Cycling in the nucleus: regulation of RNA 3' processing and nuclear organization of replication-dependent histone genes.

The histones which pack new DNA during the S phase of animal cells are made from mRNAs that are cleaved at their 3' end but not polyadenylated. Some of the factors used in this reaction are unique to it while others are shared with the polyadenylation process that generates all other mRNAs. Recent work has begun to shed light on how the cell manages the assignment of these common components to the two 3' processing systems, and how it achieves their cell cycle-regulation and recruitment to the histone pre-mRNA. Moreover, recent and older findings reveal multiple connections between the nuclear organization of histone genes, their transcription and 3' end processing as well as the control of cell proliferation.

Improving 4D plan quality for PBS-based liver tumour treatments by combining online image guided beam gating with rescanning.

Pencil beam scanned (PBS) proton therapy has many advantages over conventional radiotherapy, but its effectiveness for treating mobile tumours remains questionable. Gating dose delivery to the breathing pattern is a well-developed method in conventional radiotherapy for mitigating tumour-motion, but its clinical efficiency for PBS proton therapy is not yet well documented. In this study, the dosimetric benefits and the treatment efficiency of beam gating for PBS proton therapy has been comprehensively evaluated. A series of dedicated 4D dose calculations (4DDC) have been performed on 9 different 4DCT(MRI) liver data sets, which give realistic 4DCT extracting motion information from 4DMRI. The value of 4DCT(MRI) is its capability of providing not only patient geometries and deformable breathing characteristics, but also includes variations in the breathing patterns between breathing cycles. In order to monitor target motion and derive a gating signal, we simulate time-resolved beams' eye view (BEV) x-ray images as an online motion surrogate. 4DDCs have been performed using three amplitude-based gating window sizes (10/5/3 mm) with motion surrogates derived from either pre-implanted fiducial markers or the diaphragm. In addition, gating has also been simulated in combination with up to 19 times rescanning using either volumetric or layered approaches. The quality of the resulting 4DDC plans has been quantified in terms of the plan homogeneity index (HI), total treatment time and duty cycle. Results show that neither beam gating nor rescanning alone can fully retrieve the plan homogeneity of the static reference plan. Especially for variable breathing patterns, reductions of the effective duty cycle to as low as 10% have been observed with the smallest gating rescanning window (3 mm), implying that gating on its own for such cases would result in much longer treatment times. In addition, when rescanning is applied on its own, large differences between volumetric and layered rescanning have been observed as a function of increasing number of re-scans. However, once gating and rescanning is combined, HI to within 2% of the static plan could be achieved in the clinical target volume, with only moderately prolonged treatment times, irrespective of the rescanning strategy used. Moreover, these results are independent of the motion surrogate used. In conclusion, our results suggest image guided beam gating, combined with rescanning, is a feasible, effective and efficient motion mitigation approach for PBS-based liver tumour treatments.

An Inexpensive Alternative to CS Photoshop & Bridge: Archival Master Image Capture and Batch Derivative Creation in Canon Scan Gear & Adobe Photoshop Elements 7

This presentation was made at the Connecticut State Library Service Center, Willimantic, CT, April 14, 2009. It focused on digital capture workflows for both archival and derivative image creation using accepted current standards. Tools used were inexpensive by choice and focused towards the needs of small to mid-sized cultural heritage institutions who wish to begin digital capture in their own facilities.

NOVEL PHANTOMS AND POST-PROCESSING FOR DIFFUSION SPECTRUM IMAGING

High Angular Resolution Diffusion Imaging (HARDI) techniques, including Diffusion Spectrum Imaging (DSI), have been proposed to resolve crossing and other complex fiber architecture in the human brain white matter. In these methods, directional information of diffusion is inferred from the peaks in the orientation distribution function (ODF). Extensive studies using histology on macaque brain, cat cerebellum, rat hippocampus and optic tracts, and bovine tongue are qualitatively in agreement with the DSI-derived ODFs and tractography. However, there are only two studies in the literature which validated the DSI results using physical phantoms and both these studies were not performed on a clinical MRI scanner. Also, the limited studies which optimized DSI in a clinical setting, did not involve a comparison against physical phantoms. Finally, there is lack of consensus on the necessary pre- and post-processing steps in DSI; and ground truth diffusion fiber phantoms are not yet standardized. Therefore, the aims of this dissertation were to design and construct novel diffusion phantoms, employ post-processing techniques in order to systematically validate and optimize (DSI)-derived fiber ODFs in the crossing regions on a clinical 3T MR scanner, and develop user-friendly software for DSI data reconstruction and analysis. Phantoms with a fixed crossing fiber configuration of two crossing fibers at 90° and 45° respectively along with a phantom with three crossing fibers at 60°, using novel hollow plastic capillaries and novel placeholders, were constructed. T2-weighted MRI results on these phantoms demonstrated high SNR, homogeneous signal, and absence of air bubbles. Also, a technique to deconvolve the response function of an individual peak from the overall ODF was implemented, in addition to other DSI post-processing steps. This technique greatly improved the angular resolution of the otherwise unresolvable peaks in a crossing fiber ODF. The effects of DSI acquisition parameters and SNR on the resultant angular accuracy of DSI on the clinical scanner were studied and quantified using the developed phantoms. With a high angular direction sampling and reasonable levels of SNR, quantification of a crossing region in the 90°, 45° and 60° phantoms resulted in a successful detection of angular information with mean ± SD of 86.93°±2.65°, 44.61°±1.6° and 60.03°±2.21° respectively, while simultaneously enhancing the ODFs in regions containing single fibers. For the applicability of these validated methodologies in DSI, improvement in ODFs and fiber tracking from known crossing fiber regions in normal human subjects were demonstrated; and an in-house software package in MATLAB which streamlines the data reconstruction and post-processing for DSI, with easy to use graphical user interface was developed. In conclusion, the phantoms developed in this dissertation offer a means of providing ground truth for validation of reconstruction and tractography algorithms of various diffusion models (including DSI). Also, the deconvolution methodology (when applied as an additional DSI post-processing step) significantly improved the angular accuracy of the ODFs obtained from DSI, and should be applicable to ODFs obtained from the other high angular resolution diffusion imaging techniques.

Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.

Radiomics of NSCLC: Quantitative CT Image Feature Characterization and Tumor Shrinkage Prediction

Radiomics is the high-throughput extraction and analysis of quantitative image features. For non-small cell lung cancer (NSCLC) patients, radiomics can be applied to standard of care computed tomography (CT) images to improve tumor diagnosis, staging, and response assessment. The first objective of this work was to show that CT image features extracted from pre-treatment NSCLC tumors could be used to predict tumor shrinkage in response to therapy. This is important since tumor shrinkage is an important cancer treatment endpoint that is correlated with probability of disease progression and overall survival. Accurate prediction of tumor shrinkage could also lead to individually customized treatment plans. To accomplish this objective, 64 stage NSCLC patients with similar treatments were all imaged using the same CT scanner and protocol. Quantitative image features were extracted and principal component regression with simulated annealing subset selection was used to predict shrinkage. Cross validation and permutation tests were used to validate the results. The optimal model gave a strong correlation between the observed and predicted shrinkages with . The second objective of this work was to identify sets of NSCLC CT image features that are reproducible, non-redundant, and informative across multiple machines. Feature sets with these qualities are needed for NSCLC radiomics models to be robust to machine variation and spurious correlation. To accomplish this objective, test-retest CT image pairs were obtained from 56 NSCLC patients imaged on three CT machines from two institutions. For each machine, quantitative image features with concordance correlation coefficient values greater than 0.90 were considered reproducible. Multi-machine reproducible feature sets were created by taking the intersection of individual machine reproducible feature sets. Redundant features were removed through hierarchical clustering. The findings showed that image feature reproducibility and redundancy depended on both the CT machine and the CT image type (average cine 4D-CT imaging vs. end-exhale cine 4D-CT imaging vs. helical inspiratory breath-hold 3D CT). For each image type, a set of cross-machine reproducible, non-redundant, and informative image features was identified. Compared to end-exhale 4D-CT and breath-hold 3D-CT, average 4D-CT derived image features showed superior multi-machine reproducibility and are the best candidates for clinical correlation.

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

Comerciantes en Río de la Plata pre-virreinal. El caso de Domingo de Basavilbaso

El propósito de este texto es presentar a uno de los comerciantes más exitosos de mediados del siglo XVIII en la región Río de la Plata y una aproximación al conjunto del cual emerge. La imagen de grupo se obtiene a partir de los registros de navíos españoles, que permiten realizar un estudio pormenorizado de la participación de los individuos en los envíos a España. Al mismo tiempo, y para acercarme más a uno de los individuos en particular y su primer círculo de relaciones se utiliza, además de los registros, la bibliografía disponible y la batería habitual de fuentes consultadas en las investigaciones sobre comerciantes

Comerciantes en Río de la Plata pre-virreinal. El caso de Domingo de Basavilbaso

El propósito de este texto es presentar a uno de los comerciantes más exitosos de mediados del siglo XVIII en la región Río de la Plata y una aproximación al conjunto del cual emerge. La imagen de grupo se obtiene a partir de los registros de navíos españoles, que permiten realizar un estudio pormenorizado de la participación de los individuos en los envíos a España. Al mismo tiempo, y para acercarme más a uno de los individuos en particular y su primer círculo de relaciones se utiliza, además de los registros, la bibliografía disponible y la batería habitual de fuentes consultadas en las investigaciones sobre comerciantes

Comerciantes en Río de la Plata pre-virreinal. El caso de Domingo de Basavilbaso

El propósito de este texto es presentar a uno de los comerciantes más exitosos de mediados del siglo XVIII en la región Río de la Plata y una aproximación al conjunto del cual emerge. La imagen de grupo se obtiene a partir de los registros de navíos españoles, que permiten realizar un estudio pormenorizado de la participación de los individuos en los envíos a España. Al mismo tiempo, y para acercarme más a uno de los individuos en particular y su primer círculo de relaciones se utiliza, además de los registros, la bibliografía disponible y la batería habitual de fuentes consultadas en las investigaciones sobre comerciantes

Probability sampling protocol of classification maps from spaceborne/airborne image

To deliver sample estimates provided with the necessary probability foundation to permit generalization from the sample data subset to the whole target population being sampled, probability sampling strategies are required to satisfy three necessary not sufficient conditions: (i) All inclusion probabilities be greater than zero in the target population to be sampled. If some sampling units have an inclusion probability of zero, then a map accuracy assessment does not represent the entire target region depicted in the map to be assessed. (ii) The inclusion probabilities must be: (a) knowable for nonsampled units and (b) known for those units selected in the sample: since the inclusion probability determines the weight attached to each sampling unit in the accuracy estimation formulas, if the inclusion probabilities are unknown, so are the estimation weights. This original work presents a novel (to the best of these authors' knowledge, the first) probability sampling protocol for quality assessment and comparison of thematic maps generated from spaceborne/airborne Very High Resolution (VHR) images, where: (I) an original Categorical Variable Pair Similarity Index (CVPSI, proposed in two different formulations) is estimated as a fuzzy degree of match between a reference and a test semantic vocabulary, which may not coincide, and (II) both symbolic pixel-based thematic quality indicators (TQIs) and sub-symbolic object-based spatial quality indicators (SQIs) are estimated with a degree of uncertainty in measurement in compliance with the well-known Quality Assurance Framework for Earth Observation (QA4EO) guidelines. Like a decision-tree, any protocol (guidelines for best practice) comprises a set of rules, equivalent to structural knowledge, and an order of presentation of the rule set, known as procedural knowledge. The combination of these two levels of knowledge makes an original protocol worth more than the sum of its parts. The several degrees of novelty of the proposed probability sampling protocol are highlighted in this paper, at the levels of understanding of both structural and procedural knowledge, in comparison with related multi-disciplinary works selected from the existing literature. In the experimental session the proposed protocol is tested for accuracy validation of preliminary classification maps automatically generated by the Satellite Image Automatic MapperT (SIAMT) software product from two WorldView-2 images and one QuickBird-2 image provided by DigitalGlobe for testing purposes. In these experiments, collected TQIs and SQIs are statistically valid, statistically significant, consistent across maps and in agreement with theoretical expectations, visual (qualitative) evidence and quantitative quality indexes of operativeness (OQIs) claimed for SIAMT by related papers. As a subsidiary conclusion, the statistically consistent and statistically significant accuracy validation of the SIAMT pre-classification maps proposed in this contribution, together with OQIs claimed for SIAMT by related works, make the operational (automatic, accurate, near real-time, robust, scalable) SIAMT software product eligible for opening up new inter-disciplinary research and market opportunities in accordance with the visionary goal of the Global Earth Observation System of Systems (GEOSS) initiative and the QA4EO international guidelines.

Airborne hyperspectral image data of Heron Reef, Australia

This airborne hyperspectral (19 bands) image data of Heron Reef, Great Barrier Reef, Australia is derived from Compact Airborne Spectrographic Imager (CASI) data acquired on 1st and 3rd of July 2002, latitude -23.45, longitude 151.92. Processing and correction to at-surface data was completed by Karen Joyce (Joyce, 2004). Raw imagery consisted several images corresponding to the number of flight paths taken to cover the entire Heron Reef. Spatial resolution is one meter. Radiometric corrections converted the at-sensor digital number values to at surface spectral radiance values using sensor specific calibration coefficients and CSIRO's c-WomBat-c atmospheric correction software. Geometric corrections were done using field collected coordinates of features identified in the image. Projection used was Universal Transverse Mercator Zone 56 South and Datum used was WGS 84. Image data is in TIFF format.

In-loop Feature Tracking for Structure and Motion with Out-of-core Optimization

In this paper, a novel and approach for obtaining 3D models from video sequences captured with hand-held cameras is addressed. We define a pipeline that robustly deals with different types of sequences and acquiring devices. Our system follows a divide and conquer approach: after a frame decimation that pre-conditions the input sequence, the video is split into short-length clips. This allows to parallelize the reconstruction step which translates into a reduction in the amount of computational resources required. The short length of the clips allows an intensive search for the best solution at each step of reconstruction which robustifies the system. The process of feature tracking is embedded within the reconstruction loop for each clip as opposed to other approaches. A final registration step, merges all the processed clips to the same coordinate frame

A set of canonical problems in sloshing. Part 0: Experimental setup and data processing

In a series of attempts to research and document relevant sloshing type phenomena, a series of experiments have been conducted. The aim of this paper is to describe the setup and data processing of such experiments. A sloshing tank is subjected to angular motion. As a result pressure registers are obtained at several locations, together with the motion data, torque and a collection of image and video information. The experimental rig and the data acquisition systems are described. Useful information for experimental sloshing research practitioners is provided. This information is related to the liquids used in the experiments, the dying techniques, tank building processes, synchronization of acquisition systems, etc. A new procedure for reconstructing experimental data, that takes into account experimental uncertainties, is presented. This procedure is based on a least squares spline approximation of the data. Based on a deterministic approach to the first sloshing wave impact event in a sloshing experiment, an uncertainty analysis procedure of the associated first pressure peak value is described.