No description available.

No description available.

No description available.

The differentiation of melanocytes from multipotential neural crest cells is an ideal system for studying the processes underlying lineage determination in development. Tyrosinase is a key enzyme in melanin biosynthesis and the activation of the tyrosinase gene is characteristic of differentiated melanocytes. In order to study the mechanisms underlying activation of melanocyte-specific genes during differentiation in chick embryos, a chicken genomic DNA library was screened for tyrosinase-encoding sequences using a mouse tyrosinase cDNA probe. Two identical hybridising clones were identified. Restriction mapping and sequencing revealed that both clones contained a 4.3 kb genomic DNA fragment, CTYR4.3, that included 2125 nt of the 5' flanking region, the first exon and part of the first intron of the chicken tyrosinase gene. The 5' flanking sequence of CTYR4.3, which is the most extensive to be reported for a lower vertebrate tyrosinase gene to date, was analysed further using computer-aided homology searches and primer extension. Alignment of the promoter sequences of CTYR4.3 with those of the human, mouse, quail and turtle tyrosinase genes revealed two evolutionary conserved regions. These regions may be functionally significant as they contain regulatory elements previously reported to play a role in melanocyte-specific expression of the tyrosinase gene in mammals. These include an initiator region and an associated SP1-binding site, the M-box and an upstream enhancer element, TDE. In addition, other potential transcription factor binding motifs were identified, including an AP-1-binding site, a UV-responsive element and glucocorticoid-responsive elements. Although several TATA box motifs were identified, they were situated more than 200 bp upstream of the transcription start sites mapped by primer extension analysis and therefore are unlikely to function as TFIID-binding sites. Transcription initiation appears to occur at heterogeneous start sites, and given the absence of a functional TATA box, may be mediated via the conserved initiator region and SP1-binding site. To test the ability of the 5' flanking sequence of CTYR4.3 to drive transcription and to begin to assess the functional significance of the various conserved elements, transient transfection assays were carried out. Constructs were generated in which 2.1 kb, 1.1 kb, 0.5 kb and 0.2 kb fragments of the 5' flanking sequence were linked to a luciferase reporter gene. These constructs were introduced into cultures of chicken retinal pigment epithelial cells (RPE), immortalised quail neural crest cells (MQTNC), and human liver cells (Hep G2) by calcium phosphate-mediated transfection. Transfections with all constructs resulted in luciferase activities significantly greater than those that were observed with the promoterless luciferase construct, thus confirming that the 5' flanking sequence of CTYR4.3 does possess promoter activity. However, the level of expression from the various constructs differed markedly in the different cell types. In the tyrosinase-negative Hep G2 cells, low levels of expression were observed with all constructs. In the tyrosinase-positive RPE cells, a high level of luciferase activity was obtained specifically with the smallest (0.2 kb) promoter construct. Since the 0.2 kb promoter fragment does not include the conserved initiator region, SP1-binding site, or M-box, the role of these elements in tissue-specific transcription initiation of the chicken tyrosinase gene is now questionable. These results suggest the existence of transcription regulatory mechanisms that are unique to avians and possibly other lower vertebrates. In contrast to the results obtained for RPE cells, the highest luciferase activity was obtained with the full length 2.1 kb promoter construct in the immortalised quail neural crest-derived cells. These results may have developmental significance since they suggest that the chicken tyrosinase gene promoter is regulated differently in RPE cells and neural crest-derived cells.

Human natural killer (NK) cells are found in virtually all tissues where they act as a first line defense against malignant and virally infected cells. The development of NK cells from CD34+ hematopoietic progenitors is a complex process that involves navigating through different microenvironments and requires contact-dependent interactions with stromal cells. The molecular mediators of NK cell developmental subset trafficking, cell-cell interactions, and maturation have not been fully characterized. This thesis presents 3 studies that aim to uncover contact-dependent interactions that drive human NK cell development. Chapter 2 focuses on defining the adhesome profile of human NK cells from in vitro derived populations, tonsil, and peripheral blood. This study reveals that the tissue origin and developmental stage of NK cells influence the expression of adhesome-associated genes and proteins, as well as the content of cortical actin, which suggests a link between adhesome expression and actin regulation in NK cells. Chapter 3 presents the first comprehensive study on human NK cell development in pediatric tonsil using cyclic immunofluorescence microscopy and imaging mass cytometry. We reveal that NK cell subset localization and interactions are dependent on NK cell developmental stage and tissue residency. Chapter 4 demonstrates that neural cell adhesion molecule (NCAM) on stromal cells promotes maintenance of a mesenchymal-like state and subsequently the survival and proliferation of human NK cell precursors. Overall, this thesis provides new insights on previously unknown mediators of NK cell contact-dependent interactions and unveils the first road map of in situ NK cell development.

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No description available.

This work discusses the regulation of glycogen metabolism in Dictyostelium discoideum during its developmental cycle. Specifically, the possible cAMP dependent regulation of glycogen phosphorylase and glycogen synthase was examined. In other systems, cAMP can regulate at the level of the gene (procaryotes, CAP protein) or at the level of covalent, reversible modification of the enzyme activity (eucaryotes, cAMP-dependent protein kinase-cAMPdPK). In Dictyostelium, glycogen phosphorylase and glycogen synthase have each been found to occur in two forms; one regulated allosterically and the other independent of allosteric regulation. The regulation of the two forms of glycogen phosphorylase was examined in single-cell suspensions to which cAMP or one of several cAMP analogs were added to mimic differentiative conditions. The allosterically regulated form of glycogen phosphorylase, phosphorylase b, decreased in the presence of cAMP while a corresponding increase in phosphorylase a, the non-allosterically regulated form of glycogen phosphorylase, was observed over an 8 hr period in the same cultures. In the presence of cAMP analogs, a similar time course of regulation for the two forms of glycogen phosphorylase occurred but only 2’deoxy-cAMP gave an effect comparable to cAMP. Under these same conditions, northern blot analysis of three developmentally regulated mRNAs--PL3, D11, and D3--revealed that normal gene regulation was occurring. Under conditions where elevation of intracellular cAMP was inhibited, neither regulation of phosphorylase enzyme activity nor of the 3 genes was observed. This indicated that under these conditions intracellular elevation of cAMP was necessary for the observed effects on enzyme and gene activity. This requirement for intracellular cAMP may indicate the involvement of a cAMPdPK. The properties of a phosphorylase b kinase found in amoebal extracts are described. The kinase activity coeluted with the phosphorylase b activity on a DE·52 anion exchange column. Under the conditions described conversion of the phosphorylase b activity to the phosphorylase a activity was observed. However, an increase in molecular weight to 104 kd (as seen for purified phosphorylase a) was not observed. The characterization of a partially purified glycogen synthase I and its developmental regulation are described. Also described are in vitro attempts to convert the I form to the allosterically regulated, D form, under conditions conducive to phosphorylation. / Ph. D.

Thesis advisor: Tim Van Opijnen / Exposure to antibiotics has previously been shown to induce morphological changes to bacterial cells in Escherichia coli and Staphylococcus aureus . Response profiles to antibiotics representing various mechanisms of action provides as quick, reliable and cheap means of identifying the mechanism of action of novel antimicrobials. We evaluated whether similar cytological profiling was possible in the pathogenic bacteria Streptococcus pneumoniae and whether there were any strain specific differences in morphological changes resulting from antibiotic exposure. We evaluated antibiotics from various classes and with different mechanisms of action to develop strain specific models of phenotypic responses in order to identify clustering associated with particular mechanisms of action. Various antibiotics belonging to, cell wall synthesis inhibitors, protein synthesis inhibitors, and DNA synthesis inhibitors were evaluated using S. pneumoniae strains TIGR4 and 19F. Following exposure to high doses of antibiotics, cells were imaged for DNA and cell wall components and analyzed. Our data shows that antibiotics of the same mechanism of action induce similar morphological changes. While TIGR4 and 19F show similar changes there are strain specific differences between them. Our data shows that cytological profiling effectively indicates the mechanism of action through imaging in S. pneumoniae allowing this technique to be used to study novel antimicrobials as well as better understand bacterial responses to antibiotic stress. / Thesis (BS) — Boston College, 2019. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology.

Två huvudsakliga metodprinciper används inom cytologi för diagnostisering av cellförändringar, nämligen konventionell och vätskebaserad metod. De kan båda appliceras på såväl gynekologiska som icke-gynekologiska prover, där den senare bland annat omfattar olika sorters exsudat. Syftet med den här studien var att utveckla metoden för den vätskebaserade metoden så att etanolfixerade exsudat kunde prepareras och även påvisa bättre resultat än då de preparerats med konventionell metod. För att göra detta har 61 unika prover kategoriserade som exsudat preparerats totalt, varav 61 med konventionell metod, 54 med vätskebaserad metod och 22 med vätskebaserad metod med tillsats av ättiksyra. De färdiga glasen bedömdes sedan i mikroskop och gavs scorevärden utifrån fyra parametrar: mängden celler exklusive inflammatoriska celler; bedömbarheten av cellmorfologin; mängden inflammatorisk komponent samt mängden bakgrundsmaterial. Resultaten visade ingen förbättring mellan de glas som preparerats med konventionell eller vätskebaserad metod. Däremot visade resultaten för de ättiksyrabehandlade proverna på förbättrade scorevärden jämfört med de andra metoderna. Som slutsats drogs att vätskebaserad metod med tillsats av ättiksyra uppnår syftet eftersom det reducerar mängden bakgrundsmaterial, förekomst av ring på objektglasen samt vidhåller en god cellmorfologi, vilket gör proverna lättare att diagnostisera för cytodiagnostikerna. / Two main principles is used within cytology in order to diagnose cytological abnormalities; conventional and liquid-based cytology. Both methods can be applied on both gynaecological and non-gynaecological samples of which the later includes samples categorized as exudate. The aim of this study was to develop the method for liquid-based cytology so that exudate fixated with ethanol could be prepared and also achieve better results compared to conventional method. In order to do so, 61 unique samples were prepared of which 61 with conventional method, 54 with liquid-based method and 22 with liquid-based method with added acetic acid. The slides was then examined in microscope and was given score values within four parameters: amount of cells; cell morphology; amount of inflammatory component and amount of background. The results indicated no difference between the slides prepared with conventional or liquid-based method. However, the slides prepared with addition of acetic acid indicated more opportunistic score values when compared. The conclusion was that liquid-based method with the addition of acetic acid did satisfy the aim of this study as it reduces the amount of background, reduces “ring formation” on the slides and preserve the cells morphology well, which makes the samples easier to diagnose.