Doctor of Philosophy

dissertationLigaments and tendons are dense, fibrous connective tissue that transmit and bear loads within the musculoskeletal system. They are elastic and viscous, and thus are capable of storing and dissipating energy. Although soft and flexible, they can interface with materials that are orders of magnitude stiffer (e.g., bone) and orders of magnitude more compliant (e.g., muscle). These functions are mediated by a complex network of hierarchically organized fibrillar collagen and accessory proteins and molecules. Tissue constituents form unique structural motifs that span the nanoscale, microscale, mesoscale and macroscale. This multiscale organization enables both a robust mechanical response at the macroscopic joint level and simultaneously provides a microscale environment conducive to cellular proliferation and nutrient transport. The aim of this dissertation was to gain a deeper understanding of how the organization of tissue constituents contribute to mechanical function of tendon and ligament across scale levels. At the nanoscale, the question regarding the role of the proteoglycan decorin was addressed. A novel combination of an in vitro assay, imaging techniques and mechanical testing was used to explore how decorin acts to modify the strength of collagen fibril networks. At the microscale, computational modeling was used to examine how different fibril organizations contribute to the macroscopic volumetric response of tendon and ligament during tensile loading. The volumetric response is believed to drive fluid flux within the tissue, which may play a role in nutrient transport and the apparent viscoelastic response. This flow dependent mechanism was addressed in a study that experimentally measured the volumetric changes in mesocale fascicles during viscoelastic testing. One of the challenges in discerning structure-function relationships in tendon and ligament is the large number of uncontrolled variables, which can be difficult to account for in an experimental setting. To address this challenge, a collagen based tendon surrogate was developed for use as a physical model. The physical model was coupled to a validated micromechanical computational model. This facilitated the testing of hypotheses that would have been difficult to address experimentally. The four studies contained within this dissertation, along with a number of preliminary studies, represent a novel contribution to the field of tendon and ligament mechanics

Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces.

Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces

2018-2019 Alumni Recital - Peter Pirotte (Trumpet) and Carina Inoue (Piano)

2nd Annual Alumni Recita

Allelopathy in Echinacea Angustifoloia D.C. Roots

The purpose of this study was to examine the allelopathic effects of compounds of different molecular weights taken from Echinacea angustifolia D.C., the common purple coneflower. Roots were collected from ten plants from five geographic locations and from five fresh plants that were grown in the greenhouse. Water extracts were made from ground dried roots and crushed fresh roots. These extracts were then separated into a high and a low molecular weight fractions using a 10,000 dalton cut-off ultrafilter (Phenomenex, Torrence, CA ). Lettuce seeds (Lactuca sativa) were germinated with the high molecular weight, low molecular weight, and a crude extract from each plant. After four days the seed germination was counted and the root lengths were measured. Analyses of variance of the data were made using the general linear model procedure (SAS Institute, Cary, NC). The crude and the low molecular weight extracts exhibited the most severe allelopatic effects. High molecular weight fractions did not cause a significant inhibition of lettuce seed germination. Partitioning of the active fractions with chloroform and methylene chloride are in progress to isolate the biologically active components

Characterization of Microbial Population Shifts during Sample Storage

The objective of this study was to determine shifts in the microbial community structure and potential function based on standard Integrated Ocean Drilling Program (IODP) storage procedures for sediment cores. Standard long-term storage protocols maintain sediment temperature at 4°C for mineralogy, geochemical, and/or geotechnical analysis whereas standard microbiological sampling immediately preserves sediments at −80°C. Storage at 4°C does not take into account populations may remain active over geologic time scales at temperatures similar to storage conditions. Identification of active populations within the stored core would suggest geochemical and geophysical conditions within the core change over time. To test this potential, the metabolically active fraction of the total microbial community was characterized from IODP Expedition 325 Great Barrier Reef sediment cores prior to and following a 3-month storage period. Total RNA was extracted from complementary 2, 20, and 40 m below sea floor sediment samples, reverse transcribed to complementary DNA and then sequenced using 454 FLX sequencing technology, yielding over 14,800 sequences from the six samples. Interestingly, 97.3% of the sequences detected were associated with lineages that changed in detection frequency during the storage period including key biogeochemically relevant lineages associated with nitrogen, iron, and sulfur cycling. These lineages have the potential to permanently alter the physical and chemical characteristics of the sediment promoting misleading conclusions about the in situ biogeochemical environment. In addition, the detection of new lineages after storage increases the potential for a wider range of viable lineages within the subsurface that may be underestimated during standard community characterizations

Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species

ICA model order selection of task co-activation networks

Independent component analysis (ICA) has become a widely used method for extracting functional networks in the brain during rest and task. Historically, preferred ICA dimensionality has widely varied within the neuroimaging community, but typically varies between 20 and 100 components. This can be problematic when comparing results across multiple studies because of the impact ICA dimensionality has on the topology of its resultant components. Recent studies have demonstrated that ICA can be applied to peak activation coordinates archived in a large neuroimaging database (i.e., BrainMap Database) to yield whole-brain task-based co-activation networks. A strength of applying ICA to BrainMap data is that the vast amount of metadata in BrainMap can be used to quantitatively assess tasks and cognitive processes contributing to each component. In this study, we investigated the effect of model order on the distribution of functional properties across networks as a method for identifying the most informative decompositions of BrainMap-based ICA components. Our findings suggest dimensionality of 20 for low model order ICA to examine large-scale brain networks, and dimensionality of 70 to provide insight into how large-scale networks fractionate into sub-networks. We also provide a functional and organizational assessment of visual, motor, emotion, and interoceptive task co-activation networks as they fractionate from low to high model-orders

Ciliary Transport, Gamete Interaction, and Effects of the Early Embryo in the Oviduct: Ex Vivo Analyses Using a New Digital Videomicroscopic System in the Cow

Using a digital videomicroscopic analysis system in the bovine, we showed that the mechanisms of transport caused by ciliary beating are distinctly different in ampulla and isthmus of the oviduct. The average particle transport speed (PTS) in the oviduct (mean, 133 μm/sec) does not differ in the cycle (metestrus) and during pregnancy after implantation, but it is locally modulated at the site of the embryo. Using videomicroscopy, we were able to document that after entering the ampulla, the cumulus-oocyte complex (COC) is not transported by ciliary beating down the oviduct, but firmly attaches to the ampullar epithelium. This attachment is mediated by the cumulus cells. However, when a COC is degenerated, it is floating in the oviductal lumen. As soon as a vital COC is in the ampulla, the sperm bound in the sperm reservoir of the ampullar isthmic junction leave the reservoir and hurry to the oocyte. When a sperm has penetrated the zona pellucida, the COC detaches and continues its migration. Quantitative measurements showed that the early embryo is able to locally downregulate PTS during its migration down the oviduct. It locally changes the pattern of vascularization and induces the formation of secretory cells. Our studies imply that the oviductal epithelium is able to select vital oocytes. The early embryo is able to induce the formation of secretory cells, modify vascularization, and downregulate speed of transport, thus creating the prerequisite for the first embryo-maternal communication in the oviduct

Vascular changes in diabetic retinopathy-a longitudinal study in the Nile rat.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies

2010-2011 Master Class - Marc Reese and Ken Amis (Brass)

https://spiral.lynn.edu/conservatory_masterclasses/1079/thumbnail.jp