The Functional Assessment Of Fluorecently Tagged Adenosine A2a And Dopamine D2 Receptors And Qualitative Analysis Of Dimerization Of Adenosine A2a And Dopamine D2 Receptor By Using FretAkkuzu, Selin 01 January 2013 (has links) (PDF)
Recently, several studies have demonstrated that G protein coupled receptors exist as homo/heterodimers or oligomers. Adenosine A2A receptors and dopamine D2 receptors are present as both homo- and heterodimer. In the GABAergic striatopallidal neurons A2AR are co- localized with D2 receptors (D2R), and establish functional A2AR-D2R heteromers, which modulates dopaminergic activity. Due to be involved in physiological processes, these receptors bear critical roles. Dopamine receptors play critical role in dopaminergic pathways in regulation of memory, food intake and psychomotor activity, etc. On the other hand, adenosine A2A receptors are involved in the regulations of neurotransmission, immune response and cardiovascular systems. Dopamine D2R andadenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity The purpose of this study is to assess the functionality of EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) tagged adenosine A2A and dopamine D2 receptors and to detect homo/ hetero-dimerization of these receptors in live cells via Fluorescence Resonance Energy Transfer (FRET). Understanding the mechanisms of the interaction between adenosine and dopamine signaling will help us to figure out some molecular mechanism of neurophysiological disorders. Furthermore, the fluorescence based live cell model could be used to observe the effects of potential anti-psychotic drugs on the interaction of these two receptors.
01 July 2007
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Human telomerase is a ribonucleoprotein complex that adds hexameric TTAGGG repeats to the ends of chromosomes in order to prevent their shortening. Telomerase activity has been evaluated for its diagnostic and prognostic value in cancer since it is observed in most malignancies but not in most normal somatic tissues. In this study telomerase activity was examined in tumor specimens obtained from cervix, endometrium and their non-cancerous regions by an improved telomeric repeat amplification protocol (TRAP) &ndash / silver staining assay. Appearance of characteristic TRAP leader with 6 base pair increments indicate a positive result and was observed in all cancerous and some of the non-cancerous tissues. Telomerase activities of carcinoma tissues and normal counterparts were compared by densitometric analysis after PCR. Significantly higher telomerase activity was observed in cervical carcinoma samples compared to normal adjacent tissue. No significant difference was observed between endometrium carcinomas and normal endometrial tissue in terms of telomerase activity. High telomerase activity in normal endometrium restricts the use of assay for detection of carcinogenesis. However, in cervical tissues an accurate quantification of telomerase activity by TRAP &ndash / silver stain assay may be valuable as a confirmatory assay.
Aydin, Ozge Zelal
01 September 2009
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Bladder cancer is one of the most common urogenital cancers worldwide. Two techniques commonly used for bladder cancer diagnosis are urine cytology and cystoscopy. Cytology is not sensitive for detecting tumors. Cystoscopy is an invasive technique which disturbs patient comfort. In the current study, we used Fourier transform infrared (FT-IR) spectroscopy as a novel method which is rapid and non-invasive to investigate the bladder tumor recurrence using the bladder wash samples collected in the course of control cystoscopy. This study is unique since it is the first one to use the bladder wash sample in the diagnosis of the bladder tumor by using FT-IR spectroscopy. Molecular investigation of the FT-IR spectra revealed many differences between control and tumor samples such as a considerable increase in protein, carbohydrate and nucleic acids content, and changes in protein and carbohydrate structure. On the basis of the spectral differences, cluster analysis was performed to differentiate between the control and tumorous spectra and we reached to an overall sensitivity (including all individuals with tumor) of 91.8%, a PUNLMP sensitivity of 83.3% and a papilloma sensitivity of 77.8% in spectral range 1444-1457 cm-1. Other spectral ranges also gave similar results. Our results showed that FT-IR spectroscopy can be used to detect the bladder tumors in bladder wash sample with higher sensitivity compared to cytology. In summary, we propose the utilization of the FT-IR spectroscopy for the detection of bladder tumors since specific spectral regions might be used as effective markers for the diagnosis.
Development Of A Sandwich-type Dna Array Platform For The Detection Of Label-free Oligonucleotide TargetsCansiz, Sena 01 October 2010 (has links) (PDF)
DNA arrays have become a major bioanalytical method as they enable high-throughput screening and they can be manufactured on different surfaces depending on the nature of diagnostic purpose. However, current technologies to produce and detect arrays of DNA probes are expensive due to the requirement of specialized instrumentation. In this study we have established an array platform in sandwich hybridization format for the detection of label-free nucleic acid targets. Unlike direct hybridization, which is the main microarray hybridization principle, sandwich assay enables unlabeled target detection, lowering the cost and assay time. To this end, sequence specific signal development was achieved by a sandwich complex which is composed of a surface immobilized capture DNA probe (Probe1) and a fluorescein-tagged signal DNA probe (Probe 2), which are partially complementary to the sequence to be analyzed (target oligonucleotide). As the solid support of the array platform both 3-aminopropyl-3-methoxysilane (APTMS) activated and commercially purchased poly-L lysine coated glass slides were used and due to the less background noise property the latter one was preferred. Similarly, for the immobilization of the capture Probe (P1) onto the solid support two different methods were tried / heat immobilization and immobilization via a heterobifunctional cross-linker (HBCL). In regard to the experiments, it is observed that using a cross-linker instead of heat immobilization reduces the ratio of false negative control results in a significant manner. Following the solid support and immobilization method choice comparative optimization studies which include cross-linker type, probe concentration, sensitivity of the platform and hybridization conditions (sequence, temperature and duration) were conducted. Optimum hybridization signal was obtained with a 32.5
Development Of An Oligonucleotide Based Sandwich Array Platform For The Detection Of Transgenic Elements From Plant Sources Using Label-free Pcr ProductsGul, Fatma 01 October 2010 (has links) (PDF)
Advances in DNA micro and macroarray technologies made these high-throughput systems good candidates for the development of cheaper, faster and easier qualitative and quantitative detection methods. In this study, a simple and cost effective sandwich hybridization-based method has been developed for the rapid and sensitive detection of various unmodified recombinant elements in transgenic plants. Attention was first focused on the optimization of conditions such as time, concentration and temperature using commercial ssDNA, which in turn could be used for real sample detection. In this sandwich-type DNA chip platform, capture probes complementary to the first half of recombinant element (target adapter) were immobilized onto poly-L-lysine covered conventional microscope slides. PCR-amplified un-purified target adapter and biotin labeled detection probe, which is complementary to the second half of target adapter, were hybridized in solution-phase to complementary capture probes to create a sandwiched tripartite complex. Later, hybridization signal was visualized by the attachment of streptavidin conjugated Quantum Dot to the sandwiched complex under UV illumination. Sandwich based array system that has been developed in this study allows multiplex screening of GMO events on a single DNA chip platform. 35S promoter, NOS terminator, CRY1Ab and BAR target sequences were successfully detected on the same DNA chip platform. The platform was able to detect unlabeled PCR amplified DNA fragments of CaMV 35S promoter sequence and NOS terminator and BAR transgene sequences from transgenic potato plants and NK603 Certified GMO Reference material, respectively. The DNA-chip platform developed in this study will allow multiple detection of label-free PCR-amplified transgenic elements from real GMO samples on a single slide via a cost effective, fast, reliable and sensitive sandwich hybridization assay.
Optimization Of Fret Method To Detect Dimerization Of Dopamine D2 And Adenosine A2a Receptors In Live CellsUnlu, Gokhan 01 July 2011 (has links) (PDF)
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.
01 June 2012
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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® / 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.
01 September 2012
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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.
Similarity Search And Analysis Of Protein Sequences And Structures: A Residue Contacts Based ApproachSacan, Ahmet 01 August 2008 (has links) (PDF)
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.
Camera Trapping Large Mammals In Yenice Forest Habitats: A Feasibility Study For Camera Trapping Large Mammals In Yenice Forests, TurkeyCan, Ozgun Emre 01 September 2008 (has links) (PDF)
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 Karabü / 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.
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