Highly efficient selection, enumeration, enrichment, and molecular profiling of low-abundance biological cells

After brief overviews of low-abundance cell selection techniques in chapter 1 and circulating tumor cells in chapter 2, this dissertation initially focuses on the development of aptamer incorporated high-throughput microfluidic techniques to select rare circulation prostate cancer cells (LNCaP) directly from whole blood with subsequent quantification of these rare cells using a non-labeling approach. Then, I extended the technology to environmental samples in an effort around time, sensitivity, and portability of traditional groundwater assessment. As a model bio- pathogen, E. coli O157:H7 was chosen due to its toxicity and its adverse impact on recreational waters. Low-abundance (\u3c100 cells mL-1) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip that its capture beds were covalently decorated with E.coli O157:H7 specific polyclonal antibodies. The selected cells were enumerated using RT-qPCR technique. Finally, I have integrated HTMSU with electrokinetic enrichment microfluidic unit for performance of single recombinant low-abundance CTC cell-based assay. A series of analytical processes were carried out, including immunoaffinity selection of rare CTCs, quantification of selected cells via conductivity impedance and electrophoretic enrichment of selected cells for PCR/LDR/CE interrogation for detection of low-abundance point mutations in genomic DNA

Joint Prediction of Depths, Normals and Surface Curvature from RGB Images using CNNs

Understanding the 3D structure of a scene is of vital importance, when it comes to developing fully autonomous robots. To this end, we present a novel deep learning based framework that estimates depth, surface normals and surface curvature by only using a single RGB image. To the best of our knowledge this is the first work to estimate surface curvature from colour using a machine learning approach. Additionally, we demonstrate that by tuning the network to infer well designed features, such as surface curvature, we can achieve improved performance at estimating depth and normals.This indicates that network guidance is still a useful aspect of designing and training a neural network. We run extensive experiments where the network is trained to infer different tasks while the model capacity is kept constant resulting in different feature maps based on the tasks at hand. We outperform the previous state-of-the-art benchmarks which jointly estimate depths and surface normals while predicting surface curvature in parallel

Auxin Action in a Cell-Free System

The plant hormone auxin regulates diverse aspects of plant growth and development [1]. Despite its importance, the mechanisms of auxin action remain poorly understood. In particular, the identities of the auxin receptor and other signaling proteins are unknown. Recent studies have shown that auxin acts by promoting the degradation of a family of transcriptional regulators called the Aux/IAA proteins 2, 3 and 4. These proteins interact with another large family of plant-specific transcription factors called Auxin Response Factors (ARF) and negatively regulate their activity [5]. Auxin stimulates Aux/IAA degradation by promoting the interaction between a ubiquitin protein ligase (E3) called SCFTIR1 and the Aux/IAA protein [2]. In this report, we demonstrate that auxin promotes the interaction between the Aux/IAA proteins and SCFTIR1 in a soluble extract free of membranes, indicating that this auxin response is mediated by a soluble receptor. In addition, we show that the response is not dependent on protein phosphorylation or dephosphorylation but rather is prevented by an inhibitor of peptidyl-prolyl isomerases

Regional stability changes in oxidized and reduced cytochrome c located by hydrogen exchange and mass spectrometry

Amide hydrogen exchange rates are highly sensitive to protein structure and may, therefore, be used to detect and characterize structural changes in proteins. Specific regions within folded proteins undergoing structural change can often be identified if localized amide hydrogen exchange rates are determined by nuclear magnetic resonance (NMR). The ability to measure localized amide hydrogen exchange rates by proteolytic fragmentation followed by mass spectrometric analysis opens the possibility to also identify localized structural changes in proteins by mass spectrometry. If successful, this approach offers considerable advantage over NMR in speed, sensitivity, protein solubility, and ability to study large proteins. This possibility has been investigated by determining the amide hydrogen exchange rates in oxidized and reduced cytochrome c by protein fragmentation/mass spectrometry. The fundamental difference in these forms of cytochrome c is the oxidation state of the iron, which other studies have shown results in only minor structural changes in the protein. In the present study, the largest differences in hydrogen exchange rates were found for peptide amide hydrogens located distant from the Nand C-termini, indicating that the structure in these regions is most affected by the oxidation state of the iron. These results are consistent with previous studies of oxidized and reduced cytochrome c, suggesting that hydrogen exchange and mass spectrometry may be generally useful for locating subtle changes in protein structure

Utilization of Stockpiled Perennial Forages in Winter Feeding Systems for Beef Cattle

Two experiments were conducted to determine the effects of grazing stockpiled perennial forage in field paddocks relative to feeding similar quality round bale hay in drylot pens on rumen degradation characteristics of forage; beef cow performance, cow reproductive efficiency, estimated dry matter intake and forage utilization, forage yield and quality, soil nutrients and system costs. Winter feeding systems were (i) stockpiled perennial forage (TDN = 58.9%; CP = 8.5%) grazing (SPF) and (ii) drylot feeding (DL) of round bale hay (TDN = 57.9%; CP = 8.4%). Experiment I was an in situ study, where five Hereford heifers (398 ± 14 kg) fitted with rumen cannulae were fed a grass hay (DM = 93.2%; TDN = 50.8%; CP = 9.8%; NDF = 66.2%) diet. In situ degradability of both stockpiled forage (SPF) and round bale hay (BH) samples collected at start (October) and end (December) of the field study were determined. The soluble fraction (S) of DM was greater (P = 0.01) in SPF October forage compared to SPF December, BH October and BH December forages. The potentially degradable fraction (D) of CP was lowest (P = 0.04) in BH December forage than in SPF October, SPF December and BH October forages suggesting that hay quality declined more rapidly than stockpiled forage and method of preservation may have affected overall hay quality. Furthermore, D fraction of both ADF and NDF was higher in SPF samples suggesting stockpiled forage may be more digestible than hay. However, the D fraction of NDF in both SPF and BH forages declined with later sampling date possibly due to effect of weathering and leaf loss. In Experiment II, 6, 4-ha paddocks consisting of meadow bromegrass (Bromus riparius Rehm) and alfalfa (Medicago sativa), were randomly assigned to 1 of 2 replicated (n = 3) winter feeding systems. In this study 58 dry pregnant (120 ± 16 d) Angus cows (675 kg ± 51 kg), stratified by body weight (BW; corrected for conceptus gain), were allocated to either the SPF or DL systems. Cows in winter feeding systems were provided additional energy supplement (rolled barley) (TDN = 86.4%; CP = 12.4%) depending on environmental conditions to maintain body condition, with no weight gain above that of conceptus growth. Dry matter intake (DMI) and forage utilization were estimated using the herbage weight disappearance method. The effects of winter feeding systems on soil nutrients were determined the following spring after winter grazing. Forage yield in DL (4683 ± 495 kg ha-1) and SPF (4032 ± 495 kg ha-1) systems was not different (P = 0.18) between treatments. However, forage utilization was lower (P 0.05) between winter feeding systems. Reproductive performance of beef cows was not affected (P > 0.05) by either winter feeding methods as cows in both systems maintained body condition score (BCS) at 2.5 to 3.0 throughout the study. Average total production cost was 19% lower in SPF system compared to DL system. In conclusion, the rumen degradation characteristics of stockpiled perennial forages focused in this study support the utilization these forages in a winter feeding system to meet the nutrient requirements of dry beef cows in early to mid-gestation. It may be cost effective to manage beef cows in field grazing of stockpiled perennial forages in western Canada, without any negative impact on beef cow performance or reproductive efficiency

Tsunami disaster 2004: lessons from resettlement

The tsunami disaster of 26 December 2004 killed over 30,000 people, displaced 1,000,000 persons and affected over two thirds of the island’s coastline lying in 12 districts. Besides the loss of life and injuries, the tsunami caused extensive damage to infrastructure and property and disruptions of fisheries and other livelihood activities and business assets. After the emergency repairs were attended the government has entered into the phase of rehabilitation and reconstruction of national infrastructure in the affected areas with the assistance of many development partners. However, the national construction industry has not been able to cater to the demand or need of tsunami reconstruction work. Further challenges include procurement delays, ensuring environmental safeguards, security concerns in the uncleared areas and capacity constraints. As far as the new settlements for the beneficiaries are concerned, development of infrastructure such as water, electricity and internal roads remain the biggest challenge to the government

Priority for agriculture over housing, water supply and sanitation of Mahaweli settlers

The Mahaweli Ganga Development Scheme (MGDS) has been the largest multipurpose development project ever implemented in Sri Lanka, using the water resources of the river Mahaweli and related adjacent river basins. Of the thirteen independent development areas identified for integrated development and human settlement, System – H, System –B, System- C, System-G and System-L are either fully or partly developed and operational. The uses of water such as irrigation, power generation and domestic consumption are inter-linked within a basin. As both the quality and quantity of water available for downstream users depend on the activities of the upstream users. However, during the last three decades, the emphasis has been to increase crop production and productivity of land and water, over the development of essential infrastructure. This trend has been so established in agriculture based settlement projects, even the international funding agencies do not attach much importance to the provision or improvement of infrastructure facilities

Application of Remote Sensing and Geographic Information System for Assessment of Flood Risk on the Major Downstream Areas of Gombe Metropolis, Nigeria

The world‘s population is rapidly becoming more urbanized as the world seen a swift urban population increase. Gombe Metropolis is among the urban areas of Nigeria affected by the consequence of frequent seasonal floods leading to a unpropitious effect on the flood communities in many parts of the metropolis. These urbanization dynamics has caused a rapid urban growth through the transformation of many different land uses into the built-up environment. As a result flood risk in the metropolis has been rising in recent years and efforts by the people and government to mitigate the flood risk have not been entirely successful. Thus, this paper attempts to examine the nature of flood risk on the major downstream areas of Gombe metropolis. Fundamentally, this study applied Geographic Information System and Remote Sensing as a tool for integration of spatiotemporal data for modeling and comparison of urban development scenarios and its consequential effect in creating flood risk on the downstream areas of Gombe Metropolis. Therefore, GIS and Remote Sensing have been applied to detect land use/land cover changes, by looking at the trend in Land use/Land Cover Change from 2003 to 2014 in the Gombe Metropolis. In addition, the study identified Land use/Land cover types in different residential areas of the metropolis with more emphasis on the building density in each area. Finally, Gombe Metropolis Flood Risk Zones were detected and developed into Gombe Metropolis Flood Risk Map. The paper was able to reveal a significant growth of built-up environment and the occupation of floodplains in the downstream areas as the main factors for flood risk in Gombe Metropolis. However, the flood risk index established that the residential areas found on the very high to high flood risk zones include Barunde, Government Residential Areas/Gabuka, Pantami, Dawaki, and Tudun Wada. It was further reveal that the residential areas in the moderate flood risk zone are, Jankai, Jekadafari, Herwagana, Bolari/Madaki and MUAK. Keywords: Geographic Information System, Flood Risk, Gombe Metropolis, Remote Sensing