Analyse der Kreuzreaktivität monoklonaler Antikörper mit Leukozyten von Equiden und Klonierung von CD28

Title, table of contents, abbreviations. I. REVIEW OF LITERATURE 6 II. AIM OF THE STUDY 19 III. MATERIAL AND METHODS 20 IV. RESULTS (4.1) 43 IV. RESULTS (4.2) 81 IV. RESULTS (4.3) 92 IV. RESULTS (4.4) 100 V. DISCUSSION 104 VI. SUMMARY 126 VII. REFERENCES 130 VIII. Appendix 148 PUBLICATIONS 156 AKNOWLEDGMENTS 157 SELBSTÄNDIGKEITSERKLÄRUNG 158Monoclonal antibodies identifying leukocyte antigens are highly important tools for studying equine leukocyte biology. The rational of the present study was to provide domestic and wild equine immunology with new antibodies to leukocyte markers filling gaps of available reagents and establish a leukocyte tool-box for some members of family Equidae. To achieve the target, a large panel of mostly commercially available anti-leukocyte mAbs was analyzed for reactivity with equid leukocytes and cell lines. Analysis of 534 mAbs demonstrated for 31 mAbs a clear positive reactivity, for 2 mAbs a weak positive reactivity; for 23 mAbs a positive "+"; but possibly valuable staining; for 15 mAbs an alternate expression pattern from that expected from human immunology; for 6 mAbs a questionable staining; for 45 mAbs a weak- positive reactivity and alternate expression pattern, and 356 mAbs remained negative. In 53 cases, more appropriate target cells, such as thymocytes or bone marrow stem cells, were not available for screening. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD34, CD44, CD45, CD49d, CD68, CD83, CD91, CD163, CD172a, CD206, CD283, MHCI, MHCII, and canine B cell molecule. 22 mAbs spanning 16 different CD antigens were analyzed with Somali wild ass (Equus africanus somalicus), Grevy s zebra (Equus grevyi), and Hartmann s mountain zebra (Equus zebra hartmannae) leukocytes using one colour flow cytometry. Most but not all mAbs cross reacted with the three species. Cross-reactive mAbs analyzed at this part of the study may be considered the basis to establish an immune tool-box for these wild equids but require further analysis. In addition, a broad panel of mAbs predominantly reactive with equine PBMC were applied on equine cell lines (EqT8888 and eCAS). A limited number of these mAbs reacted positively with the equine cell lines. Based on the obtained data, eCAS may represent a very early stage of a myeloid cell line and EqT8888 may be a B cell lymphoma. Analysis of CD28 was performed for some members of the order Perissodactyla, including three members of family Equidae: Somali wild ass, Hartmann s mountain zebra, and Grevy s zebra. In addition, a member of family Rhinocerotidae, the Greater one-horned rhinoceros (Rhinoceros unicornis) was included beside the Asian elephant (Elephas maximus) representing order Proboscidae, family Elephantidae and members of order Artiodactyla, family Bovidae, such as European bison (Bison bonasus) and African Buffalo (Syncerus caffer) and one member of family Giraffidae, Nubian Giraffe (Giraffa camelopardalis). Comparison of CD28 aa sequence revealed a high degree of interspecies conservation with aa identity ranging from 68-99%. Animal sequences were more closely related between each other than to humans and mice represented the out-group. 18 aa mismatches between equids and human were detected in the extracellular domain and could explain the inability of six human CD28 specific clones to stain horse leukocytes. Anti-human polyclonals were used as another tool to detect important equine CD antigens like CD28 and CD25 to which none of the submitted mAbs were positive with horse leukocytes. The anti-human CD28 polyclonal Ab detected a protein likely to be EqCD28 on a small population of lymphocytes in flow cytometry only, but precipitated it as two proteins of 57 and 46 kDa from surface biotinylated horse lymphocytes. Cloning and expression of EqCD28 in insect cells also resulted in two proteins of 57 and 46 kDa. Equine CD25 expression was detected using a goat anti-human IL-2R polyclonal antibody in flow cytometry. In addition, equine CD25 was immunoprecipitated using this Ab as a molecule of approximately 55 kDa molecular weight which is analogous to human CD25. Although just a few mAbs were positive on equine cells (31/534), the approach may be considered useful, especially as their specificity has been resolved in this study. In addition, this study supports the use of highly purified anti-human polyclonal antibodies as an alternative approach in the analysis of equine leukocyte if their cross-reactivity can be confirmed and background signals eliminated.Monoklonale Antikörper (mAk), die Leukozyten Antigene identifizieren sind wichtig um die Biologie von equinen Leukozyten studieren zu können. Das Ziel dieser Studie war es, neue Antikörper, die sich als Marker für equine Leukozyten eignen, zu etablieren, um vorhandene Lücken in dem schon bestehenden Sortiment der equinen Immunologie zu füllen. Um dieses Ziel zu erreichen, wurde ein großes Feld von weitgehend kommerziell erhältlichen anti- Leukozyten mAk auf ihre Reaktionsfähigkeit mit equinen Leukozyten und Zelllinien untersucht. 534 mAk wurden untersucht, davon zeigten 31 mAk eine deutliche positive Reaktion, 2 mAk eine schwach positive Reaktion; 23 mAk eine positive, möglicherweise anwendbare Färbung; 15 mAK ein anderes Expressionsmuster als aus der human Immunologie erwartet. Für 6 mAk zeigte sich eine fragwürdige Färbung, für 45 mAk eine schwach positive Reaktion und abweichendes Expressionsmuster und 356 mAk blieben negativ. In 53 Fällen standen geeignete Zielzellen, wie Thymozyten oder Stammzellen nicht zur Verfügung. Positive mAk waren gegen CD2, CD5, CD11a, CD14, CD18, CD21, CD34, CD44, CD45, CD49d, CD68, CD83, CD91, CD163, CD172a, CD206, CD283, MHCI, MHCII, und ein canines B Zell Molekül gerichtet. 22 mAk, die 16 unterschiedliche CD Antigene umfassten, wurden auf Leukozyten von Somali Wild Esel (Equus africanus somalicus), Grevy s Zebra (Equus grevyi), und Hartmann s Zebra (Equus zebra hartmannae) mittels Durchflusszytometrie getestet. Die meisten Antikörper haben mit den drei Spezies kreuzreagiert. Die in diesem Teil der Studie untersuchten mAk können als Basis für die immunologische Untersuchung dieser wilden Equiden dienen. Sie benötigen aber weitergehende Untersuchungen um die Ergebnisse zu bestätigen. Zusätzlich wurde eine breite Auswahl von mAk, die zumeist mit equinen PBMC reagiert haben, auf equinen Zelllinien (EqT8888 und eCAS) getestet. Eine geringe Zahl dieser mAk zeigte eine positive Reaktion auf diesen. Basierend auf den erhaltenen Daten wäre es möglich dass eCAS einem sehr frühen Stadium einer myeloiden Zelllinie entspricht und EqT8888 der eines B-Zell Lymphoms. Die Analyse von CD28 wurde bei einigen Tieren der Ordnung Perissodactyla, einschließlich dreier Mitglieder der Familie Equidae, Somali Wild Esel, Hartmann s Zebra, und Grevy s Zebra durchgeführt. Zusätzlich wurde das Panzernashorn (Rhinoceros unicornis) als ein Angehöriger der Familie Rhinocerotidae einbezogen. Weiterhin wurden ein asiatischer Elefant (Elephas maximus) als Vertreter der Ordnung Proboscidae, Familie Elephantidae, ein europäisches Bison (Bison bonasus) und ein afrikanischer Büffel (Syncerus caffer) der Ordnung Artiodactyla, Familie Bovidae und eine Nubische Giraffe (Giraffa camelopardalis) als Angehörige der Familie Giraffidae einbezogen. Der Vergleich von CD28 Aminosäure Sequenz zeigte eine hohe Identität zwischen den Aminosäuren (68-99%) der einzelnen Spezies. Das Unvermögen der sechs human- spezifischen CD28 Ak-Klone, Pferde-Leukozyten zu färben, lässt sich möglicherweise darauf zurückführen, dass 18 Aminosäuren in der extrazellulären Domäne abweichen. Die verschiedenen tier-spezifischen Sequenzen zeigten untereinander eine größere Ähnlichkeit als zur humanen oder Maussequenz, letztere bildete die Außengruppe. Polyklonale anti-humane AK wurden als ein weiteres Werkzeug eingesetzt, um andere wesentliche CD-Antigene wie CD28 und CD25 nachzuweisen, da keiner der bereits getesteten mAk mit den Pferde Leukozyten reagiert hatte. Der polyklonale anti-humane CD28 Ak konnte ein Protein, welches wahrscheinlich EqCD28 ist, auf einer kleinen Population der Lymphozyten mittels Durchflusszytometer nachweisen, präzipitierte aber in zwei Proteine der Größe von 57 kDa und 46 kDa von der Oberfläche biotinylierter Pferde Lymphozyten. Die Klonierung und Expression von CD28 in Insektenzellen ergab ebenfalls zwei Proteine von 57 und 46kDa. Die Expression von CD25 wurde mittels einem polyklonalen Ziege anti-human IL-2R alpha Ak im Durchflusszytometer nachgewiesen. Zusätzlich wurde equines CD25 als ein Molekül mit dem ungefähren Gewicht von 55kDa mittels Immunpräzipitation dargestellt, analog zu dem humanen CD25. Obwohl nur einige der monoklonalen Antikörper (31/534) auf equinen Zellen positiv reagiert haben, kann dieser Ansatz als wertvoll erachtet werden, vor allem da ihre Spezifität in dieser Studie geklärt wurde. Weiterhin, unterstützt diese Studie die Verwendung von gereinigten anti-human polyklonalen Ak als einen alternativen Ansatz für die Analyse equiner Leukozyten, sofern ihre Kreuzreaktivität bestätigt werden kann und Hintergrund-Signale eliminiert werden können

Probing the resistance switching mechanisms in SiOₓ/Ag RRAM devices

Resistive random access memory (RRAM) devices represent promising candidates for emerging non-volatile data storage applications and neuromorphic computing. In those devices, the resistance of a dielectric -often a binary oxide- is switched between a low resistance state (LRS) and one or more high resistance states (HRS) by the application of an appropriate external electrical bias. This resistance switching could be filamentary, i.e., involves the formation of a conductive filament. This filament can be thought of as chains of conductive oxygen vacancies (intrinsic resistance switching) or metallic atoms from an active device electrode (extrinsic resistance switching). In this thesis, the relationship between device electrode material and its resistance switching mechanism in SiOx (x∼1.9)-based RRAM devices was studied. Although it’s widely reported that RRAM devices with electrochemically active top electrodes, such as Ag, switch extrinsically, I show that both mechanisms and their associated conductive filaments can be triggered during device switching in ambient conditions. Resistance vs temperature measurements and conduction mechanism analysis were used to probe the nature of the formed filaments within device oxide layer. Results show that the two mechanisms can coexist within the device during switching. The type of filament generated by the initial electroforming of the device, however, depends on the polarity of the applied voltage during the electroforming step. This finding could help in optimising those RRAM devices for the different storage applications. Although the two mechanisms were observed under ambient conditions, SiOx/Ag devices showed extrinsic switching behaviour only under vacuum. In such an oxygen-poor environment, the contribution of intrinsic resistance switching mechanism appears to be reduced or probably eliminated. In extrinsically electroformed RRAM devices with Ag top electrodes, a metallic filament is likely to form within the switching layer. Using conductance tomography technique, the metallic filament of those SiOx/Ag devices was partially imaged using a conductive AFM (CAFM) tip

Contractors’ Perspectives towards Factors Affecting Cost Estimation in Palestine

Construction projects have certain characteristics that determine the appropriate actions to estimate their costs accurately. A key factor for a successful project is the preparation of an accurate estimate, which can influence factors ranging from project feasibility to profitability. The success or failure of a project is dependent on the accuracy of several estimates throughout the course of the project. Construction estimating is the compilation and analysis of many items that influence and contribute to the cost of a project. The purpose of this paper is to identify the essential factors related to cost estimation and to show the degree of importance of these factors in cost estimation of construction projects in the Gaza strip from contractors’ perspective. The results of analyzing fifty one factors considered in a questionnaire survey concluded that the main factors are: location of the project, segmentation of the Gaza strip and limitation of movements between areas, political situation and closure of the Gaza strip, financial status of the owner and increase in unit costs of construction materials. It is recommended to review cost estimation practice in terms of education and training required for those responsible for the estimating process. Contracting companies of various classifications are advised to provide training programs for their estimators to gain experience in cost estimation

Reflective Practices and Level of Technological Acceptance and their Relationship to Developing Teaching Performance of Arabic Language Teachers in The Light of Digital Transformation

The current research aimed at identifying the reflective practices of Arabic language teachers in the light of digital transformation and estimating the level of technological acceptance and their relationship to developing the teaching performance of Arabic language teachers. This research adopted the analytical descriptive approach. The procedures followed by the two researchers to apply this approach are represented in using the Reflective Practice Scale for Arabic language teachers in the light of digital transformation, and the Technological Acceptance Scale for Arabic language teachers, as well as using the teaching Performance Development Observation Card To identify the relationship of each of the reflective practices and the level of technological acceptance to the development of the teaching performance of Arabic language teachers in the light of the digital transformation, the current research was delimited to selecting a simple random sample of Arabic language teachers (412) who represent 30% of the research group in the city of Dammam. The research concluded that reflective practices apply to Arabic language teachers to a (large) degree. The most reflective practice that applies to Arabic language teachers in the light of digital transformation is to ensure that the information is correct before transmitting it to students . The level of technological acceptance of Arabic language teachers is (medium). The most technologically acceptable level for Arabic language teachers is their confidence in using electronic platforms. There is a positive, statistically significant correlation at the significance level (0.05) of the reflective practices in developing the teaching performance of Arabic language teachers in the light of digital transformation. The most correlative relationship was between reflective practices and evaluation, and the least relationship was between reflective practices and development. There was a positive, statistically significant correlation at the significance level (0.05) of technological acceptance in developing the teaching performance of Arabic language teachers in the light of digital transformation. The most correlative relationship was between the level of technological acceptance and evaluation, while the least relationship was between the level of technological acceptance and development