Texture-Based Segmentation and Finite Element Mesh Generation for Heterogeneous Biological Image Data

The design, analysis, and control of bio-systems remain an engineering challenge. This is mainly due to the material heterogeneity, boundary irregularity, and nonlinear dynamics associated with these systems. The recent developments in imaging techniques and stochastic upscaling methods provides a window of opportunity to more accurately assess these bio-systems than ever before. However, the use of image data directly in upscaled stochastic framework can only be realized by the development of certain intermediate steps. The goal of the research presented in this dissertation is to develop a texture-segmentation method and a unstructured mesh generation for heterogeneous image data. The following two new techniques are described and evaluated in this dissertation: 1. A new texture-based segmentation method, using the stochastic continuum concepts and wavelet multi-resolution analysis, is developed for characterization of heterogeneous materials in image data. The feature descriptors are developed to efficiently capture the micro-scale heterogeneity of macro-scale entities. The materials are then segmented at a representative elementary scale at which the statistics of the feature descriptor stabilize. 2. A new unstructured mesh generation technique for image data is developed using a hierarchical data structure. This representation allows for generating quality guaranteed finite element meshes. The framework for both the methods presented in this dissertation, as such, allows them for extending to higher dimensions. The experimental results using these methods conclude them to be promising tools for unifying data processing concepts within the upscaled stochastic framework across biological systems. These are targeted for inclusion in decision support systems where biological image data, simulation techniques and artificial intelligence will be used conjunctively and uniformly to assess bio-system quality and design effective and appropriate treatments that restore system health

Disease-specific gene repositioning in breast cancer

The nuclear repositioning of specific genes may be a novel diagnostic strategy to distinguish malignant from normal tissue

Allele-specific nuclear positioning of the monoallelically expressed astrocyte marker GFAP

Chromosomes and genes are nonrandomly arranged within the mammalian cell nucleus. However, the functional significance of nuclear positioning in gene expression is unclear. Here we directly probed the relationship between nuclear positioning and gene activity by comparing the location of the active and inactive copies of a monoallelically expressed gene in single cell nuclei. We demonstrate that the astrocyte-specific marker GFAP (glial fibrillary acidic protein) is monoallelically expressed in cortical astrocytes. Selection of the active allele occurs in a stochastic manner and is generally maintained through cell division. Taking advantage of the monoallelic expression of GFAP, we show that the functionally distinct alleles occupy differential radial positions within the cell nucleus and differentially associate with intranuclear compartments. In addition, coordinately regulated astrocyte-specific genes on distinct chromosomes spatially associate in their inactive state and dissociate upon activation. These results provide direct evidence for function-related differential positioning of individual gene alleles within the interphase nucleus

Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility

Genes are transcribed in a discontinuous pattern referred to as RNA bursting, but the mechanisms regulating this process are unclear. Although many physiological signals, including glucocorticoid hormones, are pulsatile, the effects of transient stimulation on bursting are unknown. Here we characterize RNA synthesis from single-copy glucocorticoid receptor (GR)-regulated transcription sites (TSs) under pulsed (ultradian) and constant hormone stimulation. In contrast to constant stimulation, pulsed stimulation induces restricted bursting centered around the hormonal pulse. Moreover, we demonstrate that transcription factor (TF) nuclear mobility determines burst duration, whereas its bound fraction determines burst frequency. Using 3D tracking of TSs, we directly correlate TF binding and RNA synthesis at a specific promoter. Finally, we uncover a striking co-bursting pattern between TSs located at proximal and distal positions in the nucleus. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration. Stavreva et al. reveal a delay between glucocorticoid receptor (GR) binding and RNA synthesis and link GR mobility modulations in time- and treatment-dependent manner to the size and frequency of transcriptional bursts based on single molecule experiments. By reconstructing GR signaling dynamics on timescales ranging from days to milliseconds, they relate single-cell and single-molecule phenomena to glucocorticoid physiology.Fil: Stavreva, Diana A.. National Institutes of Health; Estados UnidosFil: Garcia, David A.. National Institutes of Health; Estados UnidosFil: Fettweis, Gregory. National Institutes of Health; Estados UnidosFil: Gudla, Prabhakar R.. National Institutes of Health; Estados UnidosFil: Zaki, George F.. National Institutes of Health; Estados UnidosFil: Soni, Vikas. National Institutes of Health; Estados UnidosFil: McGowan, Andrew. National Institutes of Health; Estados UnidosFil: Williams, Geneva. National Institutes of Health; Estados UnidosFil: Huynh, Anh. National Institutes of Health; Estados UnidosFil: Palangat, Murali. National Institutes of Health; Estados UnidosFil: Schiltz, R. Louis. National Institutes of Health; Estados UnidosFil: Johnson, Thomas A.. National Institutes of Health; Estados UnidosFil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Ferguson, Matthew L.. Boise State University; Estados UnidosFil: Pegoraro, Gianluca. National Institutes of Health; Estados UnidosFil: Upadhyaya, Arpita. University of Maryland; Estados UnidosFil: Hager, Gordon L.. National Institutes of Health; Estados Unido

Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior.

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism