yes / There are various techniques to enhance tissue regeneration via the application of growth factors to the site of regeneration to induce cells to proliferate, differentiate and regenerate. Generally, direct application of growth factors has little effect [1] because the growth factor diffuses out from the site of regeneration very quickly. This is a problem that can be solved by a controlled release of growth factor at the site of action over a long period of time by use of a bioabsorbable scaffold. Growth factors are protein based molecules in the body which are produced by cells and attach to the cell surface. Growth factors bind to membrane receptors, which in turn activate an intracellular signalling pathway. This will activate or inhibit a gene causing either an up regulation or down regulation of a gene product, which then alters the cells behaviour.

In patients with acute myeloid leukemia (AML), cytarabine-based chemotherapy usually achieves remission, but this is commonly followed by relapse and chemo-resistance. In this study, we aim to establish next-generation sequencing (NGS)-based microRNA expression profiling and pathway analysis to identify pathways regulated differentially between chemo-sensitive and -resistant AML as potential therapeutic targets. MicroRNA expression profiles differ significantly between chemo-sensitive and chemo-resistant AML cells and reflect differences in the activity of intracellular signaling cascades. Alterations in signaling pathway activities contribute to treatment resistance and thus represent potential drug targets. Our microRNA-led approach indicates a role for activin receptor type 2A in ARA-C resistance of AML cells and suggests activin receptor signaling to be a candidate pathway for targeted therapy.

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Articular cartilage lesions are often caused by joint trauma and can progress to osteoarthritis (OA) if left untreated. Cartilage tissue engineering is a promising approach for chondral lesion repair, involving the cultivation of cell-seeded scaffolds to generate neocartilage tissues recapitulating composition, structure, and function of native cartilage. Transforming growth factor beta (TGF-β) is widely utilized in cartilage tissue engineering for its ability to promote chondrogenesis and extracellular matrix (ECM) biosynthesis. Conventionally, TGF-β is supplemented in culture medium at supraphysiologic doses (10-100 ng/mL) during in vitro cultivation to regenerate neocartilage with native-matched sGAG content and mechanical properties. However, these doses are 10-1000-fold higher than the physiologic range, promoting undesirable tissue features that are detrimental to the functional behavior of hyaline cartilage. Additionally, TGF-β gradients from media supplementation can induce pronounced heterogeneities in ECM distribution, potentially compromising the survival of engineered cartilage under physiologic loading. The dissertation aims to enhance cartilage regeneration quality using bio-inspired latent TGF-β (LTGF-β) conjugated scaffolds. We hypothesize that LTGF-β scaffolds can achieve uniform delivery of moderated, near-physiologic doses of TGF-β through cell-mediated activation, inducing homogeneous and more hyaline cartilage-like tissue growth. We first evaluated the impact of physiologic TGF-β doses on tissue growth. To address issues related to TGF-β concentration gradients and tissue heterogeneities, we employed a reduced-size construct model. Our findings demonstrate that physiologic doses of TGF-β promote significant enhancements in tissue properties for reduced-size tissues, while also mitigating undesirable outcomes associated with excessive TGF-β. Subsequently, we developed bio-inspired LTGF-β-conjugated scaffolds to deliver physiologic doses of TGF-β. We established a quantification platform based on TGF-β autoinduction to accurately measure the bioactivity level of delivered TGF-β, confirming conjugated LTGF-β can be activated in physiologic range. Further, this quantification platform exhibits versatility for applications in native tissue studies and other TE platforms. Lastly, we determined that LTGF-β conjugation led to enhancements in tissue functional properties comparable to native tissue, while mitigating the abnormal features of neocartilage associated with TGF-β excesses. Moreover, LTGF-β conjugation significantly improves tissue spatial homogeneities in composition and mechanical properties, offering promising implications for enhancing clinical regeneration outcomes.

The emergence of pesticide-resistant mosquitoes and drug-resistant parasites in the last twenty years has made control of malaria more difficult. One novel strategy to better control malaria is the development and release of transgenic mosquitoes whose enhanced immunity prevents transmission of the parasite to the mammalian host. One candidate effector gene is Anopheles stephensi nitric oxide synthase (AsNOS), whose inducible expression and subsequent synthesis of nitric oxide (NO) limits Plasmodium development in A. stephensi. In mammals, one of the most potent physiological regulators of NOS gene expression and catalytic activity is transforming growth factor-β (TGF-β). Moreover, human TGF-β can activate Drosophila melanogaster Smads, the proteins responsible for TGF-β signal transduction. We have determined that following a bloodmeal, active human TGF-β1 (hTGF-β1) persists in the midgut of A. stephensi for up to 48 hours. My data demonstrate that the midgut epithelium recognizes hTGF-β1 as an immunomodulatory cytokine. Specifically, induction of AsNOS by hTGF-β1 occurs in the midgut within minutes of bloodfeeding. Moreover, hTGF-β1 limits development of the human malaria parasite Plasmodium falciparum in the midgut. In other experiments, provision of the AsNOS catalytic inhibitor L-NAME partially reverses the effect of hTGF-β1 on Plasmodium development. These results suggest that AsNOS is a target of hTGF-β1 signaling and additional effectors that impact parasite development may be regulated by hTGF-β1 as well. The fact that hTGF-β1 signals mosquito cells to limit malaria parasite development suggests that there is an endogenous TGF-β signaling network in place. An analysis of the A. gambiae genome database revealed the presence of six TGF-β ligands, including gene duplication in the 60A gene, the first evidence of ligand gene duplication outside of chordates. In addition to five receptors, three Smads were identified in the A. gambiae genome predicted to support TGF-β/Activin- and BMP-like signaling. Midgut epithelial cells and an immunocompetent A. stephensi cell line express all three Smads, confirming that a signaling pathway is in place to support signaling by divergent exogenous and endogenous TGF-β superfamily proteins. The results presented here provide the first evidence of immunological crosstalk between divergent free living hosts of a single parasite. Further, these results imply that the interface between mammals and the mosquitoes that feed on them provide a unique opportunity for circulating molecules in the blood, including TGF-β and other cytokines, to alter the mosquito immune response. / Master of Science

No / Bone repair and wound healing are modulated by different stimuli. There is evidence that Transforming Growth Factor-beta (TGF-β) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules influence cell behaviour. In this study cell surface attachment was examined via a trypsinization assay using various TGF-β isomers in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. Three TGF-β isomers (TGF-β1, 2 and 3), four combined forms (TGF-β(1 + 2), TGF-β(1 + 3), TGF-β(2 + 3) and TGF-β(1 + 2 + 3)) along with four different controls (BSA, HCl, BSA/HCl and negative control) were investigated in this study. The results indicated that treatment with TGF-β1, 2, 3 and HCl decreased cell attachment, however, this effect was significantly greater in the case of TGF-β3 (p < 0.001) indicating perhaps that TGF-β3 does not act alone in cell detachment, but instead functions synergistically with signalling pathways that are dependent on the availability of hydrogen ions. Widefield Surface Plasmon Resonance (WSPR) microscope was also used to investigate cell surface interactions.