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The ability to control cellular behavior at the single-cell level is of great importance for gaining a nuanced understanding of cellular machinery. This dissertation focuses on the development of novel hard nanoparticle (NP) bioconjugate materials, specifically gold nanoparticles (AuNPs) and quantum dots (QDs), for the controlled modulation of cellular behavior. These hard NPs offer advantages such as small size on the order of 1 – 100 nm, high stability, unique optical properties, and the ability to load cargo on a large surface area to volume ratio, making them ideal tools for understanding and controlling cell behavior. In Aim 1, we demonstrate the use of AuNPs to manipulate cellular biological functions, specifically the modulation of membrane potential. We present the conception of anisotropic-shaped AuNPs, known as gold nanoflowers (AuNFs), which exhibit broad absorption extending into the near-infrared region of the spectrum. We demonstrate the effectiveness of utilizing the plasmonic properties AuNFs for inducing plasma membrane depolarization in rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells. Importantly, this is achieved with temporal control and without negatively impacting cellular viability. Aim 2 explores the use of QDs as an optical, trackable scaffold for the multivalent display of growth factors, specifically erythropoietin (EPO), for the enhanced induction of protein expression of aquaporin-4 (AQPN-4) within human astrocytes. This results in enhanced cellular water transport within human astrocytes, a critical function in the brain's glymphatic system. We show that EPO-QD-induced augmented AQPN-4 expression does not negatively impact astrocyte viability and augments the rate of water efflux from astrocytes by approximately two-fold compared to cells treated with monomeric EPO, demonstrating the potential of EPO-NP conjugates as research tools and prospective therapeutics for modulating glymphatic system function. Overall, the body o

Differentiation of naive CD4+ T cells into effector T cells is a dynamic process in which the cells are polarized into T helper (Th) subsets. The subsets largely consist of four fundamental categories: Th1, Th2, Th17, and regulatory T cells. We show that human memory CD4+ T cells can produce hepatocyte growth factor (HGF), a pleiotropic cytokine which can affect several tissue types through signaling by its receptor, c-Met. In vitro differentiation of T cells into Th-like subsets revealed that HGF producing T cells increase under Th1 conditions. Enrichment of HGF producing cells was possible by targeting cells with surface CD30 expression, a marker discovered through single-cell RNA-sequencing. Furthermore, pharmacological inhibition of PI3K or mTOR was found to inhibit HGF mRNA and protein, while an Akt inhibitor was found to increase these levels. The findings suggest that HGF producing T cells could play a role in disease where Th1 are present.

Sepsis manifests as a dysregulated immune response to infection, damaging organs. Skin has a critical role in protecting the body. In sepsis, skin wound healing is impaired. The mechanisms behind it have been poorly studied. In this study, suction blister wounds were induced on intact abdominal skin in 15 septic patients. A single blister wound was biopsied from each patient and from 10 healthy controls. Immunohistochemical staining of growth factors and extracellular matrix (ECM) proteins was performed. Significance (p < 0.05) of the differences was calculated. The following growth factors were overexpressed in the skin of septic patients compared with healthy controls: epithelial growth factor (intact epithelium p = 0.007, migrating epithelium p = 0.038), vascular epithelial growth factor (intact epithelium p < 0.001, migrating epithelium p = 0.011) and transforming growth factor beta (migrating epithelium p = 0.002). The expression of syndecan-1 was upregulated in the skin of septic patients compared with healthy controls (intact epithelium p = 0.048, migrating epithelium p = 0.028). The following ECM proteins had lower expression in the epithelium in septic patients than in healthy controls: tenascin-C (migrating epithelium p = 0.03) and laminin-332 (intact epithelium p = 0.036). In sepsis, growth factor and syndecan expression was enhanced, while ECM and basement membrane proteins were mostly depressed.

Psoriasis, an immune-mediated inflammatory disease, is characterized by keratinocyte hyperproliferation. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-23, and IL-17A play critical roles in the pathogenesis of psoriasis. IL-17A secreted by T-helper 17 acts more directly against keratinocytes than TNF-alpha or IL-23 do. Regarding the receptors of cytokines, fibroblasts also express receptors against IL-17A and TNF-alpha, and induce the production of growth factors. Epiregulin (EREG), an epidermal growth factor receptor ligand, is produced by both keratinocytes and fibroblasts. EREG enhances keratinocyte proliferation and differentiation. We hypothesized that fibroblasts stimulated with IL-17A and/or TNF-alpha may play a role in epidermal hyperproliferation through the production of epidermal growth factors in psoriasis. The mRNA expression of EREG was found to be significantly upregulated by co-stimulation with IL-17A and TNF-alpha (mean, 49.2-fold). Furthermore, the stimulation with TNF-alpha alone, but not IL-17A alone, induced significant increases. Immunofluorescent staining demonstrated that the protein expression level of EREG was also increased in fibroblasts stimulated with these cytokines. Stimulation with EREG significantly enhanced keratinocyte proliferation in vitro. In human psoriatic patients' skin, immunofluorescence staining of EREG showed significantly high intensity in the dermis of lesional skin. In conclusion, cytokine stimulation with TNF-alpha and IL-17A induces the overexpression of EREG from dermal fibroblasts in the lesional skin of psoriasis, and plays a role in epidermal hyperproliferation.

Psoriasis, an immune-mediated inflammatory disease, is characterized by keratinocyte hyperproliferation. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-23, and IL-17A play critical roles in the pathogenesis of psoriasis. IL-17A secreted by T-helper 17 acts more directly against keratinocytes than TNF-alpha or IL-23 do. Regarding the receptors of cytokines, fibroblasts also express receptors against IL-17A and TNF-alpha, and induce the production of growth factors. Epiregulin (EREG), an epidermal growth factor receptor ligand, is produced by both keratinocytes and fibroblasts. EREG enhances keratinocyte proliferation and differentiation. We hypothesized that fibroblasts stimulated with IL-17A and/or TNF-alpha may play a role in epidermal hyperproliferation through the production of epidermal growth factors in psoriasis. The mRNA expression of EREG was found to be significantly upregulated by co-stimulation with IL-17A and TNF-alpha (mean, 49.2-fold). Furthermore, the stimulation with TNF-alpha alone, but not IL-17A alone, induced significant increases. Immunofluorescent staining demonstrated that the protein expression level of EREG was also increased in fibroblasts stimulated with these cytokines. Stimulation with EREG significantly enhanced keratinocyte proliferation in vitro. In human psoriatic patients' skin, immunofluorescence staining of EREG showed significantly high intensity in the dermis of lesional skin. In conclusion, cytokine stimulation with TNF-alpha and IL-17A induces the overexpression of EREG from dermal fibroblasts in the lesional skin of psoriasis, and plays a role in epidermal hyperproliferation.

BackgroundWhether race/ethnicity plays a role in hematopoietic stem/progenitor cells (HSPC) mobilization in autologous donors has not been studied. We hypothesize that donor characteristic including race/ethnicity, age, sex, body mass index, and diagnostic groups influences HSPC mobilization. Diagnostic groups include healthy allogeneic donors, autologous multiple myeloma (MM) and non-MM donors.Study design and methodsHere, we conducted a single-center retrospective study in 64 autologous patients and 48 allogeneic donors. Autologous donors were patients diagnosed with MM or non-MM. All donors were grouped as African American (AA), White (W), or "Other"(O).ResultsMultivariate analysis demonstrated diagnostic group differences for CD34+ cell yields between race/ethnicity. Specifically, non-MM patients had the lowest CD34+ cell yields in AA and O, but not in W. For pre-apheresis peripheral blood (PB) CD34+ cell numbers, race/ethnicity had a significant effect both in bivariate and multivariate analyses. Non-MM patients had the lowest, and AA patients had the highest PB CD34+ cells. The results support the view that past therapies used in MM are likely more conducive of recovery of HSPC.ConclusionsOur study shows that race/ethnicity and diagnostic group differences influenced CD34+ cell mobilization response across donor types. Interestingly, autologous MM donors with the aid of plerixafor displayed comparable CD34 yields to allogeneic donors. Even though both MM and non-MM donors received plerixafor, non-MM donors had significantly lower CD34 yields among AA and O donors but not in W donors. Larger studies would be required to validate the role of diagnostic groups and race/ethnicity interactions.

With 1.5-2.0 million new cases annually worldwide, corneal injury represents a common cause of vision loss, often from irreversible scarring due to surface corneal defects. In this study, we assessed the use of hepatocyte growth factor (HGF) loaded into an in situ photopolymerizable transparent gelatin-based hydrogel for the management of corneal defects. In vitro release kinetics showed that, in regard to the total amount of HGF released over a month, 55 ± 11% was released during the first 24 h, followed by a slow release profile for up to one month. The effect of HGF was assessed using an ex vivo model of pig corneal defect. After three days of organ culture, epithelial defects were found to be completely healed for 89% of the corneas treated with HGF, compared to only 11% of the corneas that had fully re-epithelialized when treated with the hydrogel without HGF. The thickness of the epithelial layer was found to be significantly higher for the HGF-treated group compared to the group treated with hydrogel without HGF (p = 0.0012). Finally, histological and immunostaining assessments demonstrated a better stratification and adhesion of the epithelial layer in the presence of HGF. These results suggest that the HGF-loaded hydrogel system represents a promising solution for the treatment of persistent corneal defects at risk of scarring.

Evidence from multiple scientific disciplines indicates that aerobic exercise could be effective in enhancing brain health and function in aging, and reducing the risk for cognitive impairment and dementia. Animal models have provided convincing evidence that exercise increases the expression of growth factors involved in neuroplasticity. By triggering signaling cascades in the brain, these growth factors promote proliferation, neurogenesis, neural differentiation, and cell survival in the hippocampus, and angiogenesis and/or synaptic plasticity and neural growth throughout the brain more generally. These growth factor-induced effects partly explain why exercise improves memory. Furthermore, they engender neuroprotective effects by influencing regeneration, remyelination, cell survival, and waste removal. Whereas human intervention evidence supports hippocampal plasticity, cross-sectional studies highlight effects in the cortex too, indicating that exercise-induced gross morphological changes may be regionally specific. Aerobic exercise is a promising nonpharmacological therapy to maintain brain health, although knowledge about the effects of specific exercise parameters (i.e., intensity) on different populations is still lacking.

Psoriasis, an immune-mediated inflammatory disease, is characterized by keratinocyte hyperproliferation. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-23, and IL-17A play critical roles in the pathogenesis of psoriasis. IL-17A secreted by T-helper 17 acts more directly against keratinocytes than TNF-alpha or IL-23 do. Regarding the receptors of cytokines, fibroblasts also express receptors against IL-17A and TNF-alpha, and induce the production of growth factors. Epiregulin (EREG), an epidermal growth factor receptor ligand, is produced by both keratinocytes and fibroblasts. EREG enhances keratinocyte proliferation and differentiation. We hypothesized that fibroblasts stimulated with IL-17A and/or TNF-alpha may play a role in epidermal hyperproliferation through the production of epidermal growth factors in psoriasis. The mRNA expression of EREG was found to be significantly upregulated by co-stimulation with IL-17A and TNF-alpha (mean, 49.2-fold). Furthermore, the stimulation with TNF-alpha alone, but not IL-17A alone, induced significant increases. Immunofluorescent staining demonstrated that the protein expression level of EREG was also increased in fibroblasts stimulated with these cytokines. Stimulation with EREG significantly enhanced keratinocyte proliferation in vitro. In human psoriatic patients' skin, immunofluorescence staining of EREG showed significantly high intensity in the dermis of lesional skin. In conclusion, cytokine stimulation with TNF-alpha and IL-17A induces the overexpression of EREG from dermal fibroblasts in the lesional skin of psoriasis, and plays a role in epidermal hyperproliferation.

The epidermal growth factor receptor (EGFR) has served as the founding member of the large family of growth factor receptors harboring intrinsic tyrosine kinase function. High abundance of EGFR and large internal deletions are frequently observed in brain tumors, whereas point mutations and small insertions within the kinase domain are common in lung cancer. For these reasons EGFR and its preferred heterodimer partner, HER2/ERBB2, became popular targets of anti‐cancer therapies. Nevertheless, EGFR research keeps revealing unexpected observations, which are re-viewed herein. Once activated by a ligand, EGFR initiates a time‐dependent series of molecular switches comprising downregulation of a large cohort of microRNAs, up‐regulation of newly syn-thesized mRNAs, and covalent protein modifications, collectively controlling phenotype‐determin-ing genes. In addition to microRNAs, long non‐coding RNAs and circular RNAs play critical roles in EGFR signaling. Along with driver mutations, EGFR drives metastasis in many ways. Paracrine loops comprising tumor and stromal cells enable EGFR to fuel invasion across tissue barriers, survival of clusters of circulating tumor cells, as well as colonization of distant organs. We conclude by listing all clinically approved anti‐cancer drugs targeting either EGFR or HER2. Because emergence of drug resistance is nearly inevitable, we discuss the major evasion mechanisms.

Psoriasis, an immune-mediated inflammatory disease, is characterized by keratinocyte hyperproliferation. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-23, and IL-17A play critical roles in the pathogenesis of psoriasis. IL-17A secreted by T-helper 17 acts more directly against keratinocytes than TNF-alpha or IL-23 do. Regarding the receptors of cytokines, fibroblasts also express receptors against IL-17A and TNF-alpha, and induce the production of growth factors. Epiregulin (EREG), an epidermal growth factor receptor ligand, is produced by both keratinocytes and fibroblasts. EREG enhances keratinocyte proliferation and differentiation. We hypothesized that fibroblasts stimulated with IL-17A and/or TNF-alpha may play a role in epidermal hyperproliferation through the production of epidermal growth factors in psoriasis. The mRNA expression of EREG was found to be significantly upregulated by co-stimulation with IL-17A and TNF-alpha (mean, 49.2-fold). Furthermore, the stimulation with TNF-alpha alone, but not IL-17A alone, induced significant increases. Immunofluorescent staining demonstrated that the protein expression level of EREG was also increased in fibroblasts stimulated with these cytokines. Stimulation with EREG significantly enhanced keratinocyte proliferation in vitro. In human psoriatic patients' skin, immunofluorescence staining of EREG showed significantly high intensity in the dermis of lesional skin. In conclusion, cytokine stimulation with TNF-alpha and IL-17A induces the overexpression of EREG from dermal fibroblasts in the lesional skin of psoriasis, and plays a role in epidermal hyperproliferation.

Healing in soft biological tissues is a chain of events on different time and length scales. This work presents a computational framework to capture and couple important mechanical, chemical and biological aspects of healing. A molecular-level damage in collagen, i.e., the interstrand delamination, is addressed as source of plastic deformation in tissues. This mechanism initiates a biochemical response and starts the chain of healing. In particular, damage is considered to be the stimulus for the production of matrix metalloproteinases and growth factors which in turn, respectively, degrade and produce collagen. Due to collagen turnover, the volume of the tissue changes, which can result either in normal or pathological healing. To capture the mechanisms on continuum scale, the deformation gradient is multiplicatively decomposed in inelastic and elastic deformation gradients. A recently proposed elasto-plastic formulation is, through a biochemical model, coupled with a growth and remodeling description based on homogenized constrained mixtures. After the discussion of the biological species response to the damage stimulus, the framework is implemented in a mixed nonlinear finite element formulation and a biaxial tension and an indentation tests are conducted on a prestretched flat tissue sample. The results illustrate that the model is able to describe the evolutions of growth factors and matrix metalloproteinases following damage and the subsequent growth and remodeling in the respect of equilibrium. The interplay between mechanical and chemo-biological events occurring during healing is captured, proving that the framework is a suitable basis for more detailed simulations of damage-induced tissue response. © 2021, The Author(s).

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors (GFs) to influence neighboring cells and promote migration, proliferation, or differentiation. Despite promising results in preclinical models, the use of inductive biomacromolecules has achieved limited success in translation to the clinic. The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages, which commonly result in detrimental side effects. Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix (ECM), which acts as a reservoir for GFs via electrostatic interactions. Advances in the manipulation of extracellular proteins, decellularized tissues, and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs. Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules. This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone, cartilage, and muscle.

[Background] During melanogenesis, melanocytes produce melanin through enzymatic reactions. Microphthalmia‐associated transcription factor (MITF) is a major regulator in controlling the expressions of melanogenic enzymes tyrosinase (TYR), tyrosine‐related protein‐1 (TRP1), and dopachrome tautomerase (DCT). Self‐Growth Colony (SGC) is prepared from human platelet‐rich plasma (PRP) having an enrichment of growth factors, and which has claimed skin regeneration function. [Aim] In this study, we aim to identify and investigate the novel role of SGC in skin melanogenesis. [Methods] MTT assay was performed to determine the cytotoxicity of applied SGC. Melanin assay was adopted to quantify the intracellular melanin after SGC treatment. Promoter‐driven luciferase assay, real‐time PCR, and Western blotting were performed to determine the expressions of melanogenic enzymes and MITF in SGC‐treated cultured Melan‐A cells, being treated with or without UV induction. Ex vivo mouse skin was treated with SGC, and then was subjected to Western blotting and histochemical staining. [Results] We identified that SGC inhibited melanogenesis in cultured melanocytes and ex vivo mouse skin. The phenomena were attributed to a reduction of MITF expression, which subsequently down‐regulated the melanogenic enzymes, that is, TYR, TRP1, and DCT. Moreover, ERK signaling was activated in the SGC‐inhibited melanogenesis. [Conclusions] The findings suggest that SGC extracting from human blood can be a safe and potential agent in promoting skin whitening.

Exogenous electrical fields have been explored in regenerative medicine to increase cellular expression of pro-regenerative growth factors. Adipose-derived stem cells (ASCs) are attractive for regenerative applications, specifically for neural repair. Little is known about the relationship between low-level electrical stimulation (ES) and ASC regenerative potentiation. In this work, patterns of ASC expression and secretion of growth factors (i.e., secretome) were explored across a range of ES parameters. ASCs were stimulated with low-level stimulation (20 mV/mm) at varied pulse frequencies, durations, and with alternating versus direct current. Frequency and duration had the most significant effects on growth factor expression. While a range of stimulation frequencies (1, 20, 1000 Hz) applied intermittently (1 h × 3 days) induced upregulation of general wound healing factors, neural-specific factors were only increased at 1 Hz. Moreover, the most optimal expression of neural growth factors was achieved when ASCs were exposed to 1 Hz pulses continuously for 24 h. In evaluation of secretome, apparent inconsistencies were observed across biological replications. Nonetheless, ASC secretome (from 1 Hz, 24 h ES) caused significant increase in neurite extension compared to non-stimulated control. Overall, ASCs are sensitive to ES parameters at low field strengths, notably pulse frequency and stimulation duration.

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors (GFs) to influence neighboring cells and promote migration, proliferation, or differentiation. Despite promising results in preclinical models, the use of inductive biomacromolecules has achieved limited success in translation to the clinic. The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages, which commonly result in detrimental side effects. Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix (ECM), which acts as a reservoir for GFs via electrostatic interactions. Advances in the manipulation of extracellular proteins, decellularized tissues, and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs. Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules. This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone, cartilage, and muscle.

[ES] El daño hepático inducido por fármacos es un evento clínico que puede acarrear daños hepáticos potencialmente mortales y de alto coste y que genera la necesidad del desarrollo de plataformas de cultivo celular capaces de predecirlo. Para ello, los hepatocitos en cultivo deben mantener el fenotipo y la funcionalidad hepática similar a las condiciones in vivo. Esto se consigue con el uso de biomateriales que proporcionan las condiciones fisiológicas más relevantes a las células, como son la organización espacial tridimensional, el contacto célula-célula y la interacción célula-matriz. Los hidrogeles inyectables basados en componentes de la matriz extracelular del tejido son candidatos ideales para este propósito, puesto que permiten encapsular las células homogéneamente en el material por reacciones no citotóxicas y mantenerlas en cultivos 3D de forma biomimética. Además, al ser inyectables, permiten incorporar factores de crecimiento en el momento de encapsulación celular, presentando los factores a las células desde fase sólida si la composición del hidrogel los retiene y, por tanto, siendo más eficientes para el mantenimiento del fenotipo y la funcionalidad celular. En el presente Trabajo Fin de Grado se ha estudiado la optimización de la síntesis y gelificación de hidrogeles inyectables basados en mezclas de gelatina y ácido hialurónico con aplicación en el diseño de plataformas para predecir eventos de hepatotoxicidad. Se ha buscado estrategias que permiten obtener hidrogeles de gelatina gelificables en medio de cultivo y con propiedades mecánicas adecuadas para mantener la integridad en cultivo. Se ha optimizado el proceso de síntesis del ácido hialurónico para obtener un rendimiento mayor en cada lote de fabricación. Además, se ha puesto a punto un método para la cuantificación de la liberación del factor de crecimiento hepático encapsulado en los hidrogeles optimizados y de distinta composición. Ello ha requerido el establecimiento de un método de marcado, [EN] Drug-induced liver injury is a clinical condition that can carry life-threatening and costly hepatic failures and that creates the need for the development of cell culture platforms capable of predicting it. To achieve this, cultured hepatocytes must maintain liver phenotype and functionality similar to in vivo conditions. This is achieved through the use of biomaterials that provide the most relevant physiological conditions for the cells, such as three-dimensional spatial organisation, cell-cell contact and cell-matrix interaction. Injectable hydrogels based on tissue extracellular matrix components are ideal candidates for this purpose as they allow cells to be homogeneously encapsulated in the material by non-cytotoxic reactions and maintained in 3D cultures in a biomimetic way. Moreover, as they are injectable, they allow the incorporation of growth factors at the moment of cell encapsulation, presenting the factors to the cells from the solid phase if the composition of the hydrogel retains them and, therefore, being more efficient for the maintenance of the cell phenotype and functionality. In this Bachelor¿s Thesis we have studied the optimisation of the synthesis and gelation of injectable hydrogels based on mixtures of gelatin and hyaluronic acid with application in the design of platforms for predicting hepatotoxicity events. Strategies have been sought to obtain gelatin hydrogels that can be gelled in culture medium and with suitable mechanical properties to maintain integrity in culture. The hyaluronic acid synthesis has been optimised to obtain a higher yield in each manufacturing batch. In addition, a method has been developed for the quantification of the release of hepatocyte growth factor encapsulated in the optimised hydrogels of different composition. This has required the establishment of a method for fluorescent labelling of the growth factor and decisions about the size of the hydrogel, the amount of growth factor and the appropriate rene

The prevalence of rheumatic and musculoskeletal diseases (RMDs) including osteoarthritis (OA) and low back pain (LBP) in aging societies present significant cost burdens to health and social care systems. Intervertebral disc (IVD) degeneration, which is characterized by disc dehydration, anatomical alterations, and extensive changes in extracellular matrix (ECM) composition, is an important contributor to LBP. IVD cell homeostasis can be disrupted by mitochondrial dysfunction. Mitochondria are the main source of energy supply in IVD cells and a major contributor to the production of reactive oxygen species (ROS). Therefore, mitochondria represent a double-edged sword in IVD cells. Mitochondrial dysfunction results in oxidative stress, cell death, and premature cell senescence, which are all implicated in IVD degeneration. Considering the importance of optimal mitochondrial function for the preservation of IVD cell homeostasis, extensive studies have been done in recent years to evaluate the efficacy of small molecules targeting mitochondrial dysfunction. In this article, we review the pathogenesis of mitochondrial dysfunction, aiming to highlight the role of small molecules and a selected number of biological growth factors that regulate mitochondrial function and maintain IVD cell homeostasis. Furthermore, molecules that target mitochondria and their mechanisms of action and potential for IVD regeneration are identified. Finally, we discuss mitophagy as a key mediator of many cellular events and the small molecules regulating its function.

[Background] During melanogenesis, melanocytes produce melanin through enzymatic reactions. Microphthalmia‐associated transcription factor (MITF) is a major regulator in controlling the expressions of melanogenic enzymes tyrosinase (TYR), tyrosine‐related protein‐1 (TRP1), and dopachrome tautomerase (DCT). Self‐Growth Colony (SGC) is prepared from human platelet‐rich plasma (PRP) having an enrichment of growth factors, and which has claimed skin regeneration function. [Aim] In this study, we aim to identify and investigate the novel role of SGC in skin melanogenesis. [Methods] MTT assay was performed to determine the cytotoxicity of applied SGC. Melanin assay was adopted to quantify the intracellular melanin after SGC treatment. Promoter‐driven luciferase assay, real‐time PCR, and Western blotting were performed to determine the expressions of melanogenic enzymes and MITF in SGC‐treated cultured Melan‐A cells, being treated with or without UV induction. Ex vivo mouse skin was treated with SGC, and then was subjected to Western blotting and histochemical staining. [Results] We identified that SGC inhibited melanogenesis in cultured melanocytes and ex vivo mouse skin. The phenomena were attributed to a reduction of MITF expression, which subsequently down‐regulated the melanogenic enzymes, that is, TYR, TRP1, and DCT. Moreover, ERK signaling was activated in the SGC‐inhibited melanogenesis. [Conclusions] The findings suggest that SGC extracting from human blood can be a safe and potential agent in promoting skin whitening.

The skin is the largest organ of the body. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The protective function of the skin is compromised in sepsis, and septic patients are prone to problems in tissue repair, which causes increased morbidity. However, there are only a few human studies exploring wound healing in sepsis. In this study we observed skin wound healing and the molecular mechanisms behind it in 44 septic patients and 14 healthy controls. From sepsis and healthy sera, metabolites were analyzed with proton nuclear magnetic spectroscopy, and cytokines using multiplex assay. The effect of sepsis and healthy sera was tested in vitro on keratinocyte proliferation, migration and viability. Suction blister wounds were induced on 15 patients and 10 controls and biopsied for immunohistochemical analyses. Serum levels of glucose, glycine, 3-hydroxybutyrate, creatinine and glycoprotein acetyls were significantly higher in sepsis patients compared to healthy controls, whereas levels of histidine and citrate were significantly lower in sepsis. Keratinocyte viability, proliferation and migration were reduced in sepsis in vitro. Sepsis sera supplemented with epithelial growth factor (EGF) or tumor necrosis factor alpha (TNF-α) improved and EGF receptor inhibition reduced keratinocyte migration in healthy and septic wounds. Extracellular vesicles added to healthy or sepsis serum media inhibited keratinocyte migration. Higher serum concentrations of TNF-α and vascular endothelial growth factor (VEGF) and lower levels of EGF were detected in sepsis sera compared to healthy sera. In skin immunohistochemistry samples, the levels of EGF, VEGF and transforming growth factor beta as well as syndecan-1 were increased in septic patients compared to healthy controls. Extracellular matrix (ECM) components tenascin-C and laminin-332 had lower expression in septic skin compared to healthy skin. The results of t, Tiivistelmä Elimistön väärin mitoitettu vaste tulehdukseen vaurioittaa sen omia kudoksia sepsiksessä. Iho on ihmisen suurin elin ja suoja ulkoisia haittatekijöitä vastaan. Kudosparanemisesta sepsiksessä on vain vähän tietoa. Tutkimuksen tarkoituksena on selvittää ihohaavan paranemista ja taustalla vaikuttavia molekyylitason mekanismeja sepsiksessä. Tutkimusta varten otettiin verinäytteet 44 sepsispotilaalta ja 14 terveeltä verrokilta. Lisäksi 15 potilaalle ja 10 terveelle verrokille tehtiin imurakkulahaavat iholle, ja yksi paraneva haava poistettiin kudosnäytteeksi. Aineenvaihduntatuotteiden määrää veressä tutkittiin ydinmagneettisella resonanssispektroskopialla. Haavan paranemista sepsiksessä tutkittiin mittaamalla keratinosyyttien elinkykyisyyttä, lisääntymistä ja liikkumista soluviljelymenetelmin. Lisäksi mitattiin veren kasvutekijäpitoisuuksia. Imurakkulahaavanäytteille tehtiin immunohistokemialliset tutkimukset. Veren glukoosin, glysiinin, 3-hydroksibutyraatin, kreatiniinin ja glykoproteiiniasetyylien pitoisuudet olivat sepsispotilailla korkeammat ja sitraatin ja histidiinin pitoisuudet matalammat terveisiin verrattuna. Keratinosyyttien elinvoimaisuus, liikkuminen ja lisääntyminen keinotekoisessa haavassa olivat alentuneet sepsiksessä terveisiin verrokkeihin verrattuna. Solunulkoiset vesikkelit estivät ja epiteliaalisen kasvutekijän (EGF) tai tuumorinekroositekijä alfan (TNF-α) lisäys haavoille paransi keratinosyyttien liikkumista. Veren TNF-α:n ja verisuonikasvutekijän (VEGF) pitoisuudet olivat sepsiksessä korkeammat, kun taas EGF:n pitoisuus oli matalampi. Paranevan ihohaavan solunulkoisen väliaineen komponenttien tenaskiini-C:n sekä laminiini-332:n määrä oli sepsiksessä vähäisempi ja solun pintareseptori syndekaani-1:n, transformoivan kasvutekijä beetan (TGF-β), VEGF:n ja EGF:n määrä puolestaan korkeampi. Tässä tutkimuksessa saadut tulokset tukevat aiempia kliinisiä havaintoja heikentyneestä haavan paranemisesta sepsiksessä sekä antavat lisäti

[ES] El daño hepático inducido por fármacos es un evento clínico que puede acarrear daños hepáticos potencialmente mortales y de alto coste y que genera la necesidad del desarrollo de plataformas de cultivo celular capaces de predecirlo. Para ello, los hepatocitos en cultivo deben mantener el fenotipo y la funcionalidad hepática similar a las condiciones in vivo. Esto se consigue con el uso de biomateriales que proporcionan las condiciones fisiológicas más relevantes a las células, como son la organización espacial tridimensional, el contacto célula-célula y la interacción célula-matriz. Los hidrogeles inyectables basados en componentes de la matriz extracelular del tejido son candidatos ideales para este propósito, puesto que permiten encapsular las células homogéneamente en el material por reacciones no citotóxicas y mantenerlas en cultivos 3D de forma biomimética. Además, al ser inyectables, permiten incorporar factores de crecimiento en el momento de encapsulación celular, presentando los factores a las células desde fase sólida si la composición del hidrogel los retiene y, por tanto, siendo más eficientes para el mantenimiento del fenotipo y la funcionalidad celular. En el presente Trabajo Fin de Grado se ha estudiado la optimización de la síntesis y gelificación de hidrogeles inyectables basados en mezclas de gelatina y ácido hialurónico con aplicación en el diseño de plataformas para predecir eventos de hepatotoxicidad. Se ha buscado estrategias que permiten obtener hidrogeles de gelatina gelificables en medio de cultivo y con propiedades mecánicas adecuadas para mantener la integridad en cultivo. Se ha optimizado el proceso de síntesis del ácido hialurónico para obtener un rendimiento mayor en cada lote de fabricación. Además, se ha puesto a punto un método para la cuantificación de la liberación del factor de crecimiento hepático encapsulado en los hidrogeles optimizados y de distinta composición. Ello ha requerido el establecimiento de un método de marcado, [EN] Drug-induced liver injury is a clinical condition that can carry life-threatening and costly hepatic failures and that creates the need for the development of cell culture platforms capable of predicting it. To achieve this, cultured hepatocytes must maintain liver phenotype and functionality similar to in vivo conditions. This is achieved through the use of biomaterials that provide the most relevant physiological conditions for the cells, such as three-dimensional spatial organisation, cell-cell contact and cell-matrix interaction. Injectable hydrogels based on tissue extracellular matrix components are ideal candidates for this purpose as they allow cells to be homogeneously encapsulated in the material by non-cytotoxic reactions and maintained in 3D cultures in a biomimetic way. Moreover, as they are injectable, they allow the incorporation of growth factors at the moment of cell encapsulation, presenting the factors to the cells from the solid phase if the composition of the hydrogel retains them and, therefore, being more efficient for the maintenance of the cell phenotype and functionality. In this Bachelor¿s Thesis we have studied the optimisation of the synthesis and gelation of injectable hydrogels based on mixtures of gelatin and hyaluronic acid with application in the design of platforms for predicting hepatotoxicity events. Strategies have been sought to obtain gelatin hydrogels that can be gelled in culture medium and with suitable mechanical properties to maintain integrity in culture. The hyaluronic acid synthesis has been optimised to obtain a higher yield in each manufacturing batch. In addition, a method has been developed for the quantification of the release of hepatocyte growth factor encapsulated in the optimised hydrogels of different composition. This has required the establishment of a method for fluorescent labelling of the growth factor and decisions about the size of the hydrogel, the amount of growth factor and the appropriate rene

[ES] El daño hepático inducido por fármacos es un evento clínico que puede acarrear daños hepáticos potencialmente mortales y de alto coste y que genera la necesidad del desarrollo de plataformas de cultivo celular capaces de predecirlo. Para ello, los hepatocitos en cultivo deben mantener el fenotipo y la funcionalidad hepática similar a las condiciones in vivo. Esto se consigue con el uso de biomateriales que proporcionan las condiciones fisiológicas más relevantes a las células, como son la organización espacial tridimensional, el contacto célula-célula y la interacción célula-matriz. Los hidrogeles inyectables basados en componentes de la matriz extracelular del tejido son candidatos ideales para este propósito, puesto que permiten encapsular las células homogéneamente en el material por reacciones no citotóxicas y mantenerlas en cultivos 3D de forma biomimética. Además, al ser inyectables, permiten incorporar factores de crecimiento en el momento de encapsulación celular, presentando los factores a las células desde fase sólida si la composición del hidrogel los retiene y, por tanto, siendo más eficientes para el mantenimiento del fenotipo y la funcionalidad celular. En el presente Trabajo Fin de Grado se ha estudiado la optimización de la síntesis y gelificación de hidrogeles inyectables basados en mezclas de gelatina y ácido hialurónico con aplicación en el diseño de plataformas para predecir eventos de hepatotoxicidad. Se ha buscado estrategias que permiten obtener hidrogeles de gelatina gelificables en medio de cultivo y con propiedades mecánicas adecuadas para mantener la integridad en cultivo. Se ha optimizado el proceso de síntesis del ácido hialurónico para obtener un rendimiento mayor en cada lote de fabricación. Además, se ha puesto a punto un método para la cuantificación de la liberación del factor de crecimiento hepático encapsulado en los hidrogeles optimizados y de distinta composición. Ello ha requerido el establecimiento de un método de marcado, [EN] Drug-induced liver injury is a clinical condition that can carry life-threatening and costly hepatic failures and that creates the need for the development of cell culture platforms capable of predicting it. To achieve this, cultured hepatocytes must maintain liver phenotype and functionality similar to in vivo conditions. This is achieved through the use of biomaterials that provide the most relevant physiological conditions for the cells, such as three-dimensional spatial organisation, cell-cell contact and cell-matrix interaction. Injectable hydrogels based on tissue extracellular matrix components are ideal candidates for this purpose as they allow cells to be homogeneously encapsulated in the material by non-cytotoxic reactions and maintained in 3D cultures in a biomimetic way. Moreover, as they are injectable, they allow the incorporation of growth factors at the moment of cell encapsulation, presenting the factors to the cells from the solid phase if the composition of the hydrogel retains them and, therefore, being more efficient for the maintenance of the cell phenotype and functionality. In this Bachelor¿s Thesis we have studied the optimisation of the synthesis and gelation of injectable hydrogels based on mixtures of gelatin and hyaluronic acid with application in the design of platforms for predicting hepatotoxicity events. Strategies have been sought to obtain gelatin hydrogels that can be gelled in culture medium and with suitable mechanical properties to maintain integrity in culture. The hyaluronic acid synthesis has been optimised to obtain a higher yield in each manufacturing batch. In addition, a method has been developed for the quantification of the release of hepatocyte growth factor encapsulated in the optimised hydrogels of different composition. This has required the establishment of a method for fluorescent labelling of the growth factor and decisions about the size of the hydrogel, the amount of growth factor and the appropriate rene

Receptor tyrosine kinases (RTKs) are single-span transmembrane receptors in which relatively conserved intracellular kinase domains are coupled to divergent extracellular modules. The extracellular domains initiate receptor signaling upon binding to either soluble or membrane-embedded ligands. The diversity of extracellular domain structures allows for coupling of many unique signaling inputs to intracellular tyrosine phosphorylation. The combinatorial power of this receptor system is further increased by the fact that multiple ligands can typically interact with the same receptor. Such ligands often act as biased agonists and initiate distinct signaling responses via activation of the same receptor. Mechanisms behind such biased agonism are largely unknown for RTKs, especially at the level of receptor-ligand complex structure. Using recent progress in understanding the structures of active RTK signaling units, we discuss selected mechanisms by which ligands couple receptor activation to distinct signaling outputs.

Growth factors and their receptor tyrosine kinases (RTKs), a group of transmembrane molecules harboring cytoplasm-facing tyrosine-specific kinase functions, play essential roles in migration of multipotent cell populations and rapid proliferation of stem cells' descendants, transit amplifying cells, during embryogenesis and tissue repair. These intrinsic functions are aberrantly harnessed when cancer cells undergo intertwined phases of cell migration and proliferation during cancer progression. For example, by means of clonal expansion growth factors fixate the rarely occurring driver mutations, which initiate tumors. Likewise, autocrine and stromal growth factors propel angiogenesis and penetration into the newly sprouted vessels, which enable seeding micro-metastases at distant organs. We review genetic and other mechanisms that preempt ligand-mediated activation of RTKs, thereby supporting sustained cancer progression. The widespread occurrence of aberrant RTKs and downstream signaling pathways in cancer, identifies molecular targets suitable for pharmacological intervention. We list all clinically approved cancer drugs that specifically intercept oncogenic RTKs. These are mainly tyrosine kinase inhibitors and monoclonal antibodies, which can inhibit cancer but inevitably become progressively less effective due to adaptive rewiring processes or emergence of new mutations, processes we overview. Similarly important are patient treatments making use of radiation, chemotherapeutic agents and immune checkpoint inhibitors. The many interfaces linking RTK-targeted therapies and these systemic or local regimens are described in details because of the great promise offered by combining pharmacological modalities.

Ischemic heart disease is one of the most frequent causes of death worldwide. When an imbalance occurs between myocardial oxygen supply and demand, such as in angina or myocardial infarction, interventional and surgical revascularization strategies can restore blood flow in the ischemic region. However, many patients could benefit from an additional therapeutic stimulation of micro-vascular growth. First, we combined the cell and gene therapy approaches for a controlled and sustained expression of growth factors, showing the potential to stabilize an infarction in a rodent model. Then, we moved to an optimized fibrin-based delivery platform, which provides a convenient, clinically approved and off-the-shelf product to deliver the growth factors as recombinant proteins and investigated its feasibility in the heart., (MED - Sciences médicales) -- UCL, 2020

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1' position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

We investigate the impact of granulocyte-colony stimulating factor (G-CSF) primary prophylaxis (G-PP, N = 83) versus no G-PP (N = 579) on safety and efficacy of brentuximab vedotin plus doxorubicin, vinblastine, and dacarbazine (A + AVD) in the ECHELON-1 study of previously untreated stage III/IV classical Hodgkin lymphoma. G-PP was associated with lower incidence of ≥ grade 3 neutropenia (29% versus 70%) and febrile neutropenia (11% versus 21%). Fewer dose delays (35% versus 49%), reductions (20% versus 26%), and hospitalizations (29% versus 38%) were observed. Seven neutropenia-associated deaths occurred in the A + AVD arm; none received G-PP. A + AVD with G-PP was associated with decreased risk of a modified progression-free survival event by 26% compared with A + AVD alone (95% CI: 0.40–1.37). G-PP reduced the rate and severity of adverse events, including febrile neutropenia, reduced treatment delays, dose reductions, and discontinuations, and may thus improve efficacy outcomes. These data support G-PP for all patients treated with A + AVD.

Alzheimer's disease (AD) is a neurodegenerative disorder for which there is no cure. Here, we test a dual GLP-1/GIP receptor agonist (DA4-JC) that has a cell penetrating sequence added to enhance blood-brain barrier penetration. We show in a receptor activity study that DA4-JC has balanced activity on both GLP-1 and GIP receptors but not on GLP-2 or Glucagon receptors. A dose-response study in the APP/PS1 mouse model of AD showed both a dose-dependent drug effect on the inflammation response and the reduction of amyloid plaques in the brain. When comparing DA4-JC with the GLP-1 analogue liraglutide at equal doses of 10nmol/kg bw ip. once-daily for 8 weeks, DA4-JC was more effective in reversing memory loss, enhancing synaptic plasticity (LTP) in the hippocampus, reducing amyloid plaques and lowering pro-inflammatory cytokine levels in the brain. The results suggest that DA4-JC may be a novel treatment for AD.

The COVID-19 pandemic poses a unique challenge to the care of patients with haematological malignancies. Viral pneumonia is known to cause disproportionately severe disease in patients with cancer, and patients with lymphoma, myeloma and chronic lymphocytic leukaemia are likely to be at particular risk of severe disease related to COVID-19. This statement has been developed by consensus among authors from Australia and New Zealand. We aim to provide supportive guidance to clinicians making individual patient decisions during the COVID-19 pandemic, in particular during periods that access to healthcare resources may be limited. General recommendations include those to minimise patient exposure to COVID-19, including the use of telehealth, avoidance of non-essential visits and minimisation of time spent by patients in infusion suites and other clinical areas. This statement also provides recommendations where appropriate in assessing indications for therapy, reducing therapy-associated immunosuppression and reducing healthcare utilisation in patients with specific haematological malignancies during the COVID-19 pandemic. Specific decisions regarding therapy of haematological malignancies will need to be individualised, based on disease risk, risks of immunosuppression, rates of community transmission of COVID-19 and available local healthcare resources.Copyright © 2020 Royal Australasian College of Physicians

The COVID-19 pandemic poses a unique challenge to the care of patients with haematological malignancies. Viral pneumonia is known to cause disproportionately severe disease in patients with cancer, and patients with lymphoma, myeloma and chronic lymphocytic leukaemia are likely to be at particular risk of severe disease related to COVID-19. This statement has been developed by consensus among authors from Australia and New Zealand. We aim to provide supportive guidance to clinicians making individual patient decisions during the COVID-19 pandemic, in particular during periods that access to healthcare resources may be limited. General recommendations include those to minimise patient exposure to COVID-19, including the use of telehealth, avoidance of non-essential visits and minimisation of time spent by patients in infusion suites and other clinical areas. This statement also provides recommendations where appropriate in assessing indications for therapy, reducing therapy-associated immunosuppression and reducing healthcare utilisation in patients with specific haematological malignancies during the COVID-19 pandemic. Specific decisions regarding therapy of haematological malignancies will need to be individualised, based on disease risk, risks of immunosuppression, rates of community transmission of COVID-19 and available local healthcare resources.Copyright © 2020 Royal Australasian College of Physicians

We investigate the impact of granulocyte-colony stimulating factor (G-CSF) primary prophylaxis (G-PP, N = 83) versus no G-PP (N = 579) on safety and efficacy of brentuximab vedotin plus doxorubicin, vinblastine, and dacarbazine (A + AVD) in the ECHELON-1 study of previously untreated stage III/IV classical Hodgkin lymphoma. G-PP was associated with lower incidence of ≥ grade 3 neutropenia (29% versus 70%) and febrile neutropenia (11% versus 21%). Fewer dose delays (35% versus 49%), reductions (20% versus 26%), and hospitalizations (29% versus 38%) were observed. Seven neutropenia-associated deaths occurred in the A + AVD arm; none received G-PP. A + AVD with G-PP was associated with decreased risk of a modified progression-free survival event by 26% compared with A + AVD alone (95% CI: 0.40–1.37). G-PP reduced the rate and severity of adverse events, including febrile neutropenia, reduced treatment delays, dose reductions, and discontinuations, and may thus improve efficacy outcomes. These data support G-PP for all patients treated with A + AVD.

Alzheimer's disease (AD) is a neurodegenerative disorder for which there is no cure. Here, we test a dual GLP-1/GIP receptor agonist (DA4-JC) that has a cell penetrating sequence added to enhance blood-brain barrier penetration. We show in a receptor activity study that DA4-JC has balanced activity on both GLP-1 and GIP receptors but not on GLP-2 or Glucagon receptors. A dose-response study in the APP/PS1 mouse model of AD showed both a dose-dependent drug effect on the inflammation response and the reduction of amyloid plaques in the brain. When comparing DA4-JC with the GLP-1 analogue liraglutide at equal doses of 10nmol/kg bw ip. once-daily for 8 weeks, DA4-JC was more effective in reversing memory loss, enhancing synaptic plasticity (LTP) in the hippocampus, reducing amyloid plaques and lowering pro-inflammatory cytokine levels in the brain. The results suggest that DA4-JC may be a novel treatment for AD.

Objective: The aim of the systematic review was to analyze the current clinical evidence concerning the use of tissue engineering as a treatment strategy for reconstruction of segmental defects of the mandible and their clinical outcomes using individual patient data. Methods: A systematic review of the literature was conducted using PubMed and Cochrane Library on May 21, 2019. The eligibility criteria included patients in whom segmental mandibular reconstruction was carried out using tissue engineering as the primary treatment strategy. After screening and checking for eligibility, individual patient data were extracted to the extent it was available. Data extraction included the type of tissue engineering strategy, demographics, and indication for treatment, and outcomes included clinical and radiographic outcome measures, vitality of engineered bone, dental rehabilitation, and patient-reported outcome measures and complications. Results: Out of a total of 408 articles identified, 44 articles reporting on 285 patients were included, of which 179 patients fulfilled the inclusion criteria. The different tissue engineering treatment strategies could be broadly classified into 5 groups: "prefabrication," "cell culture," "bone morphogenetic protein (BMP) without autografts," "BMP with autografts," and "scaffolds containing autografts." Most included studies were case reports or case series. A wide variety of components were used as scaffolds, cells, and biological substances. There was not a single outcome measure that was both objective and consistently reported, although most studies reported successful outcome. Discussion: A wide variety of tissue engineering strategies were used for segmental mandibular reconstruction that could be classified into 5 groups. Due to the low number of treated patients, lack of standardized and consistent reporting outcomes, lack of comparative studies, and low evidence of reported literature, there is insufficient evidence to recommen

This project investigated the role of FGFR2 isoforms (FGFR2b/FGFR2c) and their cognate FGF ligands in endometrial cancer development, prognosis, and treatment response via designing and validating an innovative BaseScope RNA in-situ hybridization assay and generating patient tumour-derived organoids. FGFR2c and high FGF18 expression were significantly associated with aggressive tumour characteristics and poor survival outcome. It was also noted FGFR2c expression is associated with progestin treatment failure in atypical hyperplasia and well-differentiated endometrial cancers. Overall, FGFR2c and FGF18 are independent prognostic biomarkers that could improve our ability to predict patient prognosis and predict response to FGFR inhibitor treatment in endometrial cancer.

Die regenerative Medizin ist ein multidisziplinäres Forschungsfeld mit dem Ziel, beschädigtes Gewebe und dessen biologische Funktion wiederherzustellen oder vollständig zu ersetzen. Durch eine Kombination von Biomaterialien, Biomolekülen und Zellen werden biologisch inspirierte Umgebungen geschaffen sowie Zellfunktionen gezielt gesteuert. Der Einsatz von nanoporösen Silica-Nanopartikeln (NPSNPs) als Freisetzungssystem zur Verbesserung der derzeitigen Ansätze hat im Laufe der Jahre zunehmend Aufmerksamkeit erlangt. Mit ihnen als Plattform können maßgeschneiderte Freisetzungssysteme, die eine kontrollierte und ortsspezifische Freisetzung von Biomolekülen ermöglichen, für verschiedenste Anwendungen entwickelt werden. Den Schwerpunkt des ersten Teils dieser Arbeit stellt die Synthese zweier verschiedener Sorten NPSNPs sowie deren Oberflächenmodifizierung mit unterschiedlichen funktionellen Gruppen mittels der post-grafting-Methode dar. Die Variation der Oberflächeneigenschaften ermöglichte unterschiedliche Wechselwirkungen mit Biomolekülen, sodass die Immobilisierungsmenge und das Freisetzungsverhalten eingestellt werden konnten. Darauf aufbauend widmet sich der zweite Teil der Entwicklung eines intrazellulären Drug Delivery-Systems für short interfering-Ribonukleinsäuren (siRNAs) mittels der synthetisierten und modifizierten Silica-Nanopartikel. Mithilfe von siRNAs lassen sich einzelne Genexpressionen herunterregulieren, wodurch Zelleigenschaften gezielt beeinflusst werden können. Um eine effektive Aufnahme in Zellen zu gewährleisten, wurden die siRNA-beladenen Nanopartikel mit einer Lipidhülle versehen. Neben der Zytotoxizität der entwickelten Nanoträger wurde die Fähigkeit, die siRNAs innerhalb von Zellen wieder freizusetzen, über die Herunterregulierung eines Testproteins untersucht. Die in vitro-Experimente lieferten vielversprechende Ergebnisse und es konnte eine Herunterregulierung (Knockdown) bis zu 45 % verzeichnet werden. Darüber hinaus zeigte sich, dass die Z

This review article focuses on the current state-of-the-art cellular and molecular biotechnology for the over-production of clinically relevant therapeutic and anabolic growth factors. We discuss how the currently available tools and emerging technologies can be used for the regenerative treatment of osteoarthritis (OA). Transfected protein packaging cell lines such as GP-293 cells may be used as “cellular factories” for large-scale production of therapeutic proteins and pro-anabolic growth factors, particularly in the context of cartilage regeneration. However, when irradiated with gamma or x-rays, these cells lose their capacity for replication, which makes them safe for use as a live cell component of intra-articular injections. This innovation is already here, in the form of TissueGene-C, a new biological drug that consists of normal allogeneic primary chondrocytes combined with transduced GP2-293 cells that overexpress the growth factor transforming growth factor β1 (TGF-β1). TissueGene-C has revolutionized the concept of cell therapy, allowing drug companies to develop live cells as biological drug delivery systems for direct intra-articular injection of growth factors whose half-lives are in the order of minutes. Therefore, in this paper, we discuss the potential for new innovations in regenerative medicine for degenerative diseases of synovial joints using mammalian protein production platforms, specifically protein packaging cell lines, for over-producing growth factors for cartilage tissue regeneration and give recent examples. Mammalian protein production platforms that incorporate protein packaging eukaryotic cell lines are superior to prokaryotic bacterial expression systems and are likely to have a significant impact on the development of new humanized biological growth factor therapies for treating focal cartilage defects and more generally for the treatment of degenerative joint diseases such as OA, especially when injected directly into the, Simple summary Osteoarthritis (OA) is the most common form of arthritis across the world. Most of the existing drugs for OA treat the symptoms of pain and inflammation. There are no drugs that can dure the disease. There are a number of new treatments for OA including cell therapy and gene therapy. This articles outlines the concept behind TissueGene-C, a new biological drug for OA. This new treatment includes cartilage cells mixed with a genetically modified cell line called GP2-293, which is effectively a “drug factory”, over-producing the growth factors that are important for cartilage regeneration and changing the environment inside joints. The mixture is injected into the affected knee joint. These cells are designed to be short-lived and cannot reproduce. Therefore, after they have done their job, they die and are cleared by immune cells. This is a new and modern approach to treating OA and TissueGene-C is the prototype cell therapy for OA. In the future, it is entirely possible to combine different clones of genetically engineered cells like GP2-293 that have been designed to over-produce a growth factor or biological drug with cells from the cartilage endplate of the intervertebral disc to treat degeneration in the spine.

Background: Pubertal timing is known to be influenced by interactions among various genetic, nutritional, environmental and socio-economic factors, although the ultimate mechanisms underlying the increase in pulsatile GnRH secretion at puberty have yet to be fully elucidated. The aim of our research was to verify the role of KISSR1 (previously named GPR54) and MKRN3 genes on pubertal timing. Methods: We analyzed the DNA sequence of these genes in 13 girls affected by central precocious puberty (CPP) who showed onset of puberty before 8 years of age, and in 6 girls affected by early puberty (EP) between 8 and 10 years of age. Results: Direct sequencing of the KISS1R (GPR54) gene revealed two SNPs. One SNP is a missense variant (rs 350,132) that has been previously reported in connection to CPP in Korean girls. The other variant that we found in the GPR54 gene (rs764046557) was a missense SNP located in exon 5 at position 209 of the aminoacid. We identified this variant in only one CPP patient. Automatic sequencing of MKRN3 in all patients revealed three variants in eight subjects. In 6 out of 19 (31.5%) patients (3/13 CPP patients and 3/6 EP patients) we found the synonymous variant c.663C > T (rs2239669). Another synonymous variant (rs140467331) was found in one of our CPP patients, as well as one missense variant (rs760981395) in another CPP patient. Conclusion: In conclusion, we identified sequence variations of the KISS1R and MKRN3 genes, two of the most frequent genetic causes of ICPP. Our results suggest that these variants might be inducible factors in the pathogenesis of CPP.

A technique developed by Snider and Petters in 2008 is used to determine hygroscopic growth factors of aerosol particles. Simultaneous measurements from two optical particle-size spectrometers are used, where one measures the particles in the ambient atmosphere, and the other after drying them out. From 8–26 July 2019, aerosol data was collected west of Boca Chica Airfield, Florida. The Naval Postgraduate School’s Aviation Facility Twin Otter aircraft, equipped with a forward scattering spectrometer probe (FSSP), passive cavity aerosol spectrometer probe (PCASP), and an aerosol and precipitation spectrometer (CAPS) was used to measure dry and ambient aerosol particles. Comparisons of particle diameters at different ambient relative humidities (RH) were used to estimate the aerosol growth factors (GF). In general, the calculated GF increased as RH increased. GF values obtained varied wildly and ranged from 0.2 to 12.5 between 27 and 95% RH, but averaged into 5% RH ranges, they ranged from 2.0 to 5.5. Using wind direction as an indication of the source of the aerosol suggests some of the variability may be attributed to the aerosol composition., Lieutenant Commander, United States Navy, Approved for public release. distribution is unlimited

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have “performed the R&D”, developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly e ective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have “performed the R&D”, developing highly e ective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with effcacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics

Development of brain circuitry requires precise regulation and timing of proliferation and differentiation of neural progenitor cells. The p75 neurotrophin receptor (p75NTR) is highly expressed in the proliferating granule cell precursors (GCPs) during development of the cerebellum. In a previous paper, we showed that proNT3 promoted GCP cell cycle exit via p75NTR. Here we used genetically modified rats and mice of both sexes to show that p75NTR regulates the duration of the GCP cell cycle, requiring activation of RhoA. Rats and mice lacking p75NTR have dysregulated GCP proliferation, with deleterious effects on cerebellar circuit development and behavioral consequences persisting into adulthood. In the absence of p75NTR, the GCP cell cycle is accelerated, leading to delayed cell cycle exit, prolonged GCP proliferation, increased glutamatergic input to Purkinje cells, and a deficit in delay eyeblink conditioning, a cerebellum-dependent form of learning. These results demonstrate the necessity of appropriate developmental timing of the cell cycle for establishment of proper connectivity and associated behavior.SIGNIFICANCE STATEMENT The cerebellum has been shown to be involved in numerous behaviors in addition to its classic association with motor function. Cerebellar function is disrupted in a variety of psychiatric disorders, including those on the autism spectrum. Here we show that the p75 neurotrophin receptor, which is abundantly expressed in the proliferating cerebellar granule cell progenitors, regulates the cell cycle of these progenitors. In the absence of this receptor, the cell cycle is dysregulated, leading to excessive progenitor proliferation, which alters the balance of inputs to Purkinje cells, disrupting the circuitry and leading to functional deficits that persist into adulthood.

Tendon injuries significantly impact quality of life and are often career ending. Mesenchymal stem cell (MSC) therapy is known to augment intrinsic tendon healing, however, little is known of the stem cells endogenous to tendon, the microenvironmental cues that induce tendon differentiation, and whether individual cells in an inflammatory milieu respond differently to these cues. To address these questions, a three-dimensional tenogenesis assay was developed as an efficient and reproducible metric of cellular capacity to differentiate toward tendon. In contrast to more complex assays of tenogenesis, this design incorporates a simple apparatus using commercially available plasticware for the application of uniaxial static strain in in a type I collagen cell-seeded hydrogel construct. Tendon-related gene expression, glycosaminoglycan levels, elongated cell morphologies and parallel cell alignments are enhanced with BMP-12 induction over ten days of culture. This dissertation provides novel insight to the roles of growth factors in MSC tenogenesis. Tendon healing in vivo is dependent on endogenous tendon stem cells (TSC) that mediate the inflammatory response to injury and promote synthesis of collagen and matrix remodeling, among other extracellular processes. Recent evidence suggests that these cells exist on a spectrum of differentiation potencies, and may be differently committed to the tendon fate. Individual stem cells were isolated from the tendon, and their capacities for proliferation, tri-lineage differentiation and tenogenesis were evaluated. Three distinct TSC phenotypes were revealed, and significant, positive correlations were found in quadra-differentiation potency (toward four lineages) and the expression of a strong, composite tendon phenotype. These studies have important implications in the current standard-of-care in regenerative therapies for tendon. Our benchtop tenogenesis assay can be used to determine the therapeutic potential of allogeneic MSC

Growth factors have recently gained clinical importance for wound management. Application of recombinant growth factors has been shown to mimic cell migration, proliferation, and differentiation in vivo, allowing for external modulation of the healing process. Perioperative drug delivery systems can enhance the biological activity of these growth factors, which have a very short in vivo half-life after topical administration. Although the basic mechanisms of these growth factors are well understood, most have yet to demonstrate a significant impact in animal studies or small-sized clinical trials. In this review, we emphasized currently approved growth factor therapies, including a sustained release system for growth factors, emerging therapies, and future research possibilities combined with surgical procedures. Approaches seeking to understand wound healing at a systemic level are currently ongoing. However, further research and consideration in surgery will be needed to provide definitive confirmation of the efficacy of growth factor therapies for intractable wounds.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

Silk fibroin is a protein that has been shown to be a biomaterial with great potential in regenerative medicine, due to its biocompatibility characteristics and its wide possibility of structural modification can be used as scaffold, favoring growth processes, cell differentiation and the regeneration of affected tissue. Bombix mori L. silkworm cocoons were used to make fibroin films, the silk fibroin were degummed using 0.02M Na2CO3, the obtained fibroin was dissolved with 9.3M LiBr, which was eliminated by dialysis and finally the fibroin solution was concentrated to 17% by counterdialysis. The fibroin was served in polystyrene boxes, dried at 90°C/24 hours and sterilized with 70% ethanol. The mesenchymal stem cells were seeded on fibroin films and induced differentiation using a specific chondrogenic medium. Differentiation was assessed in triplicate at 14 and 21 days by total RNA extraction, DNA synthesis copy and PCR amplification of a group of cartilage-specific genes using specific primers. Stable and resistant fibroin films that allowed cell growth and multiplication were fabricated, as well as the chondrogenic differentiation evidenced by the expression of chondrogenic genes, the viability and the cell count were not affected, the cells interacted with the scaffolding evidenced by the area of upholstery formed on the surface of the fibroin film. Finally, it is concluded that silk fibroin is a biomaterial that can serve as a potential scaffold for the regeneration of joint injuries., La fibroína de seda es una proteína que ha demostrado ser un biomaterial con gran potencial en medicina regenerativa, por sus características de biocompatibilidad y su amplia posibilidad de modificación estructural permite ser usada como andamio favoreciendo procesos de crecimiento, diferenciación celular y la regeneración del tejido afectado. En este estudio se utilizaron capullos de gusano de seda Bombyx mori L., para la fabricación de películas de fibroína, los capullos fueron desgomados utilizando Na2CO3 0,02M, la fibroína obtenida se disolvió con LiBr 9,3M, el cual fue eliminado mediante diálisis y finalmente la solución de fibroína fue concentrada mediante contradiálisis. La fibroína fue servida en cajas de poliestireno, secadas a 90°C/24 horas y esterilizadas con etanol al 70%. Células madre mesenquimales fueron sembradas sobre estas películas de fibroína e inducidas a diferenciación utilizando un medio condrogénico especifico. La diferenciación fue evaluada por triplicado a los 14 y 21 días mediante extracción de ARN total, síntesis de ADN copia y amplificación por PCR de un grupo de genes específicos de cartílago empleando cebadores específicos. Se fabricaron películas de fibroína estables y resistentes que permitieron el crecimiento y la multiplicación celular, así como la diferenciación condrogénica evidenciada por la expresión de genes condrogenicos, no se afectó la viabilidad ni el recuento celular, las células interactuaron con el andamio evidenciado por el área de tapizado formado sobre la superficie de la película de fibroína. Finalmente se concluye que la fibroína de seda es un biomaterial que puede servir de andamio potencial para la regeneración de lesiones articulares.

Silk fibroin is a protein that has been shown to be a biomaterial with great potential in regenerative medicine, due to its biocompatibility characteristics and its wide possibility of structural modification can be used as scaffold, favoring growth processes, cell differentiation and the regeneration of affected tissue. Bombix mori L. silkworm cocoons were used to make fibroin films, the silk fibroin were degummed using 0.02M Na2CO3, the obtained fibroin was dissolved with 9.3M LiBr, which was eliminated by dialysis and finally the fibroin solution was concentrated to 17% by counterdialysis. The fibroin was served in polystyrene boxes, dried at 90°C/24 hours and sterilized with 70% ethanol. The mesenchymal stem cells were seeded on fibroin films and induced differentiation using a specific chondrogenic medium. Differentiation was assessed in triplicate at 14 and 21 days by total RNA extraction, DNA synthesis copy and PCR amplification of a group of cartilage-specific genes using specific primers. Stable and resistant fibroin films that allowed cell growth and multiplication were fabricated, as well as the chondrogenic differentiation evidenced by the expression of chondrogenic genes, the viability and the cell count were not affected, the cells interacted with the scaffolding evidenced by the area of upholstery formed on the surface of the fibroin film. Finally, it is concluded that silk fibroin is a biomaterial that can serve as a potential scaffold for the regeneration of joint injuries., La fibroína de seda es una proteína que ha demostrado ser un biomaterial con gran potencial en medicina regenerativa, por sus características de biocompatibilidad y su amplia posibilidad de modificación estructural permite ser usada como andamio favoreciendo procesos de crecimiento, diferenciación celular y la regeneración del tejido afectado. En este estudio se utilizaron capullos de gusano de seda Bombyx mori L., para la fabricación de películas de fibroína, los capullos fueron desgomados utilizando Na2CO3 0,02M, la fibroína obtenida se disolvió con LiBr 9,3M, el cual fue eliminado mediante diálisis y finalmente la solución de fibroína fue concentrada mediante contradiálisis. La fibroína fue servida en cajas de poliestireno, secadas a 90°C/24 horas y esterilizadas con etanol al 70%. Células madre mesenquimales fueron sembradas sobre estas películas de fibroína e inducidas a diferenciación utilizando un medio condrogénico especifico. La diferenciación fue evaluada por triplicado a los 14 y 21 días mediante extracción de ARN total, síntesis de ADN copia y amplificación por PCR de un grupo de genes específicos de cartílago empleando cebadores específicos. Se fabricaron películas de fibroína estables y resistentes que permitieron el crecimiento y la multiplicación celular, así como la diferenciación condrogénica evidenciada por la expresión de genes condrogenicos, no se afectó la viabilidad ni el recuento celular, las células interactuaron con el andamio evidenciado por el área de tapizado formado sobre la superficie de la película de fibroína. Finalmente se concluye que la fibroína de seda es un biomaterial que puede servir de andamio potencial para la regeneración de lesiones articulares.

Silk fibroin is a protein that has been shown to be a biomaterial with great potential in regenerative medicine, due to its biocompatibility characteristics and its wide possibility of structural modification can be used as scaffold, favoring growth processes, cell differentiation and the regeneration of affected tissue. Bombix mori L. silkworm cocoons were used to make fibroin films, the silk fibroin were degummed using 0.02M Na2CO3, the obtained fibroin was dissolved with 9.3M LiBr, which was eliminated by dialysis and finally the fibroin solution was concentrated to 17% by counterdialysis. The fibroin was served in polystyrene boxes, dried at 90°C/24 hours and sterilized with 70% ethanol. The mesenchymal stem cells were seeded on fibroin films and induced differentiation using a specific chondrogenic medium. Differentiation was assessed in triplicate at 14 and 21 days by total RNA extraction, DNA synthesis copy and PCR amplification of a group of cartilage-specific genes using specific primers. Stable and resistant fibroin films that allowed cell growth and multiplication were fabricated, as well as the chondrogenic differentiation evidenced by the expression of chondrogenic genes, the viability and the cell count were not affected, the cells interacted with the scaffolding evidenced by the area of upholstery formed on the surface of the fibroin film. Finally, it is concluded that silk fibroin is a biomaterial that can serve as a potential scaffold for the regeneration of joint injuries., La fibroína de seda es una proteína que ha demostrado ser un biomaterial con gran potencial en medicina regenerativa, por sus características de biocompatibilidad y su amplia posibilidad de modificación estructural permite ser usada como andamio favoreciendo procesos de crecimiento, diferenciación celular y la regeneración del tejido afectado. En este estudio se utilizaron capullos de gusano de seda Bombyx mori L., para la fabricación de películas de fibroína, los capullos fueron desgomados utilizando Na2CO3 0,02M, la fibroína obtenida se disolvió con LiBr 9,3M, el cual fue eliminado mediante diálisis y finalmente la solución de fibroína fue concentrada mediante contradiálisis. La fibroína fue servida en cajas de poliestireno, secadas a 90°C/24 horas y esterilizadas con etanol al 70%. Células madre mesenquimales fueron sembradas sobre estas películas de fibroína e inducidas a diferenciación utilizando un medio condrogénico especifico. La diferenciación fue evaluada por triplicado a los 14 y 21 días mediante extracción de ARN total, síntesis de ADN copia y amplificación por PCR de un grupo de genes específicos de cartílago empleando cebadores específicos. Se fabricaron películas de fibroína estables y resistentes que permitieron el crecimiento y la multiplicación celular, así como la diferenciación condrogénica evidenciada por la expresión de genes condrogenicos, no se afectó la viabilidad ni el recuento celular, las células interactuaron con el andamio evidenciado por el área de tapizado formado sobre la superficie de la película de fibroína. Finalmente se concluye que la fibroína de seda es un biomaterial que puede servir de andamio potencial para la regeneración de lesiones articulares.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

This clinical trial investigates the efficacy of PRGF-Endoret® injections for the treatment of temporomandibular joint osteoarthritis by providing patient evaluations at a one-year follow-up. Fifty-two patients with a diagnosis of osteoarthritis according to the American Society of Temporomandibular Joint Surgeons' guidelines underwent a cycle of 3 injections (1 per month) of platelet-derived growth factors (PRFG). Two clinical parameters, pain at rest and maximum non-assisted mouth opening, were assessed by the same blinded operator at the time of the diagnosis (baseline), at each appointment during the treatment and at a 1-year follow-up visit. Both the parameters showed improvements that were maintained over time. Statistical analyses evidenced significant changes within the first two injections. Data from the present investigation support findings from studies on other joints, which show the efficacy of PRGF-Endoret injections to reduce symptoms of osteoarthritis and to maintain improvements over time.