Current status and future directions of Levy walk research

Lévy walks are a mathematical construction useful for describing random patterns of movement with bizarre fractal properties that seem to have no place in biology. Nonetheless, movement patterns resembling Lévy walks have been observed at scales ranging from the microscopic to the ecological. They have been seen in the molecular machinery operating within cells during intracellular trafficking, in the movement patterns of T cells within the brain, in DNA, in some molluscs, insects, fish, birds and mammals, in the airborne flights of spores and seeds, and in the collective movements of some animal groups. Lévy walks are also evident in trace fossils (ichnofossils) – the preserved form of tracks made by organisms that occupied ancient sea beds about 252-66 million years ago. And they are utilised by algae that originated around two billion years ago, and still exist today. In September of 2017, leading researchers from across the life sciences, along with mathematicians and physicists, got together at a Company of Biologists' Workshop to discuss the origins and biological significance of these movement patterns. In this Review the essence of the technical and sometimes heated discussions is distilled and made accessible for all. In just a few pages, the reader is taken from a gentle introduction to the frontiers of a very active field of scientific enquiry. What emerges is a fascinating story of a truly inter-disciplinary scientific endeavour that is seeking to better understand movement patterns occurring across all biological scales

Integrative Computational Modeling of the Lymph Node Stromal Cell Landscape

Adaptive immune responses develop in secondary lymphoid organs such as lymph nodes (LNs) in a well-coordinated series of interactions between migrating immune cells and resident stromal cells. Although many processes that occur in LNs are well understood from an immunological point of view, our understanding of the fundamental organization and mechanisms that drive these processes is still incomplete. The aim of systems biology approaches is to unravel the complexity of biological systems and describe emergent properties that arise from interactions between individual constituents of the system. The immune system is greater than the sum of its parts, as is the case with any sufficiently complex system. Here, we review recent work and developments of computational LN models with focus on the structure and organization of the stromal cells. We explore various mathematical studies of intranodal T cell motility and migration, their interactions with the LN-resident stromal cells, and computational models of functional chemokine gradient fields and lymph flow dynamics. Lastly, we discuss briefly the importance of hybrid and multi-scale modeling approaches in immunology and the technical challenges involved

On the Biomechanics of Cell Nuclei: Insights from Combined Force and Light Microscopy

Cell nuclei are multifunctional. Not only do they house and protect the genome, but they additionally provide mechanical stability to the surrounding cell and convert extracellular mechanical cues into biochemical responses. For example, forces exerted upon integrins at the cell surface propagate through the cytoskeleton and into the nucleus, resulting in local stretching of chromatin and transcription upregulation. The mechanical integrity of the nucleus, however, is often compromised in an array of diseases ranging from cancers to laminopathies. These diseases have targeted effects on specific nuclear constituents, and in turn lead to altered cellular migration properties, increases in DNA damage and genomic instability, and compromised nuclear mechanotransduction. Consequently, the mechanobiology of the cell nucleus has garnered increasing attention over the past several decades. Studies in nuclear biomechanics primarily make use of force probes and/or light microscopy to quantify mechanical properties of nuclei and their responses to physical perturbations. In the first half of this thesis, I describe two novel methodologies for studying nuclear mechanobiology. The first is a side-view, light-sheet fluorescence microscope combined with an atomic force microscope (AFM-LS) that enables time-correlated, multi-color, 3D light-sheet imaging coupled with AFM. The second method is a unique combination of light-sheet microscopy and fluorescence recovery after photobleaching (FRAP) known as SPIM-FRAP, which I used to simultaneously quantify diffusion across an entire 2D image. In the latter half of this thesis, I describe how I have used both AFM-LS and SPIM-FRAP to study nuclear mechanobiology. SPIM-FRAP is used to show how intranuclear diffusion of NLS-GFP is slowed in nucleoli, but overall uncorrelated with chromatin structure on the length scale of single fluorophores. Additionally, I use SPIM-FRAP to show that sites of DNA damage are more stable than the surrounding diffuse repair proteins. AFM-LS is used to separate of the roles of chromatin and lamin A/C in nuclear compression, regarding both the mechanical response of the nucleus as well as local nuclear curvature. I also use multi-channel, 3D AFM-LS to show how compression alone via AFM, independent of nuclear rupture, is sufficient to induce DNA damage in nuclei. This indicates a novel mechanism by which nuclei incur double-stand DNA breaks. Finally, I provide the first review of mechanical models of nuclei, along with the development of a continuum mechanical model for AFM indentation. Together, these developments in methodologies along with the coupled insights into intranuclear dynamics, nuclear mechanics, and DNA damage improve both our means of studying and our current understanding of nuclear mechanobiology.Doctor of Philosoph

Targeting therapeutic T cells to the bone marrow niche

Anti-cancer immunotherapies aim to mediate a specific response targeting malignant cells without accompanying damage to normal tissue associated with conventional therapies, but induction of T cell differentiation and exhaustion enables successful tumour progression. In this thesis I will explore different means of enhancing the accumulation and function of therapeutic CD8 T cells, as a means of achieving functional cure through persisting immunological memory. I will show that the key features of T cell memory can be imprinted upon CD8+ T cells by enhancing homing to specific organs, enabling privileged access to cell-mediated factors. The interaction between the chemokine receptor CXCR4 and the ligand CXCL12/SDF-1 is required for successful homing of haematopoietic stem cells (HSCs) to stromal niches within the bone marrow (BM). The bone marrow is known to be a unique organ for immunological memory, including memory T cells. I hypothesised that replicating this bone marrow homing interaction in CD8+ T cells would preferentially generate memory T cells. I demonstrate through in vivo imaging and flow cytometric analyses that T cells over-expressing CXCR4 accumulate preferentially in the BM near vascular-associated CXCL12+ cells, retain a less differentiated central memory phenotype despite repeated antigenic stimulation, and produce enhanced effector cytokines on restimulation. Compared to control T cells, these cells demonstrate lower expression of exhaustion and senescence markers, suggesting the capacity for long-term persistence after activation. I go on to show that numerical accumulation and many of these functional attributes are dependent upon cell-extrinsic expression of IL-15Rα. TCXCR4 demonstrate heightened graft-versus-tumour effects in allogeneic bone marrow transplant models of B-cell lymphoma in comparison to control T cells. I provide evidence that this anti-tumour effect is mediated by enhanced functional capacity rather than numerical accumulation or out-competing immunosuppressive populations. In summary, this strategy offers a tractable means of enhancing T cell engraftment, persistence and function, with potential for cross-platform therapeutic applications including anti-cancer immunotherapy