Bindungsstudien zur Komplexierung von Nukleotiden und Polynukleotiden

Die molekulare Erkennung von biologisch relevanten Substraten wie Polynukleotiden, Nukleotiden, Aminosäuren und Carboxylaten unter physiologischen Bedingungen ist immer noch eine komplexe Aufgabe auf dem Forschungsgebiet der Supramolekularen Chemie. Ziel der vorliegenden Arbeit war die Entwicklung, kleiner künstlicher, poly-kationischer Verbindungen, welche in der Lage sind, an Polynukleotide, an deren Bausteine die Nukleo-tide, sowie an weitere biologisch relevante Substrate zu binden. Dabei stellte die ausschließ-liche Verwendung von nicht-kovalenten Wechselwirkungen für die Komplexierung unter physiologischen Bedingungen eine besondere Herausforderung dar. Ein Großteil der Arbeit beschäftigte sich mit Untersuchungen zu den Komplexierungseigen-schaften der poly-kationischen Rezeptoren gegenüber Nukleotiden und weiteren physiolo-gisch relevanten Substraten. Überraschenderweise zeigte einer der poly-kationischen Rezep-toren bei UV/Vis-spektroskopischen Untersuchungen, in einem Gemisch aus 60 % Methanol in Wasser bei pH 6, eine deutliche Präferenz für AMP gegenüber seinen höhergeladenen Homologen dem ADP und dem ATP. Durch die Kombination unterschiedlicher Analyseme-thoden, wie UV/Vis-, NMR-Spektroskopie und computergestützte Kraftfeldrechnungen konnte ein Erklärungsmodel für dieses selektive Bindungsverhalten aufgestellt werden. Alle Ergebnisse der strukturanalytischen Untersuchungen zeigten, dass für die bevorzugte Bin-dung des AMPs die Ausbildung einer hydrophoben Kavität, in welche die Nukleobase des AMPs hineinragt, ausschlaggebend ist. Bindungsstudien in Wasser bei pH 7 konnten zeigen, dass die hydrophoben Wechselwir-kungen unter physiologischen Bedingungen noch verstärkt werden. Dies konnte auch durch die Synthese eines analogen poly-kationischen Rezeptors bewiesen werden, welcher auf-grund einer zusätzlichen aromatischen Struktureinheit ausgeprägtere hydrophobe Wech-selwirkungen ausbildet und somit die Nukleobasen stärker binden kann. Neben der Synthese neuer poly-kationischer Moleküle lag ein weiterer Schwerpunkt dieser Arbeit in der Entwicklung und Etablierung neuer analytischer Methoden zur Untersuchung von Polynukleotid-Komplexen hinsichtlich ihrer Struktur und Eigenschaften. Sowohl die Ergebnisse der UV/Vis- und CD-Spektroskopie, als auch die der isothermalen Titrationska-lorimetrie zeigten eine deutliche Wechselwirkung der poly-kationischen Verbindungen mit den Furchen der Polynukleotide aufgrund von elektrostatischen Wechselwirkungen. Zur genaueren strukturellen Untersuchung der gebildeten Polynukleotid-Komplexe wurde das bildgebende Verfahren der Rasterkraftmikroskopie eingesetzt. Hierbei zeigte sich eine signifikante Abhängigkeit der Morphologie der ausgebildeten Aggregate von der Konzentra-tion an zugegebener poly-kationischer Verbindung. Mit Hilfe der in dieser Arbeit durchgeführten Bindungsstudien an Nukleotiden und Poly-nukleotiden konnte gezeigt werden, dass es beim Übergang von den strukturell kleinen Nu-kleotiden zu den wesentlich größeren Polynukleotiden zu einem Wechsel der für die Kom-plexierung verantwortlichen supramolekularen Wechselwirkung kommt. So ist im Falle der Nukleotide die Ausbildung einer hydrophoben Kavität der entscheidende Faktor, wohinge-gen die Bindung an die Polynukleotide hauptsächlich auf elektrostatische Wechselwirkun-gen zurückzuführen ist

Morphological characterization of the human corneal epithelium by in vivo confocal laser scanning microscopy

Background: Regarding the growing interest and importance of understanding the cellular changes of the cornea in diseases, a quantitative cellular characterization of the epithelium is becoming increasingly important. Towards this, the latest research offers considerable improvements in imaging of the cornea by confocal laser scanning microscopy (CLSM). This study presents a pipeline to generate normative morphological data of epithelial cell layers of healthy human corneas. Methods: 3D in vivo CLSM was performed on the eyes of volunteers (n=25) with a Heidelberg Retina Tomograph II equipped with an in-house modified version of the Rostock Cornea Module implementing two dedicated piezo actuators and a concave contact cap. Image data were acquired with nearly isotropic voxel resolution. After image registration, stacks of en-face sections were used to generate full-thickness volume data sets of the epithelium. Beyond that, an image analysis algorithm quantified en-face sections of epithelial cells regarding the depth-dependent mean of cell density, area, diameter, aggregation (Clark and Evans index of aggregation), neighbor count and polygonality. Results: Imaging and cell segmentation were successfully performed in all subjects. Thereby intermediated cells were efficiently recognized by the segmentation algorithm while efficiency for superficial and basal cells was reduced. Morphological parameters showed an increased mean cell density, decreased mean cell area and mean diameter from anterior to posterior (5,197.02 to 8,190.39 cells/mm²; 160.51 to 90.29 µm²; 15.9 to 12.3 µm respectively). Aggregation gradually increased from anterior to posterior ranging from 1.45 to 1.53. Average neighbor count increased from 5.50 to a maximum of 5.66 followed by a gradual decrease to 5.45 within the normalized depth from anterior to posterior. Polygonality gradually decreased ranging from 4.93 to 4.64 sides of cells. The neighbor count and polygonality parameters exhibited profound depth-dependent changes. Conclusions: This in vivo study demonstrates the successful implementation of a CLSM-based imaging pipeline for cellular characterization of the human corneal epithelium. The dedicated hardware in combination with an adapted image registration method to correct the remaining motion-induced image distortions followed by a dedicated algorithm to calculate characteristic quantities of different epithelial cell layers enabled the generation of normative data. Further significant effort is necessary to improve the algorithm for superficial and basal cell segmentation

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition.

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones

Совершенствование системы нормирования труда рабочих

Объект исследования - ЗАО "СИБУР-Транс". Предмет исследования – система нормирования труда на предприятия. Цель ВКР ? совершенствование системы нормирования труда на предприятии для активизации трудового потенциала на повышение производительности труда работников.The object of study - CJSC "SIBUR-TRANS". Subject of research - the system of regulation of labor in the enterprise. The purpose of WRC-improving the system of regulation of labor in the enterprise to enhance the labor potential to increase productivity of workers

Carcinoma cells misuse the host tissue damage response to invade the brain

The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis

Pre–B cell receptor–mediated cell cycle arrest in Philadelphia chromosome–positive acute lymphoblastic leukemia requires IKAROS function

B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre–B cell receptor–dependent stages. The Philadelphia chromosome–positive (Ph+) subtype of ALL accounts for 25–30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre–B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph+ ALL cells. Pre–B cell receptor–mediated cell cycle arrest in Ph+ ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre–B cell receptor signaling pathway, even if expression of the pre–B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre–B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph+ ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre–B cell receptor–mediated tumor suppression

BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKIs). However, unless CML patients receive life-long TKI treatment, leukemia will eventually recur; this is attributed to the failure of TKI treatment to eradicate leukemia-initiating cells (LICs). Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells; however, the mechanism of FoxO-dependent leukemia initiation remained elusive. Here, we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. BCL6 represses Arf and p53 in CML cells and is required for colony formation and initiation of leukemia. Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia initiation in transplant recipients and selectively eradicates CD34+ CD38− LICs in patient-derived CML samples. These findings suggest that pharmacological inhibition of BCL6 may represent a novel strategy to eradicate LICs in CML. Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation

Optimization and scale-up of inulin extraction from Taraxacum kok-saghyz roots

The optimization and scale-up of inulin extraction from Taraxacum kok-saghyz Rodin was successfully performed. Evaluating solubility investigations, the extraction temperature was fixed at 85°C. The inulin stability regarding degradation or hydrolysis could be confirmed by extraction in the presence of model inulin. Confirming stability at the given conditions the isolation procedure was transferred from a 1 L- to a 1 m3-reactor. The Reynolds number was selected as the relevant dimensionless number that has to remain constant in both scales. The stirrer speed in the large scale was adjusted to 3.25 rpm regarding a 300 rpm stirrer speed in the 1 L-scale and relevant physical and process engineering parameters. Assumptions were confirmed by approximately homologous extraction kinetics in both scales. Since T. kok-saghyz is in the focus of research due to its rubber content side-product isolation from residual biomass it is of great economic interest. Inulin is one of these additional side-products that can be isolated in high quantity (~ 35% of dry mass) and with a high average degree of polymerization (15.5) in large scale with a purity of 77%

T-cell proliferation assay for the detection of SARS-CoV-2-specific T-cells

Both infection with and vaccination against SARS-CoV-2 trigger a complex B-cell and T-cell response. Methods for the analysis of the B-cell response are now well established. However, reliable methods for measuring the T-cell response are less well established and their usefulness in clinical settings still needs to be proven. Here, we have developed and validated a T-cell proliferation assay based on 3H thymidine incorporation. The assay is using SARS-CoV-2 derived peptide pools that cover the spike (S), the nucleocapsid (N) and the membrane (M) protein for stimulation. We have compared this novel SARS-CoV-2 lymphocyte transformation test (SARS-CoV-2 LTT) to an established ELISA assay detecting Immunoglobulin G (IgG) antibodies to the S1 subunit of the SARS-CoV-2 spike protein. The study was carried out using blood samples from both vaccinated and infected health care workers as well as from a non-infected control group. Our novel SARS-CoV-2 LTT shows excellent discrimination of infected and/or vaccinated individuals versus unexposed controls, with the ROC analysis showing an area under the curve (AUC) of > 0.95. No false positives were recorded as all unexposed controls had a negative LTT result. When using peptide pools not only representing the S protein (found in all currently approved vaccines) but also the N and M proteins (not contained in the vast majority of vaccines), the novel SARS-CoV-2 LTT can also discriminate T-cell responses resulting from vaccination against those induced by infection