Hard tissue engineering holds the promise of restoring the function of failed hard tissues and involves growing specific cells on extracellular matrix (ECM) to develop &bdquo / &bdquo / tissue-like&rdquo / structures or organoids. Chitosan is a linear amino polysaccharide that can provide a convenient physical and biological environment in tissue regeneration attempt. To improve chitosan&amp / #8223 / s mechanical and biological properties, it was blended with another polymer gelatin. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink the chitosan-gelatin matrix to produce stable structures. These natural polymers are mechanically weak especially to serve as a bone substitude and therefore, an inorganic calcium phosphate ceramic, hydroxyapatite, was incorporated to improve this aspect. The objective of this study was to develop chitosan-gelatin/hydroxyapatite scaffolds for a successful hard tissue engineering approach. For this reason, two types of hydroxyapatite, as-precipitated non-sintered (nsHA) and highly crystalline sintered (sHA) were synthesized and blended into mixtures of chitosan (C) and gelatin (G) v to produce 2-D (film) and 3-D (sponge) structures. The physicochemical properties of the structures were evaluated by scanning electron microscopy, X-Ray Diffraction (XRD), Fourier Transform Infrared-Attenuated Total Reflectance spectrometer (FTIR-ATR), differential scanning calorimetry, contact angle and surface free energy measurements and swelling tests. Mechanical properties were determined through tensile and compression tests. In vitro cell affinity studies were carried out with SaOs-2 cells. MTS assays were carried out to study cell attachment and proliferation on the 2-D and 3-D scaffolds. Several methods such as confocal, fluorescence and scanning electron microscopy were used to examine the cell response towards the scaffolds. Cell affinities of the samples were observed to change with changing chitosan-gelatin ratio and hydroxyapatite addition into the matrices. XRD and FTIR results confirmed the purity of the hydroxyapatite synthesized. Mechanical test results showed that 2-D and 3-D chitosan-gelatin/hydroxyapatite constructs have similar properties as bones, and in vitro studies demonstrated that the prepared matrices have the potential to serve as scaffold materials in hard tissue engineering applications.

In this study, regulatory gene effects on recombinant human growth hormone (rhGH) production by Bacillus subtilis were investigated. For this purpose, firstly Bacillus strains, which are deficient in abrB, aprE, degQ, degS, degU, scoC, sinI, sinR, and spo0A genes, were selected according to the regulatory gene network of aprE gene (serine alkaline protease gene of B. subtilis) since due to the degQ promoter and the pre-signal sequence of subC gene cloned in front of the hGH gene, hGH is produced by mimicking the serine alkaline protease synthesis. R-Bacillus strains were constructed by transformation of pMK4::pre(subC)::hGH plasmid to the selected strains. Thereafter, by the laboratory scale experiments, strains having the highest hGH production capacity were determined as scoC, aprE, sinR, and degU knockout strains. Using these strains, fermentation experiments were carried out in pilot-scale bioreactor in defined medium. Effect of pH control was also investigated and the highest cell and hGH concentration was obtained by scoC knockout strain in pH controlled operation as 1.62 kg m-3 and 126 g m-3, respectively. By this strain, the overall product and cell yield on total substrate were found as 16.12 g kg-1 and 0.15 g g-1, respectively. Furthermore, the highest total protease activity was attained by degU knockout strain as 65 U cm-3. On the other hand, maximum total organic acid secretion was determined as 1.31 kg m-3 in aprE knockout strain.

In this study we fabricated free standing multilayer films of polyelectrolyte complexes for potential use in tissue engineering of corneal stroma by using the layer-by-layer (LbL) approach. In the formation of these LbL films negatively charged, photocrosslinkable (methacrylated) hyaluronic acid (MA-HA) was used along with polycations polyethyleneimine (PEI) and poly(L-lysine) (PLL). Type I collagen (Col) was blended in with PLL for improving the water absorption and cell attachment properties of the films. It was shown that the LbL films could be easily peeled off from glass substrates due to the photocrosslinking of one of the LbL components, the hyaluronic acid. Film growth and composition were monitored with FTIR-ATR. Heights of peaks at 3383 cm-1, and 2958 cm-1increased along with the bilayer number confirming the polymer build-up. Film integrity and thickness were investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Films thicker than 5 bilayers (BLs) were found to be uniform in appearance and 10 BL (PEI/MeHA) films were calculated to be ca. 6 &mu / m thick. Atomic force microscopy (AFM) revealed that as the number of BLs increased, surface roughness decreased. Activity of methacrylated hyaluronic acid was shown by the increased resistance of photocrosslinked multilayer films against hydrolysis by hyaluronidase. Patterns could be created on the films by photocrosslinking further proving that the crosslinking step is successful. Since the ultimate goal was to construct a corneal stroma PEI/MA-HA films were tested with corneal stroma cells, keratocytes. Cell proliferation on PEI/MA-HA films was quite poor in comparison to TCPS. In order to improve the cell adhesion the tests were repeated with PLL/MA-HA. Collagen was added to decrease the hydrophilicity and introduce cell adhesion sequences (Arg-Gly-Asp, RGD) to improve cell proliferation on the films and thus PLL+Col/MA-HA films were also tested. Introduction of collagen to the PLL/MA-HA films was found to decrease water retention of the multilayer films and improve cell viability and proliferation. Col+PLL/MA-HA LbL thus appear to be a promising platform for tissue engineering, especially of corneal stroma.

Soft biological tissue material models in the literature are frequently limited to integer order constitutive relations where the order of differentiation of stress and/or strain is integer-valued. However, it has been demonstrated that fractional calculus theory applied in soft tissue material model formulation yields more accurate and reliable soft tissue material models. In this study, firstly a fractional order (where the order of differentation of stress in the constitutive relation is non-integer-valued) linear viscoelastic material model for soft tissues is fitted to force-displacement-time indentation test data and compared with two different integer order linear viscoelastic material models by using MATLAB&reg / optimization toolbox. After the superiority of the fractional order material model compared to integer order material models has been shown, the linear fractional order material model is extended to its nonlinear counterpart in finite deformation regime. The material model developed is assumed to be isotropic and homogeneous. v A user-subroutine is developed for the material model formulated to implement it into the commercial finite element software Msc.Marc 2010. The user-subroutine developed is verified by performing a small strain finite element analysis and comparing the results obtained with linear viscoelastic counterpart of the model on MATLAB&reg / . Finally, the unknown coefficients of the fractional order material model are identified by employing the inverse finite element method. A material parameter set with an amount of accuracy is determined and the material model with the parameters identified is capable of simulating the three different indentation test protocols, i.e., &ldquo / relaxation&rdquo / , &ldquo / creep&rdquo / and &ldquo / cyclic loading&rdquo / protocols with a good accuracy.

The constitutive studies of this thesis were achieved and presented in three parts. In the first part, the effects of solid state synthesis process parameters and the impurity content of primary calcium precursor on the cement-type hydration efficiency for the conversion of &alpha / -tricalcium phosphate (Ca3(PO4)2 or &alpha / -TCP) into hydroxyapatite (Ca10-xHPO4(PO4)6-x(OH)2-x x = 0&ndash / 1, or HAp) have been investigated (at 37&deg / C). &alpha / -TCP was synthesized by thermal processing of stoichiometric amounts of calcium carbonate (CaCO3) and monetite (CaHPO4) at 1150&ndash / 1350&deg / C for 2 h. Three commercial grade CaCO3 powders of different purity were used as starting materials for the synthesis process and the resultant &alpha / -TCP products for all synthesis routes were compared in terms of the material properties and their reactivities. In the second part of the studies, &alpha / -TCP and chitosan fiber (CF) composites were prepared as injectable bone cement systems which have a potential to degrade in time to be replaced by the natural bone tissue. &alpha / -TCP/CF composites were prepared in different compositions and the effect of CF addition on cement properties were examined by mechanical and injectability tests as well as microstructural and phase analysis studies. In the third part of the studies, metal chelating property of CFs was used on development of controlled zinc release systems that can be applied in local zinc deficiency therapies of bone tissue. For this purpose, CF scaffolds were prepared by wet-spinning technique and appropriate amount of zinc was loaded to these scaffolds in regard to the zinc content of a healthy human bone tissue. Zinc release studies were performed on calcium phosphate (CaP) covered and non-covered CF scaffolds and zinc ion concentrations of the release solutions were determined by ICP-MS.

In this study, the effects of oxygen transfer (OT) on beta-lactamase production and on intracellular reaction rates were investigated with Bacillus licheniformis ATCC 2597. In order to clarify the oxygen transfer effects on the production of beta-lactamase, firstly a glucose based defined medium was designed and using this medium, the effects of bioreactor operation parameters, i.e., pH and temperature, on beta-lactamase activity and cell formation were investigated in laboratory scale batch-bioreactors using shake bioreactors having V=33 ml working volumes. Among the investigated bioprocess conditions, the highest beta-lactamase activity was obtained as A=115 U cm-3, in the medium with 7.0 kg m-3 glucose, 7.1 kg m-3 (NH4)2HPO4 and the salt solution, at pH0=7.5, T=37C, N=200 min-1. At the optimum conditions found in laboratory scale the effects of OT on cell generation, substrate consumption, product (beta-lactamase) and by-products formations were investigated at three different air inlet (Q0/ VR = 0.2, 0.5 and 1 vvm) and at three agitation rates (N=250, 500, 750 min-1) in V = 3.0 dm3 batch bioreactors consisting of temperature, pH, foam, stirring rate and dissolved oxygen controls. Along with the fermentation, cell, substrate and by-product concentrations, beta-lactamase activity, yield coefficients, specific rates, oxygen uptake rates and the liquid phase mass transfer coefficient values were determined. The highest beta-lactamase activity was obtained at 0.5 vvm 500 min-1 and at 0.2 vvm 500 min-1 conditions as ca. A=90 U cm-3 while the highest cell concentration was obtained as Cx=0.67 kg m-3 at 0.5 vvm 750 min-1 and at 0.2 vvm 750 min-1 conditions. KLa, increased with the increase in the agitation and aeration rates and its values varied between 0.007-0.044 s-1 and oxygen uptake rate varied between 0.4-1.6 mol m-3 s-1. Finally, the influence of OT conditions on the intracellular reaction rates was investigated using metabolic flux analysis to evaluate the effects of oxygen on the metabolism. Keywords: beta-lactamase, production, Bacillus, oxygen transfer, metabolic flux analysis

Serine alkaline protease (SAP, EC 3.4.21.62) produced by Bacillus species, that are the microbioreactors within the bioreactors, is one of the major industrial enzymes. In this study, after production by Recombinant Bacillus subtilis (BGSC-1A751), carrying pHV1431::subc gene in the complex media and separation of solids, SAP was dried by using a spray drier. Experiments were performed to investigate the stabilization of SAP during spray drying and subsequent storage. Initially, the effect of air inlet temperature of the spray drier on SAP activity was evaluated. For this purpose, SAP solutions were spray dried in the absence of any protective agents at five different air inlet temperature (700C, 900C, 1100C, 1200C, 1300C). As a result, increasing air inlet temperature lead to an increase in activity loss of SAP during drying. Thereafter, the effect of protective additives, glucose and maltodextrin (0.5%, 1%, 2% w/v), on SAP activity was investigated during spray drying. The activity loss of SAP was completely inhibited in the presence of glucose at 70, 90 and 1100C. However, the addition of maltodextrin was better than glucose for activity preservation of SAP at 1300C. Among the obtained results, 1% glucose addition was the best to preserve activity of SAP during spray drying. Then, structural change of SAP during drying was investigated. FTIR-ATR spectrum was used to evaluate the change in physical structure of the dried SAP powders in the presence of 1% glucose. From infrared images, at 900C more native-like structure for dried SAP powders was observed. In the last stage of this study storage stability of obtained SAP powders at 40C for a long period (6 months) was investigated. When considering all conditions, 0.5% maltodextrin addition was the best for stabilizing SAP powders along storage time.

Electro-Magnetic Source Imaging (EMSI) is the estimation of the position, orientation and strength of active electrical sources within the brain from electrical and magnetic measurements. For an accurate source localization, the head model must correctly represent the electrical and geometrical properties of the head. To solve the forward problem using realistic head models numerical techniques must be used. This work uses the Boundary Element Method (BEM) for solving the forward problem. The accuracy of the existing BEM formulation is improved by using second order elements, recursive integration and the isolated problem approach (IPA). Two new formulations are developed to improve the solution speed by computing transfer matrices for EEG and MEG solutions. The IPA formulation is generalized and integrated into the accelerated BEM algorithm. Once the transfer matrices are computed, the forward solutions take about 300 ms for a 256 sensor EEG and MEG system. The head model used in the BEM solutions is constructed by segmenting three dimensional multimodal magnetic resonance images. For segmentation, a semi-automatic hybrid algorithm is developed that makes use of snakes, morphological operations, thresholding and region growing. The mesh generation algorithm allows intersecting tissue compartments. For the inverse problem solution genetic algorithm (GA) is used to search for a given number of dipoles. Source localization with simulated data show that the localization error is within 1.1 mm for EEG and 1.2 mm for MEG when SNR is 10 on a realistic model with 7 compartments. When a single-dipole source in a realistic model is explored using a best-fit spherical model, the localization errors increase up to 8.5 mm for EEG and 7 mm for MEG. Similar tests are also performed with multiple dipoles. It was observed that realistic models provide definitely more accurate results compared to spherical models. The EMSI approach is also tested using experimental EEG data to localize the sources of auditory evoked potentials. The reconstructed source locations are correctly found in the Heschl&#039 / s gyrus. In conclusion, this thesis presents a complete source localization framework for future brain research using the EMSI.

This thesis study focuses on the realization of an online cue based Motor Imagery (MI) Brain Computer Interface (BCI). For this purpose, some signal processing and classification methods are investigated. Specifically, several time-spatial-frequency methods, namely the Short Time Fourier Transform (STFT), Common Spatial Frequency Patterns (CSFP) and the Morlet Transform (MT) are implemented on a 2-class MI BCI system. Distinction Sensitive Learning Vector Quantization (DSLVQ) method is used as a feature selection method. The performance of these methodologies is evaluated with the linear and nonlinear Support Vector Machines (SVM), Multilayer Perceptron (MLP) and Naive Bayesian (NB) classifiers. The methodologies are tested on BCI Competition IV dataset IIb and an average kappa value of 0.45 is obtained on the dataset. According to the classification results, the algorithms presented here obtain the 4th level in the competition as compared to the other algorithms in the competition. Offline experiments are performed in METU Brain Research Laboratories and Hacettepe Biophysics Department on two subjects with the original cue-based MI BCI paradigm. Average prediction accuracy of the methods on a 2-class BCI is evaluated to be 76.26% in these datasets. Furthermore, two online BCI applications are developed: the ping-pong game and the electrical wheelchair control. For these applications, average classification accuracy is found to be 70%. During the offline experiments, the performance of the developed system is observed to be highly dependent on the subject training and experience. According to the results, the EEG channels P3 and P4, which are considered to be irrelevant with the motor imagination, provided the best classification performance on the offline experiments. Regarding the observations on the experiments, this process is related to the stimulation mechanism in the cue based applications and consequent visual evoking effects on the subjects.

In this study, it is aimed to develop an injectable controlled release system of PCL and P(L,DL)LA microspheres loaded with TA and/or Ral for local treatment of rheumatoid arthritis which will avoid from systemic side effects of traditional administration and eliminate problems caused by direct local injections. Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disorder that most commonly causes inflammation and tissue damage in joints and tendon sheaths. Current strategies for the disease are mainly towards relieving symptoms and increasing mobility. The microsphere form drug delivery systems were developed to enhance the treatment success of rheumatic diseases by providing these agents alone or together for long terms without causing systemic or local site effects upon injection to the RA joints. Microspheres were prepared with s/o/w solvent evaporation technique and optimized to achieve a suitable size for joint application, to sustain the delivery of the drug(s), to provide required amount of the agent with feasible amount of microsphere. In order to manage these, microspheres prepared with different combinations of polymers and drugs were examined for particle size analysis, surface and structural characterizations, time related drug release properties, and drug loading capacities. In vitro cytotoxicity tests using 3T3 fibroblast cells were done to evaluate the biocompatibility of drug loaded PCL microspheres. The degradation of polymers were conducted and evaluated by GPC analysis. In PCL:TA microspheres, as polymer:drug ratio decreased (from 10:1 towards 10:4), namely as the drug partition increased, it was seen that encapsulation efficiency and loading percentages increased. Meanwhile, percent release of the drug decreased, indicating more prolonged release. Among all microspheres, PCL:TA 10:4 and PCL:Ral 10:2 were found to be the most appropriate for dual release in terms of release values (ca 21% and 0.09%, respectively), loadings (ca 27% and ca 13%, respectively) and mean particle size values (ca 100 &mu / m and ca 95 &mu / m, respectively). After release studies, microspheres preserved their sphericity. These selected polymer:drug groups also represented no cytotoxic effect. The microspheres for dual drug study (PCL:TA:Ral 10:4:2) released app. 55% of its TA and 0.29% of Ral at the end of 4 weeks. Drug loading capacities of these microspheres were found to be ca 14% for TA and 8% for Ral. Furthermore, with dual loading case, smallest mean particle size (68 &mu / m) could be obtained among all studied groups. P(L,DL)LA microspheres caused high viscosity problems during microsphere preparation steps and resulted in the slowest release, which was unfavorable for the aim of the study. To our knowledge there is no microsphere study reported with P(L,DL)LA in literature. The TA and Ral delivery systems with PCL and P(L,DL)LA were developed and studied for the first time in literature and they were optimized for RA treatment purposes. The potential of these systems, should be further tested in experimental animal models of RA.