abstract: Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules. Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

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Temperature sensing in harsh environment is desired in many areas, such as coal gasification, aerospace, etc. Single crystal sapphire is an excellent candidate for construction of harsh environment sensors due to its superior mechanical and optical properties even at temperature beyond 1600°C. The temperature inside a coal gasifier can be as high as 1200°C. And there is dramatic temperature gradient between the inner and outer layers of the gasifier refractory. Previous work has been done at Virginia Tech's Center for Photonics Technology to design and fabricate a sapphire wafer based Fabry-Perot interferometer (FPI) sensor for temperature sensing in coal gasifiers. The sensor head is based on the use of sapphire wafer which is attached to a lead-in sapphire fiber to be applied in the ultrahigh temperature region; and the sapphire fiber is spliced to a multi-mode fused silica fiber for quality signal transmission in lower temperature areas. One of the challenges encountered by this approach is the shear force to the sapphire fiber, which is caused by the differential thermal expansion between the inner and outer layers of the gasifier refractory. This shear force may be so significant to break the sensor probe. This thesis proposed a free space based interrogation sensing system to address that problem. In this free space based interrogation sensing system, only the sensor head is placed in the inner refractory wall, while all the other parts of the system are placed in the outer refractory or outside the gasifier at the ambient room temperature. An optical frequency domain reflectometry (OFDR) based multiplexed technique is applied in the sensor design to realize temperature measurement at multiple locations along the optical path. In this work, three sapphire wafers based multiplexed temperature sensor is fabricated and calibrated in laboratory. This multiplexed high temperature sensor shows linear response in the range of 20°C ~ 1000°C, with a sensitivity of 1.602?10??/°C and a resolution of 1.3°C. / Master of Science

Gas Sensing Has Become a Very Important and Attractive Technique Because of Its Various Applications, Such as in the Increasingly Concerning Case of Environmental Issues, Automobile Emission Detection, Natural Gas Leakage Detection, Etc. It Also Has Significant Applications in Industries, Such as Safety and Health Monitoring in Underground Mines. Among Those Sensing Areas, Fiber-optic Sensors Have Drawn Considerable Attention Because of Its Small Size, Light Weight, High Sensitivity, and Remote Sensing Capability. However, Current Fiber-optic Gas Sensing Techniques Have Several Limitations on Their Potential for Multiplexed or Distributed Sensing Due to Difficulties Such as High Complexity or Large Loss. To Accomplish the Goal for Multiplexed Gas Sensing, an Off-centered Core Fiber Design Is Investigated. The Eccentric Core Can Reduce Attenuation, Keep Mechanical Strength, and Lower Fabrication Cost. To Verify the Feasibility of the Design, Fiber Field Distribution Is First Studied in Simulation, Which Will Be Discussed in Detail in Chapter 2. Then Two Fiber Samples with a Length of 10 Cm and 40 Cm Are Prepared and Placed in a Custom Methane Sensing System for Gas Absorption Testing, Which Is Detailed in Chapter 3. From Etching Analysis, Localized Surface Defects Are Found as the Main Reason for Power Loss. Performance Such as Detection Resolution and Sensitivity Are Investigated. In Chapter 4, Theoretical Evaluations Have Been Conducted for Multiplexed Sensors Performances Using the Off-centered Core Fiber to Study the Impact Fiber Parameters on Sensing System Design. The Conclusion and Summary Are Presented in Chapter 5. / Master of Science / Gas Sensing Has Become a Very Important and Attractive Technique Because of Its Various Applications, Such as in the Increasingly Concerning Case of Environmental Issues, Automobile Emission Detection, Natural Gas Leakage Detection, Etc. It Also Has Significant Applications in Industries, Such as Safety and Health Monitoring in Underground Mines. Among Those Sensing Areas, Fiber-optic Sensors Have Drawn Considerable Attention Because of Its Small Size, Light Weight, High Sensitivity, and Remote Sensing Capability. However, Current Fiber-optic Gas Sensing Techniques Have Several Limitations on Their Potential for Long Distance Distributed Sensing Due to Difficulties Such as High Fabrication Complexity. In This Work, a Fiber-optic Gas Sensor with Special Structure Was Designed. The Sensor Can Reduce Attenuation, Keep Mechanical Strength, and Lower Fabrication Cost. To Verify the Feasibility of the Design, Theory Analysis and Simulation Were Conducted, Which Will Be Discussed in Detail in Chapter 2. Then Two Samples with a Length of 10 Cm and 40 Cm Were Prepared and Placed in a Custom Methane Sensing System for Testing. And Their Performance Such as Sensitivity Is Investigated. In Chapter 4, Theoretical Evaluations Have Been Conducted for Multiplexed Sensors Performances Evaluation to Study the Impact Fiber Parameters on Sensing System Design. The Conclusion and Summary Are Presented in Chapter 5.

The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to the detection probe ZFP, which allows for generating chemiluminescent signal in streptavidin-HRP (Horseradish peroxidase) assay upon ZFPs binding to their target DNA. Our assay generates DNA-dependent signal and allows for a detection limit of 0.5 nM without DNA amplification or DNA labeling. Our system can be developed into a simple multiplexed detection diagnostic for multiplexed detection of dsDNA.

La thèse aborde le problème de l’évolution de la forme à travers l’exemple d’une différence de forme de branches ventrales mâles liée à l’isolement reproducteur chez deux espèces soeurs : Drosophila yakuba et Drosophila santomea. L’objectif de ce travail est d’identifier les gènes impliqués dans l’évolution de cette différence de forme ainsi que les causes évolutives de cette différence entre espèces. Dans une première partie, la thèse interroge la notion de “gène” et sa recherche. Puis sont caractérisées, dans un cadre formel, les questions de type "comment" et "pourquoi" et leur lien avec la distinction causes prochaines/causes ultimes ou évolutives. Ces réflexions philosophiques sont ensuite reliées à l’Evo-Devo et au projet expérimental. Dans la deuxième partie, par morphométrie géométrique et une nouvelle méthode de génotypage à haut débit, le MSG, nous identifions un locus de 2.7 méga-bases situé sur le chromosome 3L comme étant impliqué dans l’évolution de la forme des branches ventrales entre D. yakuba et D. santomea. Ces résultats sont mis en perspective avec notre analyse quantitative de la variation de forme dans plusieurs souches naturelles, souches de laboratoire et souches élevées à différentes températures qui apportent des indices sur les causes évolutives de cette différence de forme / The thesis tackles the problem of the evolution of shape through the example of a shape difference in the male ventral branches linked to reproductive isolation in two sister species: Drosophila yakuba and Drosophila santomea. The goal is to identify the genes involved in the evolution of this shape difference and the evolutionary causes of such difference. In a first part, the thesis interrogates the concept of “gene” and its search. Then are scientifically characterized the “how” and “why” questions and their link with the distinction of proximal/ultimate, or evolutionary, causes; these philosophical grounds are then linked to Evo-Devo and the experimental work presented in the thesis. In a second part, through geometric morphometrics and a new high-throughput genotyping method, MSG, we identify a loci of 2.7 mega-bases located on chromosome 3L as involved in the evolution of the shape of ventral branches between D. yakuba and D. santomea. These results are linked to our quantitative analysis of shape variation in multiple natural and laboratory strains and strains reared at different temperatures which bring light into the evolutionary causes of this shape difference

Com a evolução tecnológica há nos dias de hoje um aumento de dispositivos eletrônicos presentes ao nosso redor. Com o passar dos anos diversas funcionalidades vêm sendo agregadas a estes inclusive com maior poder de processamento. Em particular, para sistemas embarcados houve um crescimento da quantidade de sensores para diversos propósitos. Seguindo esta tendência, na área de saúde também houve um aumento significativo de aparelhos e dispositivos de monitoramento, tais como glicosimetros, oxímetros, monitoramento de pressão e batimento cardíaco por exemplo, que através de sensores realizam transdução dos dados pertinentes ao parâmetro envolvido. Este trabalho apresenta a pesquisa e o desenvolvimento de um sistema embarcado com a propriedade de multiplexação de sensores, ou seja, foi desenvolvido um dispositivo microcontrolado o qual visa multiplicar a capacidade de monitoramento de analitos, conseguindo analisar múltiplos sensores para um mesmo experimento. Ao decorrer deste desenvolvimento foram utilizados quatro sensores dispostos simetricamente em um béquer, os dados são coletados e tratados de forma sequencial e individual. Inicialmente utilizamos um sistema embarcado com um microcontrolador (PIC 18F2550) que é responsável por digitalizar a informação e pela conexão via terminal USB. Posteriormente um microprocessador (Raspberry Pi Zero, placa embarcada) fez-se necessário devido ao melhor processamento de dados. Os sensores aqui estudados tratam-se de sensores químicos, que são introduzidos a uma célula eletroquímica, onde se encontram um eletrodo de referência (Prata em uma solução de Cloreto de Prata) e os outros quatro filmes finos que irão compor o sistema multiplexado. Para este estudo em específico o material escolhido para fabricação dos filmes finos foi um polímero condutivo, mais especificamente polianilina (PANI). Esta foi depositada sobre um substrato de oxido de estanho dopado com flúor (FTO) através da eletrodeposição. Para sensores não específicos (não imobilizados para um analito alvo) os dois sistemas embarcados apresentaram respostas satisfatórias. Prosseguindo com o estudo e usando filmes finos para analitos biológicos (ureia e glicose) o microcontrolador não conseguiu separar os sinais de cada filme fino. Apenas o sistema com a Raspberry Pi obteve sucesso, devido a maior resolução no conversor analógico para digital e sua maior capacidade de processamento para determinar com uma maior precisão os valores obtidos. O sistema pode ser facilmente expandido para um maior número de sensores. / With the evolution of technology there is nowadays an increase in the number of electronic devices present around us. Over the years various functionalities have been added to these devices including the increased processing power. In particular, for embedded systems there has been an increase in the number of sensors for various purposes. Following this trend, in the area of health, there has also been a significant increase in systems and monitoring devices, such as glycosimeters, oximeters, pressure monitoring and heart rate, for example, which, through sensors, transduce data pertinent to the parameter involved. This work presents the research and development of an embedded system with the property of multiplexing sensors, that is, a microcontrolled device was developed which aims to multiply the capacity of analytes monitoring, being able to analyze multiple sensors for the same experiment. During this development four sensors were used symmetrically arranged in a beaker, the data were collected and treated sequentially and individually. Initially we used an embedded system with a microcontroller (PIC 18F2550) that is responsible for scanning the information and for the connection via USB terminal. Subsequently a microprocessor (Raspberry Pi Zero, embedded board) was made necessary due to the better processing of data. The sensors studied here are chemical sensors, which are introduced to an electrochemical cell, where a reference electrode is found (Silver in a Silver Chloride solution) and the other four thin films that will make up the multiplexed system. For this specific study the material chosen for the manufacture of thin films was a conductive polymer, more specifically polyaniline (PANI). This was deposited on a substrate of fluorine-doped tin oxide (FTO) by electrodeposition. For non-specific sensors (not immobilized for a target analyte) the two embedded systems presented satisfactory responses. Proceeding with the study and using thin films for biological analytes (urea and glucose) the microcontroller failed to separate the signals from each thin film. Only the system with Raspberry Pi has been successful, due to the higher resolution in the analog to digital converter and its greater processing capacity to determine with greater precision the obtained values. The system can be easily expanded to a larger number of sensors.

Malaria is a critical and global public health problem, affecting over 200 million people every year, resulting in over 500,000 deaths. A vaccine is not currently available and only one drug, primaquine, is effective against the dormant stages of Plasmodium vivax. Preclinical assessment of novel therapeutic drugs and vaccines is hampered by the lack of an in vitro liver model for P. falciparum and P. vivax. To provide a stable human hepatocyte-based culture platform for parasite development, we engineered a microfluidic bilayer device capable of both simple and complex culture methods, including perfusion and co-culture, to better understand the requirements of both hepatocytes as host cells and parasite liver stage development. As only the mechanical compaction of the device channels was found necessary for stable hepatocyte culture, and only a validated host cell lot was found necessary for improved parasite development rates, we present an efficient and simple Plasmodium liver model capable of supporting P. vivax dormant forms. Device liver platforms were used to generate kill curves of P. vivax and P. falciparum after three days under primaquine drug pressure. Furthermore, an anti-CSP antibody-based inhibition of development assay with P. falciparum successfully demonstrated antibody-specific liver stage inhibition. Ongoing studies aim to identify the minimal unit of the culture system necessary to be multiplexed in a fully functional and efficient drug and vaccine discovery platform.

<p>FIR (Finite-length Impulse Response) filters are the corner stone of many signalprocessing devices. A lot of research has gone into their development as wellas their effective implementation. With recent research focusing a lot on powerconsumption reduction specially with regards to FPGAs, it was found necessaryto explore FIR filters mapping on FPGAs.</p><p>Time multiplexed FIR filters are also a good candidate for examination withrespect to power consumption and resource utilization, for example when implementedin Field Programmable Gate Arrays (FPGAs). This is motivated by thefact that the usable clock frequency often is higher compared to the required datarate. Current implementations by, e.g., Xilinx FIR Compiler suffer from highpower consumption when the time multiplexing factor is low. Further, it needs tobe investigated how exploiting coefficient symmetry, scaling the coefficients andincreasing the time-multiplexing factor influences the performance.</p>

FIR (Finite-length Impulse Response) filters are the corner stone of many signalprocessing devices. A lot of research has gone into their development as wellas their effective implementation. With recent research focusing a lot on powerconsumption reduction specially with regards to FPGAs, it was found necessaryto explore FIR filters mapping on FPGAs. Time multiplexed FIR filters are also a good candidate for examination withrespect to power consumption and resource utilization, for example when implementedin Field Programmable Gate Arrays (FPGAs). This is motivated by thefact that the usable clock frequency often is higher compared to the required datarate. Current implementations by, e.g., Xilinx FIR Compiler suffer from highpower consumption when the time multiplexing factor is low. Further, it needs tobe investigated how exploiting coefficient symmetry, scaling the coefficients andincreasing the time-multiplexing factor influences the performance.