The chicken yolk sac and small intestine are connected through the yolk stalk and share many biological similarities. During the embryonic stage, the extra-embryonic yolk sac helps the embryo to absorb nutrients primarily in the last two weeks of incubation. The chicken yolk sac physically moves yolk contents from the yolk sac to the small intestine at the end of embryogenesis. This is the time when the small intestine replaces the yolk sac in assimilating nutrients for the embryo and later for the posthatch chicken. Additionally, both chicken small intestinal epithelia and the yolk sac secrete beta defensins for promoting intestinal health. Since there are heterogeneous cell types along the mammalian intestinal villus, which are derived from the intestinal stem cells in the crypts, we investigated if cells of the chicken yolk sac and small intestine have the same ontogeny as mammalian intestinal epithelial cells. In this dissertation, we mainly focused on the spatial expression of nutrient transporters (PepT1 and SGLT1), intestinal stem cell markers (Lgr5 and Olfm4), and avian beta defensins in the chicken yolk sac and small intestine during the embryonic and early posthatch stages. RNAscope in situ hybridization was used to identify the distribution of cells expressing PepT1 mRNA in both the chicken yolk sac and small intestine. PepT1 mRNA was found to be expressed by epithelial cells in both the yolk sac and small intestine. In the yolk sac, PepT1 mRNA was uniformly distributed in each endodermal epithelial cell along the villus-like structure. The pattern of PepT1 mRNA expression observed in the chicken yolk sac during the last 10 days of incubation revealed that PepT1 mRNA was increased from e11 to e13, and decreased from e15 to day of hatch. The peak of PepT1 mRNA expression was between e13 and e15, when the yolk sac reaches maximum absorptive area and the growth of the chicken embryo is at its fastest rate. However, the expression of PepT1 mRNA in the intestine was only detected in columnar enterocytes along the villus and not in goblet cells or cells in the crypts. The immunofluorescence assay confirmed that PepT1 protein was located at the brush border membrane of the enterocytes and that protein expression of PepT1 was restricted to the intestinal epithelial cells from approximately the middle to the tip of the villus. In order to identify intestinal stem cells, we used the known mammalian stem cell markers, Lgr5 and Olfm4. Both Lgr5 and Olfm4 are specifically expressed by cells in the chicken intestinal crypts, suggesting that they can be used as biomarkers for chicken intestinal stem cells. Dual labelling of PepT1 and Olfm4 mRNA on the same chicken intestinal sample revealed that there was a gap between PepT1-expressing enterocytes and Olfm4-expressing intestinal stem cells. The cells in this gap were presumably transit amplifying (TA) cells. Additionally, we also found that the TA cell zone along the intestinal villus was reduced during chicken growth. This TA cell population could be clearly detected at day of hatch and d1 posthatch but not later. The expression of SGLT1 mRNA was localized to yolk sac endodermal epithelial cells and showed a sharp increase at the end of incubation. This increase of SGLT1 mRNA coincided with the increase in glucose in the yolk, indicating that the chicken embryo needs glucose as energy for hatching. The mRNA expression profiles of various avian beta defensins have been examined by qPCR and in situ hybridization to investigate the immune function of the yolk sac and small intestine. We found that AvBD10 mRNA showed the highest expression level in the yolk sac and was expressed predominantly in the yolk sac endodermal epithelial cells. Additionally, the expression of AvBD10 mRNA showed a development-specific pattern, which increased from e9 to e11, and decreased from e13 towards day of hatch. The expression patterns of AvBD1, 2, and 7 mRNA were similar to each other. These three genes were found to be expressed by chicken heterophils distributed in the yolk sac blood islands and small intestinal blood vessels. Only a subset of heterophils, which might be activated, were able to express AvBD1, 2, and 7 mRNA. In the intestine, the expression of AvBD10 mRNA was localized to cells along the villus at e19 and day of hatch, but later to only a few cells located above the intestinal crypts. In summary, the endodermal epithelial cells are responsible for the absorptive and immune functions of the chicken yolk sac. The yolk sac mesoderm is critical for embryonic hematopoiesis and innate immunity. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which are functioning to absorb nutrients and secrete antimicrobial peptides. / Ph. D. / The chicken yolk sac and small intestine are connected to each other and share many biological similarities. Both chicken small intestinal and yolk sac epithelia play critical roles for nutrient absorption and immune defense. In this dissertation, the mRNA for nutrient transporters such as the peptide transporter, PepT1 and the sodium-glucose co-transporter, SGLT1 were found to be expressed by absorptive epithelial cells in both the yolk sac and small intestine. Additionally, both intestinal and yolk sac epithelial cells expressed avian beta defensins (AvBDs), which are important chicken host defense peptides. In the small intestine, there are a number of differentiated cell types that originate from stem cells in the crypt that express the known mammalian stem cell markers, Olfm4 and Lgr5 mRNA. However, in the chicken yolk sac, only the stem cell marker Lgr5 mRNA was expressed by endothelial cells. In summary, the yolk sac epithelial cells are responsible for the absorptive and immune functions for the embryonic stage. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which function to absorb nutrients and secrete antimicrobial peptides.

Yes / The advent of the Internet of Things has witnessed tremendous success in the application of wireless sensor networks and ubiquitous computing for diverse smart-based applications. The developed systems operate under different technologies using different methods to achieve their targeted goals. In this treatise, we carried out an inclusive survey on key indoor technologies and techniques, with to view to explore their various benefits, limitations, and areas for improvement. The mathematical formulation for simple localization problems is also presented. In addition, an empirical evaluation of the performance of these indoor technologies is carried out using a common generic metric of scalability, accuracy, complexity, robustness, energy-efficiency, cost and reliability. An empirical evaluation of performance of different RF-based technologies establishes the viability of Wi-Fi, RFID, UWB, Wi-Fi, Bluetooth, ZigBee, and Light over other indoor technologies for reliable IoT-based applications. Furthermore, the survey advocates hybridization of technologies as an effective approach to achieve reliable IoT-based indoor systems. The findings of the survey could be useful in the selection of appropriate indoor technologies for the development of reliable real-time indoor applications. The study could also be used as a reliable source for literature referencing on the subject of indoor location identification. / Supported in part by the Tertiary Education Trust Fund of the Federal Government of Nigeria, and in part by the European Union’s Horizon 2020 Research and Innovation Programme under Grant agreement H2020-MSCA-ITN-2016 SECRET-722424

Elucidating and preserving biodiversity is an essential component of biological research, yet many natural processes impede our ability to define basic patterns of biodiversity. Hybridization is a common process in flowering plants, with a range of outcomes that influence our understanding of species ecology and evolution. In many systems, factors that facilitate or prevent successful hybridization are poorly understood. In this study, I investigate various potential mechanisms driving patterns of hybridization between Triodanis biflora and T. perfoliata. Previous research in this system has documented extensive hybridization, but some work has also alluded to the potential role of the breeding system in limiting gene flow. These patterns are particularly interesting given conflicting evidence about species delimitation of this group, with some considering T. biflora a subspecies of T. perfoliata. Here I employ a large-scale field study as well as previously collected genetic data and synthesize our overall knowledge of factors influencing patterns of hybridization in this system. Specifically, I demonstrate the first potential genetic signature for hybridization in this system, and confirm morphological differences between T. biflora, T. perfoliata, and putative hybrids across multiple hybrid zones. Across multiple field sites, I found no evidence for microhabitat (i.e., soil texture, light availability) or pollinator visitation rates for consistently limiting gene flow. Congruent with previous work, variation in the breeding system between T. biflora and T. perfoliata appears to play a major role in apparent asymmetrical patterns of hybridization across multiple hybrid zones. These species exhibit dimorphic cleistogamy, with T. biflora producing relatively fewer open flowers, and thus, less potential to contribute to hybrid gene flow. Overall, this research, combined with multiple previous studies, emphasizes the importance of natural history studies for elucidating these patterns. Despite considerable potential for gene flow between T. biflora and T. perfoliata, variation in the breeding system appears to effectively drive the magnitude, as well as overall patterns of hybridization in this study system.

Pathogen surveillance and monitoring is the first line of defense in avoiding diseases and adverse outcomes. Point of care (POC) diagnostic devices have made huge strides to achieve that, however, advancements are still required in order to expand the use of portable devices in environmental, food, and clinical diagnostics. In this work, we address critical challenges in biosensing and pathogen detection through three innovative approaches: (i) enhancing the understanding of the impact of nanostructures in DNA hybridization kinetics, (ii) developing a rapid real-time detection system for Legionella pneumophila using functional nucleic acids as biorecognition elements and DNA barcodes as detection barcodes, and (iii) applying biomimicry in microfluidic designs for uniform velocity and DNA hybridization in multiplexing. We first designed a wash and reagent free in situ electrochemical assay to investigate the role of planar and nanostructured surfaces on real-time DNA hybridization kinetics in buffer and complex media (blood, urine, and saliva). We then conducted continuous measurements to understand how these surface modifications influence electroactive DNA hybridization on the surface under a wide range of probe densities (low, medium, high) and target concentrations (0.01-1 µM). The results show that the effectiveness of nanostructures in enhancing electrochemical sensing depends on the probe/target concentration regime and the medium used in biosensing. Specifically, nanostructures were most beneficial in certain target concentration ranges (0.1-1 µM), with enhancing biosensing in all complex media compared to planar surfaces. We then utilized these nanostructures in engineering a rapid and accurate system for the detection of L. pneumophila in cooling tower water - a key factor in preventing Legionnaires' disease. To overcome the limitations of existing technologies (cell culture, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR)), we designed an RNA-cleaving DNAzyme (RCD) electrochemical assay coupled with magnetic beads, fully housed within microfluidics. This system allows for real-time monitoring by programming RCDs to release an electroactive DNA barcode upon encountering L. pneumophila targets. The barcode is detected by an integrated sensor, achieving a limit of detection of 1.4 × 10³ CFU/mL in buffer and 1.9 × 10³ CFU/mL in cooling tower water in 3 hours. This system meets regulatory requirements and enables precise identification of L. pneumophila among other waterborne bacteria and L. non-pneumophila species. Finally, we leveraged biomimicry to design microchannel systems inspired by the efficient transport mechanisms found in human spinal vertebrae and leaf veins network. By replicating and scaling these natural structures, we developed the bio-inspired microfluidic designs that optimize flow uniformity and DNA capture in Silico. Our optimized designs achieved a coefficient of variation for flow velocity of 0.89% for spine-inspired and 0.86% for leaf veins-inspired microchannels compared to 14.68 % and 59.81 % for the unoptimized designs. Additionally, these designs were compared with a simple branched design for uniform DNA capture, using the kinetics parameters extracted from our first objective. The bio-inspired designs demonstrated high DNA capture uniformity, achieving stabilization up to 10 times faster under varying conditions than a simple branched design. Ultimately, this work offers significant advancements in optimizing three crucial aspects of POC diagnostics i) surface reaction kinetics, by studying and identifying the conditions best suited for planar and nanostructured surfaces in both buffer and complex media, ii) mass transport, by investigating flow effects on biorecognition and detection, and determining the optimal conditions for biosensing, ii) and electrochemical biosensing and microfluidics integration and design, by utilizing the optimized parameters for nanostructured surface and develop a rapid, continuous, and real-time microsystem for L. pneumophila detection meeting the regulatory standards. For the second generation of this microsystem, the two bio-inspired designs will enable multiplexed detection of various pathogens. These contributions collectively are pivotal to the development of next generation POC diagnostics, with broad applications in environmental, clinical, and food safety monitoring. / Dissertation / Doctor of Philosophy (PhD) / Point of care (POC) diagnostics are expected to improve the quality of healthcare by enabling early diagnostics, improved prognostics, and enhanced treatment selection and monitoring. To realize this, POC devices must be integrated, easy to use, sensitive, specific, and cost-effective. Despite research efforts a real-time continuous multiplexed system for bacterial detection is lacking. Therefore, this thesis addresses several key challenges in biosensing and real-time continuous pathogen detection by developing innovative approaches using nano engineering, RNA-cleaving DNAzymes, electrochemical microfluidic integration, and biomimetic microfluidic designs. We first explored the impact of surface structure on real-time DNA hybridization kinetics in complex media, identifying specific conditions under which nanostructures enhance sensitivity. Building on this, we developed a rapid, real-time electrochemical microfluidic system for detecting Legionella pneumophila, a dangerous pathogen found in fresh and potable water systems. Current systems either do not meet the required limit of detection or are limited to specific serotypes, precluding other pathogenic serotypes. The electrochemical microfluidic system performed highly sensitive detection across multiple serotypes, meeting regulatory standards and enabling real-time pathogen identification across a panel of other waterborne species, offering a continuous, real-time detection alternative to slow, traditional culture-based methods. The final objective was to draw inspiration from nature to design microchannels able to deliver uniform flow and molecules to the biosensing areas for multi-analyte detection in silico. Both inspired and optimized designs demonstrated great uniformity in DNA hybridization, confirming the hypothesis that these designs are inherently proficient in equal distribution. Together, these innovations contribute to the future of rapid, sensitive, and multiplexed POC diagnostic platforms.

My thesis focuses on the conservation consequences of the hybridization of Lesser Prairie-Chickens in Kansas. Specifically, examining how past land management practices altering the species ranges impact the distinctiveness of Lesser Prairie-Chickens. Each chapter is an individual publication that addresses if the Greater and Lesser Prairie-Chicken are distinct when applying the morphological and biological species concepts. Chapter 2 compares the evolutionary history and morphological construct of Lesser Prairie-Chickens and other Galliformes using morphometric analysis. Chapter 3 uses low-resolution microsatellite data to reflect recent changes at the population level. This study aims to observe the Greater and Lesser Prairie-Chicken using the morphological and biological species concepts, two of the many species concepts, to determine the distinctiveness and rate of hybridization for these closely related species.

Bacteria classified in the family Methylomonadaceae must derive their carbon from one-carbon compounds. They are characterized by the possession of internal membranes of two types. Type I membranes are layered and fill the middle of the cells while type II membranes form concentric layers around the periphery of the cells. Also, there are two metabolic pathways by which the methylobacteria assimilate one-carbon compounds. Further evidence of this dichotomy was sought by DNA-DNA saturation hybridization of DNAs from both types of methylobacteria. Very low DNA-DNA homology was seen between types I and II or within the types. It was not possible, therefore, to correlate the degree of genetic relatedness with either the nature of the internal membranes or the pathway of carbon assimilation.

The United States is the second largest producer of oranges and grapefruit. However, the US citrus industry experiences constraints in production due to pests, diseases and environmental concerns. Furthermore, due to the low diversity in current commercial scion cultivars any exotic diseases, if introduced into any of the producing states could be devastating. To maintain the US industry competitiveness it is necessary to improve cold, pest and disease resistance to allow expansion of citrus production areas in the US, and to improve fruit quality characteristics such as sweetness, vitamins and phytochemical contents and seedlessness. Sexual hybridization in most Citrus species is complicated because they are highly apomictic. Polyembryony makes it difficult to create large segregating populations for selection. Somatic hybridization by protoplast fusion circumvents sexual incompatibilities and is a powerful tool in genetic improvement. Symmetric and asymmetric hybdridization (gamma irradiation plus iodoacetamide) via protoplast fusion were performed with the objective of producing somatic hybrids of Citrus paradisi with C. sinensis and C. reticulata with C. sinensis. These hybrids could be used for grapefruit improvement and to create genetic diversity. Furthermore, irradiated Swinglea glutinosa microprotoplasts were fused with ‘Ruby Red’ grapefruit and ‘Mucott’ tangor to assess the possibility of introgression of pieces of S. glutinosa chromosomes into the recipient protoplasts, a possible first step for radiation hybrid mapping. Double-inactivated fusions (irradiation + iodoacetamide) produced tetraploid and aneuploid plants, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis. This is the first report of obtaining rooted Citrus asymmetric hybrid plants, produced by irradiation plus iodoacetamide. AFLP confirmed presence of S. glutinosa into the receptor genomes, showing a possible donor introgression.

The major objective of this study was to produce synthetic genotypes of Brassica juncea from its parental species <i> B. rapa </i> and <i> B. nigra </i> via somatic and sexual hybridization. As prerequisites for somatic hybridization experiments, methods were developed to improve the culture of mesophyll and hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i>, to obtain reliable plant regeneration from mesophyll protoplast cultures of <i> B. nigra </i>, and to fuse protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. A modified Kao's medium (1977), was found suitable for the culture of mesophyll protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. At a density of approximately $110\sp5$ protoplasts/ml within a culture plate insert surrounded by culture medium, mesophyll protoplast cultures of <i> B. nigra </i> accessions R890, R1819, R3392 and U1218 and <i> B. rapa </i> cvs. R500 and Wong Bok formed colonies. Genotypic differences in cell division and colony formation were observed. Hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i> were successfully isolated from 6 day-old seedlings cultured in a modified Kao's medium (1977). With <i> B. nigra </i> accession R890 and <i> B. rapa </i> cv. R500, cell division and colony formation were optimal when hypocotyl protoplasts were cultured at a density of 0.5 to $1.010\sp5$ protoplasts/ml within a culture plate insert surrounded by a nurse culture of 4 to 6 day-old mesophyll protoplasts of <i> B. nigra </i>. Plant regeneration was obtained from mesophyll protoplast-derived calli of <i> B. nigra </i> accession R890 originally cultured in inserts; a shoot regeneration frequency of 8.1% was obtained on a medium containing the salts and vitamins of medium K3 (Nagy and Maliga 1976) with 3 g/l sucrose, 18.2 g/l mannitol, 2 mg/l ZR, 0.1 mg/l NAA, 10 g/l agarose, pH 5.6. For somatic hybridizatian studies, methods were developed to select out parental protoplasts using iodoacetic acid and to efficiently fuse protoplasts on the bottom of a petri dish using PEG. Twenty-nine plants were recovered from fusion experiments between mesophyll protoplasts of <i> B. nigra </i> accession R890 and hypocotyl protoplasts of <i> B. rapa </i> cv. Tobin. The somatic hybrid plants resembled natural <i> B. juncea </i>, had $2n=36$ chromosomes and had pollen viabilities ranging from 30 to 45%. Twenty-one plants, derived from one callus colony, possessed the mitochondrial and chloroplast genomes of <i> B. rapa </i>, as found in natural <i> B. juncea </i>. Eight plants, derived from another callus, had a novel cytoplasmic combination consisting of the mitochondrial genome of <i> B. rapa </i> and the chloroplast genome of <i> B. nigra </i>. Synthetic genotypes of <i> B. juncea </i> were also produced from reciprocal sexual crosses between <i> B. rapa </i> and <i> B. nigra </i>. Seventy-eight interspecific hybrid plants from the cross <i> B. rapa </i> x <i> B. nigra </i> and six hybrid plants from the reciprocal cross were identified by their morphology, pollen viability and chromosome number. The colchicine-induced allotetraploids resembled natural <i> B. juncea </i> in morphology, had 18 bivalents at metaphase I, and had between 35 and 70% pollen viability.

The United States is the second largest producer of oranges and grapefruit. However, the US citrus industry experiences constraints in production due to pests, diseases and environmental concerns. Furthermore, due to the low diversity in current commercial scion cultivars any exotic diseases, if introduced into any of the producing states could be devastating. To maintain the US industry competitiveness it is necessary to improve cold, pest and disease resistance to allow expansion of citrus production areas in the US, and to improve fruit quality characteristics such as sweetness, vitamins and phytochemical contents and seedlessness. Sexual hybridization in most Citrus species is complicated because they are highly apomictic. Polyembryony makes it difficult to create large segregating populations for selection. Somatic hybridization by protoplast fusion circumvents sexual incompatibilities and is a powerful tool in genetic improvement. Symmetric and asymmetric hybdridization (gamma irradiation plus iodoacetamide) via protoplast fusion were performed with the objective of producing somatic hybrids of Citrus paradisi with C. sinensis and C. reticulata with C. sinensis. These hybrids could be used for grapefruit improvement and to create genetic diversity. Furthermore, irradiated Swinglea glutinosa microprotoplasts were fused with ‘Ruby Red’ grapefruit and ‘Mucott’ tangor to assess the possibility of introgression of pieces of S. glutinosa chromosomes into the recipient protoplasts, a possible first step for radiation hybrid mapping. Double-inactivated fusions (irradiation + iodoacetamide) produced tetraploid and aneuploid plants, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis. This is the first report of obtaining rooted Citrus asymmetric hybrid plants, produced by irradiation plus iodoacetamide. AFLP confirmed presence of S. glutinosa into the receptor genomes, showing a possible donor introgression.

In this study we use AFM-based nanolithography technique to produce nanofeatures of the single strand DNA and LNA probe molecules which are prepared via thiolated nucleic acid self-assembled monolayers (SAMs) on gold substrates. The goal is to observe the topographic changes of the DNA film structures resulting from the formation of rigid double strand DNA when the target and probe DNAs bind together. The so-called hybridization depends strongly on the probe density on the substrate surface. To find the proper probe density for hybridization, we vary the concentration of the probe DNA and search for the optimal conditions for measuring the height changes of the nanofeatures. We also monitor the topographic changes of the DNA nanofeatures in the different target DNA concentrations as a function of time, and the binding isotherms are fitted with the Langmuir adsorption model to derive the equilibrium dissociation constant and maximum hybridization efficiency. In addition, we extend the nanoscale hybridization reaction detection to mismatched DNA:DNA and LNA:DNA hybridization, and observe that topographic change of mismatched hybridization is inconspicuous and rapidly reach equilibrium. The results reveal the apparent difference between the perfect match and mismatch conditions, and validate that this approach can be applied to differentiate the situations for both perfect match and mismatch cases, demonstrating its potentials in the gene chip technology.