Chemotherapy is one of the common treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). MDR is caused by the activity of diverse ATP binding cassette (ABC) transporters that pump drugs out of the cells. There are several drugs which have been used in treatment of cancer. One of them is doxorubicin that intercalates and inhibits DNA replication. However, doxorubicin has been found to cause development of MDR in tumors. It has been reported that there is a correlation between multidrug resistance and invasiveness of cancer cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness. In this study, MCF-7 cell line (MCF-7/S), which is a model cell line for human mammary carcinoma, and doxorubicin resistant MCF-7 cell line (MCF-7/Dox) were used. The resistant cell line was previously obtained by stepwise selection in our laboratory. The main purpose of this study was to investigate changes of metastatic behaviour in MCF-7/Dox cell line, after transient silencing of vimentin gene by siRNA. In conclusion, down-regulation of vimentin gene expression in MCF-7/Dox cell lines was expected to change the characteristics in migration and invasiveness shown by migration and invasion assays.

Complexed microspheres of poly(4-vinyl pyridine) (P4VN) and alginic acid were prepared by internal gelation method and subsequent freeze drying. The 4% and 10% microspheres were loaded with Bone Morphogenetic Protein-2 (BMP-2) and Bone Morphogenetic Protein-7 (BMP-7), respectively for in vitro studies and were entrapped in PLGA foams. Foams containing only 4%, BMP-2 microspheres, only 10%, BMP-7 microspheres and both populations were prepared. Control samples of each group were prepared with drug free microspheres. Bone marrow derived stem cells from rat femur and tibia isolated by a surgical operation, were seeded onto foams. Proliferation of cells on foams containing both microsphere populations was higher at all time points regardless of the presence of BMPs. This was attributed to different porosity characteristics. Proliferation was higher at all times in control samples in comparison to their positive samples for all groups, suggesting proliferation attenuation related enhancement in osteogenic activity due to BMP supply. Alkaline phosphatase (ALP) activities were lower at all time points for foams containing both microsphere populations regardless of BMP presence. This was attributed to different physical characteristics of foams confirmed by the inverse correlation between proliferation and osteogenic differentiation. Total and specific ALP activity results demonstrated the significant positive influence of all BMP containing types in enhancing osteogenic differentiation. Best results were obtained with co-administration of sequential delivery performing 4% and 10% microspheres loaded with BMP-2 and BMP-7, respectively.

Osteoarthritis (OA) is a disease characterized by the progressive degradation of articular cartilage. Current strategies for the disease are mainly towards relieving symptoms. This study was aimed to investigate the therapeutic potentials of Bone Morphogenetic Protein-9 (BMP-9), Raloxifene (Ral) and Pluronic F-68 (PLF-68) with a three-dimensional in vitro OA model. Articular chondrocytes isolated from rats were cultured in growth media and embedded in agarose to obtain agarose-chondrocyte discs. Dynamic hydrostatic mechanical stress was applied to discs. The discs were incubated with Aza-C for 48 hours for OA development. After its removal, chondrocytes were treated with different doses of BMP-9, Ral and PLF-68 for 10 days. The efficacies of treatments were evaluated by measuring cell number, glycosaminoglycan and collagen amount, and mechanical properties of the v discs. Measurements of these properties were performed with MTT, quantitative colorimetric assays, histochemical staining and mechanical tests, respectively. According to comparative results with healthy groups and controls (osteoarthritic chondrocytes without any treatment), it was found that BMP-9 had negative effect on osteoarthritic chondrocytes. On the other hand, Ral showed positive results related with matrix synthesis and mechanical properties especially at 5 &amp / #956 / M dose suggesting that it holds promise for the treatment of OA. The therapeutic effect of Ral on OA was documented for the first time in literature. The potential of PLF-68 for treatment of OA was also supported by this study considering its positive effects on cell number, collagen synthesis and mechanical properties. Yet, further investigations are also suggested for conclusive results on this agent.

In this study, extracts from bark, leaves and catkins of Salix aegyptiaca L. were investigated for their antioxidant content by 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical quenching assay, total phenolic and total flavonoid assays. The highest antioxidant activity (19 ug/ml IC50 for inhibition of DPPH radical activity), total phenolic content (212 mg gallic acid equivalents/g of dried extract) and total flavonoid (479 mg catechin equivalents/g of dried extract) was observed in the ethanolic extract of bark. High performance liquid chromatography (HPLC) analyses revealed the presence of gallic acid, caffeic acid, vanillin and p-coumaric acid, myricetin, catechin, epigallocatechin gallate, rutin, quercetin as well as salicin. In addition, the anti-proliferative effects of the ethanolic extracts on colorectal cancer cell lines (HCT-116 and HT-29) were examined by an MTT cell viability assay while their apoptotic effects were assayed by acridine orange staining and caspase 3 activity. The results indicate that the ethanolic extract of bark of S. aegyptiaca can strongly inhibit cell proliferation and induces apoptosis in a dose dependent manner on both cell lines. We propose that extracts from this plant may be utilized as a source of health promoting antioxidants. Our data provide a perspective for more detailed study of biochemical pathways associated with the cancer preventive effects of active components of the extracts from S. aegyptiaca.

One of the major causes of death in developed countries is cardiovascular disease that affects small and medium sized blood vessels. In most cases autologous grafts have to be used which have limited availability. A functional tissue engineered vessel can be the ultimate solution for vascular reconstruction. Tissue engineered constructs with cells growing in an organized manner have been shown to have improved mechanical properties. In the present study collagen scaffolds with 650 nm, 500 nm and 332.5 nm wide channels and ridges were seeded with human vascular smooth muscle cells (VSMC) and human endothelial cells seperately and then co-cultured on tubular scaffolds. When the films were seeded with endothelial cells it was observed that nanopatterns do not affect cell proliferation or initial cell alignment / however, they significantly influenced cell retention under shear (fluid flow). While 35 &plusmn / 10 % of the cells were retained on unpatterned films, 75 &plusmn / 4 % was retained on 332.5 nm patterned films and even higher, 91 &plusmn / 5 % was retained on 650 nm patterned films. It was shown that nanopatterns as small as 332.5 nm could align the vascular smooth muscle cells (VSMC) and that alignment significantly improved mechanical properties. Presence of nanopatterns increased the ultimate tensile strength (UTS) from 0.55 &plusmn / 0.11 on Day 0 to as much as 1.63 &plusmn / 0.46 MPa on Day 75, a value within the range of natural arteries and veins. Similarly, Young&amp / #8217 / s Modulus values were ca. 4 MPa, again in the range of the natural vessels. Since the films would be ultimately rolled into tubes of collagen, nutrient transfer through the films is quite crucial. Diffusion coefficient for 4-acetaminophenol and oxygen through the collagen films were found to be 1.86 &plusmn / 0.39 x 10-7 cm2.s-1 and 5.41 &plusmn / 2.14 x 10-7 cm2.s-1, repectively in the unseeded form, and increased by 4 fold after cell seeding, which is comparable to that in natural tissues. When both cell types were co-cultured on the nanopatterned tubes (a both-side nanopatterned collagen tube), it was shown that on the outside of the tube VSMCs proliferated in an oriented manner and on the inside endothelial cells proliferated as a monolayer. Therefore, this study showed that cell guidance enhances the mechanical properties of engineered vessels, and help overcome the two most important challenges in vascular tissue engineering / the need for adequate mechanical properties and continuous lining of endothelial cells even under physiological shear stress.

Cereal loss due to fungal pathogens is an ongoing setback in agriculture. Elucidating plant&rsquo / s resistance and susceptibility mechanisms against these cereal killers, promises progress in agriculture. In the way of understanding barley resistance against fungus Blumeria Graminis we silenced FAS-Associated Factor 1 (FAF1) gene in its mRNA level with Virus Induced Gene Silencing (VIGS) technique. Previous research in our lab had shown an augmentation in mRNA levels of FAF1 gene in fungus infected wheat, suggesting a role of this gene in the resistance mechanism. We hypothesized that the apoptotic role of FAF1 protein in metazoan is conserved in plants by including FAF1 as a factor in hypersensitive response. Barley lines Pallas01 and Pallas03 which are respectively resistant and susceptible against fungus Blumeria graminis hordei 103 (Bgh103) were used for fungal inoculations after FAF1 silencing, to test if the hypersensitive response against fungus Bgh103 was prevented. In this aspect the formation of death lesions on the Pallas01 leaf due to fungal resistance was not prevented demonstrating that FAF1 silencing with VIGS in the resistant Pallas01 line of barley is not sufficient to stop apoptosis. On the other hand the FAF1-silenced barley susceptible line Pallas03 became more sensitive to fungal stress based on conidia (body part of the fungus) counting after trypan blue staining of the infected leaves. In the C-terminus of FAF1 an ubiquitin like domain-X (UBX) is found, which is the cause of stress sensitivity based on the reported data obtained about this domain&rsquo / s loss of function in other proteins. These results suggest that FAF1 is a catalyst in the hypersensitive response and its loss of function makes barley more susceptible to fungal stress. On the other hand a short mRNA homology was found among FAF1 and many pathogen disease related proteins making this homology a possible target site for VIGS of FAF1 generated siRNAs, which might cause some other protein to be responsible for the barley susceptibility against the fungus.

There are different methods used in the surgical treatment of peripheral nerve injury. In this respect, end-to-end surgical reconnection of the damaged nerve ends or autologous nerve grafts are applied as soon as possible after the injury. When autologous tissue transplant is considered, there are some medical devices available generally for relatively short nerve defects. As a solution for this problem, different tissue engineered nerve conduits have been developed. In the current study, a pHEMA hydrogel membranes were designed to mimic the tubular conduits and they were loaded with 1-6% (w/w) multiwalled carbon nanotubes (mwCNTs) to obtain electrical conductivity. The most important reason for the use of CNTs in peripheral nerve injury is their electrical conductivity. Within the context of the study, the degree of swelling, contact angles, electrical conductivity and mechanical properties of the membranes were analyzed. As the amount of mwCNTs were increased, the contact angles, indicating higher hydrophobicity and the electrical conductivity increased. The tensile test of the mwCNT-pHEMA composite membranes showed that the membranes have viscoelastic structure similar to the structure of the soft tissues. The structure of the mwCNT containing pHEMA composite membranes were analyzed with different microscopical techniques such as SEM, CSLM and microCT. MwCNTs on the hydrogels were morphologically similar to the original. SEM micrographs also showed that the mwCNTs were grouped in clumps on hydrogel surfaces. No mwCNT leaching was observed because the mwCNTs were embedded in the hydrogel, therefore, no cytotoxic effect was observed. The pHEMA hydrogels were porous which is suitable for transportation of materials, electrolytes and gas needed for cell nutrition and growth. In the in vitro studies, SHSY5Y neuroblastoma cells were seeded on the membranes to determine the sustainability and effects of the membranes on the cell growth. Electrical potential of 1 and 2 V were used to stimulate the cells. Microscopical examination with SEM and CSLM, and MTT viability assay were used. The SHSY5Y neuroblastoma cells were attached and proliferated on both the composite and the hydrogel membranes. The cells on pHEMA membranes without mwCNTs, however, were not able to survive after application of electrical potential. As a conclusion, use of composite membranes in the treatment of peripheral nerve injury as a nerve conduit is appropriate. Electrical stimulation, however, did not induce the cells to align in contrast to the expected results, indicating potential and current application regime needs to be optimized to obtain the desired results.

Sodium butyrate (NaBt) is a four-carbon short chain fatty acid, produced naturally in colon as the end product of the bacterial anaerobic metabolism on dietary fibers. It was previously shown that NaBt can induce differentiation and may inhibit proliferation. The objective of this study was to investigate the effect of NaBt-induced differentation on inflammatory pathways in HT29 colon cancer cells. For this purpose, first, cells were treated with varying concentrations of NaBt from 1-5 mM and amount required to induce differentiation was determined as 3 mM. To understand the effect of NaBt on inflammation, the NF-kappaB pathway (p50 and p65) was investigated. Immunofluorescent staining showed increased nuclear translocation of p50 subunit with no remarkable change in subcellular localization of p65 / moreover a synergistic effect was observed when cells were co-treated with NaBt and an NF-kappaB repressor, Bay 11-7085 / implying the formation of repressive p50 homodimers in the nucleus. Our preliminary chromatin immunoprecipitation results showed that p65 recruitment v to the promoters of ICAM-1 was reduced, whereas p50 recruitment was increased. However, analysis of NF-kappaB target genes showed that cells treated with 3 mM NaBt have higher expression of the cytokines IL1-&beta / and TNF-&alpha / , adhesion molecules ICAM-1 and VCAM-1 but not COX-2. These results suggest that NaBt-induced differentiation could cause the emergence of an inflammatory signal in HT29 cells as an anti-tumor mechanism, independent from the NFkappaB activity. This work will be important in understanding the role of SCFAs in the colon microenvironment and may provide alternative therapeutic options in colorectal cancer.

Some yeast strains under certain conditions secrete into the medium polypeptide toxins which are inhibitory to sensitive cells. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component &amp / #946 / -1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &amp / #946 / -1,3-glucanase which is stable at pH&rsquo / s and temperatures appropriate for its medical usage. &amp / #946 / -1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 &micro / g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.

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