A Pipeline for Automated Assessment of Cell Location in 3D Mouse Brain Image Sets

Mapping the neuronal connectivity of the mouse brain has long been hampered by the laborious and time-consuming process of slicing, staining and imaging the brain tissue. Recent developments in automated 3D fluorescence microscopy, such as serial two- photon tomography (STP) and light sheet fluorescence microscopy, now allow for automated rapid 3D imaging of a complete mouse brain at cellular resolution. In combination with transsynaptic viral tracers, this paves the way for high-throughput brain mapping studies, which could greatly advance our understanding of the function of the brain. Because transsynaptic tracers label synaptically connected cells, the analysis of these whole-brain scans requires detection of fluorescently labelled cells and anatomical segmentation of the data, which are very labour- and time-intensive manual tasks and prevent high-throughput analysis. This thesis presents and validates two software tools to automate anatomical segmentation and cell detection in serial two photon (STP) scans of the mouse brain. Automated mouse atlas propagation (aMAP) segments the scans into anatomical regions by matching a 3D reference atlas to the data using affine and free-form image registration. The fast automated cell counting tool (FACCT) then detects fluorescently labelled cells in the dataset with a novel approach of stepwise data reduction combined with object detection using artificial neuronal networks. The tools are optimised for large datasets and are capable of processing a 2.5TB STP scan in under two days. The performance of aMAP and FACCT is evaluated on STP scans from retrograde connectivity tracing experiments using rabies virus injections in the primary visual corte

The logic of single-cell projections from visual cortex

Neocortical areas communicate through extensive axonal projections, but the logic of information transfer remains poorly understood, because the projections of individual neurons have not been systematically characterized. It is not known whether individual neurons send projections only to single cortical areas or distribute signals across multiple targets. Here we determine the projection patterns of 591 individual neurons in the mouse primary visual cortex using whole-brain fluorescence-based axonal tracing and high-throughput DNA sequencing of genetically barcoded neurons (MAPseq). Projections were highly diverse and divergent, collectively targeting at least 18 cortical and subcortical areas. Most neurons targeted multiple cortical areas, often in non-random combinations, suggesting that sub-classes of intracortical projection neurons exist. Our results indicate that the dominant mode of intracortical information transfer is not based on 'one neuron-one target area' mapping. Instead, signals carried by individual cortical neurons are shared across subsets of target areas, and thus concurrently contribute to multiple functional pathways

The interplay of extracellular matrix and microbiome in urothelial bladder cancer

Many pathological changes in solid tumours are caused by the accumulation of genetic mutations and epigenetic molecular alterations. In addition, tumour progression is profoundly influenced by the environment surrounding the transformed cells. The interplay between tumour cells and their microenvironment has been recognized as one of the key determinants of cancer development and is being extensively investigated. Data suggest that both the extracellular matrix and the microbiota represent microenvironments that contribute to the onset and progression of tumours. Through the introduction of omics technologies and pyrosequencing analyses, a detailed investigation of these two microenvironments is now possible. In urological research, assessment of their dysregulation has become increasingly important to provide diagnostic, prognostic and predictive biomarkers for urothelial bladder cancer. Understanding the roles of the extracellular matrix and microbiota, two key components of the urothelial mucosa, in the sequelae of pathogenic events that occur in the development and progression of urothelial carcinomas will be important to overcome the shortcomings in current bladder cancer treatment strategies