolites in clinical studies.

Nonlinear microscopy refers to a range of laser scanning microscopy techniques that are based on nonlinear optical processes such as two-photon excited fluorescence and second harmonic generation. Nonlinear microscopy techniques are powerful because they enable the visualization of highly scattering biological samples with subcellular resolution. This capability is especially valuable for in vivo and live tissue imaging since it can provide both structural and functional information about tissues in their native environment. With the use of a range of exogenous dyes and intrinsic contrast, in vivo nonlinear microscopy can be used to characterize and measure dynamic processes of tissues in their normal environment. These advances have been particularly relevant in neuroscience, where truly understanding the function of the brain requires that its neural and vascular networks be observed while undisturbed. Despite these advantages, in vivo nonlinear microscopy still faces several major challenges. First, observing dynamics that occur in large areas over short time scales, such as neuronal signaling and blood flow, is challenging because nonlinear microscopy generally requires scanning to create an image. This limits the study of dynamic behavior to either a single plane or to a small subset of regions within a volume. Second, applications that rely on the use of exogenous dyes can be limited by the need to stain tissues before imaging, the availability of dyes, and specificity that can be achieved. Usually considered a nuisance, endogenous tissue contrast from autofluorescence or structures exhibiting second harmonic generation can produce stunning images for visualizing subcellular morphology. Imaging endogenous contrast can also provide valuable information about the chemical makeup and metabolic state of the tissue. Few methods have been developed to carefully and quantitatively examine endogenous fluorescence in living tissues. In this thesis, these two challenges in nonlinear microscopy are addressed. The development of a novel hyperspectral two-photon microscopy method to acquire spectroscopic data from tissues and increase the information available from endogenous contrast is presented. This system was applied to visualize and identify sources of endogenous contrast in gastrointestinal tissues, providing robust references for the assessment of normal and diseased tissues. Secondly, three methods for high speed volumetric imaging using laser scanning nonlinear microscopy were developed to address the need for improved high-speed imaging in living tissues. A spectrally-encoded high-speed imaging method that can provide simultaneous imaging of multiple regions of the living brain in parallel is presented and used to study spontaneous changes in vascular tone in the brain. This technique is then extended for use with second harmonic generation microscopy, which has the potential to greatly increase the degree of multiplexing. Finally, a complete system design capable of volumetric scan rates >1Hz is shown, offering improved performance and versatility to image brain activity.