Astronauts at are risk of losing 1.0-1.5% of their bone mass for every month they spend in space despite their adherence to high impact exercise training programs and diets high in nutrients, potassium, calcium, and vitamin D, all designed to preserve the skeletal system. This article reviews the basics of bone formation and resorption and details how exposure to microgravity or simulated microgravity affects the structure and function of osteoblasts, osteocytes, osteoclasts, and their mesenchymal and hematologic stem cell precursors. It details the critical roles that insulin-like growth factor-1 and its receptor insulin-like growth factor-1 receptor (GFR1) play in maintaining bone homeostasis and how exposure of bone cells to microgravity affects the function of these growth factors. Lastly, it discusses the potential of tumor necrosis factor-related apoptosis-inducing ligand, syncytin-A, sclerostin inhibitors and recombinant IGF-1 as a bone-saving treatment for astronauts in space and during their colonization of the Moon.

Gene therapy techniques delivering exogenous angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), are currently being investigated as potential treatments for ischemia resulting from a variety of conditions, such as peripheral artery disease (PAD) and chronic wounds. Despite these intense efforts, a viable clinical option to promote therapeutic neovascularization remains elusive. Electroporation is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues including skin and muscle. This study investigated intradermal injection of plasmids encoding angiogenic growth factors with electroporation as a novel therapeutic approach to increase perfusion in areas of ischemia. Two common animal models of ischemia were employed: a skin flap model, used to study wound healing, and a hindlimb ischemia model, used to investigate potential therapies for PAD. In the skin flap model, delivery of plasmid VEGF with electroporation significantly increased VEGF expression for 5 days after delivery compared to injection of the plasmid alone. While the increase in VEGF expression was short-term, it significantly increased expression of the downstream angiogenic growth factor endothelial nitric oxide synthase, as well as perfusion and healing in the distal area of the skin flap. To facilitate the translation of electroporation to the clinic, a novel electrode configuration was previously designed for cutaneous delivery of plasmids to a large surface area. The design of the Multielectrode Array allows for delivery to a large surface area without the need to increase the applied voltage. Conditions for plasmid delivery with this electrode were optimized and it was then utilized to deliver plasmid FGF-2 (pFGF) to the hindlimb ischemia model. FGF-2 expression, perfusion, and angiogenesis were assessed. FGF-2 expression was significantly higher for 10 days after treatment with pFGF with electroporation compared to injection of pFGF alone. This increase in FGF-2 expression induced a significant increase in perfusion and angiogenesis in the ischemic limb. The research presented here suggests intradermal injection of plasmids encoding angiogenic factors by electroporation is a novel potential therapeutic approach to increase perfusion to areas of ischemia and promote wound healing.

Indiana University-Purdue University Indianapolis (IUPUI) / Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a common cancer. Positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG) is a widely used imaging modality in HNSCC.PurposeTo provide evident data about associations between 18F-FDG PET and histopathology in HNSCC.Material and MethodsThe MEDLINE database was screened for associations between maximum standard uptake values (SUVmax) derived from 18F-FDG PET and histopathological features in HNSCC up to May 2020. Only papers containing correlation coefficients between SUVmax and histopathology were acquired. Overall, 23 publications were collected.ResultsThe following correlations were calculated: KI 67: 12 studies (345 patients), pooled correlation coefficient (PCC): 0.23 (95% confidence interval [CI] 0.06–0.40); hypoxia-inducible factor-1α: eight studies (240 patients), PCC: 0.24 (95% CI 0.06–0.42); microvessel density: three studies (64 patients), PCC: 0.33 (95% CI 0.02–0.65); vascular endothelial growth factor: two studies (59 cases), PCC: 0.27 (95% CI 0.02–0.51); tumor suppressor protein p53: four studies (159 patients), PCC: 0.05 (95% CI –0.41 to 0.51); epidermal growth factor receptor: two studies (124 patients), PCC: 0.21 (95% CI 0.05–0.37); tumor cell count: three studies (67 patients), PCC: 0.18 (95% CI –0.06 to 0.42); tumor cell apoptosis: two studies (40 patients), PCC: 0.07 (95% CI = –0.85 to 0.99); B-cell lymphoma-2 protein: two studies (118 patients); PCC: 0.04 (95% CI –0.65 to 0.74); glucose-transporter 1: 10 studies (317 patients), PCC: 0.20 (95% CI 0.10–0.30).ConclusionSUVmax derived from 18F-FDG PET cannot reflect relevant histopathological features in HNSCC.

A mixed self assembling monolayer (mSAM) chip with attached heparin was developed to analyze heparin-protein interactions using a Reichert Inc, SR7000, surface plasmon resonance (SPR) instrument. The heparin was attached via streptavidin-biotin linkage where the streptavidin was covalently coupled to the mSAM and biotinylated heparin bound to it. These chips were then used to quantify the interactions of fibroblast growth factor-2 (FGF-2) with the surface bound heparin. Kinetic rate constants of association and disassociation were calculated. The association data of FGF-2 with heparin was fit to a single compartment, well-mixed model as the data did not exhibit mass transfer limitations. The results suggested that rebinding was prevalent and observed disassociation rates differed significantly in the presence of competing soluble heparin during disassociation. Our results indicate that the Reichert instrument and mSAM chips can be used to analyze heparin-protein interactions but that a careful protocol, outlined in this thesis, should be followed to obtain optimal data. / Master of Science

The human pancreatic cancer cell line T3M4, is known to produce transforming growth factor-alpha (TGF-alpha); as well as overexpress the receptor for this ligand, epidermal growth factor (EGF) receptor. TGF-alpha messenger RNA (mRNA) levels were assayed using northern blot, after addition of epidermal growth factor or TGF-alpha. The level of TGF-alpha mRNA was found to increase 2-fold at 2 hours and then return to near basal levels at 10 hours, after treatment with either ligand. Both ligands were also equipotent in a 2 hour dose response assay, with half maximal stimulation seen at 1 nM and maximal stimulation reached at 4 nM. Furthermore, there appeared to be a 2-fold increase in TGF-alpha transcription as determined by nuclear runoff experiments. Induction of TGF-alpha mRNA coupled with the overexpression of the EGF receptor, may result in a potent autocrine cycle; which may be found in other cancers.

The overall objective was to elucidate the mechanism of action of ovulation-inducing factor/nerve growth factor (OIF/NGF) in the reproductive function of spontaneous and induced ovulators, using cow and llama as models. In Study 1, the dynamics of trkA, the high affinity receptor for OIF/NGF, were studied during periovulatory period in cows. Unilateral ovariectomies were performed by colpotomy on Days 2, 4 and 6 of the estrous cycle (Day 0= ovulation), and before and after LH administration. Ovarian samples were processed for immunofluorescent detection of trkA. The intensity and area of immuno-positive staining, and the proportion of immuno-positive cells in both the granulosa and theca layers were higher in dominant than in subordinate follicles (P<0.05). Dominant follicles displayed a different intracellular distribution of trkA from subordinate follicles. The number of positive cells was higher in the developing CL (Day 2 and 4) than in the mature or regressing CL (Day 6, Pre-LH, and Post-LH). In Study 2, the distribution of GnRH neurons in the hypothalamus was examined in female llamas (n = 4). Hypothalamic samples were processed for immunohistochemistry for GnRH. The distribution of GnRH neurons had no evident accumulation in specific hypothalamic nuclei. The majority of GnRH neurons were detected in the anterior and medio-basal hypothalamus (P<0.05). The GnRH neuron fibers were detected primarily in the median eminence and in the medio-basal hypothalamus. In Study 3, the relationship between trkA and GnRH neurons in the llama diencephalon was examined in llama brains (n = 4) obtained in Study 2. Samples were stained using double immunofluorescence. TrkA immuno-reactivity was present in most hypothalamic areas examined; the highest density was found in the diagonal band of Broca and the periventricular nuclei. A low percentage of GnRH cells (1%) showed immuno-reactivity to trkA. Close association between immuno-reactive cells (i.e., GnRH and trkA in the same microscopic field) was detected rarely (3/160 GnRH neurons). We concluded that: 1) the high affinity receptor for OIF/NGF is expressed in greater quantities in dominant than subordinate follicles and in the developing CL; 2) GnRH neurons of llamas are concentrated in the anterior and middle hypothalamus, in close relationship to the third ventricle; and, 3) expression of trkA receptors on GnRH neurons was rare, suggesting that the ovulatory effect of OIF/NGF is not via direct interaction with GnRH neurons.

Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous condition that affects approximately 1 in 4000 individuals worldwide. The most common presentation of RP is a rod-cone dystrophy, where the degeneration of cone photoreceptors occurs secondary to advanced rod loss, leading to a significant decline in central vision and a corresponding reduction in patient quality of life. The mechanisms underlying secondary cone loss are poorly understood, particularly in disorders where the gene defect is unknown or manifest only in rod photoreceptors. Consequently, the thesis presented herein proceeds on several fronts. First, in the long term a greater understanding of the causes underlying cone loss in RP is likely to be beneficial, and so in chapter one a dominant cone degeneration is characterized using intrinsically fluorescent cone photoreceptors to track the degenerative process. Second, as we develop a greater understanding of the genetic etiology underlying RP it is likely that the number of large genes identified as being causative will increase. As currently there is no efficient way to deliver large genes to photoreceptors, chapter two explores the use of alternate viral vectors that might be used to deliver a large therapeutic transgene. Lastly, whilst our understanding of cone loss in RP remains incomplete, it is necessary to develop a broadly applicable therapy to slow or attenuate further cone loss in RP patients regardless of the underlying cause. In chapters three and four we examine the use of low molecular weight "growth factors‟, such as ciliary neurotrophic factor (CNTF), to preserve cone photoreceptors long-term using a rhodopsin knockout model of RP.

This study explored hydrostatic pressure as a mechanobiological parameter to control in vitro endothelial cell tubulogenesis in 3-D hydrogels as a model microvascular tissue engineering approach. For this purpose, the present investigation used an endothelial spheroid model, which we believe is an adaptable microvascularization strategy for many tissue engineering construct designs. We also aimed to identify the operating magnitudes and exposure times for hydrostatic pressure-sensitive sprout formation as well as verify the involvement of VEGFR-3 signaling. For this purpose, we used a custom-designed pressure system and a 3-D endothelial cell spheroid model of sprouting tubulogenesis. We report that an exposure time of 3 days is the minimum duration required to increase endothelial sprout formation in response to 20 mmHg. Notably, exposure to 5 mmHg for 3 days was inhibitory for endothelial spheroid lengths without affecting sprout numbers. Moreover, endothelial spheroids exposed to 40 mmHg also inhibited sprouting activity by reducing sprout numbers without affecting sprout lengths. Finally, blockade of VEGFR-3 signaling abolished the effects of the 20-mmHg stimuli on sprout formation. Based on these results, VEGFR-3 dependent endothelial sprouting appears to exhibit a complex pressure dependence that one may exploit to control microvessel formation.

published_or_final_version / abstract / Medicine / Master / Master of Philosophy