Recent studies demonstrate that there are several G-protein coupled receptors (GPCRs) that dimerize with other GPCRs and form heterodimers. Adenosine A2A-Dopamine D2 receptor interaction is one of the examples for GPCR heterodimerization. Both receptors bear critical roles in physiological processes. Adenosine A2A receptor has functions in neurotransmission, cardiovascular system and immune response. On the other hand, dopamine receptors are the key point of dopaminergic system, which controls the regulation of memory, attention, food intake, endocrine regulation, psychomotor activity and positive reinforcement. Deregulation in dopamine signaling could cause neurological disorders such as Parkinson&rsquo / s disease and schizophrenia. Dopamine D2R and adenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity. The purpose of this study is to optimize Fluorescence Resonance Energy Transfer (FRET) method to detect dimerization of D2R and A2AR by tagging them with EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) in live N2a cell line using laser scanning confocal microscope. Establishing this model will pave the ways for understanding mechanisms of interaction between dopamine and adenosine signaling, thereby, contributing to the understanding molecular mechanisms of some neurophysiological events and disorders. Moreover, the fluorescence based live cell model will be used to detect effects of potential anti-psychotic drugs on the interaction of these two receptors. Indeed, follow-up studies are necessary to extend the limits of this project. Further imaging analyses and drug-receptor interaction studies can be readily applied to extract more information on dopamine-adenosine signaling by using the system developed with this thesis study.

Quantum dots are fluorescent semiconductor nanocrystals that have unique optical properties such as high quantum yield and photostability. These nanoparticles are superior to organic dyes and fluorescent proteins in many aspects and therefore show great potential for both in vivo and in vitro imaging and drug delivery applications. However, cytototoxicity is still one of the major problems associated with their biological applications. The aim of this study is in vitro characterization and assessment of biological application potential of a novel silver sulfide quantum dot coated with mercaptopropionic acid (2-MPA). In vitro studies reported in this work were conducted on a mouse fibroblast cell line (NIH/3T3) treated with Ag2S/2-MPA quantum dots in 10-600 &mu / g/mL concentration range for 24 h. Various fluorescence spectroscopy and microscopy methods were used to determine metabolic activity, proliferation rate and apoptotic fraction of QD-treated cells as well as QD internalization efficiency and intracellular localization. Metabolic activity and proliferation rate of the QD treated cells were measured with XTT and CyQUANT&reg / cell proliferation assays, respectively. Intracellular localization and qualitative uptake studies were conducted using confocal laser scanning microscopy. Apoptosis studies were performed with Annexin V assay. Finally, we also conducted a quantitative uptake assay to determine internalization efficiency of the silver sulfide particles. Correlated metabolic activity and proliferation assay results indicate that Ag2S/2-MPA quantum dots are highly cytocompatible with no significant toxicity up to 600 &mu / g/mL treatment. Optimal cell imaging concentration was determined as 200 &mu / g/mL. Particles displayed a punctuated cytoplasmic distribution indicating to endosomal entrapment. In vitro characterization studies reported in this study indicate that Ag2S/2-MPA quantum dots have great biological application potential due to their excellent spectral and cytocompatibility properties. Near-infrared emission of silver sulfide quantum dots provides a major advantage in imaging since signal interference from the cells (autofluorescence) which is a typical problem in microscopic studies is minimum in this part of the emission spectrum. The results of this study are presented in an article which was accepted by Journal of Materials Chemistry. DOI: 10.1039/C2JM31959D.

Obesity becomes an epidemic health problem in developing and developed countries, which arises due to stable life style and increase in the consumption of high fat diets. Obesity is generally accompanied with various clinical disorders, such as insulin resistance, type II diabetes, hypertension, dyslipidemia and cardiovascular diseases. This study aims to characterize and quantify different lipid classes in longissimus dorsi (LD) and quadriceps (Q) skeletal muscles of control (DBA/2J), obese Berlin fat mouse inbred (BFMI) and Berlin muscle mouse inbred (BMMI) lines, which display high fat and high muscle content, respectively. These mouse lines were special due to their phenotypes, especially BFMI lines, which displayed spontaneous and strong obesity. These lines, more specifically BFMI860 and BFMI861, were also special due to their possibility of being an animal model of cardiovascular diseases and metabolic syndrome, since they also displayed insulin resistance. For separation,identification and quantification of various lipids of these lines, a novel method was developed which gives better separation of main lipid classes via using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Addition of triethylamine (TEA) to the solvents being used, and altering the parameters of HPLC and ELSD instruments, and also the gradient elution, provided a better separation with an enhanced resolution. This method has been applied to the lipid extracts obtained from longissimus dorsi (LD) and quadriceps (Q) skeletal muscles of control (DBA/2J), obese Berlin fat mouse inbred (BFMI) and Berlin muscle mouse inbred (BMMI). In this method, a binary gradient elution composed of n-Hexane, isopropanol, methanol, acetic acid and triethylamine was applied to the samples. All interested lipid classes, namely triglyceride (TG), cholesteryl ester (CO), cholesterol (C), 1-oleoyl-rac-glycerol (MG), phosphatidylcholine (PC) and cardiolipin (CLPN), all of which have been known to have a role in obesity, insulin resistance, and cardiovascular diseases, were separated, identified and quantified via this novel method. According to the results, among BFMI lines, BFMI860 and BFMI861 lines and BMMI806, among BMMI lines, are worth to study obesity. Especially, the former ones may also become animal models for cardiovascular diseases and metabolic syndrome.

No description available.

Wildlife tracking is an essential task to gain better understanding of the migration pattern and use of space of the wildlife. Advances in computer technology and global positioning systems (GPS) have lowered costs, reduced processing time, and improved accuracy for tracking wild animals. In this thesis, a wildlife tracking extension is developed for ArcGIS 9.x, which allows biologists and ecologists to effectively track, visualize and analyze the movement patterns of wild animals. The extension has four major components: (1) data import; (2) tracking; (3) spatial and temporal analysis; and (4) data export. Compared with existing software tools for wildlife tracking, the major features of the extension include: (1) wildlife tracking capabilities using a dynamic data layer supported by a file geodatabase with 1 TB storage limit; (2) spatial clustering of wildlife locations; (3) lacunarity analysis of one-dimensional individual animal trajectories and two-dimensional animal locations for better understanding of animal movement patterns; and (4) herds evolvement modeling and graphic representation. The application of the extension is demonstrated using simulated data, test data collected by a GPS collar, and a real dataset collected by ARGOS satellite telemetry for albatrosses in the Pacific Ocean.

The advent of high-throughput sequencing and structure determination techniques has had a tremendous impact on our quest in cracking the language of life. The genomic and protein data is now being accumulated at a phenomenal rate, with the motivation of deriving insights into the function, mechanism, and evolution of the biomolecules, through analysis of their similarities, differences, and interactions. The rapid increase in the size of the biomolecular databases, however, calls for development of new computational methods for sensitive and efficient management and analysis of this information. In this thesis, we propose and implement several approaches for accurate and highly efficient comparison and retrieval of protein sequences and structures. The observation that corresponding residues in related proteins share similar inter-residue contacts is exploited in derivation of a new set of biologically sensitive metric amino acid substitution matrices, yielding accurate alignment and comparison of proteins. The metricity of these matrices has allowed efficient indexing and retrieval of both protein sequences and structures. A landmark-guided embedding of protein sequences is developed to represent subsequences in a vector space for approximate, but extremely fast spatial indexing and similarity search. Whereas protein structure comparison and search tasks were hitherto handled separately, we propose an integrated approach that serves both of these tasks and performs comparable to or better than other available methods. Our approach hinges on identification of similar residue contacts using distance-based indexing and provides the best of the both worlds: the accuracy of detailed structure alignment algorithms, at a speed comparable to that of the structure retrieval algorithms. We expect that the methods and tools developed in this study will find use in a wide range of application areas including annotation of new proteins, discovery of functional motifs, discerning evolutionary relationships among genes and species, and drug design and targeting.

Widely applicable, quantitative field methods are needed to gather wildlife data for conservation and management initiatives in Turkey. In order to evaluate the use of camera traps in forest habitats of Turkey, we conducted a 5 phase camera trap survey by using 16 passive infrared-triggered cameras with a total sampling effort of 1200 camera trap days in Yaylacik Research Forest, a 50 km2 forest patch of Yenice Forest in Karab&uuml / k during January-May 2006. The camera trap survey confirmed the presence of grey wolf (Canis lupus), brown bear (Ursus arctos), wildcat (Felis silvestris), red fox (Vulpes vulpes), badger (Meles meles), pine marten (Martes martes), roe deer (Capreolus capreolus) and wild boar (Sus scrofa) in the study area. The camera trap survey also revealed the presence of jackal (Canis aureus) and brown hare (Lepus europaeus), whose presence were not known by people living and working in the area. Contrary to the local belief, neither camera trapping survey nor ground survey confirmed the presence of lynx (Lynx lynx) in Yaylacik Research Forest. The wolf was observed to be crepuscular and the wildcat showed a diurnal activity pattern. Wildcat seemed to avoid other carnivores spatially and temporally. Simulation studies suggested that camera trap surveys should last 14 days for wolf, 13 days for wildcat, 10 days for pine marten, and 11 days for roe deer, while it is advisable to conduct longer surveys, probably 15-20 days, for wild boar, red fox and brown bears. The estimated population size for wildcat was 9 (SE=2.28227) with 95% confidence interval of 9 to 25 in the study area. A minimum of 6 brown bears were present in the study area. Our study indicated that the local knowledge about the presence of wildlife should be considered by researchers, but it cannot replace scientific surveys conducted by field biologists. This study was the first attempt to assess the presence, relative abundance, activity patterns and diversity of multiple mammal species by the use of camera trapping methodology in Turkey. The results suggest that camera trap surveys have the potential for gathering wildlife data at larger scales in Turkey, where information gap on large mammals is an obstacle for effective management and conservation of mammals.

COLLAGEN IN SITU GROWN CALCIUM PHOSPHATE SCAFFOLDS FOR OSTEOGENIC DIFFERENTIATION OF WHARTON&rsquo / S JELLY AND MENSTRUAL BLOOD STEM CELLS Karadas, &Ouml / zge M.Sc., Department of Biotechnology Supervisor : Prof. Dr. Vasif Hasirci Co-Supervisor: Assoc. Prof. Dr. Gamze Torun K&ouml / se February 2011, 91 pages The importance of developing new techniques for the treatment of bone and joint diseases is increasing continuosly together with the increase of human population and the average life span. Especially bone fractures as a result of osteoporosis are often seen in humans older than 50 years old. The expenses of bone and joint disease operations are very high and the duration of recovery is long. Because of these reasons World Health Organization, The United Nations and 37 countries announced that the years 2000-2010 is the Bone and Joint Decade. Tissue engineering is an alternative approach to clinically applied methods. In this study collagen scaffolds crosslinked with genipin, to improve the stability of foams in culture media, were prepared by lyophilization. To mimic the natural bone structure calcium phosphate mineral phase in the foam was formed by wet chemical precipitation. Collagen concentration (0.75% and 1%, w/v), freezing temperature (-20 oC and -80 oC) of the collagen solution before lyophilization and immersion duration (2x4 h and 2x48 h) of the foams in calcium and phosphate solutions for wet chemical precipitation were changed as process v parameters of foam production. Pore size distribution and porosity analysis as well as compression test were performed for characterization of the scaffolds. The foam with 1% w/v collagen concentration, frozen at -20 oC before lyophilization and immersed for 2x4 h in calcium and phosphate solution was chosen for in vitro cell culture studies. The defined foam had 70% porosity and pore sizes varying between 50 and 200 &mu / m. The elastic modulus and compressive strength of the foam was calculated as 127.1 kPa and 234.5 kPa, respectively. Stem cells isolated from Wharton&rsquo / s jelly (WJ) and menstrual blood (MB) were seeded to foams to compare their osteogenic differentiation. Both cells are isolated from discarded tissues and used in this study as an alternative to the commonly used cells which are isolated by invasive techniques such as bone marrow stem cells. Cells were seeded to collagen foams with and without calcium phosphate (CaP). It was observed that WJ cells proliferated during 21 days on collagen foams without CaP, but MB cell number decreased after day 14. Collagen foams with CaP supported the alkaline phosphate (ALP) activity compared to tissue culture polystyrene (TCPS) and foams without CaP. Contrarily lower cell numbers achieved on CaP containing collagen foams, possibly because of the calcium and phosphate concentration changes in the medium and as the result of osteogenic differentiation. ALP activity of both cell types increased almost 10 times and specific ALP activity (activity per cell) increased 40 times and 150 times for WJ and MB cells, respectively on the CaP containing foams compared to TCPS. Therefore, in this study it was shown that in situ CaP formed collagen foams induce osteogenic differentiation of WJ and MB cells, and these cells isolated from discarded tissues can be used as alternative cell sources in bone tissue engineering applications.

The aim of this study was to develop drug delivery system based on poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles (NPs) to achieve personalized treatment of selected skin disorders, like photo-aging, psoriasis and atopic dermatitis. Dead Sea Water (DSW) and Retinyl Palmitate (RP) were used as active agents and they were loaded in PLGA NPs prepared either as spheres or capsules by o/w or w/o/w methods. MgCl2 and bovine serum albumin (BSA) served as model active compounds. The diameter of the NPs was found to be in the range of 280 - 550 nm. The entrapment efficiency (E.E.) was less than 1% for RP, DSW and MgCl2, and 41% for BSA. Loading of Cl- together with BSA doubled the E.E. value of Cl- . In situ release studies showed a burst in the first day and more than 85% of the chloride content was released within a week. When the macromolecule BSA was encapsulated, a much slower and triphasic release profile was observed which continued for up to 80 days. In vitro tests were performed using L929 fibroblast cells. Results of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test revealed that none of the NPs were cytotoxic. Additionally, all particles were hemocompatible with hemolytic activity &lt / 1.5%. L929 fibroblast and Saos 2 human osteosarcoma cells were used to study the uptake of NPs by the cells. Particles accumulate near the nucleus. The characterization and cell viability tests, and drug release behavior indicate the suitability of these NPs for further testing to develop a patient specific skin diseases treatment approach.

The ability of a genetically-engineered <i>Erwirzia carotovora</i> subsp. <i>carotovora</I> (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or <i>Pseudomonas fluorescens</i> was tested on filters, within soil microcosms, and <i>in planta</i>. Ecc was engineered by chromosomal insertion of a disarmed <i>endo</i>-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10⁻² transconjugants per donor (TPD) and to P. <i>fluorescens</i> at a frequency of 2.4 X 10⁻⁵ TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10⁻³ and 2.0 X 10⁻³, while matings on potato slices yielded frequencies of 4.7 X 10⁻² and 2.3 X 10⁻². In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10³ and 8.4 X 10⁻⁵ TPD. / Master of Science