Theses supervised by Yrd. Doç. Dr. Ayşe Begüm Tekinay
13 theses · İhsan Doğramacı Bilkent University
Biyoaktif peptit amfifiller ile çizgili kas ve kalp kası oluşumu için mikroçevresel sinyalleri programlama
The extracellular matrix (ECM) is crucial for the coordination and regulation of various cellular processes, including cell adhesion, recruitment, differentiation and death. ECM components structurally support tissue function and regeneration by acting as a substrate for cell migration and differentiation. In addition, by facilitating the fine localization of signals within their structural framework, these components activate receptors on the cell membrane for the initiation of signal transduction cascades. As such, cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for natural or artificially assisted tissue regeneration. In keeping with this ECM-centric approach, we designed and synthesized peptide amphiphiles with bioactive epitopes to resemble the native microenvironment of muscle tissue and to examined their potential in the induction of progenitor cell differentiation into skeletal myotubes and cardiac myocytes. The formation of skeletal myotubes was promoted through the use of basal lamina-mimetic peptide nanofibers inspired by the chemical structures of laminin and fibronectin, two proteins strongly represented in the skeletal muscle extracellular matrix. We demonstrated that our basal lamina mimetic peptide nanofiber system actively interacts with the cells it contains and enhances their differentiation within 3 days. Morphological analysis and immunocytochemical stainings indicated the formation of differentiated myotubes. We also designed glycosaminoglycan-mimetic peptide amphiphiles to mimic the glycosaminoglycans found in the myocardium. Glycosaminoglycans have been reported to play substantial roles in growth factor binding and the induction of angiogenesis, and their mimicry through peptide amphiphile nanofibers is promising as a combined approach for generating multifunctional cardiovascular tissue engineering scaffolds. We demonstrated that peptide nanofibers enhance the adhesion of cells to the surface and induce cardiac myoblast cells to differentiate into cardiomyocytes through both gene expression analysis and immunostainings. In summary, myogenic platforms were developed by programming signal rich environment from self-assembled peptide nanofibers inspired from the components of the ECM to induce the differentiation of cells. These bioactive nanofiber systems serve as promising platforms for muscle tissue engineering applications.
Doku rejenerasyonunda biyoaktif peptit nanofiberler
Defects in the tissues or organs caused by trauma or diseases can have detrimental effects on all aspects of patients' life quality. During the last three decades, considerable developments have been made in tissue engineering and regenerative medicine in order to find alternative treatment methods to recover tissue function after injury. These methods are based on the development of materials that are uniquely suited to the specific requirements of the tissue type and the repair process itself. Consequently, the implanted biomaterial must be compatible with biological systems and capable of delivering the signals necessary to facilitate tissue repair. In the present thesis, peptide amphiphile molecules were used to meet these requirements and develop next-generation biomaterials that are able to enhance the repair process while minimally affecting the integrity of surrounding tissues. Peptide amphiphiles are molecules that naturally self-assemble into nanofibrous hydrogel structures that closely emulate the composition of the extracellular matrix. As peptide amphiphiles contain amino acid sequences, bioactive signals can also be integrated into their structure to create a biocompatible environment and enhance the survival and proliferation of the resident cell population. In the scope of the present thesis, peptide amphiphile systems were utilized in three distinct applications. The first chapter covers the fundamentals of regenerative medicine and tissue engineering, the interactions between biomaterials and cells and extracellular materials, and the materials that are commonly used for these applications. The second chapter details the use of fibronectin- and laminin-derived peptide amphiphiles for the regeneration of corneal injuries. The third chapter investigates the ability of heparin-mimetic peptide hydrogels to facilitate the survival of pancreatic islets in vitro and demonstrates that islets transplanted in tandem with peptide gels trigger a local angiogenic response, decrease blood glucose levels and retain these functionalities even after 28 days of observation. The fourth chapter concerns the application of heparin-mimetic peptide amphiphile molecules for the recovery of acute wound injuries through the establishment of a well-ordered collagen matrix and the enhancement of the re-epithelialization process. Distinct peptide amphiphiles bearing bioactive signals conductive to tissue development were developed and utilized in all three studies, and the use of these materials has been demonstrated to serve as an adequate means of enhancing tissue repair.
Ailesel esansiyel tremor için aday genlerin tespit edilmesi
Essential tremor (ET) is one of the most common movement disorders in humans and is characterized by action tremors that occur during voluntary motion. However, due to the strong heterogeneity exhibited by ET patients at etiological, clinical and pathological levels, the genetic architecture and pathophysiology of the disease remain largely unknown. In this thesis, whole exome sequencing and pedigree analysis were performed in 3 ET families with histories consistent with an autosomal dominant pattern of inheritance. In two independent families, we observed a rare variant that cosegregated with the disease and was predicted to affect the function of the protein. In one of these families, a homozygous variant was identified in one affected patient and a heterozygous variant was determined in five affected family members. In a second, four-generation Turkish family, the same heterozygous variant was identified in three ET cases while remaining absent in unaffected family members. In addition, whole exome sequencing allowed us to demonstrate that other missense mutation segregate with essential tremor in a different consanguineous Turkish family. Both variants were observed to involve amino acid substitutions of highly conserved domains. Furthermore, both of the affected genes are expressed in the brain and function as regulatory elements of the central nervous system. Consequently, we propose that these variants are risk factors involved in the etiology of hereditary ET, and suggest that whole exome sequencing can serve as an effective means of identifying other alleles associated with the disease.
NGF'e bağlanan peptit nanofiberlerin dizaynı ve nöral rejenerasyon çalışmalarında uygulanması
Promotion of neurite outgrowth is an important limiting step for the regeneration of nerve injury and depends strongly on the local expression of nerve growth factor (NGF). Rational design of bioactive materials is a promising approach for the development of novel therapeutic methods for nerve regeneration, and biomaterials capable of presenting NGF to nerve cells are especially suitable for this purpose. This thesis describes development of nanofibrous peptide amphiphile (PA) nanofibers capable of promoting neurite outgrowth by displaying high density binding epitopes for NGF. The high-affinity NGF-binding sequence was identified by phage display and combined with a beta-sheet forming motif to produce a self-assembling PA molecule. Our results revealed that the bioactive nanofiber had higher affinity for NGF compared to control nanofiber and in vitro studies showed that the NGF binding peptide amphiphile nanofibers (NGFB-PA nanofiber) significantly promote the neurite outgrowth of PC-12 cells. In addition, the nanofibers induced differentiation of PC-12 cells into neuron-like cells by enhancing NGF/high-activity NGF receptor (TrkA) interactions and activating MAPK pathway elements. The first time with this study a seven amino acid phage display peptide library was utilized for high affinity epitope screening for NGF, the NGF binding sequence was incorporated into peptide amphiphile structure, and the effect of NGF binding material on differentiation pathway of NGF was analyzed. This material will pave the way for development of new therapeutic agents for nervous system injuries.
Biyoesinlenilmiş malzemelerin rejeneratif tıp ve ilaç taşınımı alanlarında uygulamaları
The structural organization and functional capabilities of natural materials have inspired many technological and scientific developments. Biological systems are under constant pressure for innovation due to the constraints imposed by natural selection, which has allowed various organisms to surmount engineering challenges in ways that can scarcely be matched by modern science. Biomimetics or bioinspiration is a field that focuses on the adaptation of engineering principles observed in biological models to fabricate materials capable of circumventing longstanding problems in fields such as energy and medicine. This transition from biological systems has facilitated the design of effective materials, structures or processes within the range of nature's adaptations and strategies. In the first study of this thesis, I describe the development of a bioactive scaffold composed of adamantyl-conjugated, laminin-derived bioactive IKVAV peptide molecules enmeshed in electrospun cyclodextrin nanofiber (CDNFs). Accordingly, host-guest interactions between adamantyl groups on peptide termini and cyclodextrin molecules on electrospun nanofiber surfaces were utilized to produce a composite material for the treatment of neurodegenerative disorders. Electrospun CDNFs provided a 3-dimensional environment conductive for the growth of PC12 cells and expressed functionalized bioactive epitopes on their surfaces to enhance the differentiation of neural progenitors. In addition, CDNFs further supported neural growth through their highly aligned mesh structure. Neural III tubulin and synaptophysin I gene expression levels significantly increased when PC12 cells were cultured on aligned and IKVAV-functionalized CDNFs. Neurite extension of PC12 cells also increased significantly when cultured on aligned and IKVAV-functionalized CDNFs when compared to random and unfunctionalized electrospun CDNFs. As such, these nanofibers are able to effectively induce the neural differentiation of PC-12 cells through the physical and biochemical signals provided by their structure and bioactive sequence. The second part of the present thesis focuses on the local delivery of gemcitabine, a cytotoxic cancer drug that is rapidly degraded in plasma and cannot be encapsulated in conventional delivery vesicles due to its highly hydrophobic nature. In order to overcome these limitations, gemcitabine was coupled with Fmoc-Gly and integrated into a peptide-based nanocarier system in order to control drug concentration within the therapeutic range and minimize the adverse effects. Two oppositely-charged amyloid inspired peptides (Fmoc-AIPs) were chosen as drug carrier systems. These molecules self assemble into nanofiber structures at physiological conditions through non-covalent interactions. Overall, the present thesis demonstrates the significance of peptide-based materials for the purpose of designing functional bioinspired/biomimetic materials for various cellular applications such as tissue engineering and drug delivery. The complexity of nature necessitates the design of biomaterials that can mimic the cellular microenvironment for the treatment of diseases, and further insight into natural processes will no doubt enhance our ability to overcome the engineering challenges presented by modern medicine.
Biyoaktif peptit nanofiberlerin akut kas hasarı üzerindeki etkilerinin incelenmesi
Skeletal muscle constitutes a large part of the human body. It is a hierarchically organized heterogeneous tissue and is composed of muscle fiber bundles, muscle fibers, myofibrils and myofilaments. Since muscle cells are terminally differentiated, they have limited capacity to renew themselves. Only new cells can fuse with muscle fibers and increase the size and volume of skeletal muscle. Myosatellite cells or satellite cells are small, mononuclear progenitor cells with virtually no cytoplasm. They are located in between the sarcolemma and basement membrane of terminally-differentiated muscle fibers. Satellite cells are precursors to skeletal muscle cells, and are able to give rise to satellite cells or differentiated skeletal muscle cells. They are normally found in silent state in adult muscle, but act as a reserve cell population that is able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. Formation of the new muscular tissue is called myogenesis. During this event, satellite cells differentiate into myoblasts, and then myoblasts fuse with each other in order to form myofibers. There are many genes that regulate the myogenesis process and each of them is activated in a different step of myogenesis. Increased or decreased levels of gene expression determine the differentiation capacity. Peptide nanofibers are supramolecular structures formed via self-assembly and they are promising molecules in regenerative medicine and tissue engineering. Peptide-based molecules for tissue regeneration is a widely studied area and currently used in the treatment-investigation of many different tissues such as bone, cartilage, skin and nerve. Since laminin is one of the most abundant proteins found in the basal membrane of the skeletal muscle; in this thesis, we designed and synthesized a laminin-mimetic bioactive (LM/E-PA) molecule functionalized with bioactive groups for mimicking laminin activities and capable of accelerating satellite cell activation. Our research group had previously shown that LM/E-PA containing nanofibers can support muscle differentiation in vitro. In this thesis, the clinical relevance was demonstrated further by assessing laminin-mimetic bioactive scaffold in acute muscle injury in an in vivo rat model. Our findings revealed that this scaffold system significantly promotes satellite cell activation in skeletal muscle and accelerates regeneration following acute muscle injury. In addition, our findings show that the regeneration capacity of the skeletal muscle was increased and consequently regeneration time was reduced. This study is one of the first examples of molecular level and tissue level regeneration of skeletal muscle by using bioactive peptide nanofibers following acute muscle injury, and shows that laminin mimetic nanofiber system is a promising material for development of new therapeutic curatives for acute skeletal muscle injuries.
Kendinliğinden biraraya gelen nanofibröz yapının yeni aşı adjuvanı olarak kullanımı
Vaccination is the most effective and cost-efficient way of protection against the major infectious diseases but ideal vaccine formulation has not been found. Recent vaccine systems are mainly composed of two major substitutes that are antigen and adjuvant. Recently it was demonstrated that widely used adjuvants exhibit some safety problems that affect the neural system such as neurotoxicity and autoimmune diseases. Therefore, there are increased concerns about side effects of the adjuvants and many researchers focus on developing new adjuvants that are effective and safe. Peptide amphiphiles are chemically defined molecules that are able to self-assemble into nanofibrous structures. The nanofibrous structures are biocompatible, biodegradable, and biosafe and thereby they are ideal for vaccine systems. Also, nanofibrous structures don't contain any substance that are potentially dangerous for neural system such as metals. Thus, nanofibrous structures are promising candidates to be alternative novel vaccine adjuvants. In this thesis, I investigated the potential of a biotinylated nanofibrous structure as a novel vaccine adjuvant that is potentially safe. Briefly, biotinylated peptide amphiphiles were synthesized, purified and characterized to analyze the features of the novel material. The peptide amphiphiles were induced to form nanofibrous structures by self-assembly and antigens (ovalbumin) were bound to the biotinylated nanofibrous ii structures through streptavidin linkers. Splenocytes were treated with the nanofibrous structures to demonstrate the effects of the nanofibrous structures on the immune response. After the confirmation of efficient immune response that are induced by the nanofibrous structure in vitro, as enhancing release of stimulatory cytokines, inducing dendritic cell maturation and triggering the cross-presentation of the antigen, mice were immunized with the nanofibrous structure in the presence of antigen for further analysis of the nanofibrous structure efficiency as adjuvant in vivo. Both in vivo and in vitro results showed that the nanofibrous structure is able to effectively trigger the antigen specific immune response and thereby exhibit adjuvant properties. Overall, I suggest that the nanofibrous structure is able to be used as a new vaccine adjuvant that induces effective antigen specific adoptive immune response and thereby it could be a good alternative of recently used adjuvants that are suspected to contribute some impairments in neural system.
Yanık yara iyileşmesinin hızlandırılmasında biyoaktif peptit nanofiberlerin kullanımı
Burn injuries are one of the most typical types of trauma worldwide, and the unique physiology of burn injuries requires the use of specialized therapeutic materials for treatment and makes the development of such materials especially challenging. Here, we report the use of synthetic, functional and biodegradable peptide nanofiber gels for improved healing of burn wounds to alleviate the progressive loss of tissue function at the post-burn wound site. These bioactive nanofiber gels form scaffolds which recapitulate the morphology and function of the natural extracellular matrix through peptide epitopes, which can trigger angiogenesis through significant affinity to basic growth factors. In this study, the angiogenesis-promoting properties of the bioactive scaffolds were utilized for the treatment of thermal burn model. Following the excision of necrotic tissue, bioactive gels and control solutions were applied topically onto the wound area. The wound healing process was evaluated at 7, 14 and 21 days following injury through histological observations, immunostaining and marker RNA / protein analysis. Bioactive peptide nanofiber treated burn wounds formed well-organized and collagen-rich granulation tissue layers, developed a greater density of newly formed blood vessels, and exhibited increased re-epithelialization and skin appendage formation with minimal crust formation. Overall, the heparin-mimetic peptide nanofiber gels increased the rate of repair of burn injuries and can be used as effective means of facilitating wound healing.
Katyonik peptitler ve supramoleküler nanoyapılarının antimikrobiyal amaçla kullanılması
Many organisms including mammalians use Antimicrobial Peptides (AMPs) which are also called Host Defense Peptides against microbial organisms. AMPs are among one of the ancient and successful strategies for both plant and animal kingdoms. Even though AMPs vary among closely related species and despite they have different sequences, many of the natural AMPs share similar properties. They are mostly short sequenced, structurally amphipathic and they carry overall net positive charge. Cationic AMPs target bacterial membranes because of the electrostatic attractions between positively charged peptides and negatively charged membranes. Due to the electrostatic attractions, cationic AMPs might work on membrane disruption by passing a certain threshold concentration for hydrophobic groups to penetrate into membrane. Noncovalent interactions and electrostatic interactions can create molecular attractions and may cause molecular self-assembly which is a common mechanism used by nature for several tasks. Self-assembling peptide amphiphiles are a group of molecules which can form nanofibrous structures and may contain bioactive epitopes depending on the target of the peptide amphiphile molecule. This thesis describes the presentation of antimicrobial sequences on supramolecular nanofibers which are formed by self-assembling peptides. The comparison of self-assembling peptides and single soluble peptides without self-assembling capacity, resulting significant improvement for peptide nanofiber systems for antimicrobial therapeutic purposes is reported.
Nanoyapılar kullanılarak immün yanıt tetiklenmesi
The ability of dendritic cells to coordinate innate and adaptive immune responses makes them essential targets for vaccination strategies. Presentation of specific antigens by dendritic cells is required for the activation of the immune system against many pathogens and cancer, and nanoscale materials can be functionalized for active targeting of dendritic cells. In this work, we integrated an immunogenic, carbohydrate melanoma-associated antigen-mimetic GM3-lactone molecule into mannosylated peptide amphiphile nanofibers to target dendritic cells through DC-SIGN receptor. Based on morphological and functional analyses, when dendritic cells were treated with peptide nanofiber carriers, they showed significant increase in antigen internalization and a corresponding increase in the surface expression of the activation and maturation markers CD86, CD83 and HLA-DR, in addition to exhibiting a general morphology consistent with dendritic cell maturation. These results indicate that mannosylated peptide amphiphile nanofiber carriers are promising candidates to target dendritic cells for antigen delivery. Overall these structures are proven to be effective in terms of dendritic cell activation and maturation and hold high potential to be used with a variety of antigens for different immunotherapy purposes.
Ailesel esansiyel tremor ile ilişkili ekzonik varyantların belirlenmesi ve karakterizasyonu
Essential tremor (ET) is the most common movement disorder in humans. Despite its high heritability and frequency, the genetic basis and pathophysiology of ET is not well understood. In this study, whole exome sequencing and pedigree analyses were performed in unrelated ET families from Anatolia. Whole exome sequencing analysis of family members resulted in the identification of MMP19 p.R456Q in families ET-5 and ET-49. Expression analysis in mice showed a possible developmental pattern for expression of MMP-19 as well as a tissue-specific expression pattern showing high levels of expression in the brain for this gene. Two other families, ET-17 and ET-19 were also analyzed; however the results were not able to identify variant cosegregating with ET in these families. Identification of the new genes related with ET will provide invaluable insights into the underlying mechanism of thıs most common movement disorder and will potentially open new avenues for its treatment.
Hücreler arası matrisin taklit edilmesi için sentetik iskele olarak peptit tabanlı malzemelerin geliştirilmesi
Biomaterials obtained through self-assembling process of peptide amphiphile (PA) molecules provide great potential to introduce new therapeutic approaches in regenerative medicine through mimicking the natural environments of different types of tissues. The ability of self-assembled PA nanofibers to mimic natural extracellular matrix (ECM) renders them attractive for regenerative medicine applications. The materials-cell interactions can be modulated through the surface modification of the materials such as introducing the bioactivity via short bioactive peptide sequences derived from natural ECM proteins, which regulate cell behavior through controlling of cellular activities such as proliferation and differentiation. Herein, I described my studies on the development of PA nanofibers in order to mimic natural ECM with differentiation and regeneration purposes. Heparan sulfate mimetic and laminin mimetic PA nanofibers were used as a potential therapeutic approach in Parkinson's disease (PD). These bioactive PA nanofibers were found to reduce the progressive cell loss in SH-SY5Y cells caused by 6-hydroxydopamine treatment in vitro, and improve neurochemical and behavioral consequences of Parkinsonism in rats and provide a promising new strategy for treatment of PD. These nanofibers also proved to be effective in enhancing the viability of Schwann cells and increase nerve growth factor (NGF) release from these cells in vitro. Since NGF has a crucial role in nerve injury repair and myelination in the regenerating nerve, the bioactive epitopes used in this study present also a promising approach as guidance cues for regenerating axons. Tenascin-C is another multifunctional ECM glycoprotein common in both nerve and bone tissue. By decorating peptide nanofibers with tenascin-C derived epitope and using in three-dimensional (3D) system, this tenascin-C mimetic 3D cell culture system was found to provide both the biochemical and physical aspects of the native environment of neural cells, thereby filling the gap between 2D cell culture models and in vivo environments and contributing to more tissue-like structure and more predictive approaches to organogenesis and tissue morphology. Within the scope of this thesis, tenascin-C mimetic nanofibers were also used for osteogenic differentiation of mesenchymal stem cells (MSCs). They were found to significantly enhance the attachment, proliferation, and osteogenic differentiation of MSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation.
Biyomühendislik ve biyotıp uygulamaları için hücreler arası matriksten esinlenilen peptit nanoyapıların tasarımı ve geliştirilmesi
Advances in understanding of cell-matrix interactions and the regulation of cellular behaviors through nanobiotechnological tools have presented new perspectives for regenerative medicine. Peptide amphiphiles have been used as building blocks for development of bioactive synthetic nanofiber scaffolds for regenerative medicine applications. Biocompatibility, tailorable characteristics, and mechanical stability as well as bioactivitiy of these peptide nanostructures make them ideal candidates for biomedical applications. To guide natural cellular activities, biomaterials should provide a microenvironment similar to that experienced by cells under natural conditions. The native extracellular matrix (ECM) not only provides a suitable physical environment but also incorporates the necessary set of biochemical and mechanical signals to ensure the normal function of cells, as well as mediating their differentiation, morphogenesis and homeostasis by providing biological, physical, and chemical recognition signals that can trigger specific interactions with cell surface receptors. In this thesis, different ECM-mimetic peptide nanofiber formulations were designed and developed, which were shown to have superior chondrogenic and therapeutic effect on stem cell differentiation in vitro and cartilage regeneration in vivo. Hence, the synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as therapeutic agents and model systems.