Pseudomonas aeruginosa is one of the most studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. The transition from a planktonic to a biofilm mode of growth results in diverse changes in gene expression, which causes the attaching cells to become phenotypically and metabolically distinct from their planktonic counterparts. In this study, a proteomic approach was used to study differences in protein profiles obtained from 18-h old P. aeruginosa PAO1 (DSM 1707) planktonic, surface influenced planktonic (SIP) and biofilm populations grown in batch in the absence or presence of a glass wool substratum. Glass wool as an attachment substratum not only supported growth of biofilms, but it also allowed for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterisation. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells and several uniquely expressed proteins were seen on the 2-DE protein maps of the respective populations. Whereas a general down-regulation of protein expression was seen in the biofilm cells, in SIP cells, expression of the proteins was generally up-regulated. The results confirmed that the biofilm population differs from the planktonic population and indicated that the SIP population is not merely a mixture of planktonic and biofilm cells but rather a unique phenotype. Several differentially expressed protein spots were selected and identified using a combination of N-terminal protein sequencing and peptide mass fingerprinting. The proteins comprised mostly of outer membrane or membrane-associated proteins. Based on these analyses, a mutant P. aeruginosa strain, deficient in outer membrane protein OprG, was generated and its ability to form biofilms on a glass wool substratum was compared with that of the wild-type P. aeruginosa strain. The mutant strain was attachment-proficient but biofilm-deficient, suggesting that OprG plays a role in P. aeruginosa biofilm development under the culturing conditions used in this study. / Thesis (PhD (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Background: Healthcare associated infection (HCAI) is a global issue and one of the most frequent adverse events in healthcare. HCAI is a major burden for patients’ and leads to added pain and higher cost for the society. The most important measure to prevent the transmission of HCAI is hand hygiene. In developing countries can insufficient equipment and supplies, lack of financial support and understaffed hospital units have a negative effect for the patients safety and makes it difficult to reduce HCAI. Aim: The aim of the study was to get knowledge about Kenyan nurses’ experiences of healthcare-associated infections. Method: The author used a qualitative method. Individual semi-structured interviews were made with four nurses at a government financial hospital in Eldoret, Kenya. The interviews were analyzed with a manifest analysis. Result: The analysis of the transcribed text made five categories, Education for healthcare workers, students and patients, Lack of equipment and supplies, The issues with crosscontamination, Prevention of HCAI and Understaffed hospital. The results were discussed with Dorothea Orem’s Self-care Theory with a focus on the Theory of Nursing system and other studies relating to the results. Conclusion: Knowledge about HCAI is one of the first steps to be able to reduce infections. Cross-contamination is the main risk factor for HCAI and multiple interventions are an effective strategy to successfully increase hand hygiene.

Immunotherapy has revolutionized the treatment of certain cancers by facilitating the antitumor immune response and represents today one of the mainstays of cancer therapy. However, only a subset of patients responds to immunotherapy, which can also lead to serious complications. The tumor microenvironment is composed of multiple and complex cellular and molecular interactions providing to cancer cells not only a supportive framework but promoting also many steps of immunosuppression and tumor progression. To date, the mechanisms that drive the acquisition of these immunosuppressive features are still poorly defined. Tumor-associated macrophages can be highly represented in the tumor microenvironment where they are shaped and become key players in the innate and adaptive immune escape of the tumor cells.Heme oxygenase-1 is the rate-limiting enzyme that catabolizes heme into three major biologically active byproducts which display cytoprotective, antioxidant and immunomodulatory effects. We hypothesized that tumor-associated macrophages might suppress anti-tumor T-cell response through heme oxygenase-1 induction in the tumor microenvironment and macrophage polarization. We showed that heme oxygenase-1 is highly expressed in tumor-associated macrophages. By using a subcutaneous EG7-OVA lymphoma model on genetically engineered mice with a conditional deletion of heme oxygenase-1 in macrophages, our data show that myeloid-restricted heme oxygenase-1 deficiency improves the effect of a therapeutic antitumor immunization by enhancing tumor-infiltrating antitumor CD8+ T-cell proliferation and cytotoxicity and represses tumor growth. Our data suggest a major role of myeloid heme oxygenase-1 in the differentiation and the phenotypic, functional, transcriptional and epigenetic reprograming of tumor-associated macrophages. Myeloid HO-1 inhibition might be considered as a new myeloid HO-1-mediated immune checkpoint blockade. Targeting myeloid compartment could reprogram the tumor microenvironment and synergize with other cancer therapies. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

No description available.

Thesis advisor: Abhishek Chatterjee / In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. We describe the application of this technology to the modification of adeno-associated virus (AAV) for the first time, enabling the generation of vectors with precisely re-engineered cell-targeting for gene therapy. Our UAA-AAV production platform enables the incorporation of UAAs bearing bio-orthogonal reactive handles into multiple specific sites on the virus capsid and their subsequent functionalization with various labeling molecules. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. Next, we used our unique UAA-AAV vector as a tool for the directed evolution of more active UAA incorporation machinery in mammalian cells. It is well known that the efficiency of unnatural amino acid mutagenesis in mammalian cells is limited by the suboptimal activity of the suppressor tRNAs currently in use. The ability to improve their performance through directed evolution can address this limitation, but no suitable selection system was previously available to achieve this. We have developed a novel platform for virus-assisted directed evolution of enhanced suppressor tRNAs (VADER) in live mammalian cells. Our system applies selective pressure for tRNA activity via the nonsense suppression-dependent production of UAA-AAV, and selectivity for the specific incorporation of interest comes from a novel virus purification strategy based on the unique chemistry of the UAA. We demonstrated > 10,000-fold selectivity for active tRNAs out of mock libraries and used this system to evolve libraries generated from the commonly used archaeal pyrrolysyl suppressor tRNA, ultimately identifying a variant which is three times as active as the original tRNA. Finally, we used next-generation sequencing to analyze the fate of every library member over the course of the selection and found that our VADER selection scheme is indeed selective for the enrichment of more active tRNA variants. This work provides a general blueprint for the evolution of better orthogonal suppressor tRNAs in mammalian cells. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Mucin 1 (MUC1), the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern in many cancers. The presence of truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play key roles in tumor initiations, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with escape of immune defenses. Human macrophage galactose-type lectin (hMGL, HML, CD301 or CLEC10A), a C-type lectin expressed by antigen presenting cells (APC), is a receptor of mucin-type TACAs, -GalNAc (Thomsen nouvelle antigen; Tn; CD175) and its 2,6-sialylated derivative (sTn; CD175s). To date, the relative contributions of these glycans, as well as underlying peptide backbone, and different degrees of valency, on binding thermodynamics and kinetics with hMGL remains elusive. In order to discern the subtle utility of these distinct features, chemical syntheses of the MUC1, HGVTSAPDTRPAPGSTAPPA tandem repeat sequence, and its site-specific serine (Ser) and threonine (Thr) glycosylated analogs were carried out. Circular dichroism (CD) spectroscopy experiments detected increasing structural order of the Thr glycopeptides compared to its nonglycosylated analogs. Isothermal titration calorimetry (ITC) data analysis of lectin binding to the Thr glycopeptides invariably showed enthalpy-driven processes. Affinity enhancement of the Thr glycopeptides for hMGL occurred relative to free GalNAc, revealing an increasing trend in affinity by one order of magnitude, for mono- (KD = 6-8 μM) to triglycosylated (KD = 600 nM) MUC1 peptides. To delineate the relevance of the solvent structure in the protein carbohydrate recognition process, experiments in D2O were performed, exposing enthalpy-entropy compensation differences. KinITC analysis highlighted prolonged complex lifetimes. Furthermore, atomic force microscopy (AFM) based dynamic single-molecule force spectroscopy (SMFS) provided molecular level insight into the energy landscapes governing recognition of the MUC1(Tn)-hMGL complexes. In summary, our results suggest that contact with hMGL critically depends on the type of TACA, nature of the vicinity surrounding the glycan, and its density. This highlights the importance and current efforts in design of prophylactic and therapeutic cancer vaccines with special emphasis on the synthetic glycopeptide vaccines. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Chromatin associated RNAs play a key role in mediating epigenetic mechanism. To improve our comprehension of how chromatin-associated RNAs influence gene expression, it is crucial to identify the genomic locus that RNA interacts with on a genome-wide scale. Emerging technologies were developed to capture binding sites of lncRNAs globally. Such techniques rely on the concept of Proximity ligation via a biotinylated adapter to ligate DNA and RNA on each end, such as MARGI, GRID-seq, ChAR-seq and the most recent technology, devised in our lab RNA And DNA Complexes Ligated & Sequenced (RADICL-seq). RADICL-seq has several advantages over the other methods. However, while this method produced a fairly global picture of the chromatin associated “RNA-ome”, the specific association with specific regulatory factors is missing, in addition all emerging technologies lack the sensitivity to capture low-expressed RNAs. Thus, we set out a strategy to address this issue by enriching global RNA-chromatin interactions mediated by a specific factor which will be achieved by combining RADICL with Chromatin Immunoprecipitation (ChIP) method. Since our lab interest is investigating the role of lncRNAs in muscle differentiation, RADICL-ChIP has been performed using the C2C12 mouse skeletal muscle. In particular, the role of identification of RNA moieties interacting with PRC2 PcG proteins in stress response and their genome wide distribution in chromatin.

The oncogenic role of many of inflammatory genes in prostate cancer (PCa) remains unexplored despite the increasing association of chronic inflammation with PCa initiation, progression, and therapy resistance. The overarching goal of this project was to identify dysregulated inflammatory genes that correlate with PCa progression and seek to understand their molecular mechanisms and the therapeutic potential of targeting them. To achieve this, we utilized cutting-edge integrative (epi) genomic and transcriptomic techniques to identify and characterize inflammatory genes whose deregulation or (epi) genetic alterations correlate with PCa progression. Weighted Gene Co-expression Network Analysis and other multivariate analysis techniques identified IRAK1 as one of the inflammatory signatures found to be overexpressed in over 80% of prostate adenocarcinoma (PRAD) samples. We also explored the diagnostic and prognostic potential of IRAK1 as a biomarker using Kaplan Meier Survival Analysis and AUROC Analysis. DNA methylation analysis showed that IRAK1 is hypomethylated and found to negatively correlate with its overexpression in PRAD patients. We also found some missense and truncated mutations in some patients and reported a high level of IRAK1 gene amplification in castration-resistant and neuroendocrine PCa patients. / Includes bibliography. / Dissertation (PhD)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Upon sensing pathogens, plants initiating defense responses typically resulting in programmed cell death (PCD). PCD effectively subdues biotrophic pathogens but is hijacked by necrotrophs that colonize the resulting dead tissues. We showed that barley wall associated kinase (WAK) genes, underlying the rcs5 QTL, are manipulated by the necrotrophic fungal pathogen Bipolaris sorokiniana to cause spot blotch disease. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four WAK genes, designated HvWak2, Sbs1, Sbs2, and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in the susceptible barley cultivars Steptoe and Harrington resulted in resistance, suggesting a dominant susceptibility function. Sbs1&2 expression is undetectable in barley prior to pathogen challenge; however, specific upregulation of Sbs1&2 occurred in the susceptible lines post inoculation. Promotor sequence polymorphisms were identified in the allele analysis of Sbs1&2 from eight resistant and two susceptible barley lines, which supported the possible role of promotor regulation by virulent isolates contributing to susceptibility. Apoplastic wash fluids from virulent isolates induced Sbs1expression, suggesting regulation by an apoplastic-secreted effector. Thus, the Sbs1&2 genes are the first susceptibility/resistance genes that confer resistance against spot blotch, a disease that threatens barley and wheat production worldwide. The nec3 mutants of barley are hyper-susceptible to many necrotrophs and show distinctive cream to orange necrotic lesions that are induced by infection, representing aberrant PCD. The γ- irradiation induced necrotic mutant, nec3-γ1 (Bowman) was confirmed as a nec3 mutant by allelism tests. The F2 progeny of a cross of nec3 x Quest inoculated with B. sorokiniana segregated as a single recessive gene fitting a 3 WT: 1 mutant ratio. The homozygous F2 mutant progeny were genotyped with four SSR and 25 SNP markers at nec3 locus on chromosome 6H, a physical region spanning ~ 16.96 Mb containing 91 high and low confidence annotated genes. Exome capture sequencing of nec3 mutants failed to identify a candidate gene, however, RNAseq analysis identified two candidates in the nec3 region with >three-fold downregulation. We hypothesize that the underlying aberrant PCD mechanism in the nec3 barley mutant facilitates extreme susceptibility to multiple adapted fungal pathogens of barley.

African horsesickness, a disease of equines caused by African horsesickness virus (AHSV), is often fatal, although the pathogenic effect in different animals is variable. Current AHSV vaccines are live attenuated viruses generated by serial passage in cell culture. This process affects virus plaque size, which has been considered an indicator of AHSV virulence (Erasmus, 1966; Coetzer and Guthrie, 2004). The most likely AHSV proteins to be involved in viral virulence and attenuation are the outer capsid proteins, VP2 and VP5, due to their role in attachment of viral particles to cells and early stages of viral replication. Nonstructural protein NS3 may play an equally important role due to its function in release of viral particles from cells. Two viruses were obtained for this study, AHSV-4(1) and AHSV-4(13). The thirteenth passage virus, AHSV-4(13), originated from the primary isolate AHSV-4(1). The three most variable AHSV proteins are VP2, VP5 and NS3. The question of sequence variation of these proteins between AHSV-4(1) and AHSV-4(13) arising during the attenuation process was addressed. The subject of plaque size variation between these viruses was also investigated. Some of the sequence variation observed in NS3, VP2 and VP5, between AHSV-4(1) and AHSV-4(13), occurred in protein regions that may be involved in virus entry into and exit from cells. The sequence information also indicated that AHSV-4(1) and AHSV-4(13) consist of genetically heterogeneous viral pools. The plaque size of AHSV-4(1) was variable, with small to relatively large plaques, whereas the plaques of AHSV-4(13) were mostly large. During serial plaque purification of AHSV-4(1) plaque size increased and became homogenous in size. No sequence variation in NS3 or VP5 of any of the plaque variants could be linked to variation or change in plaque size. NS3 and VP5 have a possible role in the AHSV virulence phenotype, and exhibit cytotoxic properties in bacterial and insect cells. As these proteins have not been studied in mammalian cells, an aim of this study was to express them in Vero cells and investigate their cytotoxic and membrane permeabilization properties within these cells. The NS3 and VP5 genes of AHSV-4(1) and AHSV-4(13) were successfully inserted into a mammalian expression vector and transiently expressed in Vero cells. The transfection procedure was optimized using eGFP, but expression levels were still low. When NS3 and VP5 were expressed, no obvious signs of cytotoxicity were observed. Cell viability and membrane integrity assays were performed and expression of NS3 and VP5 in Vero cells had no detectable effect on cell viability or membrane integrity. Low expression levels may have resulted in protein levels too low to cause membrane damage or affect cell viability. As Vero cells support AHSV replication, low levels of NS3 and VP5 may not be cytotoxic in these cells. NS3 was further investigated by expressing an NS3-eGFP fusion protein in Vero cells. Putative localization with membranous components and possible perinuclear localization of the fusion protein was observed. These observations may be confirmed with more sensitive microscopic techniques for a better assessment of the localization. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted