Functional analysis of Nopp140 and P5CDh1 in Drosophila melanogaster

Nopp140 is a nucleolar protein with several purported roles in ribosome biogenesis. To further characterize Nopp140 in Drosophila, we used FLP-FRT recombination to delete the Nopp140 gene. Genomic PCR, RT-PCR, and immuno-fluorescence microscopy confirmed the loss of Nopp140 and its products. Compared to embryos bearing the transposons used to generate the deletion, Nopp140-/- embryos displayed similar hatching rates, but resulting larvae died after 8 days, still in the second instar stage. Nucleoli were apparent in Nopp140-/- cells with no observable morphological defects, but the rRNA methyl-transferase, fibrillarin, redistributed partially to the nucleoplasm. BrU-labeling indicated that rDNA transcription is reduced overall in Nopp140-/- larvae, while Northern analysis showed that pre-RNA cleavage was un-affected. Surprisingly, Northern analyses of Nopp140-/- larvae showed an unusually large pre-rRNA bearing the R2 retro-transposon sequence and the processed R2 transcript. The observation suggests a fundamental shift in rDNA chromatin structure and expression upon loss of Nopp140. Transmission electron microscopy of Nopp140-/- cells showed excess nuclear virus-like copia particles, reduced cytoplasmic ribosomes, autophagosome-like structures, and many electron dense cytoplasmic granules that are likely to be stress bodies and proccessing (P) bodies. Because the downstream piggyBac element used to delete Nopp140 resides in CG7145, phenotypes caused by this insertion were compared to those caused by Nopp140 deletion. Delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) is a nuclear-encoded mitochondrial enzyme that catalyzes the second step in proline degradation. Mutations in human P5CDh cause type II hyperprolinemia, a complex syndrome displaying increased serum proline and mental disabilities. Conceptual gene CG7145 in Drosophila melanogaster encodes the orthologous DmP5CDh1. The mutant allele, CG7145f04633, contains a piggyBac transposon that truncates the enzyme by 83 residues. Heterozygous (CG7145f04633/TM3) individuals developed normally, while homozygous (CG7145f04633/CG7145f04633) individuals displayed proline levels twice that of normal, swollen mitochondria, and ultimately larval and pupal lethality. These mutants showed normal ribosomal enrichment in cytoplasm, and normal rRNA gene transcription, processing, and pre-rRNA modifications. We conclude that the Nopp140 plays a critical role in ribosomal biosynthesis, and that the phenotypes in Nopp140-/- can be distinguished from the phenotypes caused solely by the CG7145 mutation

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond

Center detection algorithm for printed circuit board circular marks based on image space and parameter space

Key Project of Shaanxi Province Industrial Innovation Program, China (Grant No. 2017ZDCXL-GY-11-02-02)

Investigation of Lung Structure-Function Relationships Using Hyperpolarized Noble Gases

Magnetic Resonance Imaging (MRI) is an application of the nuclear magnetic resonance (NMR) phenomenon to non-invasively generate 3D tomographic images. MRI is an emerging modality for the lung, but it suffers from low sensitivity due to inherent low tissue density and short T2*. Hyperpolarization is a process by which the nuclear contribution to NMR signal is greatly enhanced to more than 100,000 times that of samples in thermal equilibrium. The noble gases 3He and 129Xe are most often hyperpolarized by transfer of light angular momentum through the electron of a vaporized alkali metal to the noble gas nucleus (called Spin Exchange Optical Pumping). The enhancement in NMR signal is so great that the gas itself can be imaged via MRI, and because noble gases are chemically inert, they can be safely inhaled by a subject, and the gas distribution within the interior of the lung can be imaged. The mechanics of respiration is an elegant physical process by which air is is brought into the distal airspaces of the lungs for oxygen/carbon dioxide gas exchange with blood. Therefore proper description of lung function is intricately related to its physical structure, and the basic mechanical operation of healthy lungs -- from pressure driven airflow, to alveolar airspace gas kinetics, to gas exchange by blood/gas concentration gradients, to elastic contraction of parenchymal tissue -- is a process decidedly governed by the laws of physics. This dissertation will describe experiments investigating the relationship of lung structure and function using hyperpolarized (HP) noble gas MRI. In particular HP gases will be applied to the study of several pulmonary diseases each of which demonstrates unique structure-function abnormalities: asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Successful implementation of an HP gas acquisition protocol for pulmonary studies is an involved and stratified undertaking which requires a solid theoretical foundation in NMR and hyperpolarization theory, construction of dedicated hardware, development of dedicated software, and appropriate image analysis techniques for all acquired data. The author has been actively involved in each of these and has dedicated specific chapters of this dissertation to their description. First, a brief description of lung structure-function investigations and pulmonary imaging will be given (chapter 1). Brief discussions of basic NMR, MRI, and hyperpolarization theory will be given (chapters 2 and 3) followed by their particular methods of implementation in this work (chapters 4 and 5). Analysis of acquired HP gas images will be discussed (chapter 6), and the investigational procedures and results for each lung disease examined will be detailed (chapter 7). Finally, a quick digression on the strengths and limitations of HP gas MRI will be provided (chapter 8)

Single-molecule techniques in biophysics : a review of the progress in methods and applications

Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft matter behaviour, active biological matter lives far from thermal equilibrium, and typically covers multiple length scales from the nanometre level of single molecules up several orders of magnitude to longer length scales in emergent structures of cells, tissues and organisms. Biological molecules are often characterized by an underlying instability, in that multiple metastable free energy states exist which are separated by energy levels of typically just a few multiples of the thermal energy scale of kBT, where kB is the Boltzmann constant and T the absolute temperature, implying complex, dynamic inter-conversion kinetics across this bumpy free energy landscape in the relatively hot, wet environment of real, living biological matter. The key utility of single-molecule biophysics lies in its ability to probe the underlying heterogeneity of free energy states across a population of molecules, which in general is too challenging for conventional ensemble level approaches which measure mean average properties. Parallel developments in both experimental and theoretical techniques have been key to the latest insights and are enabling the development of highly-multiplexed, correlative techniques to tackle previously intractable biological problems. Experimentally, technological developments in the sensitivity and speed of biomolecular detectors, the stability and efficiency of light sources, probes and microfluidics, have enabled and driven the study of heterogeneous behaviours both in vitro and in vivo that were previously undetectable by ensemble methods..

Image statistical frameworks for digital image forensics

The advances of digital cameras, scanners, printers, image editing tools, smartphones, tablet personal computers as well as high-speed networks have made a digital image a conventional medium for visual information. Creation, duplication, distribution, or tampering of such a medium can be easily done, which calls for the necessity to be able to trace back the authenticity or history of the medium. Digital image forensics is an emerging research area that aims to resolve the imposed problem and has grown in popularity over the past decade. On the other hand, anti-forensics has emerged over the past few years as a relatively new branch of research, aiming at revealing the weakness of the forensic technology. These two sides of research move digital image forensic technologies to the next higher level. Three major contributions are presented in this dissertation as follows. First, an effective multi-resolution image statistical framework for digital image forensics of passive-blind nature is presented in the frequency domain. The image statistical framework is generated by applying Markovian rake transform to image luminance component. Markovian rake transform is the applications of Markov process to difference arrays which are derived from the quantized block discrete cosine transform 2-D arrays with multiple block sizes. The efficacy and universality of the framework is then evaluated in two major applications of digital image forensics: 1) digital image tampering detection; 2) classification of computer graphics and photographic images. Second, a simple yet effective anti-forensic scheme is proposed, capable of obfuscating double JPEG compression artifacts, which may vital information for image forensics, for instance, digital image tampering detection. Shrink-and-zoom (SAZ) attack, the proposed scheme, is simply based on image resizing and bilinear interpolation. The effectiveness of SAZ has been evaluated over two promising double JPEG compression schemes and the outcome reveals that the proposed scheme is effective, especially in the cases that the first quality factor is lower than the second quality factor. Third, an advanced textural image statistical framework in the spatial domain is proposed, utilizing local binary pattern (LBP) schemes to model local image statistics on various kinds of residual images including higher-order ones. The proposed framework can be implemented either in single- or multi-resolution setting depending on the nature of application of interest. The efficacy of the proposed framework is evaluated on two forensic applications: 1) steganalysis with emphasis on HUGO (Highly Undetectable Steganography), an advanced steganographic scheme embedding hidden data in a content-adaptive manner locally into some image regions which are difficult for modeling image statics; 2) image recapture detection (IRD). The outcomes of the evaluations suggest that the proposed framework is effective, not only for detecting local changes which is in line with the nature of HUGO, but also for detecting global difference (the nature of IRD)