A Theoretical Framework for Effective Online Learning

A key, overarching goal for any committed educator is to ensure that the learner has a meaningful and memorable learning experience while achieving the desired learning outcomes. In this paper it is argued that in order to achieve such a goal, a strategy needs to be put in place that is capable of providing students with a fully integrated, all-encompassing learning environment. The reasoning, simply, is that learning will not necessarily emanate from one specific source and when it happens, it will occur through different means, for different people. One of the great strengths of the online learning space is that, harnessing the power of the various information and communication technologies (ICTs), there is greater scope for catering for individual learning needs. With this in mind, the paper puts forward a framework that comprises a number of overlapping \u27sub-environments\u27 which, together, provide the scaffolding considered essential for the construction of a truly holistic learning environment. To illustrate how this framework for effective online learning can be operationalised, the authors refer to the case of Universitas 21 Global, an institution which offers completely online programs to post-graduate students in more than 40 countries around the world

Patient specific finite volume modeling for intraosseous PMMA cement flow simulation in vertebral cancellous bone

Ph.DDOCTOR OF PHILOSOPH

Stiffness of Extracellular Matrix Components Modulates the Phenotype of Human Smooth Muscle Cells in Vitro and Allows for the Control of Properties of Engineered Tissues

AbstractSmooth muscle cells (SMCs) play a significant role in the pathogenesis of atherosclerosis. 2D cultures elucidated valuable information about the interaction between SMCs and extracellular matrix (ECM) components. However, 3D constructs better represent the native vascular environment. Furthermore, a limited number of studies addressed the effect of ECM stiffness on SMCs phenotype. We investigated the effect of stiffness of different ECM substrates by modulating their concentrations, including the effect on morphology, proliferation, expression of the contractile protein α-smooth muscle actin (α-SMA) and deposition of collagen type I (Col I) and collagen type III (Col III) proteins. At low concentrations of Col I gels and Col I gels supplemented with 10% fibronectin (Fn), SMCs exhibited non-elongated, ‘hill-and-valley’ shape and large mean cellular area, indicating a hypertrophic morphology, characteristic of the synthetic phenotype. However, with increasing concentration, mean cellular area and proliferation relative to cells cultured in 2D dropped. Whole protein secretion into the culture media and deposition of Col I and Col III generally decreased with increasing stiffness. Moreover, percentage of α-SMA+ SMCs decreased with increasing gel concentration, pointing to a shift towards the synthetic phenotype. Supplementing Col I with 10% Laminin (Ln) maintained higher cellular area and aspect ratio at all gel concentrations and did not change α-SMA expression significantly, compared to Col I alone or Col I + Fn. Overall, these results demonstrate that ECM components and stiffness could provide the tools to modulate the phenotype and function of SMCs in vitro, which allows for the control of properties of engineered tissues

Biomimetic 3D models for investigating the role of monocytes and macrophages in atherosclerosis

Atherosclerosis, the inflammation of artery walls due to the accumulation of lipids, is the most common underlying cause for cardiovascular diseases. Monocytes and macrophages are major cells that contribute to the initiation and progression of atherosclerotic plaques. During this process, an accumulation of LDL-laden macrophages (foam cells) and an alteration in the extracellular matrix (ECM) organization leads to a local vessel stiffening. Current in vitro models are carried out onto two-dimensional tissue culture plastic and cannot replicate the relevant microenvironments. To bridge the gap between in vitro and in vivo conditions, we utilized three-dimensional (3D) collagen matrices that allowed us to mimic the ECM stiffening during atherosclerosis by increasing collagen density. First, human monocytic THP-1 cells were embedded into 3D collagen matrices reconstituted at low and high density. Cells were subsequently differentiated into uncommitted macrophages (M0) and further activated into pro- (M1) and anti-inflammatory (M2) phenotypes. In order to mimic atherosclerotic conditions, cells were cultured in the presence of oxidized LDL (oxLDL) and analyzed in terms of oxLDL uptake capability and relevant receptors along with their cytokine secretomes. Although oxLDL uptake and larger lipid size could be observed in macrophages in a matrix dependent manner, monocytes showed higher numbers of oxLDL uptake cells. By analyzing major oxLDL uptake receptors, both monocytes and macrophages expressed lectin-like oxidized low-density lipoprotein receptor-1 (LOX1), while enhanced expression of scavenger receptor CD36 could be observed only in M2. Notably, by analyzing the secretome of macrophages exposed to oxLDL, we demonstrated that the cells could, in fact, secrete adipokines and growth factors in distinct patterns. Besides, oxLDL appeared to up-regulate MHCII expression in all cells, while an up-regulation of CD68, a pan-macrophage marker, was found only in monocytes, suggesting a possible differentiation of monocytes into a pro-inflammatory macrophage. Overall, our work demonstrated that collagen density in the plaque could be one of the major factors driving atherosclerotic progression via modulation of monocyte and macrophages behaviors

Investigation of a Compressor Coupling Failure by Torsional Excitation

Case StudyThis case study examines a repeat compressor coupling failure that was a result of a complex, dynamic interaction of components that excited the 2nd torsional natural frequency of the train. The mature unit had operated successfully for 17 years until a seemingly insignificant series of compromising events created degradation which triggered the excitation of the natural frequencies. The couplings exhibited evidence of fatigue fractures leading to catastrophic failure due to high alternating torsional stress. A natural frequency modal analysis showed that a mode shape of the compressor train bull gear enabled it to interact within the total system torsional modal response to create an excitation frequency at the 2nd torsional natural frequency. Further, discussion on practical design, operation and equipment monitoring shows limitations in guarding against the vulnerabilities mentioned

Architected poly(lactic acid)/poly(ε-caprolactone)/halloysite nanotube composite scaffolds enabled by 3D printing for biomedical applications

Herein, we report the physicochemical, thermal, mechanical and biological characteristics, including bioactivity, biodegradation and cytocompatibility of additive manufacturing-enabled novel nanocomposite scaffolds. The scaffolds comprise a blend of polylactic acid (PLA) and poly-ε-caprolactone (PCL) reinforced with halloysite nanotubes (HNTs). The nanoengineered filaments were developed by melt blending, and the nanocomposite scaffolds were manufactured by fused filament fabrication. Uniform dispersion of HNTs in the PLA/PCL blend is revealed via scanning electron microscopy. Mechanical property loss due to the addition of PCL to realize a suitable biodegradation rate of PLA was fully recovered by the addition of HNTs. Bioactivity, as revealed by the fraction of apatite growth quantified from XRD analysis, was 5.4, 6.3, 6.8 and 7.1% for PLA, 3, 5 and 7 wt% HNT in PLA/PCL blend, respectively, evidencing enhancement in the bioactivity. The degradation rate, in terms of weight loss, was reduced from 4.6% (PLA) to 1.3% (PLA/PCL) upon addition of PCL, which gradually increased to 4.4% by the addition of HNTs (at 7 wt% HNT). The results suggest that the biodegradation rate, mechanical properties and biological characteristics, including cytocompatibility and cell adhesion, of the 3D printed, microarchitected PLA/PCL/HNT composite scaffolds can be tuned by an appropriate combination of HNT and PCL content in the PLA matrix, demonstrating their promise for bone replacement and regeneration applications

Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber

The migration of cells such as leukocytes, tumor cells, and fibroblasts through 3D matrices is critical for regulating homeostasis and immunity and for driving pathogenesis. Interstitial flow through the extracellular matrix, which can substantially increase during inflammation and in the tumor microenvironment, can influence cell migration in multiple ways. Leukocytes and tumor cells are heterogeneous in their migration responses to flow, yet most 3D migration studies use endpoint measurements representing average characteristics. Here we present a robust new microfluidic device for 3D culture with live imaging under well-controlled flow conditions, along with a comparison of analytical methods for describing the migration behavior of heterogeneous cell populations. We then use the model to provide new insight on how interstitial flow affects MDA-MB-231 breast cancer cell invasion, phenomena that are not seen from averaged or endpoint measurements. Specifically, we find that interstitial flow increases the percentage of cells that become migratory, and increases migrational speed in about 20% of the cells. It also increases the migrational persistence of a subpopulation (5-10% of cells) in the positive or negative flow direction. Cells that migrated upstream moved faster but with less directedness, whereas cells that migrated in the direction of flow moved at slower speeds but with higher directedness. These findings demonstrate how fluid flow in the tumor microenvironment can enhance tumor cell invasion by directing a subpopulation of tumor cells in the flow direction; i.e., towards the draining lymphatic vessels, a major route of metastasis

Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fc gamma receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

Background: KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies