Image processing methods for human brain connectivity analysis from in-vivo diffusion MRI

In this PhD Thesis proposal, the principles of diffusion MRI (dMRI) in its application to the human brain mapping of connectivity are reviewed. The background section covers the fundamentals of dMRI, with special focus on those related to the distortions caused by susceptibility inhomogeneity across tissues. Also, a deep survey of available correction methodologies for this common artifact of dMRI is presented. Two methodological approaches to improved correction are introduced. Finally, the PhD proposal describes its objectives, the research plan, and the necessary resources.

Study and analysis of the Leap Motion sensor and the Software Development Kit (SDK) for the implementation of visual Human Computer Interfaces

En este proyecto, se presenta un informe técnico sobre la cámara Leap Motion y el Software Development Kit correspondiente, el cual es un dispositivo con una cámara de profundidad orientada a interfaces hombre-máquina. Esto es realizado con el propósito de desarrollar una interfaz hombre-máquina basada en un sistema de reconocimiento de gestos de manos. Después de un exhaustivo estudio de la cámara Leap Motion, se han realizado diversos programas de ejemplo con la intención de verificar las capacidades descritas en el informe técnico, poniendo a prueba la Application Programming Interface y evaluando la precisión de las diferentes medidas obtenidas sobre los datos de la cámara. Finalmente, se desarrolla un prototipo de un sistema de reconocimiento de gestos. Los datos sobre la posición y orientación de la punta de los dedos obtenidos de la Leap Motion son usados para describir un gesto mediante un vector descriptor, el cual es enviado a una Máquina Vectores Soporte, utilizada como clasificador multi-clase.

Image processing methods for human brain connectivity analysis from in-vivo diffusion MRI

The structural connectivity of the brain is considered to encode species-wise and subject-wise patterns that will unlock large areas of understanding of the human brain. Currently, diffusion MRI of the living brain enables to map the microstructure of tissue, allowing to track the pathways of fiber bundles connecting the cortical regions across the brain. These bundles are summarized in a network representation called connectome that is analyzed using graph theory. The extraction of the connectome from diffusion MRI requires a large processing flow including image enhancement, reconstruction, segmentation, registration, diffusion tracking, etc. Although a concerted effort has been devoted to the definition of standard pipelines for the connectome extraction, it is still crucial to define quality assessment protocols of these workflows. The definition of quality control protocols is hindered by the complexity of the pipelines under test and the absolute lack of gold-standards for diffusion MRI data. Here we characterize the impact on structural connectivity workflows of the geometrical deformation typically shown by diffusion MRI data due to the inhomogeneity of magnetic susceptibility across the imaged object. We propose an evaluation framework to compare the existing methodologies to correct for these artifacts including whole-brain realistic phantoms. Additionally, we design and implement an image segmentation and registration method to avoid performing the correction task and to enable processing in the native space of diffusion data. We release PySDCev, an evaluation framework for the quality control of connectivity pipelines, specialized in the study of susceptibility-derived distortions. In this context, we propose Diffantom, a whole-brain phantom that provides a solution to the lack of gold-standard data. The three correction methodologies under comparison performed reasonably, and it is difficult to determine which method is more advisable. We demonstrate that susceptibility-derived correction is necessary to increase the sensitivity of connectivity pipelines, at the cost of specificity. Finally, with the registration and segmentation tool called regseg we demonstrate how the problem of susceptibility-derived distortion can be overcome allowing data to be used in their original coordinates. This is crucial to increase the sensitivity of the whole pipeline without any loss in specificity.

Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

Oxygen Isotope Analysis of Human Bone Phosphate Evidences Weaning Age in Archaeological Populations

Peer reviewed

Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF2α—F2-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF2α-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF2α-VI in hypercholesterolemia and of 8,12-iso-iPF2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

Genomic analysis of human and mouse TCL1 loci reveals a complex of tightly clustered genes

TCL1 and TCL1b genes on human chromosome 14q23.1 are activated in T cell leukemias by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T cell receptor genes. In this report we present the cloning, mapping, and expression analysis of the human and murine TCL1/Tcl1 locus. In addition to TCL1 and TCL1b, the human locus contains two additional genes, TCL1-neighboring genes (TNG) 1 and 2, encoding proteins of 141 and 110 aa, respectively. Both genes show no homology to any known genes, but their expression profiles are very similar to those of TCL1 and TCL1b. TNG1 and TNG2 also are activated in T cell leukemias with rearrangements at 14q32.1. To aid in the development of a mouse model we also have characterized the murine Tcl1 locus and found five genes homologous to human TCL1b. Tcl1b1–Tcl1b5 proteins range from 117 to 123 aa and are 65–80% similar, but they show only a 30–40% similarity to human TCL1b. All five mouse Tcl1b and murine Tcl1 mRNAs are abundant in mouse oocytes and two-cell embryos but rare in various adult tissues and lymphoid cell lines. These data suggest a similar or complementary function of these proteins in early embryogenesis.

Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16

Rhinoviruses are a frequent cause of the common cold. A series of antirhinoviral compounds have been developed that bind into a hydrophobic pocket in the viral capsid, stabilizing the capsid and interfering with cell attachment. The structures of a variety of such compounds, complexed with rhinovirus serotypes 14, 16, 1A, and 3, previously have been examined. Three chemically similar compounds, closely related to a drug that is undergoing phase III clinical trials, were chosen to determine the structural impact of the heteroatoms in one of the three rings. The compounds were found to have binding modes that depend on their electronic distribution. In the compound with the lowest efficacy, the terminal ring is displaced by 1 Å and rotated by 180° relative to the structure of the other two. The greater polarity of the terminal ring in one of the three compounds leads to a small displacement of its position relative to the other compounds in the hydrophobic end of the antiviral compound binding pocket to a site where it makes fewer interactions. Its lower efficacy is likely to be the result of the reduced number of interactions. A region of conserved residues has been identified near the entrance to the binding pocket where there is a corresponding conservation of the mode of binding of these compounds to different serotypes. Thus, variations in residues lining the more hydrophobic end of the pocket are primarily responsible for the differences in drug efficacies.

Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: Induction of interferon-responsive RNAs

We used differential display analysis to identify mRNAs that accumulate to enhanced levels in human cytomegalovirus-infected cells as compared with mock-infected cells. RNAs were compared at 8 hr after infection of primary human fibroblasts. Fifty-seven partial cDNA clones were isolated, representing about 26 differentially expressed mRNAs. Eleven of the mRNAs were virus-coded, and 15 were of cellular origin. Six of the partial cDNA sequences have not been reported previously. All of the cellular mRNAs identified in the screen are induced by interferon α. The induction in virus-infected cells, however, does not involve the action of interferon or other small signaling molecules. Neutralizing antibodies that block virus infection also block the induction. These RNAs accumulate after infection with virus that has been inactivated by treatment with UV light, indicating that the inducer is present in virions. We conclude that human cytomegalovirus induces interferon-responsive mRNAs.

Screening for human T cell leukaemia/lymphoma virus among blood donors in Sweden: cost effectiveness analysis

Objective: To analyse the cost effectiveness of a national programme to screen blood donors for infection with the human T cell leukaemia/lymphoma virus.

Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5

Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning Ache to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by Ache and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse–human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.

Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs

The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.