Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.

Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 64-80).

The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations. The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation. DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xii, 239 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Cytotoxic T lymphocytes (CTL) kill virally infected and tumourigenic cells via the regulated secretion of specialised secretory lysosomes. These secretory lysosomes contain cytolytic effector molecules, such as perforin and granzymes, which are able to induce apoptosis in target cells. Secretion occurs at the contact point between the CTL and its target, in a highly structured region termed the immunological synapse (IS). Upon formation of the IS, CTL undergo polarisation of their microtubule cytoskeleton and movement of the microtubule organising centre (MTOC) to the IS. Secretory lysosomes are then able to polarise along microtubules, fuse with the plasma membrane and deliver their effector molecules to the IS. The Adaptor Protein 3 complex (AP-3) sorts transmembrane proteins to lysosomes and deficiency in AP-3 results in missorting of proteins from the lysosomal to plasma membrane. CTL from AP-3 deficient patients, who suffer from Hermansky-Pudlak Syndrome Type 2 (HPS2), show reduced killing of target cells. This thesis describes two new patients with HPS2, both with homozygous mutations in the AP3B1 gene, which codes for the β3A subunit of the AP-3 complex. CTL from the new HPS2 patients show reduced cytotoxicity, which is shown here to be due to impaired secretory lysosome polarisation towards the IS. This impairment is common to HPS2 CTL, but varies between patients. In order to determine differences between HPS2 and wild type CTL, the localisation of a range of lysosomal, cytolytic, transmembrane, inhibitory and activation marker proteins is examined. This shows that in HPS2 CTL, LAMP1, CD63 and CD9 are potential AP-3 cargos. In addition, a possible effect on the key lytic effector perforin is identified. Preliminary experiments to allow proteomic comparison of HPS2 and wild type CTL are also presented. Further investigation of these results will help to shed light on the mechanisms involved in secretory lysosome polarisation in CTL.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Lic.-avh. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.