Several pathogenic Gram-negative bacteria invest in sophisticated type III secretion systems (T3SS) to incapacitate their eukaryotic hosts. T3SSs can secrete protein cargo outside the bacterial cell and also target many of them into the eukaryotic cell interior. Internalized proteins promote bacterial colonization, survival and transmission, and can often cause severe disease. An example is the Ysc-Yop T3SS apparatus assembled by pathogenic Yersinia spp. A correctly assembled Ysc-Yop T3SS spans the Yersinia envelope and also protrudes from the bacterial surface. Upon host cell contact, this system is competent to secrete hydrophobic translocators that form a translocon pore in the host cell membrane to complete the delivery channel bridging both bacterial and host cells. Newly synthesized effector Yops may pass through this channel to gain entry into the host cell cytosol.As type III secretion (T3S) substrates function sequentially during infection, it is hypothesized that substrate export is temporally controlled to ensure that those required first are prioritized for secretion. On this basis three functional groups are classified as early (i.e. structural components), middle (i.e. translocators) and late (i.e. effectors). Factors considered to orchestrate the T3S of substrates are many, including the intrinsic substrate secretion signal sequences, customized chaperones, and recognition/sorting platforms at the base of the assembled T3SS. Investigating the interplay between these elements is critical for a better understanding of the molecular mechanisms governing export control during Yersinia T3S.To examine the composition of the N-terminal T3S signals of the YscX early substrate and the YopD middle substrate, these segments were altered by mutagenesis and the modified substrates analyzed for their T3S. Translational fusions between these signals and a signalless β-Lactamase were used to determine their optimal length required for efficient T3S. This revealed that YscX and YopD export is most efficiently supported by their first 15 N-terminal residues. At least for YopD, this is a peptide signal and not base upon information in the mRNA sequence. Moreover, features within and upstream of this segment contribute to their translational control. In parallel, bacteria were engineered to produce substrate chimeras where the N-terminal segments were exchanged between substrates of different classes in an effort to examine the temporal dynamics of T3S. In several cases, Yersinia producing chimeric substrates were defective in T3S activity, which could be a consequence of disturbing a pre-existing hierarchal secretion mechanism.YopN and TyeA regulatory molecules can be naturally produced as a 42 kDa YopN-TyeA hybrid, via a +1 frame shift event somewhere at the 5’-end of yopN. To study this event, Yersinia were engineered to artificially produce this hybrid, and these maintained in vitro T3S control of both middle and late substrates. However, modestly diminished directed targeting of effectors into eukaryotic cells correlated to virulence attenuation in vivo. Upon further investigation, a YopN C-terminal segment encompassing residues 278 to 287 was probably responsible, as this region is critical for YopN to control T3S, via enabling a specific interaction with TyeA.Investigated herein were molecular mechanisms to orchestrate substrate export by the T3SS of Yersinia. While N-terminal secretion signals may contribute to specific substrate order, the YopN and TyeA regulatory molecules do not appear to distinguish between the different substrate classes.

The thesis is a study of the effects of radiation on the salivary glands in an experimental and a clinical study. Irradiation is a cornerstone in the management of head and neck cancer and is as other modalities of cancer treatment, afflicted with adverse reactions. An optimal radiotherapy regime is limited by the sensitivity of the normal tissues with regard to early and late effects. In certain cases the early effects can be so troublesome that it will cause interruption in the irradiation and questioning of the curative intention. Although DNA is the lethal target, other parts of the cell have been proposed as sensitive targets to irradiation. Different in vitro secretory models and quantitative morphological characterization and immunohistochemical evaluation of neuropeptides were performed in rat salivary glands after irradiation. The irradiation was given unilaterally or bilaterally once a day for a five-day schedule with 6 MV photons (total dose 20, 30, 35, 40, 45 Gy) or a two fractions regime in five days with a total dose of 24 or 32 Gy. The contralateral gland served as a control for unilaterally treated animals and parallel analyses were done 10 days or 180 days following the last irradiation dose. An early, dose-dependent effect of fractionated irradiation on noradrenaline-stimulated potassium fluxes (86Rb+ fluxes) was demonstrated. In contrast, the exocytotic amylase release displayed no obvious alterations, and morphologically no changes were seen. Regarding late effects (180 days) the noradrenaline-stimulated electrolyte secretion was decreased at least for the higher doses of irradiation. Amylase content and loss of acini was also dose-dependently decreased. At 10 days after bilateral irradiation there was a marked increase in the expression of the neuropeptides substance P, leu-enkephalin and bombesin in the ganglionic cells associated with the submandibular glands and in nerve fibers of the glandular parenchyme. In addition, a clinical prospective evaluation of 25 patients was performed before, during radiotherapy and 6, 12 and 18 months after the end of treatment. A great interindividual variation in the recovery was demonstrated with regard to salivary flow rate. Irradiation doses about 40-50 Gy caused generally reversible changes; sometimes salivary secretion was almost completely restored 6-18 months after the end of radiotherapy. Doses exceeding 65 Gy induced almost irreversible alterations. Even if DNA is the target for the lethal effect of irradiation, other constituents, such as the cell membrane or neuropeptide expression can be significantly affected by irradiation and cause important physiological changes. / <p>S. 1-43: sammanfattning, s. 47-164: 6 uppsatser</p> / digitalisering@umu

Transplantation of the islets of Langerhans is a promising technique for restoring the impairedinsulin production in brittle type 1 diabetics. The downside is that the patient will have to takeimmunosuppressant drugs in order to protect the islet cells from the immune system. Donorsare also sparse, making the quest of finding sufficient amounts of islets for transplantationhard. Encapsulation of the islets of Langerhans has been proposed as a means of protectingthe cells from the immune system taking away the need for immunosuppresives. The mostcommon encapsulation technique is extravascular capsules, which are categorized into micro-and macrocapsules. The microcapsules hold only one or a small set of islet whereas themacrocapsules hold a large quantity of islets.This thesis investigates the encapsulation impact on the beta-cells rapid insulin response torising plasma glucose levels. This was done by simulating the glucose-insulin system inMATLAB with included encapsulation of the islets. Two current macro-encapsulation set upswere used in the model, Beta-Air and ViaCyte devices, and they were compared against anormal case. The results showed that the Beta-Air device would not be able to restorenormoglycemia in a T1DM patient but rather showed a delay in insulin response, while theViaCyte device could mimic the normal case well.

Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.

A thorough understanding of beta-cell proliferation, function, death and regeneration under normal condition as well as in the progression of diabetes is crucial to the conquest of this disease. The work presented in this thesis aimed to investigate the expression and role of a novel transcription factor, Zinc finger BED domain-containing protein 6 (ZBED6), in beta-cells. ZBED6 was present in mouse βTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. Lentiviral shRNA-mediated stable silencing of ZBED6 in βTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell cycle arrest, increased expression of beta-cell specific genes, and higher rates of apoptosis. ChIP sequencing of human islets showed that ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis and apoptosis. We proposed that ZBED6 supported proliferation and survival of beta-cells, possibly at the expense of specialized beta-cell function, i.e. insulin production. To further investigate the role of ZBED6 in beta-cells, ChIP sequencing and whole transcriptome analysis were performed using MIN6 cells. More than 4000 putative target genes of ZBED6 were identified, including Pdx1, MafA and Nkx6.1. ZBED6-silencing resulted in differential expression of more than 700 genes, which was paralleled by an increase in the content and release of insulin in response to a high glucose concentration. Altered morphology/growth patterns as indicated by increased cell clustering were observed in ZBED6 silenced cells. We found also that ZBED6 decreased the ratio between N- and E-cadherin. A lower N- to E-cadherin ratio may hamper the formation of three-dimensional beta-cell clusters and cell-to-cell junctions with neural crest stem cells, and instead promote efficient attachment to a laminin support and monolayer growth. Thus, by controlling beta-cell adhesion and cell-to-cell junctions, ZBED6 might play an important role in beta-cell differentiation, proliferation and survival.

F. tularensis is a Gram-negative bacterial pathogen and it is the causative agent of tularemia. It has the ability to replicate to high numbers within a variety of host cells, including macrophages. Little is known of its virulence mechanisms; however, all species of Francisella contain a cluster of virulence genes known as the Francisella Pathogenicity Island (FPI), which is thought to encode a type 6 secretion system. While 14 of the 18 FPI genes encode products required for intracellular growth in macrophages, the functions of most of these proteins remain to be determined. Therefore, further work is required to understand the role played by the FPI in Francisella pathogenesis. In this thesis, the localization of the core FPI proteins IglA, IglB, IglC and IglD, was examined in order to further elucidate of the structure and activities of the FPI-encoded secretion system. Deletion mutagenesis of pdpA was performed to determine how host intracellular signalling might be affected by secretion of the putative FPI effector protein PdpA. In addition, variations in virulence between different biotypes of Francisella were investigated with respect to the role played by the FPI protein PdpD. Considering the highly infectious nature of Francisella and the absence of a quality vaccine, it is clear that this organism represents an excellent model for proof of principle investigations focussing on new vaccine technologies for intracellular pathogens. The second half of this thesis describes the construction and characterization of live attenuated temperature-sensitive vaccines. These vaccines were created in the intracellular pathogen F. novicida through allelic replacement of essential genes with naturally-occurring, cold-adapted, thermolabile homologues isolated from Arctic bacteria. Thus, the objectives of this work were twofold: to provide further characterization of the structural components and effector proteins associated with the FPI-encoded secretion system, and to develop a new and effective vaccine technology for use against intracellular bacteria. / Graduate

This thesis presents evidence for a novel mechanism of regulation of pyruvate dehydrogenase (PDH) kinase by fatty acids and also results of a study of muscle triacylglycerol concentration. In animals regulation of PDH complex activity is central to the selection of respiratory fuels and to the conservation of glucose during carbohydrate deprivation. The principal means of regulation of PDH complex is interconversion of phosphorylated (inactive) and dephosphorylated (active) forms effected by PDH kinase and PDH phosphatase. Earlier in vitro studies by others had identified both shorter term (min) and longer term (hours) mechanisms of activation of PDH kinase by fatty acid. In the present study PDH kinase activity (as measured by rates of ATP-dependent inactivation of PDH complex in extracts) was shown to be increased when rat heart mitochondria were incubated with palmitoyl-L-carnitine [PC] (and other CoA utilising respiratory substrates). The activation of PDH kinase persisted through removal of respiratory substrate following incubation with CCCP. A comparable effect of PC was also demonstrable in heart mitochondria from 48h-starved rats (i.e. the mechanism may be distinct from that which increases PDH kinase activity in starvation). Rates of ATP-dependent inactivation of PDH complex were also increased when extracts of rat heart mitochondria were incubated with palmitoyl-CoA (PCoA); the increase was comparable with that seen on incubation of intact mitochondria with PC. The PC effect in intact mitochondria and the PCoA effect in mitochondrial extracts may not be identical as PCoA further increased PDH kinase activity in extracts from mitochondria incubated with PC. Rates of incorporation of ³²P from [γ-<sup>32</supP]ATP into PDH complex were unaltered by pnor incubation of mitochondria with PC or by pnor incubation of mitochondrial extracts with PCoA. Three lines of evidence confirmed that the effect of PC to accelerate ATP-dependent inactivation involved phosphorylation of the PDH complex (viz; use of a non-phosphorylatmg ATP analogue; use of known inhibitors of PDH kinase; and use of known activators/inhibitors of PDH phosphatase). Earlier studies had shown that phosphorylation in punfied bovine and porcine PDH complexes is half site (involves only one α-chain in E1 (α2β2) and had suggested that phosphorylation in rat heart complex may be full site (i.e. involves both α-chains). The present study suggests the possibility that elevation of fatty acyl CoA under slaughter house conditions might be a determinant of half site phosphorylation. A method was developed and evaluated for measurement of triacylglycerol in rat soleus muscle strips with the object of investigating factors that may regulate triacylglycerol synthesis in this muscle. This study was abandoned because, although the method was highly reproducible, great variation was found in the triacylglycerol concentration of individual muscles suggesting the possibility of variable contamination with small amounts of adipose tissue.

v, 132 p. leaves : photos., graphs, tables ; 31 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Biochemistry, University of Adelaide, 1978

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Titel på diss.-titelbl.: Islet glucose metabolism and insulin secretion in two animal models of glucose intolerance. Härtill 5 uppsatser.