published_or_final_version / Computer Science / Master / Master of Philosophy

Molecular diagnostics offer quick access to information for healthcare decision-making towards personalized therapeutics, but complicated procedures requiring extensive labor and infrastructure restrict their use. Droplet-based technologies can expand the accessibility of molecular diagnostics by miniaturizing devices, shortening sample-to-answer times, decreasing costs and increasing throughput. Methods for droplet manipulation are central to the automation of molecular diagnostics protocols. The innovative method, wire-guided droplet manipulation (WDM), is the actuation of liquid droplets in a hydrophobic milieu with a wire, or needle, guide. In this work, WDM is demonstrated for the automation of the polymerase chain reaction (PCR) on reprogrammable platforms for the diagnosis of cardiovascular infections. WDM is used to minimize thermal resistance by convective heat transfer for PCR amplification at a maximum speed of 8.67 s/cycle. The oil-water interfacial boundary is shown to passively partition molecular contaminants from sample matrices, including blood and heart valve tissue. Molecular self-assembly at the oil-water interface is used to increase PCR efficiency with blood in situ and is used as an innovative sensing modality for real-time monitoring of PCR amplification. Temperature feedback controlled droplet actuation is achieved by using a thermocouple loop as a functionalized wire-guide. Our novel methodology for real-time PCR, droplet-on-thermocouple silhouette real-time PCR (DOTS qPCR), utilizes interfacial effects to achieve droplet actuation, relief from PCR inhibitors and amplification sensing, for a sample-to-answer time as short as 3 min 30 s. DOTS qPCR addresses three major issues for rapid PCR—sample preparation, rapid thermocycling and sensitive real-time detection—on an inexpensive, disposable device with smartphone-based detection. In contrast, commercially available real-time PCR systems rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection we anticipate extending this technology towards trending biological research applications such as single cell, single nucleus, and single DNA molecule analyses, especially in droplet microfluidic platforms.

Rapid diagnosis of infectious disease and timely initiation of proper clinical antibiotic treatment is the determinant in obtaining the optimal clinical outcomes and reducing emergences of multidrug-resistant organisms. In particular, acute infections require the detection to be accomplished in limited time with high sensitivity due to the low concentration of organisms causing the infections. Real-time Polymerase Chain Reaction can provide quantitative identification of specific genetic materials and has revolutionized clinical microbiology laboratory diagnosis. It is becoming a standard for infectious disease detection. However, most real-time PCR instruments on the market are bulky, fragile and costly due to their delicate optical components, which restricted their use to point-of-care application. Modern microfluidic and sensing technology provide a transition from benchtop real-time PCR to miniaturizable, robust, and portable real-time PCR devices to achieve rapid, low-cost, and efficient point-of-care diagnosis. In this work, an innovative electrokinetic PCR (EK-PCR) platform that combines AC electrothermal flow (ACEF) and Joule heating induced temperature gradient to implement thermal cycling for DNA amplification is discussed. In addition, in situ electrochemical sensing is incorporated in the EK-PCR chamber for real-time monitoring of the DNA concentration toward quantification of the initial copies of the DNA template. EK-PCR can improve the energy efficiency with minimized total thermal mass and remain high amplification efficiency. More importantly, it represents a highly integrated strategy for portable point-of-care devices.

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Το πρότυπο IEC61499 ορίζει το Function Block ως νέο τρόπο ανάπτυξης συστημάτων ελέγχου και αυτοματισμού. Τα συστήματα αυτά αποτελούνται από κατανεμημένες, ενσωματωμένες συσκευές οι οποίες διασυνδέονται μέσω βιομηχανικών δικτύων πραγματικού χρόνου. Λόγω του κατανεμημένου χαρακτήρα των συστημάτων αυτών, η εύρεση και επιδιόρθωση σφαλμάτων και ο έλεγχος της ορθής λειτουργίας τους θα πρέπει να γίνεται στο περιβάλλον των ενσωματωμένων αυτών συστημάτων. Στα πλαίσια αυτής της διπλωματικής δίνεται μία υλοποίηση για την κάλυψη της παραπάνω ανάγκης. Ο χρήστης μέσω μίας γραφικής διεπαφής, έχει την δυνατότητα να εκτελεί βασικές λειτουργίες απασφαλμάτωσης στο περιβάλλον των ενσωματωμένων συστημάτων, σε πραγματικό χρόνο, με την χρήση του RTAI και του RTnet. / The IEC 61499 standard defines Function Block as a new way of developing control and automation systems. These systems consist of distributed embedded devices which interconnect via real time industrial networks. Due to the distributive character of these systems, debugging and operation integrity check, should be done on target environment. This dissertation provides a tool for covering this need. Via a graphical user interface the user has the ability to perform basic real time debugging operations in the target enviroment, using RTAI and RTnet.

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Smoking is a worldwide problem and it is the second major cause of death. People often try to quit, but few succeed mainly because of withdrawal symptoms such as irritability, anxiety, increased appetite, hyperventilation and difficulty concentrating. The overall aim of this project was to study neurochemical changes in the brain following sensitization to nicotine which could give more information about what causes an individual to go from using drugs to abusing the drugs. Therefore, we investigated messenger ribonucleic acid (mRNA) expression of several genes known to be involved in the mesolimbic dopamine pathway in the nucleus accumbens, caudate putamen, prefrontal cortex and medial prefrontal cortex using real-time polymerase chain reaction (real-time PCR). The results showed that in the nucleus accumbens, mRNA expression of gamma-aminobutyric acid (GABA) Aα1 subunit receptor and GABA transporter 3 (GAT-3) were significantly increased following nicotine administration, while in the caudate putamen no difference in expression was observed. In prefrontal cortex, the expression of adrenergic subunit receptor α2A was significantly increased following hexamethonium administration. In medial prefrontal cortex a significant decrease of expression of GAT-1 was shown following nicotine and hexamethonium administration, while a decrease of CART expression only was shown following nicotine administration. Overall, these changes in the GABA system may help to explain the mechanism of nicotine sensitization.

The purpose of this study was to evaluate variations of the Natural Resources Conservation Service (NRCS) curve number (CN) method for estimating near real-time runoff for naturalized flow, using high resolution radar rainfall data for watersheds in various agro-climatic regions of Texas. The CN method is an empirical method for calculating surface runoff which has been tested on various systems over a period of several years. Many of the findings of previous studies indicate the need to develop variations of this method to account for regional and seasonal changes in weather patterns and land cover that might affect runoff. This study seeks to address these issues, as well as the inherent spatial variability of rainfall, in order to develop a means of predicting runoff in near real-time for water resource management. In the past, raingauge networks have provided data for hydrologic models. However, these networks are generally unable to provide data in real-time or capture the spatial variability associated with rainfall. Radar networks, such as the Next Generation Weather Radar (NEXRAD) of the National Weather Service (NWS), which are widely available and continue to improve in quality and resolution, can accomplish these tasks. In general, a statistical comparison of the raingauge and NEXRAD data, where both were available, shows that the radar data is as representative of observed rainfall as raingauge data. In this study, watersheds of mostly homogenous land cover and naturalized flow were used as study areas. Findings indicate that the use of a dry antecedent moisture condition CN value and an initial abstraction (Ia) coefficient of 0.1 produced statistically significant results for eight out of the ten watersheds tested. The urban watershed used in this study produced more significant results with the use of the traditional 0.2 Ia coefficient. The predicted results before and during the growing season, in general, more closely agreed with the observed runoff than those after the growing season. The overall results can be further improved by altering the CN values to account for seasonal vegetation changes, conducting field verification of land cover condition, and using bias-corrected NEXRAD rainfall data.

The feasibility of using real-time functional magnetic resonance imaging (fMRI) feedback regarding the level of activation in rostromedial prefrontal cortex (rMPFC) to learn improved regulation of this brain area was examined in a group of 5 young adults. Subjects received real-time feedback from the target brain region while engaging in a blocked-design task involving alternating blocks of attempted up-regulation and down-regulation of the target brain region. A transient negative emotional state was induced prior to each scanning session. Subjects completed 6 scanning sessions (a pre-training session, 4 feedback sessions and a post-training session - no feedback was provided for pre and post-training sessions). The guideline strategy provided to subjects of engaging in emotional awareness during up-regulation and bodily awareness during down-regulation was found to consistently regulate the region in the pre-training session prior to the fMRI feedback sessions. This finding is in line with the previously proposed role of the rMPFC in emotional awareness. In contrast to previous real-time fMRI findings, greater recruitment of the region was observed in the pre-training session compared to the post-training session, with a non-significant negative trend observed across feedback sessions. These results suggest that there may be limitations to which the feedback techniques successfully employed for other brain regions extend to yet unexplored brain regions.