Using CRISPR/Cas9 to Identify Gene Interactions with Hexosamine Biosynthesis and N-Glycan Remodeling Pathway Enzymes

Genetic studies by classical mutagenesis and screening methods have revealed many molecular interactions and regulatory relationships in animal models. The hexosamine biosynthesis pathway and N-glycosylation are upregulated in most cancers and have shown to play a role in many cancer cell phenotypes. In this thesis, a CRISPR/Cas9 genome-wide targeted mutagenesis approach was employed to identify gene interactions with chosen genes-of-interest from these two pathways: NAGK, GFPT1, MGAT1 and MGAT5. The gene interactions identified suggest relationships between our genes-of-interest and cell-cell adhesion, cytoskeleton, and folate and nucleotide metabolism. Further characterization of metabolite levels in the gene-of-interest knockout cells was done to help understand potential gene interactions from the screen. For example, metabolic imbalance in mutant cells likely indicates cell stress and reactive oxygen species, consistent with PRDX1, an antioxidant, being a suggested genetic interactor in multiple screens. These findings provide new insight on vulnerabilities and genomic redundancies in cancer cells.M.Sc

Metal-support interactions regulate substrate binding in Fe/Co/Se cluster catalysts

Here, we investigate the stereo-electronic requirements of a family of Fe/Co6Se8 molecular clusters to achieve a Goldilocks regime of substrate affinity for the catalytic coupling of tosyl azide and tert-butyl isocyanide. The reactivity of a catalytically competent iron-nitrenoid intermediate, observed in situ, is explored towards nitrene transfer and hydrogen atom abstraction. The dual role of isocyanide, which on one hand prevents catalyst degradation, but, in large amounts, slows down reactivity is exposed. The impact of distal changes (number of neighboring active sites and identity of supporting ligands) on substrate affinity, electronic properties, and catalytic activity is investigated. Overall, the study reveals that the dynamic, push-pull interactions between the substrate (tBuNC), active site (Fe), and support (Co6Se8) create a regime where increased substrate activation occurs concomitantly with expedited dissociation

Probing Edge/Support Electronic Cooperativity in Single Edge Fe/Co6Se8 Clusters

This study provides insights into the electronic structure of an atomically precise Fe/Co6Se8 cluster and the extent of redox cooperativity between the Fe active site and the non-innocent Co6Se8 support. Chemical oxidation studies enable the isolation of two types of oxidized Fe/Co6Se8 clusters, in which the counterion is either directly coordinated to the Fe, or completely dissociated. Experimental characterization by single crystal X-ray diffraction, 57Fe Mössbauer spectroscopy, and 31P NMR spectroscopy is complemented by computational analysis. In aggregate, the study reveals that upon oxidation, the charge is shared between the Fe edge site and the Co6Se8 core, and that anion coordination perturbs the density of unpaired electrons on Co6Se8

Pathogenic E. coli from Cattle as a Reservoir of Resistance Genes to Various Groups of Antibiotics

Antimicrobial resistance has become a worldwide concern in all public health domains and reducing the spread has become a global priority. Pathogenic E. coli is responsible for a number of illnesses in humans and outbreaks in the past have been correlated with the consumption of contaminated bovine products. This is why surveillance in all the steps of production is essential. This study focused on identifying the pathogenic strains of E. coli in two large bovine abattoirs from Romania and France, and on associating them with the antimicrobial resistance patterns. A total of 250 samples from intestinal content were aseptically collected during the evisceration step of the cattle slaughtering process, from which 242 E. coli strains were isolated. Seventeen percent of all samples tested positive to at least one E. coli isolate carrying eaeA, stx1 and stx2 genes. The most prevalent genetic profile found in the E. coli strains tested was Stx1-positive and Stx2/eaeA-negative. More than 68% of the pathogenic E. coli isolated in Romania showed multi-drug resistance (MDR) and in France, the percentage was significantly lower (38%). The MDR profiles showed a high gene diversity for antibiotic resistance, which represents a great risk for environmental spread and human health. Our results indicate that in Romania, bovines can represent a reservoir for MDR E. coli and, hence, a surveillance system for antimicrobials usage in farm animals is highly needed

CLINICAL AND MICROBIOLOGICAL ASPECTS IN DOG PERIODONTAL DISEASE

The purpose of this study is the identification of microflora present in the dog oral pathology, establishing connections between it and the clinical manifestations, and to make a correlation between clinical and microbiological aspects and the stages of periodontal disease. During this research 14 cases were diagnosed with periodontal disease: stage I on 3 patients (age of 3 years), stage II on 3 patients (age of 7, 6 years), stage III on 5 patients (age of 7 years) and stage IV on 2 patients (age of 10 years). In the initial stages (I, II) of periodontal disease we found gram positive bacillus arranged in a filament shape and in stages III and IV we encountered the growth of oral cocobacilli G-. The most effective antibiotics in our study is different depending of the stage of periodontal disease and was represented by Amoxiclav and Enroxil for stages I, III and IV, Amoxiclav and Ceftiofur for stage II. The oral microbiota is changing with evolution of periodontal disease, increasing the gram negative cocobacil population, especially Pseudomonas spp. Every individual presents a different sensitivity with an specific antibiogram

Metal-support interactions in molecular single-site cluster catalysts

This study provides atomistic insights into the interface between a single-site catalyst and a transition metal chalcogenide support and illustrates how their cooperativity can be harnessed to modulate catalytic activity. A molecular platform MCo6Se8(PEt3)4(L)2 (1-M, M = Cr, Mn, Fe, Co, Cu, Zn) was designed in which the active site (M)/support (Co6Se8) interactions are interrogated by sys-tematically probing the electronic and structural changes that occur as the identity of the metal varies. All 3d transition metal 1-M clusters display remarkable catalytic activity for coupling tosyl azide and tert-butyl isocyanide, with Mn and Co derivatives show-ing the fastest turnover in the series. Detailed structural, electronic, and magnetic characterization of the clusters was performed using single crystal X-ray diffraction, 1H and 31P nuclear magnetic resonance spectroscopy, electronic absorption spectroscopy, cyclic voltammetry, and computational methods. Distinct metal/support redox regimes can be accessed in 1-M based on the energy of the edge metal’s frontier orbitals with respect to those of the cluster support. As the degree of electronic interaction between the edge and the support increases, a cooperative regime is reached wherein the support can deliver electrons to the catalytic site, in-creasing the reactivity of key metal-nitrenoid intermediates

Comparative Assessment of the Shaping Ability of Reciproc Blue, WaveOne Gold, and ProTaper Gold in Simulated Root Canals

Maintaining the original trajectory of the root canal is a major challenge in endodontic therapy, especially in narrow and curved root canals. The present study aims to assess the shaping capacity of three endodontic systems made of different nickel–titanium alloys on simulated curved root canals. Thirty-six endodontic resin blocks (Ref. V040245, VDW) divided into three groups, each of twelve blocks (n = 12), were shaped, photographed, and analyzed: Group 1-Protaper Gold (PTG) (Dentsply Maillefer, Ballaigues, Switzerland) F2 25/08; Group 2-Reciproc Blue (RB), RB 25/08 (VDW, Munich, Germany); Group 3-WaveOne Gold (WOG) (Dentsply Maillefer), WOG 25/07. Each block was standardized and photographed before and after shaping in the same position, with the foramen oriented to the left. Post-shaping images were superimposed onto the initial ones. Thirteen measurement points were used for evaluation, spaced with 1 mm distance from one another, from level 0, apical foramen, to level 12, coronal orifice. The amount of removed resin from inner (X1) and outer (X2) walls, the direction of transportation (X1 − X2), and the centering ability (X1 − X2)/Y were measured, calculated, and comparatively analyzed. Statistical differences (p < 0.05) were observed between the shaping capacity of the considered systems in the middle and coronal thirds. PTG had a better centering ability than WOG and RB in the coronal third, while RB was more centered in the middle third in comparison to both WOG and PTG. In the apical third, the centering capacity of WOG was higher, without being statistically significant. WOG 25/07 and PTG 25/08 tend to cut more on the inner wall of the root canals, and RB 25/08 on the external one

Metal–Support Interactions in Molecular Single-Site Cluster Catalysts

This study provides atomistic insights into the interface between a single-site catalyst and a transition metal chalcogenide support and reveals that peak catalytic activity occurs when edge/support redox cooperativity is maximized. A molecular platform MCo6Se8(PEt3)4(L)2 (1-M, M = Cr, Mn, Fe, Co, Cu, and Zn) was designed in which the active site (M)/support (Co6Se8) interactions are interrogated by systematically probing the electronic and structural changes that occur as the identity of the metal varies. All 3d transition metal 1-M clusters display remarkable catalytic activity for coupling tosyl azide and tert-butyl isocyanide, with Mn and Co derivatives showing the fastest turnover in the series. Structural, electronic, and magnetic characterization of the clusters was performed using single crystal X-ray diffraction, 1H and 31P nuclear magnetic resonance spectroscopy, electronic absorption spectroscopy, cyclic voltammetry, and computational methods. Distinct metal/support redox regimes can be accessed in 1-M based on the energy of the edge metal’s frontier orbitals with respect to those of the cluster support. As the degree of electronic interaction between the edge and the support increases, a cooperative regime is reached wherein the support can deliver electrons to the catalytic site, increasing the reactivity of key metal-nitrenoid intermediates

Metal–Support Interactions in Molecular Single-Site Cluster Catalysts

This study provides atomistic insights into the interface between a single-site catalyst and a transition metal chalcogenide support and reveals that peak catalytic activity occurs when edge/support redox cooperativity is maximized. A molecular platform MCo6Se8(PEt3)4(L)2 (1-M, M = Cr, Mn, Fe, Co, Cu, and Zn) was designed in which the active site (M)/support (Co6Se8) interactions are interrogated by systematically probing the electronic and structural changes that occur as the identity of the metal varies. All 3d transition metal 1-M clusters display remarkable catalytic activity for coupling tosyl azide and tert-butyl isocyanide, with Mn and Co derivatives showing the fastest turnover in the series. Structural, electronic, and magnetic characterization of the clusters was performed using single crystal X-ray diffraction, 1H and 31P nuclear magnetic resonance spectroscopy, electronic absorption spectroscopy, cyclic voltammetry, and computational methods. Distinct metal/support redox regimes can be accessed in 1-M based on the energy of the edge metal’s frontier orbitals with respect to those of the cluster support. As the degree of electronic interaction between the edge and the support increases, a cooperative regime is reached wherein the support can deliver electrons to the catalytic site, increasing the reactivity of key metal-nitrenoid intermediates