Investigation of polymorphism in some native cattles for lactoferri̇n gene providing resistance to masti̇ti̇s

Bu çalışma, Aydın ilinde yetiştirilen yerli sığırlarda Laktoferrin gen polimorfizminin belirlenmesi amacıyla yapılmıştır. DNA, farklı lokasyonlarda yer alan 6 sürüde bulunan 100 bireyden alınan kan örneklerinden elde edilmiştir.Genotiplerin belirlenmesi için PCR-RFLP metodu kullanılmıştır. Sığır Laktoferrin genine ait 1050 bç'lik bölge PCR ile çoğaltılmış ve ardından HinfI restriksiyon enzimi ile kesime tabi tutulmuştur. HinfI enzim kesimi sonucu 635, 480, 180, 120 bç'lik bantlardan oluşan bir karışım elde edilmiştir. Bu çalışmada, daha önce literatürde bahsedilmemiş farklı bant uzunluklarına sahip olan (480, 180 ve 120 bç) ve "C" olarak adlandırılan bir allel tanımlanmıştır. Çalışmada, gen frekansları A ve C allelleri için sırası ile 0.435 ve 0.565, genotip frekansları ise AA, AC ve CC genotipleri için sırası ile 0.17, 0.53 ve 0.30 olarak belirlenmiştir. Konuyla ilgili yapılan çalışmalarda yaygın olarak gözlemlenen B alleli bu çalışmada gözlenmemiştir. Daha somut bilgilerin ortaya konabilmesi amacıyla bu gen bölgesinin DNA dizi analizinin yapılması faydalı olacaktır. Bu çalışmadan elde edilen bulgular literatüre önemli katkı sağlayacak düzeydedir. This study was carried out to determine Laktoferrin gene polymorphism in Native cattle raised in Aydın province. DNA was extracted from 100 individual blood samples collected from cattle raised in 6 flocks at different locations. The PCR-RFLP methods were used to determine genotypes. A 1050 bp DNA fragment within bovine Laktoferrin gene was amplified with PCR and then digested with restriction endonuclease enzyme HinfI. The HinfI digestion produced a mixture containing of 635, 480, 180 and 120 bp. In this study we have detected a new allele as ''C'' with different fragments (480, 180 and 120 bp) which has not been observed in literature yet. In present study, A and C allele frequencies were identified with 0.435 and 0.565; AA, AC and CC genotype frequencies were identified with 0.17, 0.53 and 0.30, respectively. The B allele of Laktoferrin gene has not been observed. In order to reveal more concrete information, sequencing for the Laktoferrin gene may be useful. Results obtained from this study will provide a significant contribution to the literature

KARACABEY MERİNOSU KUZULARINDA MELANOKORTİN-4 RESEPTÖR (MC4R) GEN POLİMORFİZMİNİN BÜYÜME ÖZELLİKLERİNE ETKİSİ VE SELEKSİYONDA KULLANIM OLANAKLARININ ARAŞTIRILMASI

KARACABEY MERİNOSU KUZULARINDA MELANOKORTİN-4 RESEPTÖR (MC4R) GEN POLİMORFİZMİNİN BÜYÜME ÖZELLİKLERİNE ETKİSİ VE SELEKSİYONDA KULLANIM OLANAKLARININ ARAŞTIRILMASI Semih SEVİM Doktora Tezi, Zootekni Anabilim Dalı Tez Danışmanı: Prof.Dr. Orhan KARACA 2020, 85 Sayfa Karacabey Merinosu kuzularında yapılan bu çalışmada, Melanokortin-4 Reseptör (Mc4R) geni bakımından polimorfizmin tanımlanması ve kuzuların büyüme özellikleri bakımından genotipler arası farklılıkların ortaya konması amaçlanmıştır. Bu çalışma Koyunculuk Araştırma Enstitüsü Müdürlüğü bünyesindeki koyunculuk şubesinde bulunan 300 baş Karacabey Merinosu kuzuda yürütülmüştür. Kuzuların büyüme özelliklerinin genotiplere göre değişimini saptamak üzere doğum ağırlığı, 45., 90. ve 180. gün canlı ağırlıkları, 0-90.gün, 90-180.gün ve 0-180 gün ortalama günlük canlı ağırlık artışları, 90 ve 180. gün vücut ölçümleri ve ultrason ölçümleri yapılmıştır. Mc4R geni, dizi bilgileri temin edildikten sonra tasarlanan primer çiftleri vasıtasıyla Mc4R1 ve Mc4R2 bölgelerine ayrılmış ve sırasıyla 825 ve 590 bç uzunluğundaki bu bölgeler sorunsuz olarak dizilenmiştir. DNA dizilimi çıkartılan bu bölgelerden Mc4R1 bölgesi içerisinde 9.T>C, 12.G>C, 93.G>A ve 381.G>A SNP’leri; Mc4R2 bölgesi içerisinde ise 681.G>C ve 1016.G>A SNP’leri tespit edilmiştir. 9.T>C SNP’inin 90. gün vücut ölçümlerinden sağrı genişliği (pC SNP’inin 90. gün canlı ağırlığı ve 0-90.gün ortalama günlük canlı ağırlık artışı (pA SNP’inin 90. gün vücut ölçümlerinden sağrı genişiliği (pA SNP’inin 90. gün ultrason ölçümlerinden yağ kalınlığı (pC SNP’sinin 90. gün vücut ölçümlerinden cidago yüksekliği ile but çevresi (p<0.05), 90. gün ultrason ölçümlerinden yağ kalınlığı (p<0.01), 1016.G>A SNP’inin 180. gün canlı ağırlığı (p<0.05), sütten kesim öncesi ortalama günlük canlı ağırlık artışı (p<0.05), sütten kesim sonrası günlük canlı ağırlık artışı (p<0.01), 0-180.gün ortalama günlük canlı ağırlık artışı (p<0.05), 90. gün ultrason ölçümlerinden deri kalınlığı (p<0.05), 180. gün ultrason ölçümlerinden kas derinliği viii (p<0.05) ile ilişkili olduğu bulunmuştur. Sonuç olarak Mc4R geninin koyunlarda büyüme özellikleri için moleküler markör olarak kullanılabileceği düşünülmektedirİÇİNDEKİLER KABUL VE ONAY SAYFASI.............................................................................. iii BİLİMSEL ETİK BİLDİRİM SAYFASI ................................................................v ÖZET..................................................................................................................... vii ABSTRACT............................................................................................................ix ÖNSÖZ ...................................................................................................................xi KISALTMALAR VE SİMGELER DİZİNİ........................................................ xvii ŞEKİLLER DİZİNİ...............................................................................................xix ÇİZELGELER DİZİNİ .........................................................................................xxi 1. GİRİŞ ...................................................................................................................1 2. KAYNAK ÖZETLERİ ........................................................................................6 2.1. Karacabey Merinosu Irkı...................................................................................6 2.2. Koyun Yetiştiriciliğinde MLD (Musculus Longissimus Dorsi) Kasına ait Ölçümlerin Önemi............................................................................................7 2.3. DNA Temelli Markörler....................................................................................8 2.3.1. Rastgele Çoğaltılmış Polimorfik DNA (RAPDs)...........................................8 2.3.2. Çoğaltılmış Parça Uzunluk Polimorfizmi (AFLPs) .......................................9 2.3.3. Tek Zincirli Konformasyon Polmorfizmi (SSCP) .........................................9 2.3.4. Sınırlandırılmış Parça Uzunluk Polimorfizmi (RFLPs) ...............................10 2.3.5. Mikrosatelitler..............................................................................................10 2.3.6. Tek Nükleotid Polimorfizmi (SNP) .............................................................10 2.4. DNA Dizi Analizi Yöntemleri ........................................................................11 2.4.1. Maxam Gilbert Yöntemi ..............................................................................12 2.4.2. Sanger Yöntemi............................................................................................12 2.5. Mc4R Geninin Yapısı ve İşlevi.......................................................................13 2.6. Koyunlarda MLD kası üzerine yapılan çalışmalar..........................................14 xiv 2.7. Çiftlik Hayvanlarında Mc4R Gen Polimorfizmi ile İlgili Yapılmış Çalışmalar ................................................................................................... 16 2.7.1. Koyunlarda yapılan Çalışmalar................................................................... 16 2.7.2. Farklı Türlerde yapılan çalışmalar............................................................... 17 3. MATERYAL VE YÖNTEM ............................................................................ 20 3.1. Materyal.......................................................................................................... 20 3.2. Yöntem........................................................................................................... 20 3.2.1. Hayvanlara Ait Verim Özelliklerinin Belirlenmesi..................................... 20 3.2.2. Kan Örneklerinin Alınması ve Saklanması ................................................. 23 3.2.3. Kan Örneklerinden DNA İzolasyonu .......................................................... 23 3.2.4. DNA’nın Saflık ve Miktar Ölçümü............................................................. 24 3.2.5. Mc4R Geninin PCR ile Yükseltgenmesi..................................................... 24 3.2.6. Dizi Analizi ................................................................................................. 26 3.2.6.1. PCR ürünlerinin saflaştırılması ................................................................ 26 3.2.6.2. Döngüsel dizileme protokolü (Cycle Sequencing)................................... 26 3.2.7. İstatistik Analiz............................................................................................ 27 4. BULGULAR VE TARTIŞMA.......................................................................... 29 4.1. Genomik DNA İzolasyon Sonuçları............................................................... 29 4.2. Mc4R Gen Bölgesinin PCR ile Çoğaltılması ................................................. 29 4.3. DNA Dizi Analizi Sonuçları .......................................................................... 29 4.4. Dizi Analizi ile Genotipleme Sonuçları ......................................................... 33 4.5. Canlı Ağırlık, Vücut ve Ultrason ölçümleri ................................................... 37 4.5.1. Canlı Ağırlık, Vücut ve Ultrason Ölçümlerine Ait Tanımlayıcı İstatistikler ..................................................................................................................... 37 4.6. Mc4R Gen Polimorfizmi ile Büyüme ve Verim Özellikleri Arasındaki İlişkiler ........................................................................................................ 39 4.6.1. Canlı Ağırlık Kayıtları ile Genotipler Arası İlişkiler................................... 39 xv 4.6.2. 90. Güne Ait Vücut ve Ultrason Ölçümleri ile Genotipler Arası İlişkiler....47 4.6.3. 180. Güne Ait Vücut ve Ultrason Ölçümleri ile Genotipler Arası İlişkiler..51 4.7. Haplotip Blokları ile Verim Özellikleri Arasındaki İlişkiler...........................54 4.7.1. Diplotipler ile Canlı Ağırlıklar Arasındaki İlişki .........................................57 4.7.2. Diplotipler ile 90. Güne Ait Vücut ve Ultrason Ölçümleri Arasındaki İlişki .....................................................................................................................62 4.7.3. Diplotipler ile 180. Güne Ait Vücut ve Ultrason Ölçümleri Arasındaki İlişki .............................................................................................................65 5. SONUÇ ..............................................................................................................68 KAYNAKLAR ......................................................................................................71 ÖZGEÇMİŞ ...........................................................................................................8

An Atomic Force Microscope with Dual Actuation Capability for Biomolecular Experiments

We report a modular atomic force microscope (AFM) design for biomolecular experiments. The AFM head uses readily available components and incorporates deflection-based optics and a piezotube-based cantilever actuator. Jetted-polymers have been used in the mechanical assembly, which allows rapid manufacturing. In addition, a FeCo-tipped electromagnet provides high-force cantilever actuation with vertical magnetic fields up to 0.55 T. Magnetic field calibration has been performed with a micro-hall sensor, which corresponds well with results from finite element magnetostatics simulations. An integrated force resolution of 1.82 and 2.98 pN, in air and in DI water, respectively was achieved in 1 kHz bandwidth with commercially available cantilevers made of Silicon Nitride. The controller and user interface are implemented on modular hardware to ensure scalability. The AFM can be operated in different modes, such as molecular pulling or force-clamp, by actuating the cantilever with the available actuators. The electromagnetic and piezoelectric actuation capabilities have been demonstrated in unbinding experiments of the biotin-streptavidin complex

Genotyping of the ? – casein gene by PCR – RFLP (AcuI digestion) and its relationship to milk yield traits in Anatolian buffaloes, Murrah buffaloes, and their crosses

The aim of this study was to investigate the polymorphism of the ? – casein gene (kappa casein, CSN3) in buffaloes and determine its effects on milk yield traits. Data were obtained from 2016 to 2019 from all animals in the ongoing Anatolian Buffalo Breeding Project. Genomic DNA was collected from a total of 209 buffaloes of different ages and sexes, namely 31 Murrah, 46 Anatolian buffaloes, and 132 of their crosses (Murrah × Anatolian). Genotyping of CSN3 gene by polymerase chain reaction–restriction fragment length polymorphism (PCR – RFLP) technique with restriction enzyme (AcuI) revealed three genotypes: AA (0.689), AB (0.282), and BB (0.028), and the frequencies of A and B alleles were 0.83 and 0.17, respectively. A total of 113 milk yield data and 74 milk component analyses of dairy buffaloes were used to compare CSN3 genotypes for milk yield traits. Statistical analysis showed that there was a significant difference in lactation milk yield between CSN3 genotypes: BB, 1560.3 ± 326.7 kg, AB, 1278.1 ± 111.3 kg, and AA, 1060.9 ± 85.6 kg (p 0.05). These results suggest that these variations $(135^{ThrACC} / IleATC /136^{ThrACC} / ThrACT)$ in the buffalo CSN3 gene may be used as genetic markers in dairy buffalo breeding programs

3D printing of covalent organic frameworks: a microfluidic-based system to manufacture binder-free macroscopic monoliths

Covalent organic frameworks (COFs) have witnessed outstanding developments in the past 15 years, particularly in optimizing their pore structures, linkages, and variety of monomers used in their synthesis. Yet, a significant challenge remains unaddressed: the processability of COFs into macroscopic architectures with arbitrary shapes, as they are typically obtained as unprocessable powders. This study presents a novel strategy to address this issue by developing a 3D printable ink comprising a colloidal water suspension of COF nanoparticles. A microfluidic device is engineered that provides precise control over the gelation process of the COF-based ink, allowing for a layer-by-layer fabrication. As a result, the direct production of large-scale binder-free COF architectures from digital designs is achieved at room temperature and atmospheric pressure while eliminating the use of toxic organic solventsThis work had been supported by the Spanish MINECO (PID2019- 106268GB-C32, PID2022-138908NB-C31, TED2021-129886B-C42, PDC2022-133498-I00, and PID2020-116612RB-C33). The authors acknowledge the service from the MiNa Laboratory at IMN and funding from CM (project S2018/NMT-4291 TEC2SPACE), MINECO (project CSIC13-4E-1794) and EU (FEDER, FSE). F.Z. acknowledges financial support from the Spanish Ministry of Science and Innovation, through the “María de Maeztu” Programme for Units of Excellence in R&D (CEX2018- 000805-M). S.P., J.P.-L., and F. Z. also acknowledge support from the European Innovation Council under grant Agreement 101047081 (EVA). The authors acknowledge the support from the “(MAD2D-CM)-UAM” project funded by Comunidad de Madrid, by the Recovery, Transformation and Resilience Plan, and by NextGenerationEU from the European Unio

Electrostatic Catalysis of a Click Reaction in a Microfluidic Cell

Electric fields have been highlighted as a smart reagent in nature's enzymatic machinery, as they can directly trigger or accelerate redox and/or non-redox chemical processes with stereo- and regio-specificity. In natural catalysis, controlled mass transport of chemical species in confined spaces is also key in facilitating the transport of reactants into the active reaction site. Despite the opportunities the above offers in developing strategies for a new, clean electrostatic catalysis exploiting oriented electric fields, research in this area has been mostly limited to theoretical and experimental studies at the level of single molecules or small molecular ensembles, where both the control over mass transport and scalability cannot be tested. Here, we quantify the electrostatic catalysis of a prototypical Huisgen cycloaddition in a large-area electrode surface and directly compare its performance to the traditional Cu(I)-catalyzed method of the same reaction. Mass diffusion control is achieved in a custom-built microfluidic cell, which enhances reagent transport towards the electrified reactive interface while avoiding both turbulent flow conditions and poor control of the convective mass transport. This unprecedented electrostatic continuous-flow microfluidic reactor is an example of an electric-field driven platform where clean large-scale electrostatic catalytic processes can be efficiently implemented and regulated.Comment: Main Manuscript part includes 12 pages, 4 figures, 1 table and Supporting Information part includes 20 pages, 8 figures, 1 tabl

Nanomechanics on FGF-2 and Heparin Reveal Slip Bond Characteristics with pH Dependency

Fibroblast growth factor 2 (FGF-2), an important paracrine growth factor, binds electrostatically with low micromolar affinity to heparan sulfates present on extracellular matrix proteins. A single molecular analysis served as a basis to decipher the nanomechanical mechanism of the interaction between FGF-2 and the heparan sulfate surrogate, heparin, with a modular atomic force microscope (AFM) design combining magnetic actuators with force measurements at the low force regime (1 × 101 to 1 × 104 pN/s). Unbinding events between FGF-2–heparin complexes were specific and short-lived. Binding between FGF-2 and heparin had strong slip bond characteristics as demonstrated by a decrease of lifetime with tensile force on the complex. Unbinding forces between FGF-2 and heparin were further detailed at different pH as relevant for (patho-) physiological conditions. An acidic pH environment (5.5) modulated FGF-2–heparin binding as demonstrated by enhanced rupture forces needed to release FGF-2 from the heparin-FGF-2 complex as compared to physiological conditions. This study provides a mechanistic and hypothesis driven model on how molecular forces may impact FGF-2 release and storage during tissue remodeling and repair

MoSBOTs: Magnetically Driven Biotemplated MoS2_2‐Based Microrobots for Biomedical Applications

2D layered molybdenum disulfide (MoS$_2$) nanomaterials are a promising platform for biomedical applications, particularly due to its high biocompatibility characteristics, mechanical and electrical properties, and flexible functionalization. Additionally, the bandgap of MoS$_2$ can be engineered to absorb light over a wide range of wavelengths, which can then be transformed into local heat for applications in photothermal tissue ablation and regeneration. However, limitations such as poor stability of aqueous dispersions and low accumulation in affected tissues impair the full realization of MoS$_2$ for biomedical applications. To overcome such challenges, herein, multifunctional MoS$_2$-based magnetic helical microrobots (MoSBOTs) using cyanobacterium Spirulina platensis are proposed as biotemplate for therapeutic and biorecognition applications. The cytocompatible microrobots combine remote magnetic navigation with MoS$_2$ photothermal activity under near-infrared irradiation. The resulting photoabsorbent features of the MoSBOTs are exploited for targeted photothermal ablation of cancer cells and on-the-fly biorecognition in minimally invasive oncotherapy applications. The proposed multi-therapeutic MoSBOTs hold considerable potential for a myriad of cancer treatment and diagnostic-related applications, circumventing current challenges of ablative procedures

Synthesis of 2D porous crystalline materials in simulated microgravity

Altres ajuts: the ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The GIWAXS experiments were conducted on the NCD-SWEET beamline of the ALBA synchrotron, Spain. GIWAXS experiments of Ni(HITP) and COF-TAPB-BTCA were performed at the NCD-SWEET beamline at ALBA Synchrotron with the collaboration of ALBA staff.To date, crystallization studies conducted in space laboratories, which are prohibitively costly and unsuitable to most research laboratories, have shown the valuable effects of microgravity during crystal growth and morphogenesis. Herein, an easy and highly efficient method is shown to achieve space-like experimentation conditions on Earth employing custom-made microfluidic devices to fabricate 2D porous crystalline molecular frameworks. It is confirmed that experimentation under these simulated microgravity conditions has unprecedented effects on the orientation, compactness and crack-free generation of 2D porous crystalline molecular frameworks as well as in their integration and crystal morphogenesis. It is believed that this work will provide a new "playground" to chemists, physicists, and materials scientists that desire to process unprecedented 2D functional materials and devices

Magnetic PiezoBOTs: a microrobotic approach for targeted amyloid protein dissociation

Piezoelectric nanomaterials have become increasingly popular in the field of biomedical applications due to their high biocompatibility and ultrasound-mediated piezocatalytic properties. In addition, the ability of these nanomaterials to disaggregate amyloid proteins, which are responsible for a range of diseases resulting from the accumulation of these proteins in body tissues and organs, has recently gained considerable attention. However, the use of nanoparticles in biomedicine poses significant challenges, including targeting and uncontrolled aggregation. To address these limitations, our study proposes to load these functional nanomaterials on a multifunctional mobile microrobot This microrobot is designed by coating magnetic and piezoelectric barium titanate nanoparticles on helical biotemplates, allowing for the combination of magnetic navigation and ultrasound-mediated piezoelectric effects to target amyloid disaggregation. Our findings demonstrate that acoustically actuated PiezoBOTs can effectively reduce the size of aggregated amyloid proteins by over 80% in less than 10 minutes by shortening and dissociating constituent amyloid fibrils. Moreover, the PiezoBOTs can be easily magnetically manipulated to actuate the piezocatalytic nanoparticles to specific amyloidosis-affected tissues or organs, minimizing side effects. These biocompatible PiezoBOTs offer a promising non-invasive therapeutic approach for amyloidosis diseases by targeting and breaking down protein aggregates at specific organ or tissue sites