<p> Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population.</p> <p> The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. </p> <p> The gene <i>Dual-specificity tyrosine-regulated kinase 1a </i>(<i>Dyrk1a</i>) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic <i>Dyrk1a</i> is related to the emergence of trabecular defects at P30, expression of <i>Dyrk1a</i> in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30.</p> <p> The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background.</p> These data led to the hypothesis that reduction of <i>Dyrk1a</i> gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce <i>Dyrk1a</i> gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic <i>Dyrk1a</i> does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

This diploma thesis deals with selection, design and creation of new education materials for high school teacher supporting webpage www.studiumchemie.cz. The selection of the proper theme was done on the basis of research and evaluation of recent materials on the mentioned webpage and materials created at the Department of chemical education of Faculty of Science of Charles University in Prague. Preliminarily, the themes of synthetic macromolecular materials and new materials were selected. The convenience of the selected themes was confirmed by research and evaluation concerned to the preliminarily selected theme of recent high school textbooks and webpages. Consequently, educational materials in the form of PowerPoint presentations, websites, worksheets, tests, games and experiments focused on the themes of synthetic macromolecular substances and new materials were created. Materials were processed in accordance with the Framework Education Programme for Secondary General Education (Grammar Schools) as well as in accordance with the requirements of the Catalogue of graduation exam requirements from chemistry. The PowerPoint presentation and the webpage on synthetic macromolecular materials were roughly evaluated by group of teachers and the results of the evaluation are also part of the thesis....

The development of solution-processable semiconducting polymers has brought mankind’s long-sought dream of plastic electronics to fruition. Their potential in the manufacturing of lightweight, flexible yet robust, and biocompatible electronics has spurred their use in organic transistors, photovoltaics, electrochromic devices, batteries, and sensors for wearable electronics. Yet, despite the successful engineering of semiconducting polymers, we do not fully understand their molecular behavior and how it influences their doping (oxidation/reduction) properties. This is especially true for donor-acceptor (D-A) p-systems which have proven to be very efficient at tuning the electronic properties of organic semiconductors. Historically, chain-length dependent studies have been essential in uncovering the relationship between the molecular structure and polymer properties. Discussed here is the systematic investigation of a complete D-A molecular series composed of monodispersed and well-defined conjugated molecules ranging from oligomer (n=3-21) to polymer scale lengths. Structure-property relationships are established between the molecular structure, chain conformation, and redox-active opto-electronic properties for the molecular series in solution. This research reveals a rod-to-coil transition at the 15 unit chain length, or 4500 Da, in solution. The redox-active optical and electronic properties are investigated as a function of increasing chain-length, giving insight into the nature of charge carriers in a D-A conjugated system. This research aids in understanding the solution behavior of conjugated organic materials. <br>

<p>DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While <i>dnmt3a</i> and <i>dnmt3b</i> genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme. </p> <p>DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.</p> <p>Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B. </p>

<p> </p> <p>Learning about ‘behavior’ has always been at the heart of my research endeavors. While my undergraduate work in evolution and ecology exposed me to the science behind why a behavior exists, in my graduate work, I intended to explore how to use something’s behavior to widen its applicability. In this thesis, <em>I will present three works that utilize some of the fundamental</em></p> <p><em>behaviors (i.e., properties) of elastin-like polypeptides (ELP) to improve existing protein purification methods or explore their applicability in bladder cancer imaging and immunotherapy. </em></p> <p>Bladder cancer has high recurrence rates (60-70 % annually) that necessitate multiple follow-up therapies making it one of the costliest cancers per patient. In this work, we have attempted to address two leading causes of the recurrence. First is a low sensitivity (62-84 %) and variable specificity (43-95 %) of white light cystoscopy used to diagnose and remove tumors. We aimed to address the heart of this problem, i.e., the non-specific mode of detection using white light. Only the trained eyes can discern abnormal from normal-appearing tissues even then, leaving up to 45% of tumors unresected to colonize and spread. <em>We developed and characterized near infrared dye-peptide-ligand conjugates (NIR-ELP-ligand) that undergo receptor-mediated binding and internalization to human bladder cancer cells in vitro and tissues ex vivo.</em> By using a molecular target-based probe in combination with NIR imaging, we can aid in improving the detection limit via selective binding to the tumor and reduction in background autofluorescence.</p> <p>Bacillus-Calmette Guérin (BCG) instillation in the bladder is the gold-standard</p> <p>immunotherapy used after surgical removal of bladder tumors. This was approved as a response to the inefficiency of surgery alone in improving cancer status. It has succeeded by reducing the recurrence rate to 30-50 %. But it comes with the complications of putting a live mycobacterium</p> <p>in the human body and giving a patient a urinary tract infection right after surgical tumor resection. <em>Thus, we aimed to deliver nucleic acid as immunotherapeutic cargo in a selective manner to elicit robust anti-tumor immune responses while minimizing the side effects due to its carrier.</em> Towards</p> <p>this goal, we have developed a highly modular and adaptable ELP-ligand fusion protein-based nucleic acid delivery carrier targeted toward bladder cancer. Before developing targeted peptide-based cancer imaging and nucleic acid delivery modalities, we addressed the Achilles heel of peptide-based approaches. The peptide and protein industry suffers</p> <p>through complex, time-consuming, inconsistent, and low-yielding purification methods. <em>We have developed a scalable, facile, and reproducible protein purification method that delivers ELP and ELP fusion proteins free of host cell proteins and nucleic acids and has low lipopolysaccharide</em></p> <p><em>content in just 3 h starting from a bacterial pellet. </em>Thus, for a coherent narrative, the thesis is structured as follows:</p> <p>1. Introduction</p> <p>2. ELP as a protein purification tag: Development of a rapid purification method for ELPs and ELP fusion proteins.</p> <p>3. ELP as a cancer imaging agent: Development of NIR-ELP-Ligand imaging probe targeting bladder cancer.</p> <p>4. ELP as a drug delivery agent: Utilizing ELP-ligand fusion protein in the formulation of targeted nucleic acid delivery carrier to bladder cancer.</p>

Selon leur formulation et leur mise en forme et grâce à leur complexité microstructurale induite, les polymères thermoplastiques bénéficient d’une grande diversité de propriétés thermomécaniques. Cependant, l’évolution de la microstructure de ces matériaux au cours de leur utilisation reste difficile à identifier. Afin de mieux comprendre les modifications microstructurales ayant lieu au cours de sollicitations thermomécaniques, différentes techniques non destructives de caractérisation en temps réel et in situ ont été développées. Dans ce contexte, un Poly (Ethylène Téréphtalate) (PET) amorphe et semi-cristallin a été étudié afin de mettre en évidence l’effet de la microstructure sur les propriétés macroscopiques du matériau. Pour ce faire, plusieurs couplages de techniques expérimentales de caractérisation ont été mis en œuvre tels que la spectroscopie Raman et la diffraction/diffusion des rayons X couplées au système de VidéoTraction™ ou la spectroscopie Raman couplée à la calorimétrie différentielle à balayage (DSC) pour une caractérisation des micromécanismes de déformation et du comportement thermique du matériau respectivement. Le suivi de différentes bandes vibrationnelles judicieusement identifiées a permis d’établir un nouveau critère robuste et capable de mesurer avec exactitude le taux de cristallinité du matériau ou de remonter aux températures caractéristiques de sa morphologie (Tg, Tc, Tcc, Tf) grâce aux informations extraites d’un spectre Raman. De plus, un système de caractérisation relaxationnelle par un couplage de la spectroscopie diélectrique dynamique avec un essai de traction a été utilisé afin de mettre en évidence l’effet de la mobilité moléculaire sur la déformation élasto-visco-plastique du PET. D’un point de vue mécanique, les principaux micromécanismes de déformation ont été étudiés en temps réel pendant un essai de traction à différentes températures et vitesses de déformation vraies constantes : l’orientation macromoléculaire, l’endommagement volumique, le développement de mésophase et la cristallisation induite sous contrainte, ont été observés et quantifiés in situ en utilisant les couplages précédents au synchrotron Petra III de Hambourg et au synchrotron Elettra de Trieste. En parallèle, une étude de la mobilité moléculaire (paramètre déterminant à la prédominance de tel ou tel micromécanisme de déformation) a été menée via des analyses relaxationnelles réalisées au cours de la déformation du matériau. En complément, des expériences en temps réel, des études post mortem par les techniques précédemment citées et par radiographie X, microscopie électronique à balayage et tomographie X ont été réalisées afin d’apprécier l’influence de la relaxation mécanique du PET. / According to their formulations and forming processes and thanks to the complexity of their induced microstructure, thermoplastic polymers show a wide range of thermomechanical properties. However, the identification of the evolution of the microstructure of these materials during their use remains difficult. To better understand the microstructural changes occurring during thermomechanical loadings, various in situ and non-destructive techniques of characterization have been used. In this context, a Poly (Ethylene Terephthalate) (PET) amorphous and semi-crystalline was studied in order to highlight the effect of the microstructure on the macroscopic properties of the material. This way, different coupling systems combining several experimental characterization techniques have been implemented such as Raman spectroscopy and X-rays diffraction/scattering coupled to the VidéoTraction™ system or Raman spectroscopy coupled with differential scanning calorimetry (DSC) for the characterization of the deformation micro-mechanisms and the thermal behavior of the material respectively. Monitoring specific vibrational bands thoroughly identified allowed the establishment of a new robust criterion which enables to accurately measure the crystallinity ratio of the material and the identification of the characteristic temperatures of its morphology (Tg, Tc, Tcc, Tm). In addition, a relaxational characterization system by coupling dynamic dielectric spectroscopy to a tensile test has been used in order to highlight the effect of molecular mobility on the elasto-visco-plastic deformation of PET. From a mechanical point of view, the main deformation micro-mechanisms have been studied in real time during a tensile test at different temperatures and constant true strain rates: macromolecular orientation, volume damage, development of mesophase and strain induced crystallization were observed and quantified in situ using the coupled characterization technics presented previously at Petra III (Hambourg) and Elettra (Trieste) synchrotrons. In parallel, a study of the molecular mobility (a determining parameter for the predominance of one deformation micromechanism to another) was conducted via relaxational analysis performed during the deformation of the material. In addition to in situ experiments, post mortem analysis by the previously mentioned technics and by X radiography, scanning electron microscopy and X tomography were performed to assess the influence of the mechanical relaxation of the polymer.

<p>This dissertation describes the effects of a <i>de novo</i> mutation of CaMKII found in children with learning disabilities and describes its effect on catalytic activity. We develop a malachite green assay for the measurement of CaMKII activation and use it for high-throughput chemical screening to identify CaMKII inhibitors and enhancers. We also propose a new mechanism of regulation of CaMKII activity by ADP.</p><p><br></p>