LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora

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http://hdl.handle.net/11527/19402

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Konu "cell" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora'a göz atma
  • Öge
    Biointerfacial cell/protein–polymer interactions investigated by quartz crystal microbalance with dissipation
    (Graduate School, 2023-08-01) Sert Özdabak, Ayşe Buse ; Kılıç, Abdülhalim ; 521162101 ; Molecular Biology-Genetics and Biotechnology
    Understanding cell-surface interactions is required for the development of novel and functional biomaterials. The biocompatibility and in vivo performance of these biomaterials heavily depend on processes such as protein adsorption and subsequent cell adhesion on the material surface. Common experimental approaches used to assess these processes typically involve end-point assays. However, these assays often require cell fixation or disruption and pre-or post-labeling of the cells, potentially affecting cell physiology and leading to the loss of valuable information. Furthermore, these methods cannot distinguish interfacial interactions occurring at nanometer scales between cells and the surface. The physicochemical properties of the material surface also significantly impact the performance of potential biomaterials. The interactions taking place at the interface between cells/proteins and materials are intricate and must be comprehensively understood and carefully designed to meet specific application requirements. In this context, Quartz Crystal Microbalance with Dissipation (QCM-D) emerges as an alternative and complementary method. QCM-D serves as a powerful, noninvasive technique that enables real-time and label-free monitoring of cell-surface interactions at the nanoscale. QCM-D provides distinct data regarding specific interactions at the cell - material interface, thereby offering new insights into the cell adhesion / protein adsorption behaviors. The aim of the thesis is to investigate cell-polymer interactions and to monitor the entire process in real-time using QCM-D system. For this purpose, two commonly employed polymers in the biomaterials field, Polycaprolactone (PCL) and Chitosan (CH), as well as their blends (75:25 and 25:75), were employed to investigate real-time cell adhesion behavior. As surface topography, chemical composition and wettability have significantly influence on cell adhesion process, it is important to analyze cell adhesion on well-characterized surfaces. Two types of cell lines (hFOB and 3T3) were employed to monitor cell interactions. Complementary cell culture assays were also conducted to validate the outcomes obtained from QCM-D. In the first part of the thesis, the preparation and characterization of thin films on silicon substrates and silica sensor surfaces were completed. In order to achieve homogeneous films, various parameters were investigated, i.e., polymer ratio, solvent type, substrate surface characteristics (activated with oxygen plasma or hydrophobic treatment), polymer molecular weight, and polymer ratio. The homogeneity of the films was assessed using Atomic Force Microscopy (AFM). It was founded that blends prepared with a constant amount of chitosan yielded homogeneous coatings on the silicon substrate. The chemical composition of the constructed surface was further analyzed using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR). The spectra exhibited distinct peaks at 1725 cm-1 for PCL and 1645 cm-1 and 1584 cm-1 for chitosan, confirming the successful coating of both polymers onto the surface. Analysis of AFM images over a scanned area of 100 µm2 revealed that pure polymers produced morphologically homogeneous films, whereas the blend surfaces displayed visible domains. Particularly, 75:25 PCL/CH blend exhibited micrometer-scale domains. Comparatively, thin films prepared with pure polymers displayed smoother surfaces compared to the blends. In contrast, the blends prepared with a ratio of 75:25 exhibited the highest roughness value. In terms of zeta potential, pure PCL films exhibited the highest negative value (-88 mV), whereas pure chitosan films displayed the lowest negative value (-15 mV) at pH 7.4. Blend film zeta potential values were between these two extremes. Film thickness analysis revealed that pure chitosan films had the smallest thickness (ca. 10 nm), whereas 75:25 PCL/CH blend film had the greatest thickness (ca. 55 nm). The pure PCL film and the 25:75 blend film had thicknesses of approximately 38 nm and 14 nm, respectively. In addition, in situ spectroscopic ellipsometry was employed to determine swollen polymer thicknesses. It was observed that PCL, being a hydrophobic polymer, did not swell much in aqueous solutions. Chitosan thin films exhibited the highest degree of swelling. The blend films exhibited swelling degrees between those of the pure polymer films, while higher PCL amount (75:25) resulted in reduced swelling, as expected. Before the cell adhesion studies, protein adsorption studies onto the constructed films was conducted. Bovine Serum Albumin (BSA) adsorption was monitored in real-time using both QCM-D and spectroscopic ellipsometry at various pH values at room temperature. In the case of pure PCL film, BSA adsorption onto pure PCL film showed a consistent frequency change upon adsorption with QCM-D for all pH values investigated. However, for the other investigated films, the presence of chitosan led to pH dependent adsorption behavior. At pH 4.5, both BSA and CH were positively charged, resulting in adsorption under repulsive conditions. At pH 6.0, the electrostatic attraction between the polymer chains and BSA led to higher adsorption on films containing chitosan. The lowest frequency decrease, i.e., mass load, was observed at pH 7.4 compared to pH 4.5 and 6.0. These findings indicate that blend composition, pH and ion presence in the environment have a substantial influence on protein adsorption. To compare the adsorbed protein amounts determined by QCM-D and ellipsometry methods, diverse models were applied. When two methods are assessed, the protein quantity derived from QCM-D data was consistently higher than that obtained by ellipsometry. The amount of protein calculated from ellipsometric data was similar for the blend films and pure chitosan films for all pH values investigated. However, higher values were evident in QCM-D method due to the inclusion of coupled water in the calculations. In addition, fibrinogen adsorption presented composition dependent behavior on thin films. The highest adsorbed fibrinogen amount was monitored on pure PCL films. In contrast, no significant protein adsorption was monitored on pure chitosan films. Consequently, the adsorbed amount of fibrinogen decreased with an increasing percentage of chitosan in the films, which predominantly showed an inverse correlation with the surface hydrophilicity. Following the comprehensive characterization of the films and conduction of the protein adsorption experiments, the cell adhesion behavior of two cell lines, human fetal osteoblastic (hFOB) and mouse fibroblast (NIH/3T3), was monitored on constructed films using QCM-D. For this purpose, the cells were introduced into the QCM-D chamber and allowed to flow for 1 hour. Initial cell sedimentation after 1 h resulted in reduced cell deposition as the chitosan ratio increased in the film. This trend was consistent for the both cell lines in the first hour. Subsequently, changes in frequency and dissipation were monitored over an 18-hour period. Complementary cell culture assays were performed to validate the observations of QCM-D. For this purpose, fluorescence images and live cell images at various time intervals were captured. Distinct QCM-D signal patterns were found for the investigated cell lines, indicating the influence of the varying interfacial properties on cell adhesion, which is also dependent on the specific cell type. In the case of hFOB cells, fully spreading was observed on pure PCL films, with elongated morphologies as confirmed by fluorescence microscopy and scanning electron microscopy (SEM). Corresponding QCM-D signals showed the highest frequency drop and the highest dissipation. Blend films supported hFOB cell adhesion, but with lower dissipation values compared to the PCL film. This might be attributed to higher rigidity at the hFOB cell−blend interface, because these cells did not progress to the further stages of spreading after secretion of their extracellular matrix (ECM) proteins. Variations in the QCM-D data obtained from the blend films could be attributed to differences in the morphology of the films. Pure chitosan films showed limited hFOB cell adhesion, accompanied by low frequency drop and low dissipation. The initial sedimentation of 3T3 cells onto the constructed surfaces similarly showed dependence on the surface composition. Unlike the behavior of hFOB cells, 3T3 cell lines did not adhere to pure chitosan surfaces, evident from consistent positive frequency signals. The highest frequency change was observed on reference silica surface, with dissipation gradually decreasing. This behavior indicated an average number of cells remaining in ECM remodeling stage. The ΔD signal shape was similar for 75:25 PCL/CH blend to the reference silica surface, however a slight decrease in the frequency was observed after 10 h. This suggests the stronger attachment to the surface while cells lacked further spreading stages on 75:25 PCL/CH blend. 3T3 cells on 25:75 PCL/CH blend showed substantial frequency drop after 10 h, which accompanied by an increase in dissipation. This behavior corresponded to the later stages of cell adhesion, implying that cells probably underwent actin remodeling and fully spreading on the surface. In conclusion, distinct QCM-D signal patterns were evident in the adhesion of hFOB and 3T3 cell lines. These distinctive patterns were attributed to the variations in the strength of cell adhesion, which are influenced by both cell type and surface chemical properties. The real-time and label-free data collected through QCM-D gave us a more profound comprehension of the dynamic adhesion behavior of the cells on constructed thin films. This knowledge and understanding holds the potential to provide valuable insights for the design of novel biomaterials tailored to diverse applications.
  • Öge
    Investigation of NFİB function and regulation of its putative target genes in human neural stem cell and SH-SY5Y neuroblastoma cell lines
    (Graduate School, 2023-03-13) Uluca, Betül ; Kumbasar, Aslı ; 521132101 ; Molecular Biology-Genetics and Biotechnology
    The central nervous system comprises numerous neuronal and glial subpopulations that have unique identities. Molecular mechanisms that underlie the generation of this cellular diversity have been under investigation. During development, the formation of cell subclasses with particular features is determined by tissue-specific transcription factors (TF). TFs sequence-specifically bind to DNA, interact with other proteins, and affect the expression of target genes. One of the key TFs in the developing brain is the Nuclear Factor I (NFI) family. There are four members (A, B, C, and X) in vertebrates. NFI proteins comprise a highly conserved N-terminal DNA binding and dimerization domain and bind to a TTGGC(N5)GCCAA consensus sequence as homo or heterodimers. However, they have a less conserved C-terminal transcription modulation domain which may lead to differential transcriptional regulation of target genes. In the developing mouse, Nfia, Nfib, and Nfix are expressed in an overlapping but distinct expression pattern in different regions of the embryonic brain, while their expression is restricted to stem cell niches in the adult. In the central nervous system, deletion of each member leads to delayed glial and neuronal differentiation, aberrant cell migration and increased proliferation. In the developing hindbrain, only the absence of Nfib leads to delayed development of several precerebellar nuclei, indicating that Nfib may play a unique role in this system. Dysregulated expression of NFIs have also been linked to tumor growth and progression, however, with opposing effects. For example, NFIB is oncogenic and promotes metastasis in colorectal cancer, melanoma, gastric cancer, estrogen receptor (ER)-negative breast cancer, and small cell lung cancer; while it has a tumorsuppressive function in non-small cell lung cancer, glioblastoma, osteosarcoma, and cutaneous cell carcinoma. NFIs perform their context dependent, cell-type and tissue specific functions, via regulation of specific set of downstream transcriptional targets. Despite the fact that NFI binding motifs have been found in the promoter and upstream enhancer regions of many genes, only a few of them have been so far investigated as direct NFI targets. Further identification and characterization of downstream targets of NFIs in various tissues will help elucidate molecular mechanisms that regulate embryonic development and related diseases, as well as cancer pathologies. In an attempt to investigate how NFIB regulates neurogenesis in developing precebellar nuclei, differentially expressed genes in E14 Nfib knock-out mouse precerebellar neuroepithelium have been analyzed. The RNA profiling analysis revealed putative candidates for further research. Of these putative NFIB targets, we selected Cdon (Cell adhesion molecule related, down regulated by oncogenes) and Fgf15 (Fibroblast growth factor 15), since these genes have been implicated in neural development of the cortex. We examined NFIB-mediated transcriptional regulation mechanisms of CDON and FGF19, the human ortholog of mouse Fgf15, in human neural stem cells (hNSCs derived from H9 ESC, Gibco). Neural stem cell culture systems provide an in vitro model of human neural development. Since NFIs have been reported to regulate neuron production in diverse parts of the developing brain, they may have comparable functions in vitro. Understanding these processes and the underlying molecular mechanisms in vitro will also help understand how the brain develops in vivo as well as failures in this process. Thus, in the first chapter of the thesis, we set out to examine NFI function and regulation mechanisms of potential NFIB targets, CDON and FGF19, in neuronal differentiation of hNSCs in vitro. RT-qPCR analyses revealed that mRNA expression of NFIB, NFIC, and NFIX is downregulated, whereas NFIA is upregulated in differentiating hNSCs. Since NFIA levels are quite low in these cells, overall NFI expression levels decrease during neuronal differentiation in hNSCs. These cells express NFIB at much higher levels compared to the other NFI members. Therefore, this study focuses on NFIB's role in hNSCs. We analyzed cell proliferation and differentiation by BrdU incorporation assays and immunofluorescence staining of neural stem and neuronal marker proteins. However, NFIB overexpression or knockdown did not affect the proliferation or neuronal differentiation potential of hNSCs. Nevertheless, these data cannot preclude NFIB's potential role in differentiation and/or self-renewal of hNSCs since NFIB could be silenced only by 30–50% in these cells and analyses were performed in whole cell populations that might mask possible changes induced by NFIB loss. Moreover, in NFIB overexpression experiments, we may need other proteins acting as cofactors that are not supplied along with NFIB. This study identifies FGF19 as a novel downstream target of NFIB in hNSCs. Human FGF19 is preferentially expressed in the fetal brain, among other tissues. Recombinant human FGF19 treatment has been shown to enhance neuronal differentiation in mouse neuroepithelial and cortical cells. In accordance with these data, FGF19 expression increases in differentiating hNSCs. Moreover, FGF19 expression increases in NFIB silenced hNSCs while it is reduced in NFIB overexpressing cells, indicating that NFIB regulates FGF19 transcription in hNSCs. Indeed, NFIs directly repress FGF19 promoter-driven luciferase activity, confirming that NFIs transcriptionally target FGF19. Moreover, chromatin immunoprecipitation (ChIP) assays showed that NFI proteins occupy −777 (relative to the transcription start site) in hNSCs, indicating NFI interaction with the FGF19 promoter in vivo. Since NFIB expression decreases upon neuronal differentiation, while FGF19 increases and NFIB directly represses FGF19 in hNSCs, future studies are required to address functional relevance of NFIBmediated FGF19 repression in the control of self-renewal and neural differentiation of these cells. In the absence of NFI, Cdon, a cell surface glycoprotein of the immunoglobulin (Ig) superfamily, is upregulated in the developing mouse brain. CDON is expressed in various tissues, primarily in the brain, muscle, and endocrine tissues during human and murine embryogenesis. Moreover, CDON is implicated in proliferation and differentiation control as it promotes myogenesis and neurogenesis in vitro and is essential for proper brain and skeletal-muscle development. However, in this study, CDON expression decreased in differentiating hNSCs and it did not change in NFIB overexpressed or silenced hNSCs, analyzed by RT-qPCR. These data indicate that CDON is not an NFIB target in this system. Recently, CDON has been described as a dependence receptor that induces apoptosis in the absence of its ligand SHH. During cancer progression, in an environment with limited SHH, tumorigenic tissue may downregulate CDON to eliminate its apoptotic activity. Indeed, CDON expression decreases in colon, lung, and neuroblastoma tumors, implicating a tumor suppressor role for CDON. As NFIs are involved in progression of various cancers, we examined whether NFIs regulate CDON transcription in SH-SY5Y human neuroblastoma cells. ChIP assays showed that NFIs bind to human CDON gene regulatory regions, -8 and -941 (relative to the transcription start site), in hNSCs and SH-SY5Y cells. Moreover, NFIs repress CDON promoter-driven luciferase expression via interacting with those NFI sites. Finally, CDON is upregulated in NFIB silenced SH-SY5Y cells, suggesting that the NFIB-CDON axis may be involved in neuroblastoma biology. On the other hand, silencing NFIB in SH-SY5Y cells decreases cell viability and proliferation, suggesting an oncogenic role for NFIB in neuroblastoma. Next, we tested the hypothesis that NFIB may affect SH-SY5Y cell survival by suppressing expression and thereby, pro-apoptotic activity of CDON. However, downregulation of CDON on its own could not rescue the phenotype induced by NFIB silencing, most likely because other NFIB downstream targets, which may include p21, are also involved. Further studies are required to investigate the functional consequences of NFIB mediated CDON repression in other developmental systems and disease models. NFIB's oncogenic effects in SH-SY5Y cells may involve inhibition of apoptosis and/or regulation of cell cycle components. Moreover, NFIB might promote differentiation of SH-SY5Y cells and/or contribute to the aggressive state of neuroblastoma tumorigenesis. However, these and underlying mechanisms need to be further investigated.