Moleküler Biyoloji-genetik ve Biyoteknoloji Anabilim Dalı (disiplinlerarası)

İstanbul Technical University · Fen Bilimleri Enstitüsü

Construction and characterization of solid-supported lipid bilayers to investigate cell-surface interactions

Investigation of cell-cell/biomolecule and cell-surface interactions are essential to understand basic mechanisms such as cell signaling, attachment, proliferation and differentiation. Getting insight to these mechanisms also elucidates the topics such as the development and improvement of effective medical systems. Supported lipid bilayers (SLBs) when functionalized with bioactive molecules that are responsible for specific biological interactions, allow long-term experiments for many practical applications such as investigation of the basic features of biological membranes, construction of biosensors, drug screening platforms, cancer cell detection platforms and investigation of the cell–cell/biomolecule, cell-surface interactions. These biomimetic lipid bilayer platforms allow the usage of surface-sensitive characterization techniques such as atomic force spectroscopy (AFM), surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). The aim of the thesis is to construct functional SLB platform to investigate cell-surface interactions and to monitor whole process in real time via QCM-D system. In this way, it is possible to investigate changes in cell-surface interactions (surface characteristics, cell type and time dependent) that can not be distinguished under the microscope. SLB surfaces were functionalized with RGD, and osteocalcin mimetic (OSN) peptides and their interaction with osteoblast cells were evaluated. RGD is the binding motif of various extracellular matrix (ECM) proteins so it is expected to promote cell adhesion whereas OSN is known to promote biomineralization rather than cell attachment so chosen as control. Similar platforms were used in parallel in cell culture environment for comparison. The first part of the thesis focused on the construction of an SLB platform in single step. Most of the model lipid membrane studies on gold-coated sensors involve the usage of various surface-modification strategies to rupture liposomes and induce lipid bilayer formation since liposomes with polar surfaces do not interact with bare, hydrophobic gold. For this purpose, it was aimed to form SLB on gold sensor surfaces without further modification by incorporating a thiol-modified phospholipid, 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (DPPTE) into phosphatidylcholine (PC) based liposomes. The binding kinetics of liposomes with different DPPTE ratio (0.01 to 100% mol/mol) and diameters were monitored by QCM-D. The dissipation change per frequency change, i.e., acoustic ratio, which is evaluated as a degree of the viscoelasticity, considerably decreased with the presence of DPPTE (from 162.3 GHz−1 for flattened PC liposomes to ca. 89.5 GHz−1 for 100% DPPTE liposomes) when compared to the results of two reference rigid monolayers and two viscoelastic layers. To assess the quality of SLB platform, the interpretation of QCM-D data was also complemented with Surface Plasmon Resonance. While QCM-D is sensitive to the liquid content within the layers, SPR provides liquid-independent results and thus the results of these techniques are compared to make more realistic interpretations. The optimum thiolated-lipid ratio (1%, lower thiol ratio and higher rigidity) was then used to determine the dry-lipid mass deposition, the water content and the thickness values of the SLB via viscoelastic modelling. Further, surface characterization studies were performed by Atomic Force Microscopy with high spatial resolution. The results suggested that model membrane was almost continuous with minimum defects but showed more dissipative/soft nature compared to an ideal bilayer due to partially fused liposomes/overlapped lipid bilayers/multilayer islands. These local elevations distorted the planarity and led the increase of overall membrane thickness to ∼7.0 nm. However, the construction of SLB on gold in a single step has been successfully accomplished. In the second part of the thesis, the aim was to obtain insight about how to evaluate cell – surface interactions in QCM-D system. QCM-D is one of the powerful techniques, which allow real time, quantitative and noninvasive analysis of the interaction of different cell types with various modified surfaces. For this purpose, different surfaces were prepared without using SLB system and their interaction with the cells for 18 h were interpreted. First, the dynamic adhesion behavior of human fetal osteoblastic bone (hfOB) cell lines was monitored on untreated and hydrophilically treated gold sensor surfaces as reference substrates. Adhesion was also observed under light microscopy to facilitate the evaluation. Cells increased their surface contact area and spread more on hydrophilic surfaces, and showed distinct profile with an increased rigidity at the cell-surface interfacial layer, which is assigned to extracellular matrix remodeling. Further, the adhesion strength and kinetics were characterized on cell adhesive (poly-L-lysine and fibronectin) and repellent (bovine serum albumin) surfaces. The overall results indicated that protein-mediated specific interactions contributed mostly to the dissipation changes (ΔD) or acoustic ratio (ΔD/Δf). Finally, the potential of QCM-D to distinguish healthy and cancerous cells were evaluated by comparing the results of hfOB cells with that of SaOS-2 (osteosarcoma) cancerous cells. Cancerous cells interacted more strongly with the surfaces and showed more viscoelastic characteristic than the healthy cells. In the final part of the thesis, SLB activation and functionalization with RGD and OSN peptides and examination of the interactions of these systems with cells via QCM-D were aimed. First, the stability of lipid bilayers in various medium conditions were evaluated. The structural integrity was conserved until fifth day in PBS, but hole formations started to form from the first day and increase their number and size in time under serum-supplemented medium conditions. SLBs enriched with different lipids (cholesterol, PS, DOTAP) increased the stability of SLB to pure PC. Lateral mobility of the lipid bilayer was also demonstrated via FRAP (Fluorescence Recovery after Photobleaching) method. After the SLB characterization for both stability and fluidity, the kinetics of peptide attachment to SLBs were compared to their nonspecific adsorption onto gold surfaces and the characteristic of resultant layers were analyzed via QCM-D. The results indicated that conjugation to SLB provide higher structural flexibility than passive adsorption. When cells were incubated with these surfaces, it was seen that low density of mobile RGD peptides on SLB platforms preserved their biological activity and promoted cell adhesion more efficiently than high number of immobile peptides on the gold surfaces. Cell attachment and spread on RGD-fSLB surfaces were also confirmed in cell culture. Limited number of cells were attached on bare SLB surface and they remained in rounded morphology. Cells did not spread well on OSN-coated surfaces, and acoustic ratio measured on each OSN layers probably did not originate from specific adhesion of the cells, instead the negative charge of OSN could provide electrostatic interactions between surface and biomolecules on the cell membrane, thus promoting nonspecific protein and cell attachment. In conclusion, nonfouling SLB surfaces were successfully functionalized and constructed fSLB platforms were shown to have the potential for real time and label-free monitoring of the specific cell – surface interactions via QCM-D.

Investigation of micrornas that interact with the NFIX 3′untranslated region

Nuclear factor I (NFI) transcription factor family controls many important cellular and developmental events in the embryo and the adult. This family is made up of four members in vertebrates: NFIA, NFIB, NFIC and NFIX. NFIs carry an N-terminal DNA binding-dimerization and a C-terminal transcriptional activation and repression domain which includes a proline-rich region. The N-terminal DNA binding and dimerization domain is highly conserved among NFI members, while the C-terminal domain varies. There are four conserved cysteine residues in the NFI N-terminal domain, one of which is sensitive to oxidative inactivation while the other cysteine residues are necessary for NFI DNA binding capacity. NFIs bind as homo- or heterodimers to the TTGGC(N5)GCCAA consensus sequence on duplex DNA. NFIs can regulate gene expression in a cell and tissue specific manner by various mechanisms: interaction with basal transcription factors, co-regulation with other transcription factors, binding to co-activators or co-repressors, alteration of the nucleosome structure and modification of the methylation pattern near the promoter of the target gene. NFIA, NFIB and NFIX are expressed in neural progenitors, neurons and glia of the central nervous system during embryonic development, however expression of these NFI members is restricted to stem cells in the adult brain. NFIs regulate processes such as neurogenesis and gliogenesis in different brain regions in the embryo and the survival of progenitor cells in adults. Nfia, Nfib and Nfix null mice demonstrate enlarged ventricles in the cerebral cortex, indicating misregulation of differentiation and neural progenitors. In the cerebellum, NFIs are required for neuronal migration and terminal differentiation and in cerebellar granule neurons they regulate the expression of molecules that are associated with these processes such as the extracellular signaling molecule (Wnt), cell adhesion molecules (Ephrin-B1, N-cadherin and Tag1). Nfix promotes neurogenesis by repressing of Sox9, a transcription factor involved in stem cell self-renewal in hippocampus. In Nfix null post-natal mice, progenitor cell specific markers (Pax6, Hes1, Hes5 and Sox2) are upregulated indicating expansion of progenitors while neuronal and glial differentiation is disrupted as well. In addition, Nfix also regulates neuronal migration, and one of its targets is a neurotropic factor (Gdnf) that promotes migration of progenitors in the olfactory bulb. In the spinal cord, Nfia and Nfib both induce Gfap expression, an astrocyte differentiation marker, Nfib also induces Nfix expression. Nfix, in turn, regulates astrocyte terminal differentiation by upregulation of Gfap and other astrocyte specific genes. In addition to its role in development, Nfix also involved in the regulation of stem cell quiescence and neurogenesis in the adult hippocampus. Interestingly, in cell culture, Nfix is upregulated in quiescent stem cells and is necessary and sufficient to induce quiescence. On the other hand, conditional knock-out experiments show that Nfix promotes neuroblast differentiation and represses oligodendrogenesis inhibiting expression of oligodendrocyte specific genes (Olig1, Olig2, Ptprz1 and Ntrk2) in vivo. Moreover, Nfix deletion in mice leads to fewer neurons and disrupted dendritic formation and process branching. Therefore, NFIX may have a dual role in adult neurogenesis: preserving stem cell quiescence and cell fate determination to neurons and astrocytes. Previously, we found that NFIX expression is downregulated in differentiating neural stem cells in vitro. In order to identify novel upstream regulators of NFIX, we first analyzed miRNA binding sites on the NFIX 3'UTR. We selected miRNAs which are highly expressed in neural progenitor cells for further analysis: miR25, miR92b, miR124 and miR363. Additionally, miR1915 which was reported to supress NFIX is selected as positive control. NFIX 3'UTR fragments which contain binding sites of these miRNAs were isolated, fragment I (nts ~1300-2600) was inserted in the downstream of the luciferase reporter gene. Then, supression of reporter gene translation by miRNA mimics was assayed. miR25 and miR363 repress luciferase activity from the NFIX 3'UTR I, while preliminary data indicate that miR92b and miR124 did not have such an effect. Interestingly, miR1915 could not repress translation of reporter genes fused to NFIX 3'UTR I. However, the activity of this mimic has not been validated yet. We are in the process of mutating the miR25 and miR363 binding sites on the NFIX 3'UTR to ascertain the specificity of these interactions. Future studies will explore the physiological significance of potential supression of NFIX by these miRNAs.

Ece Çağdaş

2018

İstanbul Technical University · Fen Bilimleri Enstitüsü

Bag-1L'in ubikuitin/proteazom sisteminde rolünün meme kanserinde incelenmesi

An anti-apoptotic protein BAG-1, with its adaptor function, can interact with a diverse array of molecular targets such as Hsp70/Hsc70 chaperones, proteasome machinery, Bcl-2, Raf-1 kinase, nuclear hormone receptors and DNA, and is found to involve in the regulation of a wide variety of cellular processes. In human cells, three functionally distinct and differentially localized BAG-1 isoforms originating from a single mRNA are expressed through alternative translation and these three isoforms include BAG-1S (33 kDa), BAG-1M (46 kDa) and BAG-1L (52 kDa), which have distinct N-terminus. BAG-1L, which possess a nuclear localization signal is a largely nuclear protein, whereas BAG-1M partitions between nucleus and cytoplasm. BAG-1 with its isoforms is a candidate protein to form hubs to transmit signals for survival. It has been reported that relatively high BAG-1L levels are observed in tumor cells. Protein folding process occurs in the endoplasmic reticulum. This folding process involves heat shock proteins, lectin proteins, disulfide isomerases present in the ER. In protein folding, N-glycosylation occurs which increases protein stability. N-linked glycans increase protein solubility and directly affect protein folding pathways. Calnexin (ER membrane bound) and Calreticulin (ER lumen) are lectin chaperones and support protein folding. Lectin chaperones connect to the last glucose and then control the folding process. In addition, lectin chaperones interact non-covalently with oxidoreductase PDI (ERp57), thus triggering disulfide bond formation and isomerization on the substrate. PDI is a member of the disulfide isomerase protein family and is found in the ER lumen. The formation of disulfide bonds limits the conformational flexibility of the proteins and increases the structural stiffness of the proteins. Despite the quality control mechanism in ER, when protein folds are inadequate and the protein can not fold properly, aberrant proteins accumulate in the ER. Therefore, the activation of the unfolded protein response (UPR) occurs. Through ERAD pathway, which is part of the UPR system, abnormal protein retrotranslation from the ER to the cytosol occurs. The ERAD pathway consists of a series of processes that mediate the retrotranslocation of selected aberrant proteins and the ubiquitin-dependent proteasomal degradation. The energy that drives substrate dislocation is derived from adenosine triphosphate (ATP) hydrolysis by the abundant, ubiquitin-dependent hexameric AAA+ ATPase VCP (p97). VCP is recruited to ERAD dislocation complexes through direct interactions with several ERAD components. ERAD substrate polyubiquitination functions as a binding site that facilitates extraction by VCP and acts as the signal for proteasomal degradation. The addition of ubiquitin occurs via sequential enzymatic reactions involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase that mediates the final transfer of ubiquitin to the substrate. CHIP (E3 ligase) is part of a system that directs misfolded proteins from chaperones to proteasomes. Two modulators, BAG-1 and CHIP, have implicated in cytosolic degradation pathways, xxii putting molecular chaperones at the center of the decision process between folding and degradation of non-native polypeptides. The ubiquitin/proteasome system, the interaction partner of BAG-1, degrades misfolded or unfolded proteins. Investigating the effect of BAG-1 on the estrogen receptor has found that BAG-1L promotes estrogen-dependent transcription but BAG-1M or BAG-1S do not increase transcription of ER. BAG-1L interacts with estrogen receptors and enhances receptors activity. Studies on proteasome have shown that toxic effects of proteasome inhibition in cancer cells are more sensitive than normal cells. Although the mechanism behind the higher susceptibility of cancer cells is not known, there is a high probability of using the proteasome to regulate proliferation and anti-apoptotic pathways. BAG-1, an adaptor protein in the ubiquitin/proteasome system, can be used as a drug target in cancer due to cell survival effect. Here, we adopted a proteome-based strategy comprising isolation of BAG-1L associated complexes from breast cancer and epithelial cell lines as well as breast tissues. Identification of the proteins involved in BAG-1L associated complexes to ultimately delineate the adaptor role of BAG-1L in breast cancer. Proteins involved in BAG-1L associated complexes were identified via LC-MS/MS analysis with an BAG-1L isoform-specific manner. Further analyses with bioinformatics tools hinted the involvement of BAG-1L isoform in protein quality control system, which includes the action of endoplasmic reticulum-associated degradation (ERAD) mechanism to maintain intracellular homeostasis of cellular proteins. Interaction of BAG-1L isoform with the proteasome were poorly characterized, detailed understanding of the involvement of BAG-1L isoforms in the ubiquitin mediated proteasomal degradation is lacking. Determined interactions which includes specifically VCP/p97 and Rad23, components of proteasome machinery, set a critical role for BAG-1 in the ubiquitin-dependent proteasomal degradation.

Sevilay Acar

2018

İstanbul Technical University · Fen Bilimleri Enstitüsü

Nuclear factor one (NFI) family of transcription factors regulates gene expression during embryonic development and in the adult stem cell niches. NFI family comprises four members in vertebrates: NFIA, NFIB, NFIC and NFIX. NFIs bind to the consensus sequence, TTGGC(N5)GCCAA on double-stranded DNA as homodimers or heterodimers. Members of NFI family contain an N-terminal DNA binding and dimerization domain and a C-terminal transcriptional activation and repression domain. The N-terminal domain is highly conserved among members and includes a MH1 motif also found in SMAD proteins of the TGF- signaling pathway. Proline rich C-terminal domain is less conserved and may be involved in differential protein-protein interactions between NFIs and nuclear proteins. Alternative splicing generates multiple isoforms of each member. Expression of NFIA, NFIB and NFIX is required for normal development of central nervous system, and loss of each one leads to severe defects in brain development. The fact that NFIA, NFIB and NFIX knock-out mice display distinct phenotypes suggests that each NFI has a distinct function in the brain. Interactions with different regulatory proteins may account for this specificity. Several proteins have been found to interact with NFI family members, including the transcription factors FOXA1, Sox9, Olig2. However, proteins that bind NFI and regulate NFI function in the developing brain have not been studied well. In order to identify proteins that may interact with NFIB in the developing central nervous system, yeast two-hybrid (Y2H) screen of a human fetal brain cDNA library was previously performed. PIN1 and LMO4 were identified as putative NFIB binding partners. PIN1 belongs to the peptidyl-prolyl isomerase family which catalyzes cis-trans isomerization of peptidyl prolyl bonds. PIN1 recognizes phosphorylated serine or threonine residues in pSer/Thr-Pro peptide sequences on target proteins. Cis-trans isomerization by PIN1 changes conformation of target proteins and affects their function. LMO4 belongs to the LIM-Only (LMO) subfamily of LIM proteins and contains two tandem LIM domains. LMO4 binds a variety of proteins via its LIM domain and acts as an adaptor that links proteins, including transcription factors, to form multiprotein complexes. In the developing brain, PIN1 and LMO4 are expressed in the right location to interact with NFIs: PIN1 is highly expressed within the ventricular zone and the subventricular zone in the cortex. LMO4 is also expressed within both ventricular zone and subventricular zone, the neurogenic regions of the cortex. Moreover, phenotypes of PIN1 and LMO4 null mice show that these proteins are involved in neural development: In the absence of PIN1 development of late-born neurons is impaired in the cerebral cortex. Absence of LMO4 leads to neural tube closure defects in the dorsal brain and defects in cortical neurogenesis affecting both early and late-born neurons. xxiv In this study, we investigated interactions between NFIs and two putative NFIB binding partners, PIN1 and LMO4. To this end, epitope tagged PIN1 or LMO4 and NFIs were co-expressed and binding was assayed via co-immunoprecipitation experiments. We found that each NFI member and PIN1 could be co-immunoprecipitated with each other. GST pull-down assays confirmed the interactions detected in co-immunoprecipitation experiments, bacterially expressed GST-PIN1 pulled down each NFI members. To identify NFI domains required for interaction with PIN1, we created NFIB mutants that contain only the N-terminal DNA binding and dimerization or the C-terminal transactivation domains. GST pull-down experiments with these mutants showed that NFIB C-terminal domain is not involved in PIN1 interactions while the N-terminal domain is required for PIN1 binding. Site-directed mutagenesis of conserved serine residues in pSer/Thr-Pro motifs in the NFIB C-terminal domain did not interfere with NFIB-PIN1 binding, in either co-immunprecipitation or GST pull down assays consistent with the finding that NFI N-terminal domain is necessary for NFI-PIN1 interactions. On the other hand, preliminary data from co-immunoprecipitation and GST pull down experiments indicate that only NFIX interacts with LMO4. We tested the NFIB mutant that lacks the C-terminal domain for LMO4 binding via co-immunoprecipitation assays and found that it did not interact with LMO4. As NFI members differ in their ability to bind LMO4, these data suggest that the C-terminal domain of NFIX is involved in LMO4 interactions. Further site-directed mutagenesis of NFIB and NFIX can be used to pinpoint the exact regions required for PIN1 and LMO4 binding.

Neurons

Sinem Sarıtaş

İstanbul Technical University · Institute of Graduate Studies in Science

Generation and validation of genome editing tools for mouse CatSper β & Ɣ using CRISPR/Cas9 and split-EGFP systems

Mammalian sperms swim through convoluted female reproductive system and interact with the eggs in the fallopian tube. Sperm cells have the capacity to fertilize eggs but they cannot achieve this until they go through capatitation acrosome reaction. During capacitation, plasma membrane, permeability and motility patterns and flagellar activity change and Ca2+ is increased that causes hyperactivation, an asymmetric whip-like movement of the flagellum. This entry of Ca2+ is crucial for sperm hyperactivation and Ca2+ ions during this reaction flows through a flagellar specific ion channel called as Cation channel of sperm (CatSper). CatSper channel is a Ca2+ selective ion channel and maintains the Ca2+ entry into sperm cells. CatSper channel is comprised of CatSpers1–4, CatSper β (beta), CatSper γ (gamma) and CatSper δ (delta) and the loss of these genes in mice causes infertility. CatSper channel is localized at the principal piece of the sperm flagellum CatSper 1–4 mutations show no CatSper current detection, indicating all α subunits are required for the functional CatSper channel complex. It is shown that CatSper δ null mutants are infertile and they lose hyperactivated motility and Ca2+ current in the spermatozoa. The loss of CatSper δ also reduces CatSper1 detection, showing that CatSper δ auxiliary subunit is crucial and responsible for the formation of the CatSper channel assembly. Recombinant DNA technology provides the power to modify DNA molecules at a precise and targeted fashion within a cell, offering the ability to understand the gene functions, develop innovative therapeutic drugs, and biotechnological products. With the new developments in genomic engineering, in theory, it is now possible to directly edit or regulate the functions of DNA sequences in their endogenous conditions for any organism. Sequence specific DNA nucleases can be programmed to edit any endogenous genomic sequences with high precision and efficacy at a predetermined locus in a broad range of organisms and cell types, thus effectively promoting genome editing. They can either nick single strand DNA or cleave both strands of DNA either within or nearby the targeted sequence in a non-specific manner since they have sequence specific DNA binding domains bound to a nuclease. These nucleases have sequence-specific nuclase domains that can cut DNA strands within or out of the targeted locus by nicking one strand or cutting the double strands on DNA molecules. Cells activate of of two repair mechanisms when the DNA is damaged. They can activate either non-holomogous end joining (NHEJ) or homologous recombination (HR). NHEJ can introduce indels, thus causing frame-shift mutations or knock genes out of function. In HDR repair, cells can repair the damage in by recombining donor DNA as homologous to the targeted site with explicit genomic modifications. Recent developments in genome editing has lead to a revolutionizing Clustered, Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system as it allows gene manipulations in many cell lines and organisms including human. CRISPR is an immune system for bacteria and archaea. These organisms detect and eliminate foreign DNA and viral phages with its CRISPR accociated gene product, Cas9 and add small fragments into their CRISPR cluster. Bu using this ability of Cas9, CRISPR/Cas9 gene editing system is developed. Cas9 has a recognition and nuclease site. Both domains of the nuclease cuts different DNA strands. Cas) recognizes ~20 bp sequence with NGG at the beginning of the sequence. By using this 20 nucleotide sequence with NGG and guide RNA with Cas9, almost any sequence beginning with NGG can be targeted and edited. In our study, we used pCAG-EGxxFP and pX330 plasmids containing CatSper ion channel auxiliary subunits CatSper β ve CatSper γ gene fragments and co-transfected them together in HEK293 cells to express EGFP gene. We cloned CatSper β ve CatSper γ gene fragments individually into pX330 to obtain pX330-β ve pX330-γ constructs. pX330 contains Cas9 gene and single guide RNA. We also used pCAG-EGxxFP plasmid which has CMV promoter and EGFP genes. This plasmid has 482 bp homology sequence with 5` and 3` EGFP fragments and placed under ubiquitous CAG promoter. The vector is designed that we can insert out targed sequence into EGFP gene. We cloned CatSper β ve CatSper γ gene fragments into pCAGxxFP individually to obtain pCAG-EG-β-FP and pCAG-EG-γ-FP constructs. Our aim is to co-transfect pX330-β with pCAG-EG-β-FP and pX330-γ with pCAG-EG-γ-FP into HEK293 cells to check whether guide RNAs containing CatSper β ve CatSper γ fragments can bind to targeted gene fragments in pCAG-EG-β-FP and pCAG-EG-γ-FP constructs, thus cleaving the targeted sequences with the expressed Cas9 enzyme. If Cas9 cleaves the DNA fragments, pCAGxxFP plasmid will reconstitute the EGFP and give green fluoresence. Therefore, we transfected pX330-β, pCAG-EG-β-FP, pX330-γ and pCAG-EG-γ-FP constructs individually as negative controls into HEK293 cells. And we also co-transfected pX330-β with pCAG-EG-β-FP and pX330-γ with pCAG-EG-γ-FP into HEK293 cells and incubated them at 37 °C for 24 hours. When we checked HEK293 cells under confocal microscope, we observed green flourescence in co-transfected constructs but not in individually transfected constructs. This indicates that single guide RNAs recognized CatSper β and CatSper γ gene fragments and Cas9 enzyme cleaved the targeted sequences. With this study, we demonstared that our designed CRISPR/Cas9 constructs are working in HEK293 cell lines.

Yusuf İşeri

İstanbul Technical University · Institute of Graduate Studies in Science

Optimization of cultivation conditions and engineering by random mutagenesis for high lipid production in Schizochytrium sp. S31

Schizochytrium sp. is widely studied microalgae to obtain high content of polyunsaturated fatty acids especially docosahexaenoic acid (DHA). They contain various bioactive compounds that can be used as pharmaceutical raw material, food additive, aquaculture and animal feed. They also play a crucial role in biofuel production which can be used as an alternative energy source. Schizochytrium sp. which is grown rapidly and produced high amount of DHA can be used as an alternative to fish oils. In this study, cultivation conditions were optimized to increase biomass and total lipid productivity of the Schizochytrium sp. S31 by using response surface methodology. After optimizing growth conditions, large scale production of Schizochytrium sp. S31 under favorable conditions were performed and downstream process including cell lysis, drying and lipid extraction were compared and optimized to maximize total lipid specially DHA extracted from Schizochytrium sp. S31. Finally, random mutation was applied by ultraviolet (UV) radiation or chemical mutagen and high yield of lipid accumulating mutants of a Schizochytrium sp. were selected by flow cytometric-based selection. Many factors may affect the cultivation conditions of Schizochytrium sp. such as medium composition, pH, salinity and temperature. Since polyunsaturated fatty acids have gained significance due to their role in human health, industrial and commercial usage, research on enhancement of biomass and lipid production has increased. Therefore, optimization of medium composition and environmental conditions to improve biomass and total lipid content of the organism is important. Applying a statistical strategy was an effective tool for optimization of the production and extraction process, which would also reduce the production costs through maximizing the yield. Statistical methodologies such as Plackett burman and central composite design have been extensively used to optimize several factors and their interactions. Plackett burman design was used recently as the first step in optimizing different bioprocesses to identify the factors with a significant effect on desired responses. Following the selection of the most significant factors, response surface methodology with central composite design is used to determine the optimum values of these factors. Based on contour plots and canonical analysis, a maximum biomass production of 26.86g·L-1 was obtained with 2.29 g.L-1 monosodium glutamate (MSG), pH 5.8 cultivation conditions. Maximum lipid production of 35% was obtained with 0.49 g.L- 1 MSG at 17.6 °C temperature conditions. Schizochytrium sp. S31 can be grown heterotrophically in bioreactor by supplying with alternative raw materials such as sugars, organic acids and alcohols. Thus, heterotrophic systems assure a cost effective way to obtain valuable microalgae produced DHA by using cheap substrates on large scale. Large scale heterotrophic growth technologies has lower contamination risk, lower harvesting costs, eliminated light limitation, high degree of process control and reproducible. In most instances, heterotrophic culture commonly results in much higher cell densities and lipid productivity than phototrophic culture because there is no light limitation in heterotrophic culture and controllable. According to large scale production results, 65 g/l of cell dry weight with the initial growth rate of 0.312 h-1 was obtained. C/N ratio of the Schizochytrium sp. culture was calculated as 19.32. The total lipid content was 27.5 % of the cell dry weight after applying optimized conditions. DHA percentage of the Schizochytrium sp. culture in 5 liter was calculated as 30.18%. Biomass, lipid and DHA productivity was calculated as 0.65 g/l.h, 0.17g/l.h and 0.053g/l.h respectively. Downstream processing steps required to obtain lipid from microalgal biomass once large scale production process is completed. Downstream process include harvesting, dewatering, cell disruption, lipid extraction. Harvesting microalgal culture includes centrifugation, filtration, and flocculation methods to concentrate microalgal culture. Drying step is known as post-dewatering step in which the cell pellet is completely dewatered. The cell pellet is exposed drying process for enhancing the efficiency of subsequent lipid extraction and lowering the cost of downstream process. Cell disruption is a key step in influencing lipid extraction yields. Sonication, high-pressure homogenizers, grinding, enzymatic reactions, chemical hydrolysis are the most known cell disruption methods applied for microalgae. Different types of solvents and extraction methods have been used in the literature to recover microalgal lipids. Hexane, methanol, ethanol, isopropanol are the typical solvents used for lipid extraction. According to the cell lysis and lipid extraction results, ultrasonication with hexane method increased the total lipid yield significantly with clear appearance. Sonication resulted in a 1.4-fold increase in lipid yield when compared with solvent alone. However, these traditional lipid extraction methods use large amounts of solvents that are mostly toxic. Supercritical liquid extraction (SFE) generally uses carbondioxide as a solvent at high pressure to extract lipid and/or nutraceutical products with higher selectivity in shorter extraction times. The effect of extraction temperature, pressure and time of SFE on the lipid yield and %DHA amount were investigated by using RSM. According to SFE results, pressure and temperature have significant effect (p <0.05) on total lipid yield and DHA concentration. Based on contour plot analysis, optimum extraction conditions were found to be 425 bar pressure at 40.5°C for 97.5min. After optimization of pressure, temperature and time variables of SFE, 30.2% lipid yield was obtained. Isolation of high yield lipid accumulating mutants of microalgae by flow cytometricbased selection can be performed by introducing mutations in the genome. There are mainly two mutation agents for random mutagenesis: UV radiation and chemical mutagenizing agent. Both methods do not require any genetic information about microalgae. Fluorescent activated cell sorting is a high throughput technique for selection of mutants with desired phenotype. This study highlighted that UV mutagenesis and high throughput selection improved lipid productivity in Schizochytrium sp. S31. Nutrient limitation conditions, such as nitrogen (N), phosphorus (P) starvation, and temperature limitation induced lipid accumulation in Schizochytrium sp. According to characterization results, mutants which were treated to UV for 30 second accumulate lipid faster than wild type. Time course experiment were conducted to understand the lipid accumulation profile difference between mutants and wild type. Total lipid was increased by 28.4 % for Mutant1 and 10.8% for Mutant2 comparing to wild type. The results obtained from gas chromatography mass spectrometry analyses confirmed the results obtained by flow cytometry, showing an increase in DHA in UV treated cultures (30sec) compared to untreated controls. There was a 17.9% and 12.1% increase compare to wild type in terms of DHA percentage in two different mutants respectively.

Nurcan Vardar

İstanbul Technical University · Fen Bilimleri Enstitüsü

Construction of a functional biodegradable bone tissue engineering scaffold for enhanced biomineralization

In case of accidents, injuries or diseases, a various tissue deformations may occur. Tissue regenerations can take a long time and sometimes recovery is not possible completely. The metals, polymers and ceramics are widely used in biomedical field to assist the body to address these problems. Tissue engineering aims to produce tissues by mimicing natural structures with using natural and synthetic polymeric scaffolds. This study aims to obtain composite scaffolds for bone tissue regeneration using silk fibroin and poly(l-lactide) as natural and synthetic polymers. These scaffolds were produced by freeze drying method and insulation cover was used to observe the effect of freezing conditions on properties of scaffolds. After lyophilization process, the scaffolds were treated with methanol to induce beta sheet regions from random coil structures in silk fibroin protein. To induce biomineralization, various peptides can be used. In this study, OSN peptide, which is obtained from osteocalcin protein, was used to promote hydroxyapatite (HAp) crystallization. The scaffolds were characterized with water uptake, biodegradation, biomineralization and cell proliferation tests. The water uptake capacities of scaffolds were found to be higher than 400% for both SF:PLLA (1:3 w/w) and SF:PLLA (3:1 w/w) scaffolds. The samples with high silk fibroin ratio had higher water uptake capacity (almost two fold) compared to other ones. The degradation analysis was carried out for two month. The protease XIV enyzme (0.1U/mL) was used to see its effect on fibroin degradation. After two months, the remaining weight of SF:PLLA (1:3 w/w) and SF:PLLA (3:1 w/w) scaffolds were higher than 50% with and without enzyme. As expected, sample with high SF showed more degradation in the presence of protease enzyme. The effect of methanol treatment on formation of beta sheet structures from random coils were investigated with FTIR analysis by observing shifted peaks at amide I and amide II regions for SF:PLLA (1:3 w/w) and only amide I region for SF:PLLA (3:1 w/w) scaffolds. After methanol treatment, the insoluble structures were obtained with increasing beta sheet formations in fibroin proteins and used for further analysis. The mineralization process was followed for a month and weight changes of scaffolds were reported. Two events simultaneously occur in solution: The formation of apatite crystals and degradation of polymers and weight of scaffolds was affected from both. The scaffolds that were incubated in 3xmSBF solution showed weight loss at the end of a month. On the other hand, the scaffolds incubated in 1xmSBF showed weight gains after a month. The effect of OSN peptides could not be clearly observed from weight change data. The SEM images were taken and the atomic ratios of Ca/P were determined by EDS after 2, 3 and 4th weeks of mineralization process. According to the SEM images, mineral formations can be observed after 2nd week in scaffolds with and without peptides in 1xmSBF and 3xmSBF solutions. The Ca/P ratios were calculated in scaffolds incubated in 1xmSBF solution. The calcification appeared because of the more calcium accumulation after 3rd and 4th weeks of mineralization. The Ca/P ratios of SF:PLLA (3:1 w/w) scaffolds with peptides after 4th week of mineralization and SF:PLLA (1:3 w/w) scaffolds with peptides after 2nd week of mineralization were found to be closer to the stoichiometric value of HAp (1.67). The FTIR spectra were obtained for both scaffolds without peptides incubated in 3xmSBF solution. The presence of characteristic groups of PLLA made diffucult to distinguish the groups of HAp crystals. The few absorption peaks shifted because of the apatite growth on polymer surfaces. The CO32-, PO43-, type A and B carbonate (CO3HAp) and OH- groups that belongs to HAp crystals were obtained on scaffolds especially after 1th week of mineralization according to the FTIR spectra. To investigate the proliferation of human osteoblast cells (hFOB) on composite scaffolds, MTS analysis was applied at 1, 4 and 7 day. The SF:PLLA (3:1 w/w) scaffolds showed higher cell numbers than SF:PLLA (1:3 w/w) scaffolds. Additionally, the SF:PLLA (1:3 w/w) with peptides had more hFOB cells on polymer surfaces than without peptides scaffolds. On the other hand, the SF:PLLA (3:1 w/w) scaffolds with peptides showed less cell proliferation than without peptides forms. In conclusion, the composite scaffolds with two different concentrations were produced by lyophilization process to mimic bone tissue structures and a peptide to direct the mineralization process was added for further improvement. The biodegradability of the scaffolds seemed to be appropriate for bone tissue engineering. Peptide addition affected the mineralization and cell proliferation behavior of the scaffolds but not always in positive manner. For future prospects, the labeled-peptides can be used to obtain the distribution of peptides on the surfaces of polymers. HPLC analysis can be applied to optimize the concentration of immobilized peptides. In addition, the von Kossa staining can be used to observe calcium deposition. In sum, a more comprehensive study is needed to understand and optimize the effect of peptides on the scaffolds surface.

İnas Özcan

İstanbul Technical University · Institute of Graduate Studies in Science

Molecular and physiological characterization of iron resistant Saccharomyces cerevisiae

Iron, the fourth most abundant element in earth crust, is essential for living organisms. At high concentrations, however, it is toxic. The electron transport potential between its two most common oxidation states (Fe3+ and Fe2+) makes iron crucial for all living organisms. Iron ions play critical roles in oxidative phosphorylation. Iron shows similar physical and chemical properties with other transition metals, like copper, zinc, nickel and cobalt. Transition metal ions have common transport systems or they can replace each other in some structures. Thus, it can be expected that iron resistance and resistance to other transition metals may have common features. The aim of this study was the molecular and physiological characterization of an iron-resistant Saccharomyces cerevisiae mutant that was previously obtained by evolutionary engineering, an inverse metabolic engineering strategy. For this purpose, the cross-resistance of the iron-resistant strain against other stress types (particularly other transition metal ions) were determined in detail. Growth physiological analyses were also performed, which revealed that the iron-resistant mutant produced high levels of glycerol and accumulated high amounts of trehalose. The yeast S. cerevisiae can survive toxic metals by either of the two major kinds of resistance mechanisms: sequestration or avoidance. To gain insight into the major iron resistance mechanism in the resistant strain, iron contents of the mutant and the reference strain were determined both in the presence and absence of iron stress, by using Flame Atomic Absorption Spectrophotometry (F-AAS). The results showed that the iron-resistant yeast strain had significantly higher iron level than the reference strain, already in the absence of any iron stress. When iron stress was present in the culture medium, however, the iron-resistant mutant did not take up as much iron ions as the reference strain did. These results imply that in the presence of iron stress, the iron-resistant mutant avoids iron ion uptake, to survive iron stress conditions. For molecular characterization of the iron-resistant S. cerevisiae strain, transcriptomic analyses were performed using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and microarray technology, both in the presence and absence of iron stress. Microarray analysis results showed that the iron-resistant S. cerevisiae strain had higher expression levels of the oxidative stress-related genes than the reference strain, even in the absence of any iron stress. According to the results obtained by the microarray analysis, lipid peroxidation assay and intracellular reactive oxygen species (ROS) levels determination assay were performed. The intracellular ROS level of the iron-resistant strain was found to be lower than that of the reference strain. Thus, the iron-resistant strain possibly expresses high levels of oxidative stress-related genes and produces antioxidant c which reduce intracellular ROS levels. By this means, it may survive high levels of iron stress.

Production of biodegradable polymer from microbial mixed culture (MMC)

Polyhydroxyalkanoates (PHAs) are biodegradable, biocompatible, non-toxic and cost effective polyesters that provide alternative to conventional petroleum based plastics. PHA polymers are composed of fatty acids monomers with attached hydroxyl groups and naturally produced as intracellular carbon and energy storage material by many microorganisms. Owing to its environmental-friendly characteristics and being derived from renewable sources, PHA production strategies are being a hot topic for both research-scale and industrial scale. Since Microbial Mixed Culture approach does not require aseptic conditions and can be operated by using cheaper substrates, it is preferred instead of pure culture cultivation for PHA production. The main goal of this study is increasing the PHA storage capacity of activated sludge by two steps strategy; culture enrichment and PHA production processes. For the culture enrichment, synthetic wastewater, which contains acetate as carbon source and peptone as carbon and nitrogen source, was fed to the system under aerobic dynamic feeding (ADF) conditions in SBR. Also, for the PHA production process, Volatile Fatty Acids (VFA)-rich industrial wastewater was used for 6 hours of batch operation. In order to maximize the production, different food-to-microorganism ratios (F/M) were performed with VFA-rich industrial wastewater and the results were compared with reference acetate containing synthetic wastewater of F/M=4. Additionally, investigation of microbial community structure in enrichment reactor was also aimed. In order to observe the changes in microbial community profile 16S rRNA based DGGE finger printing analyses were operated. Besides the DGGE analyses, 16S-based Next Generation Sequencing were also analysed for three samples of enrichment process. Due to the enrichment process, weight percentages of accumulated PHA were increased by nine fold, from 0.47% to 4.1% (g PHA/ g dry biomass). The efficiency of enrichment process was also proved by batch operations with VFA-rich industrial wastewater that were performed for both enriched and non-enriched cultures. While the maximum produced PHA content of non-enriched culture which fed by VFA-rich industrial wastewater was 4.44% of cell dry weight; 43% PHA of dry biomass was accumulated by enriched culture batches. The produced PHA level in VFA-rich industrial wastewater industrial wastewater after enrichment reached to a level as high as the one obtained in pure culture processes. Furthermore, enrichment process of microbial mixed culture was also analysed in taxonomic level. After the 12 months of enrichment process, SBR was become dissimilar with 76,4 % . This differentiation ratio was also proved by NGS analyses. According to the taxonomy classification of NGS analyses, Shinella genus of Alphaproteobacteria emerged as the most abundant group as they occupied 49% ofsample from the end of 20 months-enrichment process while they were not detected in inoculation sample of enrichment. Fundamentally, it can be postulated that PHA production in mixed culture has been successfully optimized. By the using of two steps enrichment-production strategy, PHA production capacity has been increased by ten times.

Büşra Ahata

İstanbul Technical University · Institute of Graduate Studies in Science

Investigating the behavior of endothelial cells and monocytes under disturbed flow conditions within a microfluidic system

Cells are capable of sensing and responding to the mechanical forces exerted by their environment. Reception of physical stimuli is followed by transmission and conversion of the signal into biological activity in a process defined as mechanotransduction. Mechanical signals combine with biochemical signals to produce multiple responses within a cell. Cumulative regulation in response to mechanical cues by a population of cells in close proximity can maintain important tissue functions. Disturbance in physiological patterns of mechanical forces may lead to abnormalities and disease. Blood vessels are particularly regulated via mechanical stimulation. Endothelial cells that line arterial, venular and capillary inner walls are under continuous strain by the hemodynamic forces as a result of blood flow. Alterations in the mechanical signals received by endothelial cells are frequent. The endothelium provides a bridge and a barrier between the bloodstream and the underlying vascular tissue. Consequently, mechanical signaling at the endothelium has an influence on overall vascular maintenance; mostly regulating the level of vessel dilation via the production of nitric oxide. The most studied mechanical force on endothelial cells is shear stress, the parallel frictional force caused by the flow of a fluid. Studies on shear stress have revealed that it has a major role in maintaining endothelial barrier and in preventing vascular diseases. Hemodynamic shear stress have a regular pattern that can locally variate in case of a damaged vessel, or in sites of branching geometries such as bifurcations and curvatures. In the branching geometries, laminar blood flow is disrupted and separated into secondary flows of oscillatory or reversing behavior, creating low shear stress in the local area. Mechanobiology is the discipline that studies how mechanical forces can affect cellular functions. Studying vascular mechanobiology is particularly challenging in that it requires establishment of the three-dimensional vascular microenvironment and generation of fluid flow. Recently, microfluidic channels have come into use to enable creating such research environments. Culturing of endothelial cells in microdevices of unique channel geometries allows to study cell behavior under controlled shear environments. Atherosclerosis, formation of fatty plaques in arteries, draws much attention because it is a common disease that has been linked to local alterations in shear stress. The cytoskeleton is at the core of mechanotransduction. Elements of the cytoskeleton are connected to each other and to membrane proteins, providing the balance and transmission of tensional forces. The shearing force initially deforms the cell surface and activates membrane ion channels, followed by transmission of the signal by secondary messengers assisted by the cytoskeleton. Then the message is converted into biochemical activity and is carried to distant regions of the cell. Transduction of the signal activates intracellular signaling pathways to promote gene transcription or regulates cell adhesion interactions. Upon exposure to laminar shear stress, endothelial cells elongate and align their cytoskeleton in the direction of flow. Low shear stress has been shown to increase the expression of endothelial adhesion molecule ICAM-1 (intercellular adhesion molecule 1), promoting endothelial permeability to leukocytes. Studies on human aortas suggest that monocytes accumulate greatly at the endothelium in the outer walls of bifurcations that are susceptible to disturbed shear stress patterns. Monocytes are phagocytic innate immune cells that remove bacteria, viruses and toxic metabolites. Monocytes circulate in the bloodstream, but they enter tissues via diapedesis and differentiate into macrophages in order to participate in homeostasis or inflammation via phagocytosis and antigen presentation. Monocytes play a central role in long-term, tissue-damaging inflammatory conditions such as atherosclerosis. Atherosclerosis is a chronic inflammatory response to endothelial injury characterized by formation of fat-laden plaques in artery walls. Progression and perchance rupture of atherosclerotic plaques can lead to cardiovascular disease. The occurrence of atherosclerosis depends on the nature of local blood flow as well as the presence of biochemical risk factors. While high laminar shear stress in straight blood vessels is atheroprotective, low shear stress and oscillatory/reversing blood flow in vessel curvatures bring about atheroprone sites. In addition, the blood vessel becomes narrower as the atherosclerotic plaque grows, changing the local shear stress at the plaque site. The significance of physiological shear stress and the response of endothelial cells to alterations in shear stress have been well studied to date. However, no studies yet have analyzed the biological effects of disturbed flow on endothelial cells applying a model that can mimic the atherosclerotic plaque microenvironment with high-to-low alteration of shear stress. In this study, a unique design of cavitated microfluidic channel mimicking the atherosclerotic plaque geometry was used for the first time to study the effects of disturbed flow on HUVECs (Human Umbilical Vein Endothelial Cells). The first part of the study describes methods to obtain a confluent endothelial monolayer within straight and cavitated microfluidic channels and a method to apply shear stress on cells. In the preliminary studies, disturbed shear effects on endothelial F-actin network and ICAM-1 expression were investigated. Low-level disturbed shear stress was shown to cause F-actin accumulation at the cell cortex and F-actin reorganization in the direction of flow. This effect was higher in the narrower channel regions where shear stress was relatively high. The more random alignment of HUVECs in the low shear-harboring cavities may be related to the local disturbance of flow within the region. In addition, low-level disturbed shear stress was found to upregulate ICAM-1 mRNA expression, although a less increase in the expression was obtained when compared to TNFα activation. Taken together, disturbed shear stress at atheroprone regions may contribute to monocyte recruitment at the endothelium via upregulation of the endothelial adhesion molecule ICAM-1, driving the effect further with local presence of TNFα. Since this study involved only static cells as a reference, further studies may include application of laminar shear stress on endothelial cells in straight channels as an additional control. The individual effect of shear stress on monocyte behavior has not been studied to date. In this study, THP-1 monocytic cells were subjected to sequential high and low shear stress in cavitated channels at an average of 4 Pa as normal, 15 Pa as high or 45 Pa as very high shear stress and changes in F-actin levels were analyzed by fluorescence microscopy and flow cytometry. High level (15 Pa) disturbed shear stress was determined to increase F-actin content, whereas very high (45 Pa) level of disturbed shear stress was concluded to decrease the F-actin content in the long term in THP-1 monocytes. Furthermore, the effect of disturbed shear on F-actin in shorter time intervals may be assessed to fully characterize time-dependent changes in the F-actin.

Rana Fuçucuoğlu

Blotting-westernCytoskeletonCellular neural networks+3

Strategies for isolation of novel enzymes by using metagenomics approach

Microorganisms have been used for many biotechnological applications. Due to their both scientific and industrial importance, they have attracted many researchers. In traditional microbiological methods, microorganisms are isolated from their environment and grown in cultures. Until 2000s, it was believed that the microorganisms could not be characterized without culturing. However, scientific observations revealed the fact that only tiny amount of microorganisms could be grown in pure cultures. At the beginning of this century, a new field called metagenomics was entered to the literature. It is defined as the culture independent study of microorganisms that live in any environment. Metagenomic studies can be accomplished by function or sequence based approaches. In function based approach, a metagenomic library, as similar to genome library, is constructed and screened for desired phenotype. In sequence based metagenomics, either the metagenome is sequenced or already known sequence information is used to amplify specific regions from the metagenome. Extremophiles, as the most interesting group of microorganisms have great potential in industrial biotechnology, due to their macromolecules that conserve their stability in extreme conditions. Among these macromolecules, extremozymes are usually stable and active in harsh conditions, which is desired for many industrial enzyme catalyzed reactions. However, due to their extreme characteristics, it is not always possible to isolate an extremophile from its natural habitat and grow in laboratory conditions. At this point, metagenomics become an efficient approach to isolate novel enzymes from extremophiles. In this study, it is aimed to isolate lipolytic enzymes from extreme environments by metagenomics approach. Both function based and sequence based approaches were applied. Environmental samples were collected from acid mine drainage site (Balıkesir, Turkey; acidic environment) and a hypersaline lake (Acıgöl, Turkey; high-saline environment). In previous studies, a metagenomic fosmid library was constructed from acid mine drainage site and two potential clones (named F1 and F3) were detected. Within the scope of this thesis, strategies that can be applied in function based metagenomics approach are presented to identify the potential lipolytic enzymes. Firstly, subcloning library of F3 fosmid was constructed. F3 fosmid was partially digested with HindIII and EcoRI and ligated to pUC19 plasmid. Resulting constructs were transformed to E. coli DH5α cells and each clone was screened for lipolytic activity. One of the transformants gave positive result but the gene could not be identified by sequence analysis. As a second strategy, preliminary functional analysis was perfomed. Crude lysates of F1 and F3 clones were prepared and tested for lipolytic activity on agar plates. Once the positive results were obtained, crude lysates of F1 and F3 were partially purified by ammnonium sulfate precipitation. Each fraction was tested for lipolytic activity using para-nitrophenol palmitate as substrate. No activity was detected for F3 but 45%-70% fraction of F1 has 1.45 fold more activity compared to control. To further analyze the proteins in cleared lysates of F1, they were fractioned according to their molecular weight. Fractions between 30-50 and 50-100 kDa were analyzed by two dimensional gel electrophoresis. Gel results for F1 and FC were compared and spots that differ between gels were cut for analysis with mass spectrometry. Although the activity was verified with the preliminary functional studies, the targeted protein could not be identified with proteomic approach. As a third alternative, fosmid sample from F3 clone was sequenced. The partial Sanger sequencing revealed that the cloned insert match with nucleotide sequences from Acidithiobacillus species, verifying the acidophilic origin of the genome library. To identify the whole fosmid sequence, sample was sent for next generation sequencing. Partial sequences belonging to acidophilic microorganisms were obtained. A potential sequence similar to alpha/beta hydrolase fold enzymes was detected. Detailed analysis is going to be conducted with further studies. In the second part of the study, sequence based metagenomics approach was applied by using environmental samples collected from hypersaline lake Acıgöl. In previous studies, the microbial diversity of the samples were identified. Within the scope of this study, nucletide sequences of the lipolytic enzymes of these microorganisms were retrieved from databases. They were grouped according to their similarities. Each group was used for degenerate primer design, targeting conserved regions among lipolytic enzymes. Primers were designed by primer4clades online tool, using CODEHOP strategies. A total of nine degenerate primer pairs could be designed for 3 groups, including lipases and lipolytic enzymes, α/β hydrolase fold enzymes and carboxylesterases. While PCR was set for all primer pairs, only one positive result was obtained with the set of primers targeting α/β hydrolase fold enzymes. The obtained amplicon was cloned to pCRTM-4TOPO® vector and transformed to E. coli TOP10 cells. Sequence analysis revealed an insert of 480 bp as expected. When compared with BLAST, the sequence matched with Halolamina sediminis strain with an identity of 75%. To identify the unknown 5' and 3' ends, genome walking strategy was applied. For each end, two sets of gene specific primers were designed according to the partial sequence obtained by degenerate PCR. Metagenomic DNA was digested with restriction enzymes and adaptors were ligated to ends of digested DNA fragments. Using gene specific and adaptor specific primers, unknown 5' and 3' ends were amplified. After sequence analysis, fragments were assembled to yield a full length gene. The gene was composed of 783 bp, which corresponds to 260 amino acids. The gene starts with ATG start codon and ends with UGA stop codon. The gene sequence best matched with Halolamina sediminis with an identity of 75%. Deduced amino acid sequence best matched with the esterase from Halomonas gudaonensis with an identity of 91%. This result showed that a novel enzyme was successfully isolated from an environmental sample. The molecular weight and pI of the protein was calculated as 28368 Da and 5.27, respectively, using the ExPASy tool. To express the novel enzyme, primers targeting 5' and 3' of the novel gene with EcoRI and HindIII restriction sites were designed. The gene was amplified using metagenomic DNA as template and cloned into pET-28a(+) expression vector. The construct was transformed to E. coli C43 (DE3) chemically competent cells. To optimize the expression, 0.1, 0.5 and 1 mM IPTG concentrations were tried. In addition, induction at 25ºC for 16 hours, 30ºC for 6 hours and 37ºC for 3 hours were tried. The optimum expression was observed with 0.1 mM IPTG at 30ºC for 6 hours. After the gene was successfully expressed, purification studies were conducted. The protein was purified using His-tag method. Analysis with SDS-PAGE showed that the purification was successful. The apparent molecular weight of the protein (28.2 kDa) was consistent with the theoritical molecular weight, which was calculated as 28.4 kDa. The protein was found to be more stable in the presence of 10% NaCl, consistent with the halophilic nature of the protein. The activity of the protein was determined with kinetic assay, using para-nitrophenyl esters as substrate. The enzyme hydrolyzes the ester bond and the increasing absorbance of released pNP is measured at 410 nm. The path length for spectrophotometric measurements and the extinction coefficient of pNP at each reaction condition was calculated before activity calculations. To determine the substrate specificity, activity towards pNP-butyrate, pNP-hexanoate, pNP-octanoate, pNP-decanoate, pNP-dodecanoate and pNP-hexadecanoate were calculated. The enzyme showed a maximum activity towards pNP-hexanoate with a kcat value of 12.30 s-1. Also, the activity towards pNP-butyrate (6.03 s-1) and pNP-octanoate (6.26 s-1) was high. Its preference for short carbon chain length substrates indicates that it is an esterase. The optimum pH of the enzyme was found to be 9 and the enzyme was stable after storage at pH 8 to 10 for 24 hours. The enzyme completely losts its activity after storage at pH values below 6. The optimum temperature of the enzyme was found to be 30ºC but it is retains more than 50% of its maximum activity between 5ºC and 40ºC. The enzyme retains 96% of its maximum activity after incubation at 55ºC but the activity was dropped to 27% of its maximum after incubation at 60ºC. As the NaCl concentration increases, the activity of the enzyme drops but storage with increasing NaCl concentrations (10% to 25%) did not affect the stability of the enzyme. The effect of organic solvents (10%) on the activity of the enzyme was also investigated. While the presence DMSO did not affect the activity of the enzyme, glycerol slightly increased the activity. Activity in the presence of methanol was 80% of its maximum and other solvents showed inhibitory effect on the enzyme. The enzyme was found to be organic solvent tolerant that when stored with different organic solvents at 15% and 30% concentrations for 24 hours, except for 30% isopropanol and 30% 1-propanol, it retains more than 80% of its maximum activity. The effect of various metal ions, detergents and inhibitors were also investigated. The esterase was found to highly tolerant to metal ions as compared with the similar enzymes in the literature. The enzyme was inhibited by PMSF, confirming that it is a serine hydrolase. The enzyme is stable in storage conditions, that it retains its activity in storage buffer until 14th day at 4ºC.

Cytoskeleton is a network which consists of an interconnected filamentous proteins and regulatory motor proteins. In addition, cytoskeleton has important cellular activity roles for surviving and functioning such as determining cell shape and cell movement. Rearrangement of cytoskeleton is responsible for environmental changes and adaptation. Neurons distinguish from other cell types in the nature with their morphology. Their unique morphology allows them to perform their specific tasks. All neurons contain common structural backbone which is microtubule. Neurons have capacity to reconfigure their morphology and it is related to microtubule scaffolding. Neuronal cells undergo major developmental changes. Complex nervous systems require cytoskeleton-based processes that coordinate neuronal activites. Neurons are highly polarized cells and the establishment and maintenance of neuronal polarization is crucial for correct development of nervous system. They develop two types of cytoplasmic extensions; axons and dendrites. In response to electrical signals from the cell body, axons release neurotransmitters at the axon terminals. Dendrites, especially dendritic spines contain receptors for neurotransmitters and important function in signaling. The motility capacities of microtubules are directly related to their length. Mostly, the parent axon is dominated by very long microtubules chopped into short microtubules for direction of axon outgrowth. Microtubule severing is an important mechanism that take role in formation of specific neuron morphology. Neurons are rich in an enzyme called p60-katanin that makes breaks in a microtubule array. p60-katanin severes microtubules from the centrosome of neurons and later develops short microtubules that move more avidly than longer microtubules. There is a model called 'cut and run', in which the longer microtubules are mobilized by enzymes that sever them into shorter polymers. p60-katanin is not phosphorylated itself, yet its severing activity is regulated by phosporylation of microtubule binding proteins. Also, it is indicated that phosphorylation of MAP4 has regulatory effect on p60-katanin microtubule-severing activity in non-neuronal cells. Neuronal cells does not contain MAP4 but in place of MAP4, it has been shown that tau protects microtubules in the axon from severing by p60-katanin. According to this model, microtubule-associated proteins (MAPs) especially the axonal MAP tau bind along the axonal microtubules and has protective activity from severing proteins on microtubules. Tau deattaches from microtubules by phosphorylation and this controls levels of short microtubules in specific axonal regions. Short microtubules can then be transported by motor proteins into collateral branches to promote their growth and stabilization. Over the last several decades, Septin protein family has been recognized as fourth cytoskeleton compenent because of their filamentous appereance and their association between other cytoskeleton elements such as microtubules. Septins are evolutionarily conserved family classified under the GTPase superclass of P-loop NTPases. Septins have important roles in maintenance of cell shape and microtubule-dependent processes. For instance, it has been demonstrated that Septin2 and MAP4 intract and this interaction results in microtubule destabilization. Septin3 (Sept3) specifically associates with neurons and nerve terminals, which is found as a neuronal spesific protein after examination of 12 different tissues. Septin3 is found enriched in synaptosomes and presynaptic terminals. Here we sought to determine Septin3 - Tau interaction from the point of neuronal process formation and branching and how this interaction would effect p60-katanin activity on Septin3 filament formation. Initially, physical interaction of Tau & Septin3 and Tau & p60-katanin proteins were determined co-immunoprecipitation (co-IP) with rat brain lysate. co-IP results indicated that Septin3 and tau physically interacted, however tau and p60-katanin did not physically interact. The studies on RFL6 fibroblasts sought seek to test Septin3 – Tau – p60-katanin interaction in non-neuronal cell which do not express Septin3 and Tau endogenously. RFL6 cells were over-expressed both with tau and Septin3 and co-localization of tau then were confirmed. Septin3 over-expression resulted in filament formation, however after over-expression of both Septin3 and p60-katanin, Septin3 filament formation was damaged. This effect of p60-katanin is considered as similar to its effect on microtubules. In addition, RFL6 cells nucleofected with Septin3, tau and p60-katanin and Septin3 filament formation was investigated. According to these results, Septin3 protein formed filamentous appearance, in spite of p60-katanin over-expression. Several studies showed that septins increase spine morphogenesis and dendritic spine formation. For instance, Septin7 is found to localize at dendritic protrusions and dendritic branch points in cultured hippocampal neurons. Primary cortical neuron studies were performed to determine Septin3 – Tau – p60 - katanin interaction from the point of neuronal processes formation. Primary rat cortical neurons over-expression studies were carried out by nucleofection and gene silencing was carried out by Accell siRNA transfection. Septin3 over-expression increased dendritic branch formation and expedited neuronal development. Also, over-expression of Septin3 resulted in long and thick bundles in microtubules. Upon down regulation of Septin3, dendritic spine morphology and exon elongation were impaired. Following over-expression of both Septin3 and p60-katanin in the same cell, Septin3 was found to be interrupted along the axon in the presence of p60-katanin. Moreover, microtubules remained partially uninterrupted. Septin3 and tau were localized predominantly along the axon and resulted in even thicker bundles of Septin3 and microtubules. Besides Tau' s localization in mainly axons, Septin3 over-expressed and Tau silenced cells still lost their axon elongation. Observation in this study showed that, in addition to other known septin' s, Septin3 forms filaments in non-neuronal cells. Also, Septin3 has a role on neuronal polarization. Similar to some septins, Septin3 interacts with Tau and this interaction results in long and thicker microtubule bundles on neuronal microtubules. Although Further experiments needs to be carried out. tau may have a protective effect on Septin3 filaments against severing, like it does on the microtubules.

Didem Baran

İstanbul Technical University · Fen Bilimleri Enstitüsü

İstanbul Technical University · Institute of Graduate Studies in Science

Molecular and physiological investigation of longevity in yeast

World population has changed in both developed and developing countries due to low birth and death ratio, resulting in relatively elder populations, which is a major challenge. Aging is a risk factor for several diseases such as Alzheimer, Parkinson, diabetes, cancer and cardiovascular diseases, due to declining cellular physiological functions with time. In recent years, researchers have been focusing on this issue due to the increasing socioeconomic concerns. The purpose is to understand the cellular aging process and to provide healthy lifespan. In aging research, the yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster, the worm Caenorhabditis elegans, rodents and primates have been used as model organisms. The yeast S. cerevisiae is a unicellular eukaryotic microorganism and it has been used in industrial processes such as baking, wine and beer production since ancient times. It has also been commonly used as a eukaryotic model organism in genetic and molecular biological research since it does not need expensive and special media and cultivation conditions. The yeast S. cerevisiae is a commonly preferred model organism in aging studies because of the shortness of its life span, availability of its genome sequence data and extensive molecular tools and techniques, and its similarity to mammalian cells. Aging studies can be performed by two ways in S. cerevisiae: by investigating the replicative life span (RLS) or the chronological lifespan (CLS). RLS has been developed because of the asymmetrical division of S. cerevisiae cells. In the method, after mitosis, the daughter cell is separated from the mother cell by using a micromanipulator, and the procedure is continued until the mother cell stops division. At the end of mitosis, the number of buds one mother cell produced are determined and the number of daughter cells is the RLS of S. cerevisiae. CLS is the length of time where non-dividing stationary phase yeast cells survive. In the method, cell viability at the beginning of the stationary phase of growth is determined by viable cell counting method on solid media. After that, viability of non-dividing cells at the stationary phase is monitored. Since (i) stationary phase cells produce energy from mitochondria, (ii) damage accumulates over time and (iii) cells exit from the cell cycle (G0), CLS method is accepted as a convenient model to study aging of non-dividing cells (e.g., neurons) in higher eukaryotic organisms. Long-lived S. cerevisiae, C. elegans and Drosophila mutants have had a major role for understanding cellular aging mechanism. The studies share a similar approach, since the long-lived mutant strain is obtained first, and then it is investigated in detail to understand its molecular basis. These strategies show similarity to inverse metabolic engineering approach. In inverse metabolic engineering approach, first, the desired phenotype is obtained, and then, its genetic mechanism is identified. This approach is a very powerful strategy to obtain complex desired microbial phenotypes with genetic complexity. So far, the approach has not been used to obtain chronologically long-lived yeast. In the present study, inverse metabolic engineering approach was used to obtain chronologically long-lived S. cerevisiae. Furthermore, since previous aging research with model organisms has shown a close relationship between stress resistance and longevity, in the current study, CLS of previously obtained stress-resistant S. cerevisiae mutants was also investigated. To isolate a chronologically long-lived yeast mutant by using inverse metabolic engineering, first, genetic diversity of the initial population for selection was increased by ethyl methane sulfonate (EMS)-mutagenesis. Successive batch cultivation of the initial mutant population was performed under gradually enhanced caloric restriction, since caloric restriction is known to extend lifespan. During successive batch cultivations, glucose concentration of the medium was decreased from 0.5% to 0.02%. In the last batch cultivation, since growth ratio of the mutant population decreased to 10%, the selection procedure was ended. After that, individual mutants were randomly picked from the last population. Semi-quantitative and quantitative CLS analyses were applied to these individual mutants. A single mutant (called SRM11) that has the highest chronological survival among the other mutants was chosen for further physiological and molecular analysis. Quantitative CLS method based on viable cell counting on solid media is tedious for large studies where multiple strains and multiple conditions are tested, as a high number of plates and flasks is required for incubation. It is also labor-intensive. Therefore, in the present study, the semi-quantitative method was used for preliminary determination of CLS in mutants. A culture density adjustment step was introduced to the semi-quantitative method, for a more reliable evaluation of CLS of cultures at significantly different culture densities. Following the semi-quantitative CLS, quantitative CLS analysis was applied to the prominent mutants. The procedure was applied successfully and correctly determined the chronologically long-lived phenotypes in a wide range of strains and conditions. Growth behavior of the mutant SRM11 was investigated by growth curve and biomass analysis. The purpose of these experiments was to determine whether any trade-off in growth physiology occurred or not. Analysis results showed that there was no trade-off in the growth fitness of the mutant SRM11 in both standard medium (2% YMM) and calorie-restricted medium (0.5% YMM). In this experiment, it was observed that 75% reduction of glucose concentration of the medium (from 2% to 0.5% glucose) did not affect growth ratio of the yeast strains when glucose was utilized. High-Pressure Liquid Chromatography (HPLC) analysis was performed to investigate the metabolic profiles of the long-lived mutant SRM11 and the reference strain. For this purpose, glucose, ethanol, glycerol and acetate concentrations in culture samples were monitored by HPLC analysis. Even though there was no significant difference in glucose consumption between the reference strain and the mutant SRM11, there was a significant difference in the production of fermentative metabolites. The mutant SRM11 produced low amounts of ethanol, glycerol and acetate in standard glucose medium (2% YMM). These findings indicated a respirative shift in the metabolism of the mutant SRM11, showing that respirative metabolism is related to chronological longevity in S. cerevisiae. In 0.5% YMM, a limited ethanol and glycerol production was observed in the cultures of the reference strain and the mutant SRM11, which indicates that at 0.5% glucose concentration, fermentation is limited. Aging studies have shown a close relationship between stress resistance and longevity in model organisms. Therefore, stress resistance analysis of the long-lived mutant SRM11 was performed, using the semi-quantitative spot assay method. The results revealed that the mutant SRM11 highly resisted to copper stress. Also, SRM11 resisted to silver, phenylethanol, ethanol and boric acid stresses, indicating that these stress types may be related with chronological longevity. Whole genome transcriptome of the long-lived mutant SRM11 was analyzed by using DNA microarray technology, to understand the molecular mechanisms of chronological longevity in yeast. DNA microarray analysis results revealed that 769 open reading frames (ORFs) were upregulated, 838 ORFs were downregulated by two-fold and higher in the long-lived SRM11 mutant, compared to the reference strain. For the interpretation of the gene expression change, Gene Ontology (GO) analysis was used. GO analysis of the upregulated genes indicated that oligosaccharide-carbohydrate metabolic biological process and response to oxidative stress were induced in SRM11. Furthermore, GO analysis of the downregulated genes revealed that ribosome subunits biogenesis, ribosome assembly and RNA processing were repressed in SRM11. These results suggest that repression of protein synthesis and activation of oligosaccharide-carbohydrate metabolic processes and response to oxidative stress are associated with the extension of chronological longevity in the yeast S. cerevisiae. CLS of various stress-resistant S. cerevisiae mutants was also investigated by semi-quantitative and quantitative methods. Among the tested stress-resistant mutants, silver-resistant, ethanol-resistant, phenylethanol-resistant, oxidative stress-resistant mutants were determined as long-lived mutants. Resistance to oxidative stress has been shown to be related with chronological survival in yeast, however; resistance to silver, ethanol and phenylethanol stress has not been shown, yet. The present study, for the first time, showed that resistance to silver, ethanol and phenylethanol may be related with chronological longevity in the yeast S. cerevisiae. In the current study, a comparative transcriptomic analysis of the long-lived stress resistant mutants (silver-resistant, phenylethanol-resistant and ethanol-resistant mutant and long-lived mutant SRM11) was also carried out to find a relationship between chronological longevity and stress resistance. The comparative transcriptomic analysis showed that 17 downregulated genes and 48 upregulated genes were overlapping in the long-lived and stress-resistant mutants. GO analysis of the overlapping upregulated genes showed that the upregulated genes were enriched in mostly oligosaccharide and carbohydrate metabolic process and generation of precursor metabolites and energy. GO analysis of the overlapping downregulated genes indicated that ribosomal subunits biogenesis and aminoacid transport biological process were repressed. As a conclusion, the present study suggests that increased respirative metabolism, resistance to copper, silver, ethanol and phenylethanol stress, repression of protein synthesis and induction of oligosaccharide and carbohydrate metabolic processes are associated with chronological longevity in the yeast S. cerevisiae. Further detailed analyses at genomic and proteomic levels will help understand the complex molecular basis of the longevity in S. cerevisiae.

Saccharomyces cerevisiae

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