ToScA North America (6 – 8 June 2017, The University of Texas, Austin, TX) Program

ToScA North America will address key areas of science, including Multi-modal Imaging, Geosciences, Forensics, Increasing Contrast, Educational Outreach, Data, Materials Science and Medical and Biological Science.University of Texas High-Resolution X-ray CT Facility (UTCT); Jackson School of Geosciences, The University of Texas at Austin; Natural History Museum (London); Royal Microscopical Society (Oxford, UK)Geological Science

Mechanisms of Pierce\u27s disease resistance in grapevine (Vitis vinifera L.): from xylem structure to whole plant function

Xylella fastidiosa (Xf) is a xylem-dwelling bacterium that causes Pierce’s disease (PD) in grapevines (Vitis vinifera L.), and disease in a range of other ecologically and economically important woody plants. To successfully colonize the xylem network, Xf cells accumulate on the vessel walls and form a biofilm. The biofilm contains cell wall-degrading enzymes, allowing the bacteria to breach the intervessel pit membranes. Thus, Xf can move from one vessel to another and colonize the xylem network. Degraded intervessel pit membranes and the production of tyloses in response to the presence of Xf likely contribute to significant declines in both hydraulic conductivity and resistance to drought-induced embolism spread. Indeed, Xf-infected grapevines typically display a range of symptoms that are often associated with water transport dysfunction.Despite the consensus that PD susceptibility is associated with Xf multiplication and systemic spread within the xylem network, there significant gaps in our understanding of the relationships between xylem structure and function that allow for Xf establishment and colonization still remain. For instance, although Xf can breach pit membranes to move from one vessel to another, the consequences of the breakdown of pit membranes within the context of embolism spread and hydraulic conductivity, and the subsequent implications for whole-plant physiological decline, remain inconclusive. Furthermore, the physical structure of the xylem network, i.e. the spatial distribution of xylem connections that might facilitate the spread of Xf, are largely unknown because of the complex, three-dimensional nature of the network. In my dissertation research, I explored the roles of the xylem structure and function related to the mechanisms of PD resistance. Throughout my work, I applied a holistic approach, coupling anatomical and physiological measurements across different grapevine genotypes with different levels of PD resistance -- from non-cultivated North American species to commercial European vinifera cultivars and their hybrids. In the first chapter, I tested the hypothesis where if the 3D structure of the xylem network connectivity plays a significant role in Xf spread, then PD resistant grapevine genotypes should have fewer total connections in the lateral and radial directions, which thereby limits the total number of pathways. Given that the Xf spread is essentially dependent on the intervessel connections, comparing the number and orientation of connections was a logical step in the fundamental understanding of this host-pathogen relationship. The chapter concludes, however, that there was limited evidence to support this hypothesis, and network connectivity does not appear to be strongly correlated with PD resistance and Xf spread. While network connectivity in the radial and lateral directions is somewhat variable within the genus Vitis, no clear trends emerged linking connectivity with resistance to PD. In the second chapter, I investigated the consequences of the extracellular cell wall-degrading enzyme released by Xf on pit membrane integrity and the downstream effects on water transport. The enzymatic breakdown of the pit membranes was relatively small, less than 10% of the pit aperture area, but enough to weaken pit membrane resistance to air-seeding by introducing pores into the membrane. Not only would larger pore diameters facilitate Xf movement, but they would subsequently increase the vulnerability of those vessels to drought-induced embolism spread. These factors would significantly affect the water transport capacity of infected grapevines and put them at greater risk to the effects of drought. In the third chapter, my objective was to determine the key physiological mechanisms that lead to mortality in the Xf infection process. This chapter reveals the mechanistic cascade of events that occur after Xf inoculation, with a coordinated decline in hydraulic conductivity, photosynthesis, and starch storage in PD susceptible grapevine genotypes. The results support the theory that hydraulic failure and carbon starvation underlie plant mortality resulting from PD. My dissertation explored the roles of the xylem structure and function on the PD mechanisms of resistance. Collectively, this work (1) identifies the variability in 3D xylem network traits in six different Vitis genotypes, representing the most complete analysis of its type for any plant group; (2) reveals that in young shoots the axial pathway appears to be the most important in determining the long-distance movement and systemic spread of Xf in the xylem network, (3) and provides a more robust, mechanistic understanding of the timing and sequence of events from initial Xf inoculation to ultimate death, as well as the variability in this mortality sequence in resistant and susceptible genotypes. As we do not have an effective remedy against the Xf bacterium, a more accurate understanding of how some grapevines resist to the infection process is one piece of this very important puzzle

The Differential Effects of Two Critical Osteoclastogenesis Stimulating Factors on Bone Biomechanics

Many skeletal diseases, such as osteoporosis and malignant bone metastases, are generally osteolytic and associated with increased bone resorption and decreased bone strength. Within a complex cytokine environment, the proteins RANKL and M-CSF are critical for osteoclast differentiation and activation, and thus fundamental effectors of osteolytic disorders. Previous studies showed that M-CSF stimulates the proliferation and early differentiation of osteoclast progenitors to osteoclast lineage, while RANKL targets the later stages of fusion and activation, and stimulates the formation of functional active osteoclasts. However, impacts of artificially elevated levels of these proteins on the skeleton system have not been fully characterized. In this project, we amplified the circulating levels of RANKL and M-CSF by injections or continuous administrations and examined the effects on bone volume and quality. We hypothesized that while M-CSF and RANKL can both stimulate osteoclastogenesis, the differences in activation stages targeted by these two cytokines would result in distinct responses on bone biomechanics. RANKL would directly stimulate osteoclast activity and increase bone resorption, while M-CSF would act anabolically through coupling between osteoblast development and the promoted osteoclastogenesis at the early stage, and promote bone formation indirectly. Data obtained in this project demonstrated that in vivo administration of RANKL and M-CSF induced general opposing effects on bone volume, architecture, mineralization and strength. RANKL directly stimulated bone resorption and reduceed bone biomechanical properties. The destructive skeleton induced by RANKL could serve as a novel animal model that exhibits a series of skeletal complications similar to those observed in osteolytic skeletal diseases, such as osteoporosis. Alternately, administrations of M-CSF markedly stimulated trabecular bone formation and had less of an influence on cortical bone. These changes demonstrated the potential of M-CSF as an anabolic agent for osteoporosis. This project has further examined the in vivo characteristics and functional effects of RANKL and M-CSF on the skeleton system. Findings in this project, such as the creation of RANKL induced bone loss model and characterization of the anabolic potential of M-CSF on the skeleton, could provide useful information and tools for further explorations on human skeletal diseases

Characterization of Posidonia Oceanica Seagrass Aerenchyma through Whole Slide Imaging: A Pilot Study

Characterizing the tissue morphology and anatomy of seagrasses is essential to predicting their acoustic behavior. In this pilot study, we use histology techniques and whole slide imaging (WSI) to describe the composition and topology of the aerenchyma of an entire leaf blade in an automatic way combining the advantages of X-ray microtomography and optical microscopy. Paraffin blocks are prepared in such a way that microtome slices contain an arbitrarily large number of cross sections distributed along the full length of a blade. The sample organization in the paraffin block coupled with whole slide image analysis allows high throughput data extraction and an exhaustive characterization along the whole blade length. The core of the work are image processing algorithms that can identify cells and air lacunae (or void) from fiber strand, epidermis, mesophyll and vascular system. A set of specific features is developed to adequately describe the convexity of cells and voids where standard descriptors fail. The features scrutinize the local curvature of the object borders to allow an accurate discrimination between void and cell through machine learning. The algorithm allows to reconstruct the cells and cell membrane features that are relevant to tissue density, compressibility and rigidity. Size distribution of the different cell types and gas spaces, total biomass and total void volume fraction are then extracted from the high resolution slices to provide a complete characterization of the tissue along the leave from its base to the apex

Post-traumatic osteoarthritis in mice following mechanical injury to the synovial joint

We investigated the spectrum of lesions characteristic of post-traumatic osteoarthritis (PTOA) across the knee joint in response to mechanical injury. We hypothesized that alteration in knee joint stability in mice reproduces molecular and structural features of PTOA that would suggest potential therapeutic targets in humans. The right knees of eight-week old male mice from two recombinant inbred lines (LGXSM-6 and LGXSM-33) were subjected to axial tibial compression. Three separate loading magnitudes were applied: 6N, 9N, and 12N. Left knees served as non-loaded controls. Mice were sacrificed at 5, 9, 14, 28, and 56 days post-loading and whole knee joint changes were assessed by histology, immunostaining, micro-CT, and magnetic resonance imaging. We observed that tibial compression disrupted joint stability by rupturing the anterior cruciate ligament (except for 6N) and instigated a cascade of temporal and topographical features of PTOA. These features included cartilage extracellular matrix loss without proteoglycan replacement, chondrocyte apoptosis at day 5, synovitis present at day 14, osteophytes, ectopic calcification, and meniscus pathology. These findings provide a plausible model and a whole-joint approach for how joint injury in humans leads to PTOA. Chondrocyte apoptosis, synovitis, and ectopic calcification appear to be targets for potential therapeutic intervention

Testing Foundations of Biological Scaling Theory Using Automated Measurements of Vascular Networks

Scientists have long sought to understand how vascular networks supply blood and oxygen to cells throughout the body. Recent work focuses on principles that constrain how vessel size changes through branching generations from the aorta to capillaries and uses scaling exponents to quantify these changes. Prominent scaling theories predict that combinations of these exponents explain how metabolic, growth, and other biological rates vary with body size. Nevertheless, direct measurements of individual vessel segments have been limited because existing techniques for measuring vasculature are invasive, time consuming, and technically difficult. We developed software that extracts the length, radius, and connectivity of in vivo vessels from contrast-enhanced 3D Magnetic Resonance Angiography. Using data from 20 human subjects, we calculated scaling exponents by four methods--two derived from local properties of branching junctions and two from whole-network properties. Although these methods are often used interchangeably in the literature, we do not find general agreement between these methods, particularly for vessel lengths. Measurements for length of vessels also diverge from theoretical values, but those for radius show stronger agreement. Our results demonstrate that vascular network models cannot ignore certain complexities of real vascular systems and indicate the need to discover new principles regarding vessel lengths

Non-invasive imaging of drought-induced cavitation in plants

The increased frequency of extreme events, associated with climate change, can lead to loss of biodiversity and forest dieback. Intense drought has disastrous effects on plants growth and physiology and maintaining hydraulic conductivity during time of water stress is necessary for plants survival. However, during drought, the blockage of the hydraulic pathway by air-seeding results in the loss of conductivity by embolism formation. Therefore, it is essential to be able to accurately measure the conductivity, leading to a better prediction of species vulnerability through vulnerability curves (VCs). The measure of vulnerability thresholds is necessary in order to evaluate the causes of forests dieback. Moreover, understanding the mechanisms behind drought-recovery and embolism repair can contribute to the advance of models used to predict the impact of drought on vegetation dynamics. This PhD was designed to test the accuracy and applicability of new visual techniques and aim to provide alternatives to invasive methods for measure of VCs. While invasive techniques measure conductivity on cut samples, visual techniques allow for measurements on intact samples. However, the question of the accuracy of visual methods are still discussed on the ground that they do not provide a direct measurement of conductivity (PLC) but instead use a proxy through the measurements of loss of vessels (PLV). In conclusion, my PhD research addresses the use of visual techniques as an accurate alternative for invasive methods and aims to provide further knowledge concerning mechanisms that regulates drought-induced embolism and recovery, under controlled conditions as well as in field-based studies. The results of this research suggest that (1) visual techniques may be used with all xylem anatomy, but need precise implementation in order to avoid erroneous results, (2) embolism repair via refilling is not a common for E. saligna and severe drought may be responsible for lagged-mortality also observed in the field and that (3) hydraulic failure driven by drought-induced embolism was one of the factors that contributed to the massive dieback of A. marina in northern Australia. Overall, drought-induced embolism may lead to forest mortality and lagged-mortality in many ecosystems, and the understanding of species dependant embolism recovery responses is necessary to determine species resistance and resilience to extreme events and climate change