In this study, the benzaldehyde lyase (BAL, EC 4.1.2.38) production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3) pLySs cells, the effect of bioprocess parameters was systematically investigated. Among the investigated media, the highest cell concentration and benzaldehyde lyase activity were obtained as 2.0 kg m-3 and 1060 U cm-3, respectively, in the medium containing 20.0 kg m-3 glucose, 11.8 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, oxygen transfer effects on benzaldehyde lyase production were investigated at air inlet v rate of QO/VR = 0.5 vvm, and agitation rates of N=500 and 750 min-1 and at QO/VR = 0.7 vvm, N=750 min-1 in pilot scale bioreactor and the highest cell concentration and volumetric BAL activity were found as 1.7 kg m-3 and 990 U cm-3, respectively, at 0.5 vvm, 750 min-1 condition. Next, the signal DNA sequence of serine alkaline protease (SAP) from B. licheniformis DSM 1969 chromosomal DNA (pre-subC) was fused in front of the bal by using PCR-based gene splicing by overlap extension (SOE) method. The fusion product of hybrid gene first cloned into pUC19 plasmid, thereafter sub-cloned into pBR374 shuttle vector and recombinant plasmid was transferred into various Bacillus species. However, no extracellular production of benzaldehyde lyase was observed in none of the developed recombinant Bacillus species, probably because of ineffective secretion system, inefficient folding of heterologous protein, degradation of enzyme due to proteolytic activity or high inactivation rate of the enzyme.

Chemotherapy is one of the most important treatments for cancer. However, systemic toxicity, drug resistance and unstable kinetics of the drug in the blood are serious problems of chemotherapy. The use of biodegradable polymers for controlled release of anticancer drugs has gained popularity in recent years. Controlled release of drugs from polymeric carriers has some advantages such as improvement in the efficiency of treatment, reduction in systemic toxicity and prevention of the drug resistance that is developed by the cancer cells. In this study, poly(D,L-lactide-co-glycolide) microparticles were used as carriers for the controlled release of all-trans-Retinoic acid, tamoxifen, tamoxifen citrate and idarubicin. It was aimed to prepare a drug carrier system for controlled release of hydrophobic anticancer drugs. The empty and drug loaded poly (D,L-lactide-co-glycolide) microparticles were prepared by solvent extraction/evaporation technique with single emulsion (oil/water). Optimized microparticles were characterized by using inverted light microscopy and scanning electron microscopy to examine their morphology and sizes. Drug content of microparticles and the amount of released drug were determined spectrophotometrically. In vitro toxicity of the microparticles on MCF-7 human breast cancer cells was investigated. It was revealed that the microparticles were smooth and spherical in shape. Their sizes differed in the range of 2-20 &micro / m. atRA-loaded microparticles showed approximately 90% encapsulation efficiency and it was confirmed that changing in drug/polymer ratio affected the extend of drug content. Increase in drug content caused a slower release pattern. Moreover, although the empty microparticles caused some toxicity, atRA-loaded PLGA microparticles showed slight cell growth inhibition.

The purpose of this thesis work is to model the gate control theory, which explains the modulation of pain signals, with a motivation of finding new possible targets for pain treatment and to find novel control algorithms that can be used in engineering practice. The difference of the current study from the previous modeling trials is that morphologies of neurons that constitute gate control system are also included in the model by which structure-function relationship can be observed. Model of an excitable neuron is constructed and the response of the model for different perturbations are investigated. The simulation results of the excitable cell model is obtained and when compared with the experimental findings obtained by using crayfish, it is found that they are in good agreement. Model encodes stimulation intensity information as firing frequency and also it can add sub-threshold inputs and fire action potentials as real neurons. Moreover, model is able to predict depolarization block. Absolute refractory period of the single cell model is found as 3.7 ms. The developed model, produces no action potentials when the sodium channels are blocked by tetrodotoxin. Also, frequency and amplitudes of generated action potentials increase when the reversal potential of Na is increased. In addition, propagation of signals along myelinated and unmyelinated fibers is simulated and input current intensity-frequency relationships for both type of fibers are constructed. Myelinated fiber starts to conduct when current input is about 400 pA whereas this minimum threshold value for unmyelinated fiber is around 1100 pA. Propagation velocity in the 1 cm long unmyelinated fiber is found as 0.43 m/s whereas velocity along myelinated fiber with the same length is found to be 64.35 m/s. Developed synapse model exhibits the summation and tetanization properties of real synapses while simulating the time dependency of neurotransmitter concentration in the synaptic cleft. Morphometric analysis of neurons that constitute gate control system are done in order to find electrophysiological properties according to dimensions of the neurons. All of the individual parts of the gate control system are connected and the whole system is simulated. For different connection configurations, results of the simulations predict the observed phenomena for the suppression of pain. If the myelinated fiber is dissected, the projection neuron generates action potentials that would convey to brain and elicit pain. However, if the unmyelinated fiber is dissected, projection neuron remains silent. In this study all of the simulations are preformed using Simulink.

In tissue engineering applications, use of biodegradable and biocompatible materials are essential. As the tissue regenerate itself on the material surface, the material degrades with enzymatic or hydrolytic reactions. After a certain time, natural tissue takes the place of the artificial support. Poly(&epsilon / -caprolactone) (PCL) is one of the preferable polymers used in the restoration of the bone defects due to its desirable mechanical properties and biocompatibility. Addition of inorganic calcium phosphate particles in PCL structures can improve the mechanical properties as well as osteoconductivity / and presence of an antibiotic can prevent infection that may occur at the defect site. In this study, three forms of biodegradable hard tissue supports which are bone fillers, bone regenerative membranes and 3D scaffolds were designed and prepared. As biodegradable bone fillers, composite microspheres containing gelatin and &beta / -tricalcium phosphate (&beta / -TCP) were prepared and characterized. Synthesized &beta / -TCP particles were coated with gelatin at different weight ratios and the effects of &beta / -TCP/Gelatin ratio on the morphology of the microspheres were evaluated. Also, a model antibiotic, gentamicin, was loaded to these microspheres and release behaviours of the drug and its antibacterial effect on E.Coli was determined. The selected composition of these microspherical bone fillers were used as additives in the preparation of bone regenerative membranes and scaffolds. For this purpose, microspheres were added into PCL solution and processed by either solvent casting or freeze-drying in order to prepare bone regenerative membranes or scaffolds, respectively. For every material, the ratio of constituents (microsphere and PCL) was altered in order to obtain optimum properties in the resulted hard tissue support structure. The effects of the ratio of the microspheres to PCL in terms of morphological, mechanical and degradation properties of composite films, as well as in vitro antibiotic release and antibacterial activities against E.Coli and S.Aureus were investigated. For scaffolds, the effects of the ratio of the microspheres to PCL on the morphological, mechanical, pore size distribution, degradation properties and in vitro antibiotic release were examined.

The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5&rsquo / end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through PCR. The resulting hybrid gene pre(subC)::skc was cloned into the pUC19 plasmid. Then, the hybrid gene was sub-cloned to pMK4 plasmid which is an E. coli-Bacillus shuttle vector with high copy number and high stability. Recombinant plasmid pMK4::pre(subC)::skc was finally transferred into B. subtilis (npr- apr-) and B. licheniformis 749/C (ATCC 25972) species. Streptokinase production capacities of these two recombinant Bacillus species were compared. The highest production was observed in recombinant B. lichenifomis 749/C (ATCC 25972) strain in a defined medium which was optimized in terms of carbon and nitrogen sources by a statistical approach, namely Response Surface Methodology (RSM). RSM evaluated the streptokinase concentration as the response and the medium components as the independent variables. The highest recombinant streptokinase concentration was found as 0.0237 kgm-3 at glucose and (NH4)2HPO4 concentrations of 4.530 and 4.838 kgm-3 respectively. The fermentation and oxygen transfer characteristics of the streptokinase production were investigated in a 3 dm3 pilot scale batch bioreactor (Braun CT2-2) equipped with temperature, pH, foam, air inlet and agitation rate controls having a working volume of VR=1.65 dm3 using the production medium optimized for the recombinant B. lichenifomis 749/C (ATCC 25972) strain. Streptokinase and &amp / #946 / -lactamase activities, cell, glucose and organic acid concentrations, dissolved oxygen, pH, oxygen uptake rate, overall liquid phase mass transfer coefficient for oxygen, maintenance coefficient for oxygen, specific cell growth rate and yield coefficients were determined through the bioprocess. The bioprocess of recombinant streptokinase production was performed at uncontrolled pH of these bioreactor operation conditions: air inlet rate of Q0/VR=0.5 vvm, and the agitation rate of N=400min-1. The resulting streptokinase volumetric activity reached its maximum as 1.16 PUml-1 (0.0026 g/l streptokinase) at t=20 h.