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Determination of platelet activation and antibody binding capacities of platelet surface receptors in patients with menstrual cycle and malnutrition based iron deficiency anemia
IDA occurs when serum iron concentration decreases under a certain threshold and hemoglobin production is disrupted. Although there are several reasons for IDA to occur such as blood loss by disorders that cause chronic gastrointestinal bleedings, IDA has a high prevalence in women in developing countries due to heavy menstrual cycle and deficiency in iron uptake. In recent years, there are a few studies trying to determine the effects of IDA on platelets but there are conflicting reports whether IDA can affect the activation of resting platelets or not. In our study, platelet activations and ABCs of platelet surface receptors were examined with flow cytometry to determine the effects of IDA on platelet functions. 35 women patients with newly diagnosed IDA (16-50 years old) and 18 healthy women without IDA in same age group were included into study. We have found out that despite the increase in activated platelet population, ABC of CD41/CD61 fibrinogen binding complex was decreased in IDA patients. Also ABC of platelet activation markers was not changed in IDA patients in spite of platelet activation. This shows us that there may be conformational changes in fibrinogen binding to CD41/CD61 complex and surface activation molecules in IDA patients.
Bone tissue formation on modified polybutylene succinate scaffolds with porcine dental germ stem cells
BONE TISSUE FORMATION ON MODIFIED POLYBUTYLENE SUCCINATE SCAFFOLDS WITH PORCINE DENTAL GERM STEM CELLS Designing and providing a scaffold are very important for the cells in Tissue Engineering. In this area, there are three important parameters that are cells, scaffolds, and chemical signals. These are also known as the basic building blocks of tissue engineering. These can lead to the repair or formation of tissue. Scaffolds have the suitable substrate for cell attachment, cell proliferation and differentiation function. They can also stimulate cellular response, possess mechanical strength and dimensional stability. Besides, scaffold should be biocompatible and biodegradable and have porous interconnected network to facilitate vascularization and growth of newly formed tissue. Surface modification is necesssary for modifying biomaterials to elicit surface properties by the combination of physics and chemistry working together. One of the suitable scaffolds for bone tissue engineering is poly(butylene succinate) (PBS) that is a family of biodegradable synthetic plastics with excellent biodegradability, non-toxic degradation products, thermoplastic processability and balanced mechanical properties. Dental germ stem cells are preferable cell source for the tissue regeneration. Dental germs contain mesenchymal stem cells (MSCs) which can be easily isolated, expanded and cryo-preserved. These cells are able to differentiate into osteo-, adipo- and neuro-genic cells. In this study, Porcine Dental Germ Stem Cells (pDGSCs) seeded polybutylene succinate (PBS) scaffolds were used to investigate the effect of surface modification on these foams in tissue engineering applications. The surface of the PBS scaffolds was modified with either fibronectin or laminin. The osteogenic potentials of pDGSCs on these modified and unmodified foams were examined by using osteogenic differentiation medium to heal bone defects. For this study, MTS cell proliferation assay was used to learn about the cytotoxic effects of modified and unmodified surfaces. For the characterization of pDGSCs, flow cytometry analysis was carried out. Besides, alkaline phosphatase (ALP) test and von Kossa staining were applied to analyze osteogenic potentials of pDGSCs. Confocal microscopy and CM-Dil staining studies were carried out to support MTS, ALP and von Kossa tests. The results of these studies demonstrated that pDGSCs were differentiated into osteogenic cells on fibronectin modified PBS foams better than on unmodified and laminin modified PBS foams.
Origanum minutiflorum as a potential therapy for the eradication of Helicobacter Pylori
Helicobacter pylori is a gram negative, spiral shaped, microaerophilic bacterium which colonizes the human stomach of more than half of the world?s population. H. pylori infection plays a crucial role in the pathogenesis of peptic ulcer, gastritis, and gastric cancer. Infection of H. pylori treatment consists of a proton pump inhibitor (PPI) with a combination of antibiotics. This study aims to investigate the potential use of commercial extracts of Siberian ginseng (Eleutherococcus senticosus), Black cohosh (Cimicifuga racemosa), Red clover (Trifolium pratense), Milk thistle (Silybum marianum) and various extracts (methanol, ethanol, aqueous, essential oil) of a Turkish endemic plant Origanum minutiflorum for the eradication of H. pylori. Methanol, ethanol, aqueous and essential oil (EO) extracts of O. minutiflorum were collected via soxhlet extractor and clevenger apparatus. The anti-bacterial activity (agar well diffusion method) and the minimum bactericidal concentration (MBC) of all extracts were determined according to CLSI protocols. The chemical composition of the extract showing the strongest antimicrobial activity was determined via gas chromotography mass spectrom (GC-MS). The cell viability effects of the chosen extract were determined on AGS (human adenocarcinoma) cell line by WST-1 assay for 24, 48, and 72 hours. H. pylori adhesion and invasion of cultured cells was carried out using a standard gentamicin assay at 1/20,000- 1/40,000 (v/v) dilutions of O. minutiflorum EO. Examination of effect of O. minutiflorum EO on H. pylori and H. pylori infected cells were done by scanning electron microscopy (SEM). The results of in vitro studies indicated that the EO extract of O. minutiflorum (92 µg/ml) displayed the strongest antimicrobial effect against H. pylori (ZI= ?90) in comparison to all other plant extracts. The major compound of the EO was determined as carvacrol (29.22 %). The MBC was observed at a concentration of 46 µg/ml (equal to 1/20,000 (v/v) dilution) EO. The MBC value of EO did not show any cytotoxic effect against AGS cells and exhibited a reduction of more than 80 % H. pylori adhered or invaded AGS cells. The findings of this study show the potential future use of O. minutiflorum EO as an alternative therapy to antibiotics for treatment of H. pylori infections.
The influence of patterned magnetic beads on the self-assembly of magnetic nanoparticles
The assembly of nanostructures has critical importance for construction of higher structures to utilize the unique properties of nanoparticles (NPs). The future applications of nanotechnology mostly depend on directing the NPs on desired location or orientation in the proposed structure. Therefore, there is a need to understand the forces governing their self-assembly and approaches to control them. The magnetic nanoparticles (MNPs) also show unique properties and can be manipulated under the influence of a magnetic field. However, there is limited information on how to control their behavior in a well-controlled manner. In this study, the influence of the presence of magnetic layer of particles and their template structure on the controlled assembly of MNPs is investigated. Several unpatterned and patterned, hydrophobic and hydrophilic surfaces are prepared to assemble the magnetic beads (MBs) on these surfaces to investigate their influences on the assembly of MNPs that are composed of iron oxide (Fe3O4). Different surfaces with different patterns are successfully used and tested. It is found that the presence of a patterned surface between a magnetic field and the substrate that the MNPs are assembled on has an influence on the finally formed structure. It is also found that the assembly of the MNPs on surfaces is influenced by the solvent that the MNPs are dispersed in. This work may offer a new and simple approach for preparation of patterned and renewable surfaces, constructed from MNPs, which find use in many fields of science and technology.
Characterization of cancer stem cells in chordoma
Chordoma is the most malignant type of bone tumors. Studies had revealed thatchordoma was originated from the remnants of notochord. It is a very rare type of tumorwith little tendency to metastasize to nearby tissues, however recurrence rate is quiet high.Although it is a very slow growing tumor, death age is around sixty and survival isbetween 5 to 10 years. Due to the rare cases of chordoma, studies performed are verylimited and especially difficult in determining molecular markers in the diagnosis ofchordoma. Since chordoma has got similar characteristics such as self-renewal, andpluripotency, with those of stem cells, they might contain a subgroup of cells called cancerstem cells. In order to detect the presence of cancer stem cells in chordoma, severalmethods can be used such as differentiation therapy, scratch, and proliferation assays.Differentiation therapy is based on arresting cells at an immature state anddifferentiating them into a more committed stage and eventually leading to death. Asdifferentiating agents, retinoic acid and osteogenic medium are used in this study to detectthe variation in the expression of genes, which are known to play important roles inmaintenance and development of cancer stem cells. Addition to this therapy, scratch assayand proliferation assays were performed in order to see the effects of differentiation on themigration and the proliferation of cells, respectively.Using the above methods, an alteration in the expression of genes involved in cancerstem cell has been found. For instance, differentiation by retinoic acid and osteogenicmedium had caused a slower migration and proliferation rate when compared to those ofuntreated ones. As a result, the detection of cancer stem cells in chordoma might become avery useful tool in targeting this tumor and providing an alternative treatment method tochordoma.
Expression profiling of microRNAs and Hsa-miR-145 targets in human prostate cancer
Prostate cancer is one of the most significant cancers of the men and there is a need to identify novel therapeutic approaches in this disease. MicroRNAs are small, approximately 18?24 nucleotides, non-coding, endogenously synthesized new class of small RNAs as regulators of gene expression post-transcriptionally. Alteration in microRNA expression may play a critical role in pathogenesis of the disease and its gain of androgen independency. Recurrence is one of the main problems in prostate cancer therapy due to lack of understanding the mechanism and insufficient treatment options. Global microRNA expression studies revealed the key regulatory microRNAs in prostate cancer. It was shown that human prostate cancer possess a widespread deregulation of microRNA expression and hsa-miR-145 is down regulated in prostate tumors as compared to normal prostatic tissues. Hsa-miR-145 is also down-regulated in prostate cancer cell lines including PC3, DU145, LNCaP and LAPC4 but has relatively higher expression in normal prostatic epithelial cells. The microRNA expression profiling studies between recurrent and non-recurrent prostate cancer samples suggest that there is not a widespread change but expression of some microRNAs is significantly altered.
Assembly of peptide coated gold nanoparticles
The assembly of nanoparticles (NPs) into the desired organizations and patterns is critically important for construction of higher structures using nanoparticles as building blocks. The future of nanotechnology mostly depends on the directing the NPs to desired locations and orientations in the construction of predetermined structures. Peptide modification of NPs has been increasingly used for this purpose. In this regard, we designed different sized and charged peptides that can stably bind to gold nanoparticles (AuNPs). Then, we have investigated how the peptide binding to the nanoparticle surface can influence the assembly of the AuNPs in suspension and on hydrophilic surfaces from a drying droplet of their colloidal suspension. We first studied the effect of anionic peptide modification on assembly of the AuNPs in suspension and on surfaces and then we studied the effect of cationic peptide modification. We found that the peptide charge along with the size has a dominating effect on the behavior of the AuNP in suspension and on surfaces. The knowledge acquired from this work may offer simple and new techniques in assembly of nanostructures.
Identification of upstream regulators of MAPK stimulated Pea3-mediated neural differentiation in PC12 cells
Pea3 transcription factor family belongs to ETS domain transcription factor super-family and act as nuclear targets of signal-transduction pathways, including cell proliferation, differentiation, development and apoptosis like the other members of ETS super-family. A wide variety of the regulatory events that involve ETS domain proteins require MAPK pathways in order to mediate the phosphorylation processes. In a previous study performed in our laboratory, it was observed that over-expression of Pea3 protein induces neuronal differentiation in PC12 cells via MAPK cascade when induced with growth factors which alone induce proliferation in the absence of Pea3. Although the phosphorylation sites for other members of Pea3 transcription family were identified and there were a few studies that investigate the post-translational modification of Pea3 protein, phosphorylation is still unknown. In this thesis project, first approach to study the upstream regulators of Pea3 mediated neuronal differentiation, is blocking suggested MAPK phosphorylation sites on Pea3, by converting suspected Serine residues into Alanine residues. MAP kinases are proline-directed Serine/Threonine kinases and phosphorylate the serine or threonine in the dipeptide S/T-P motif. Therefore, conversion of Serine to Alanine will silence phosphorylation, whereas converting Serine to Glutamate will mimic it. Comparison of the signals obtained from a mutated phosphorylation site with the wild-type will be used to identify the phosphorylation sites on Pea3. Second approach involves deletion of suggested MAPK docking sites on Pea3. MAPKs are known to interact with their targets and regulators via high affinity docking sites in order to obtain specific and efficient selectivity. To be able to phosphorylate their targets efficiently, MAPKs require the integrity of the docking site. Therefore, deletion of docking sites on Pea3 will result in decreased activity as a result of inefficient interaction with the kinases. Docking site specificity in different MAPKs will help us identify the upstream regulators of Pea3-mediated neuronal differentiation mechanism in PC12 cells. This study will permit us to better understand the regulation of ETS domain transcription factor Pea3, in neuronal differentiation. The findings from this work might be considered as a first step in the treatment of neurodegenerative disorders in the future.
Dual role of human tooth germ derived mesenchymal stem cells on cancer cells
Mesencyhmal stem cells (MSCs), important components of the tumor microenvironment, could modulate tumor growth and development in many ways. MSCs have been used as targeted-delivery vehicles for cancer gene therapy because of their potential of tumor tropism. In this study, we investigated the effects of Human Tooth Germ (HTG) derived MSCs on MCF-7 (Human breast cancer cell line) and SH-SY5Y (Human metastatic neuroblastoma cell line) cells treated with doxorubicin and paclitaxel, anticancer drugs. We also investigated the in vitro effect of genetically modified MSCs secreting apoptosis inducers (Tumor necrosis factors related apoptosis inducing ligand (TRAIL), Dickkopf-related protein-1(Dkk-1)) on SH-SY5Y cells.In the first part, conditioned medium (CM) of hTGSCs was collected when the cells reached to 70% confluency. Effects of this CM on MCF-7 and SH-SY5Y treated doxorubicin and paclitaxel were measured by MTS (cell viability assay) assay and real time PCR analysis of apoptotic markers caspase3 and p53.In the second part, TRAIL and Dkk-1 genes tagged with pkh67 (Green Fluorescent Cell Linker Kit) were transferred into MSCs by electroporation either alone or in combination. A GFP gene transfected group was used as a control. Genetically modified MSCs were co-cultured with SH-SY5Y cells for 24 hours. Thereafter the cell survival of SH-SY5Y cells was determined by using flow cytometry. The expression of Bcl-2, Bax, Stat3 (proliferation genes), FADD, caspase3 (apoptotic genes) in SH-SY5Y cells were assessed by real time PCR.The results showed CM of hTGSCs increased the survival of MCF-7 and SH-SY5Y cells treated with doxorubicin and paclitaxel by 30% and also CM reduced doxorubicin and paclitaxel induced apoptosis. On the other side, genetically modified MSCs secreting TRAIL and Dkk-1 reduced the cell survival of cancer cells by inducing apoptosis and inhibiting the proliferation. Our findings demonstrated that MSCs play roles in growth of tumor cells by decreasing the effect of anti-cancer drugs on cancer cells. On the other hand genetically modified HTG derived MSCs expressing Trail and Dkk-1 might be a potential gene and cell therapy approach to treat certain types of cancer.
DNA mediated self assembly of carbon nanotubes into nanorings
Carbon Nanotubes(CNT) are newly discovered nanomaterials with unique physical, chemical, electrical and optical characteristics and they have a variety of applications in different areas. However, due to their strong affinity towards each other to form aggregates, it becomes difficult to extend their use into a wide range of applications. DNA attracts the attention of researchers with its ability to disperse nanotubes. DNA-CNT interaction involves two different approaches. In non-covalent approach, DNA wraps around the Carbon Nanotubes. This approach protects the intrinsic properties of the carbon nanotubes but offer limited potential for self-assembly research. On the other hand, covalent DNA binding to CNTs allows the use of DNA hybridization properties and has the potential to produce complex shapes. In this work, Single Walled Carbon Nanotubes (SWCNTs) were functionalized with carboxyl acid groups and oligodeoxynucleotides(ODNs) were covalently bound to them. Nanoring structures were formed by binding two different ODN bound SWCNTs together with an ODN linker. These nanoring structures and the factors affecting them were studied under Atomic Force Microscopy(AFM). The knowledge obtained from this work shows the preparation of nanorings using DNA mediated self-assembly of SWCNTs, which can be used for designing more complex structures, photothermal therapy, nanoelectronics, gene delivery.