Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

No description available.

The intricate task of segmenting and quantifying cell nuclear pores in high-resolution 3D microscopy data is critical for cellular biology and disease research. This thesis introduces a deep learning pipeline crafted to automate the segmentation and quantification of nuclear pores from high-resolution 3D cell organelle images. Our aim is to refine computational methods capable of handling the data's complexity and size, thus improving accuracy and reducing manual labor in biological image analysis. The developed pipeline incorporates data preprocessing, augmentation strategies, random block sampling, and a three-stage post-processing algorithm. It utilizes a 3D U-Net with a VGG-16 backbone, optimized through cyclical data augmentation and random block sampling to tackle the challenges posed by limited labeled data and the processing of large-scale 3D images. The pipeline has demonstrated its capability to effectively learn and predict nuclear pore structures, achieving improvements in validation metrics compared to baseline models. Our experiments suggest that cyclical augmentation helps prevent overfitting, and random block sampling contributes to managing data imbalance. The post-processing phase successfully automates the quantification of nuclear pores without the need for manual intervention. The proposed pipeline offers an efficient and scalable approach to segmenting and quantifying nuclear pores in 3D microscopy images. Despite the ongoing challenges of computational intensity and data volume, the techniques developed in this study provide insights into the automation of complex biological image analysis tasks, with potential applications extending beyond the detection of nuclear pores. / Master of Science / This thesis outlines a computer program developed to automatically segment and count nuclear pores in 3D cell images, aiding cell and disease research. This program aims to handle large, complex image data more effectively, boost accuracy, and cut down the need for manual labor. We created a system that prepares data, applies a technique called augmentation to enrich it, selects specific image sections, and carries out a three-step final analysis. At the core of our program is a 3D U-Net model, a type of deep learning network, that has been enhanced to address the challenges of scarce labeled data and the processing of very large images. The system developed is capable of learning and identifying the structure of nuclear pores in cell images. Our experiments indicate that using augmentation in a cyclical manner during training can prevent overfitting, which is when a model learns the training data too well, and cannot suitably generalize. Selecting certain parts of the images for processing proves helpful with imbalanced data. Additionally, the program can automatically count nuclear pores in the final step. The proposed program is effective for analyzing and counting nuclear pores in 3D cell images and has the potential for broader applications in cell analysis. Despite the challenges of managing large datasets and the significant computational power required, our methods open new possibilities for automating cell studies, with uses extending beyond just nuclear pores.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

No description available.