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ÖZET Mühendislikte kuleler özel yapılar grubuna girerler. Dolayısı ile dinamik etkilere göre çözümleri de özel yöntemler gerektirmektedir. Bu çalışmada problem önce teorik olarak ele alınmış olup yapı dinamiğinin temel denklemlerinden hareket edilerek bir kulenin dinamik analizinin nasıl yapılacağı çeşitli kaynaklardan yararlanılarak derlenmiştir. Sonlu eleman yönteminin kullanılması ile kulenin davranışı, matris biçiminde yazılabilen, sadece zamana bağlı bir diferansiyel denklem haline gelmektedir. Bu diferansiyel denklem, bu çalışmada yapılan kabuller dolayısıyla lineer bir diferansiyel denklemdir. Bunun zamana göre çözümü ile kulenin davranışı incelenmektedir. Karşılaşılan diferansiyel denklemlerin yaklaşık çözümü için bu çalışmada SAP80 paket programı kullanılmıştır. S.AP80 paket programı, sonlu eleman yöntemi ile yapıların üç boyutlu statik ve dinamik hesabını yapan bir programdır bu program, elde edilen matris diferansiyel denklemin çözümünde mod süperpozisyonu yöntemini kullanmaktadır. Sonlu eleman yöntemi iTe yapılan çözümlerde, yapılan yaklaşık çözüm ile gerçek çözüm arasındaki yakınlık, kullanılan sonlu eleman sayısına bağlıdır. Teorik incelemeden sonra ilk önce çubuk elemanlardan oluşan bir çelik kulenin dinamik analizi yapılmıştır. Sonra da konik betonarme bir kule üzerinde uygun sonlu eleman ağının seçimi yapılarak elemen sayısını artırmanın sonuçlardaki yaklaşıklığa etkisi araştırılmıştır. Elde edilen sonuçlar değerlendirilerek bazı önerilerde bul unu 1 muştur. IV SUMMARY THE DYNAMIC ANALYSIS OF THE TOWERS AND THE APPROXIMATION OF THE SOLUTION The towers are special buildings in the civil engineering. In this study the dynamic analysis of the towers has been researched. In this study firstly the problem has been considered theoreticcal ly. The dynamic analysis of the towers are researched by using the basic equations of the dynamics of structures. After the use of the finite element method, it is obtained a differential equation in matrix form depend on the time for the behavior of the tower under the dynamic effects. It will be solved according to time. In this study, the differential equation has been thought linearly. In the later chapters, the approximate numerical solutions of the problem has been obtained by using the SAP80 program which is based on the finite element method. The modal superposition method is used in SAP80, in order to solve this matrix differential equation. In the finite element method the accuracy of the approximate solution increases with respect to the number of the finite elements. After the theoretically considering, firstly a steel truss tower has been analysed. Secondly a conical reinforced concrete tower has been analysed. 59

Soluble ACE2 (sACE2) decoys are promising agents to inhibit SARS-CoV-2, as their efficiency is unlikely to be affected by escape mutations. However, their success is limited by their relatively poor potency. To address this challenge, multimeric sACE2 consisting of SunTag or MoonTag systems is developed. These systems are extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates, perform better than the dimeric or trimeric sACE2, and exhibit greater than 100-fold neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag fused to a more potent sACE2 (v1) achieves a sub-nanomolar IC50, comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutations for variants of concern, including delta and omicron, are also neutralized efficiently with multimeric sACE2. Finally, therapeutic treatment of sACE2(v1)-MoonTag provides protection against SARS-CoV-2 infection in an in vivo mouse model. Therefore, highly potent multimeric sACE2 may offer a promising treatment approach against SARS-CoV-2 infections., Koç University Isbank Center for Infectious Diseases (KUISCID); Koç University Research Center for Transla-tional Medicine (KUTTAM)

One of the key challenges in engineering three-dimensional tissue constructs is the development of a mature microvascular network capable of supplying sufficient oxygen and nutrients to the tissue. Recent angiogenic therapeutic strategies have focused on vascularization of the constructed tissue, and its integration in vitro; these strategies typically combine regenerative cells, growth factors (GFs) with custom-designed biomaterials. However, the field needs to progress in the clinical translation of tissue engineering strategies. The article first presents a detailed description of the steps in neovascularization and the roles of extracellular matrix elements such as GFs in angiogenesis. It then delves into decellularization, cell, and GF-based strategies employed thus far for therapeutic angiogenesis, with a particularly detailed examination of different methods by which GFs are delivered in biomaterial scaffolds. Finally, interdisciplinary approaches involving advancement in biomaterials science and current state of technological development in fabrication techniques are critically evaluated, and a list of remaining challenges is presented that need to be solved for successful translation to the clinics., Scientific and Technological Research Council of Turkey (TÜBİTAK); ARDEB 1001 Scientific and Technological Research Projects Funding Program; 3501 National Young Researchers Career Development Program; Koç University Seed Fund

Terapötik peptitler yüksek bağlanma ve özgül bağ kurma yeteneleri, dokunun içerisine derinlemesine girebilme, düşük toksisite ve yapısal modifikasyonlarının kolay olması özelliklerinden dolayı ilaç tasarımı için gelecek vaat eden moleküller olmuşlardır. Bir ilaç adayı molekülün pazara girebilmesi için yaklaşık 15 senelik bir araştırma ve geliştirme süreci ile birlikte 2.6 trilyon dolarlık yatırım gerekmektedir. İlaç Tasarımı ve Geliştirme sürecinde aday molekülün incelenmesi için gerekli en önemli parametrelerden birisi aday molekülün hedefe olan bağlanma derecesidir. Bağlanma derecesi, ligand ve hedef molekül arasındaki kovalent olmayan bağların güçlülük değeridir. Bu tez çalışmaları IL1β-Reseptör proteinini bloke etmek için terapötik olarak kullanılan, insanda doğal olarak bulunan IL1-Reseptör antagonist molekülü ile aynı yapıya sahip ve bir rekombinant protein olan Anakinra yerine bilgisayar ortamında peptit tasarımına odaklanmıştır. Bilgisayar ortamında hesaplamalı olarak tasarımı yaılan peptitlerin geçerliliğini göstermek amacıyla da bağlanma derecesini ölçebilecek bir metot olan yüzey plazmon rezonans deneyleri yapılmıştır. IL1-Reseptör proteininin üç boyutlu yapısı incelenerek ve kovalent olmayan bağların türü ve sayısı dikkate alınarak bu yapıdan 9 farklı peptit dizilimi tasarlanmıştır. Bu peptitlerin bağlanma derecelerini inceleyebilmek için moleküler seviyede yerleştirme (docking) çalışmaları her bir peptit için aralarındaki bağları sabit tutarak 100 kez yapılmıştır. Arg – Ile – Trp – Asp – Val – Asn – Gln dizilimine sahip Peptit C, 7.91x10-8 M değerindeki KD değeri ile tasarlanan peptitler arasında en yüksek bağlanma derecesini göstermiştir. Peptit C'nin yüksek bağlanma derecesine sahip olduğunu teyit etmek için docking çalışmaları peptidin bağlarını sabitlemeden 999 defa yapılmış ve benzer sonuç elde edilmiştir. Peptit C'nin bağlanma derecesinin yüksekliğinin tutarlılığını gösterdikten sonra yapısal kararlılığını arttırmak amacıyla peptidin amino ve karboksil grup uçlarına modifikasyon yapılması amaçlanmıştır. N- terminal modifikasyonu Phe eklenmesi sonucunu verirken C- terminal modifikasyonu da karboksil grubu ucuna Lys aminoasidi eklenmesi sonucunu vermiştir. Bu modifikasyonlar peptit ve reseptör arasındaki hidrojen bağlarını tutarak aradaki hidrofobik etkileşimleri arttırmış ve hem N- hem de C- terminalleri modifiye olmuş 9 aminoasitli peptit 0.24x10-9 M KD değeri sonucunu vermiştir. Tasarlanan peptitlerin kopma yolunu anlamak ve bağlanma derecelerini daha doğru ve gerçekçi bir ortamda incelemek amacıyla SMD simülasyonları üç peptit için de yapılmıştır. SMD sonuçlarına göre üç peptit için de bağlanma dereceleri docking çalışmalarından alınan ortalama değerlerle tutarlılık göstermektedir. SMD simülasyonlarında ortamda su bulunduğu için peptit ve reseptör moleküllerinin arasına su molekülleri girerek etkileşimler ve bağlar arasındaki mesafelerin artmasına ve bu da bazı hidrojen bağlarının ve çoğu hidrofobik etkileşimlerin kaybolmasına neden olmaktadır. Bu nedenlerden dolayı da peptitler, docking çalışmalarından alınan en iyi sonuçlar yerine ortalama değerlere yakın bağlanma dereceleri göstermektedir. Tezin 3. bölümünde bilgisayar ortamında hesaplamalı olarak tasarlanan peptitler Fmoc kimyasının kullanıldığı Katı Hal Peptit Sentezi metoduyla sentezlenmişlerdir. Yüksek Performanslı Sıvı Kromatografi metodu da yapılan sentezi incelemek için kullanılmıştır. Modifiye olmamış peptit ilk sentezimiz olmuş olup o sentezin başarılı olduğunu göstermemize rağmen kromatografi kolonunda veya cihazın kendisinde daha sonradan oluşan bir problemden dolayı peptitlere ait standard örenklerin dahi kromatogramları elde edilememiştir. Daha sonra standard örneklerin kromatogramları başka bir cihazda aynı metodu kullanarak elde edilmiştir. Ancak TFA'nın peptidi parçalara ayırma özelliğinden dolayı bu bekleme sürecinde peptitler bozulmaya uğramış olabilir ve gelecek çalışmalara standard olarak kullandığımız peptitlerle devam edilmiştir. Son olarak, deneysel olarak peptitlerin bağlanma derecelerini ölçmek amacıyla Yüzey Plazmon Rezonans metodu kullanıldı. Biacore T200 cihazında CM5 sensör çipine amin birleştirme kimyası yoluyla IL1-Reseptör proteini yerleştirildi. Cihazın doğru çalıştığından emin olmak için IL1-β proteininin IL1-Reseptör Proteinine olan bağlanma derecesi ölçüldü ve KD değeri 2.8 nM olarak elde edildi. Literatürde de bu değerin 2.7 nM olarak elde edildiği gösterilmiştir. Peptitler için de konsantrasyona bağlılık elde edilmiş olsa da DMSO'nun hacim etkisinden dolayı bağlanma dereceleri ölçülememiştir. Peptitlerin bağlanma derecelerini ölçmek için yapılacak olan kinetik analiz deneyleri DMSO'nun hacim etkisini engellemek için çözücü doğrulama metodu kullanarak tekrar edilecektir. Therapeutic peptides have become promising due to their high affinity and specificity to the target, deep tissue penetration, low toxicity, convenience for structural modifications, and cost effectiveness. For a lead molecule to reach the market, approximately a 15-year period and an investment of around 2.6 billion dollars are necessary. One of the most important parameters to evaluate the lead compounds in the process of Drug Discovery and Development is binding affinity, which is the strength of the non-covalent interactions between the ligand and the target molecules. This thesis study focuses on in silico design of peptides to block IL1β – Receptor instead of therapeutically used recombinant protein, Anakinra, which has the same structure as the human native IL1 – Receptor antagonist molecule. In order to verify the validity of the computationally designed peptides, surface plasmon resonance methods were used to measure the binding affinity of them. 9 different peptide sequences were designed from the three-dimensional structure of IL1-Receptor Antagonist protein by considering the type and number of non-covalent interactions that form with the receptor. In order to evaluate the binding affinities of these peptides, molecular docking simulations were performed for 100 runs by fixing the bonds. Peptide C, which is Arg – Ile – Trp – Asp – Val – Asn – Gln, showed the highest binding affinity with KD of 7.91x10-8 M. The docking studies were repeated to confirm the binding affinity of Peptide C without fixing the bonds for 999 runs. Since Peptide C is consistent in terms of its binding affinity to the receptor, N- terminus and C- terminus modifications were applied to increase the conformational stability of the peptide. N- terminus modification resulted as Phe insertion to the amino group of Peptide C, while C- terminus modification requires Lys insertion. The modifications increased the number of hydrophobic interactions while also keeping hydrogen bonds between the peptide and the receptor that both N- and C- termini modified peptide has KD of 0.24x10-9 M. To understand unbinding pathway and binding affinities of our designed peptides in a more accurate and realistic environment, SMD simulations were performed for unmodified sequence which is Peptide C, N- terminus modified 8- amino acid peptide, and both N- and C- termini modified 9- amino acid peptide. The binding affinity values for the three of the peptides from SMD simulations are consistent with the average values obtained from the docking. Since water molecules are present in SMD simulations, they interfere between the peptide and the receptor molecules that the distance between the interactions increase thereby, causing the loss of some of the hydrogen bonds and most of the hydrophobic interactions. Due to this fact and realistic aspect of SMD, three of the peptides show binding affinities to the receptor at the average values of the docking results. In Chapter 3, three of the peptides obtained as a result of the computational studies were synthesized using Fmoc Solid Phase Peptide Synthesis methods. High Performance Liquid Chromatography was used to analyze the synthesis. Even though, we showed that our first synthesis which is for the unmodified peptide was successful, due to a problem occurrence either in the column or in the instrument, after a while, we even could not obtain the chromatograms of the standard samples of the peptides. The chromatograms of the standard samples were obtained using a different instrument. However, due to TFA fragmentation of the peptides, our synthesis products might have corrupted that further studies continued with the custom synthesis peptides.Finally, to validate the binding affinity of the peptides experimentally, Surface Plasmon Resonance (SPR) method was used. In Biacore T200 instrument, CM5 sensor chip surface was immobilized with IL1-Receptor using amine coupling chemistry. To verify the accuracy of the instrument, binding affinity of IL1-β to IL1-Receptor was measured performing kinetics analysis. Concentration dependency was obtained and KD was found out as 2.8 nM which is a consistent result with the literature value, 2.7 nM. Although concentration dependency was also obtained for the peptides, binding affinity values could not be obtained yet due to the bulk effect of DMSO. The kinetics analysis to measure binding affinity of the peptides will be repeated by performing solvent correction prior to the analysis to prevent bulk effect of DMSO. 77

Summary Cells receive time-varying signals from the environment and generate functional responses by secreting their own signaling molecules. Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems. We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically. Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs. Employing this system to analyze macrophage signal processing under pathogen inputs, we found that the dynamics of TNF secretion are highly heterogeneous and surprisingly uncorrelated with the dynamics of NF-κB, the transcription factor controlling TNF production. Computational modeling of the LPS/TLR4 pathway shows that post-transcriptional regulation by TRIF is a key determinant of noisy and uncorrelated TNF secretion dynamics in single macrophages., Graphical Abstract, Highlights • Dynamic stimulation of single immune cells with a versatile microfluidic device • Coupled longitudinal measurements of NF-κB localization and TNF secretion on the same cell • Single-cell harvesting, staining, and mRNA quantification on the same device • High-content dataset, and modeling of TRIF-based noise in TNF secretion, Junkin et al. have developed a method to quantitatively probe single-cell input-output dynamics with an automated microfluidic system. They conduct coupled measurements of transcription factor and cytokine secretion dynamics from the same single cells to enable modeling, which uncovers dynamic and noise-based roles of TRIF in the NF-κB-TNF pathway.

Dynamical analysis of single-cells allows assessment of the extent and role of cell-to-cell variability, however traditional dish-and-pipette techniques have hindered single-cell analysis in quantitative biology. We developed an automated microfluidic cell culture system that generates stable diffusion-based chemokine gradients, where cells can be placed in predetermined positions, monitored via single-cell time-lapse microscopy, and subsequently be retrieved based on their migration speed and directionality for further off-chip gene expression analysis, constituting a powerful platform for multiparameter quantitative studies of single-cell chemotaxis. Using this system we studied CXCL12-directed migration of individual human primary T cells. Spatiotemporally deterministic retrieval of T cell subsets in relation to their migration speed, and subsequent analysis with microfluidic droplet digital-PCR showed that the expression level of CXCR4 – the receptor of CXCL12 – underlies enhanced human T cell chemotaxis.

We report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step decamer using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-L-phenylalanine (pAzF) or pp-ropargyloxy-L-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were monomer coupled by copper-catalyzed azide alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained >= 63% of their specific activity (i.e., fluorescence) after coupling. Polymerization of a concentrated solution of triply substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or copolymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated kat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4 x 10(-5) s(-1). Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450 +/- 20 mu g/ml CAT109pAzF and 428 +/- 2 mu g/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe. (C) 2012 Elsevier Inc. All rights reserved.

Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells.

Cell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school laboratory course. Ninety students in a biotechnology class used CFPS to study the effects of the concentrations of amino acids, cell extract, DNA, and the energy source on accumulation of active super-folder green fluorescent protein. Students estimated product concentrations simply by comparing solution colors to a printed green color gradient. This simple and inexpensive method allows for immediate measurements, and 26 of the 30 groups observed measurable product concentrations within 60 min. These student-generated data were then discussed to illustrate concepts of data analysis such as outliers and standard deviation. We also combined the laboratory experience with a visit to a university campus that included a laboratory tour and a college-style lecture. Our overall objective was to excite the students about the scientific enterprise and to instill a sense of personal relevance and attainability so that these students could realistically consider technical careers.

The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins. (C) 2012 Elsevier Inc. All rights reserved.