A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13 derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract, oligomeric, or polymeric procyanidin-rich fractions demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components were upregulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with Nrf2 nuclear localization and expression of Nrf2 target genes, including NRF-1 and GABPA, essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and upregulation of genes critical for mitochondrial respiration, and, ultimately, enhanced glucose-stimulated insulin secretion and β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

The secretion of the acqueous component of tears is dependent on the uneven distribution and nature of ion channels across lacrimal gland acinar cell membranes. Models to describe such secretion in lacrimal glands are based primarily on one species, the rat and have been developed in conjunction with observations of channel function and secretion from salivary and pancreatic exocrine glands, which are structurally similar to lacrimal glands. The applicability of these models to all species is unknown and furthermore none takes into account volume regulation mechanisms in these acinar cells. Therefore, the aim of this thesis was to broaden the basis for such models by identifying the major membrane ionic channels, characterising their mode of activation and determining their possible physiological roles in rabbit superior lacrimal gland acinar cells. Initially, a culture system for maintaining acinar cells for short and long terms was developed. This allowed for electrophysiological techniques to be employed to study the role of ionic channel activity in the secretion of the aqueous component of tears and cell volume regulation. This study demonstrates a number of unique findings and indicates that the models currently used to describe lacrimal gland secretion are not applicable to all species and need modifications which take into account cell volume regulation / Doctor of Philosophy (PhD)

There are two main human pathogens in the family of Chlamydiaceae. Different serovars of Chlamydia trachomatis cause sexually-transmitted disease and eye infections whereas C. pneumoniae (TWAR) is a common cause of community-acquired respiratory infection. Chlamydia species are obligate, intracellular bacteria sharing a unique developmental cycle that occurs within a protected vacuole termed an inclusion. These microorganisms can be distinguished by two different forms: the infectious, metabolically inert elementary body (EB) and the reproducing non-infectious form, termed the reticulate body (RB). The cycle is terminated when re-differentiation of RBs back to infectious EBs occurs. Chlamydia possesses a type III secretion system (T3SS) essential for delivery of effector proteins into the host for host-cell interactions. This virulence system has been systematically characterized in several mammalian pathogens. Due to lack of a tractable genetic system for Chlamydia species, we have employed chemical genetics as a strategy to investigate molecular aspects of the T3SS. We have identified that the T3S-inhibitors INP0010 and INP0400 block the developmental cycle and interfere with secretion of T3S effector proteins in C. pneumoniae and C. trachomatis, without any cytotoxic effect. We have further shown that INP0010 decreases initiation of transcription in C. pneumoniae during the early mid-developmental cycle as demonstrated by a novel calculation, useful for measurement of transcription initiation in any intracellular pathogen. The mechanism regulating the signal(s) for primary as well as terminal differentiation of RBs has not been defined in Chlamydia. We show using T3S-inhibitors that INP0010 targets the T3SS and thereby arrests RB proliferation as well as RB to EB re-differentiation of C. pneumoniae as where INP0400 targets the T3SS and provokes a bacterial dissociation from the inclusion membrane presumed to mimic the natural occurrence of terminal differentiation. The effect of INP0010 on iron-responsive genes indicates a role for T3S in iron acquisition. Accordingly, our results suggest the possibility that C. pneumoniae acquires iron via the intracellular trafficking pathway of endocytosed transferrin. Moreover, we have for the first time presented data showing generalized bone loss from C. pneumoniae infection in mice. The infection was associated with increased levels of the bone resorptive cytokines IL-6 and IL-1beta. In addition, an increased sub-population of T-cells expressed RANKL during infection. Additionally, C. pneumoniae established an infection in a human osteoblast cell line in vitro with a similar cytokine profile as seen in vivo, supporting a causal linkage. Collectively, these data may indicate a previously unknown pathological role of C. pneumoniae in generalized bone loss.

The aim of the present thesis was to find a test for prediction of caries activity which would be useful in routine clinical work.Correlations between oral health, general health, food habits and socioeconomic conditions were investigated in 4- and 8-year-old children. It was found that the salivary secretion rate and the prevalence of oral lactobacilli were factors which might be useful in caries prediction.In 5- and 8-year-old children negative correlations between caries frequency and secretion rate, pH and buffer effect of saliva were demonstrated. However, these parameters showed a wide range of variation.A dip-slide test (Dentocult®), for determination of the number of lactobacilli in saliva, were investigated. The test proved to be reliable for determining of the number of lactobacilli in saliva.The clinical use of information on salivary secretion rate and number of lactobacilli in saliva in prediction of caries activity was examined in 115 14-year-old children over a period of 64 weeks. The number of lactobacilli in saliva, but not the salivary secretion rate, was correlated to caries activity. The number of lactobacilli in saliva seems to reflect the frequency of ingested fermentable carbohydrates and indirectly the risk for initiation of carious lesions. However, when the lactobacillus test is used it is important that there are no such areas of microbial retention on the teeth, as open cavities, poorly executed conservations, dentures or orthodontic bands. The lactobacillus test would make it possible to individualize prophylactic caries treatment. / <p>Annan ISSN på omslaget och titelblad (ISSN 0934-7532).</p><p>Härtill 5 delarbeten.</p> / digitalisering@umu

A cloned esterase and several cytoplasmic proteins which lack a classical cleavable signal-peptide were secreted during late stationary phase in B. subtilis. Several lines of evidence indicate that secretion of enolase, SodA, and Est55 is not due to cell lysis. The extent of possible release of these proteins mediated by membrane vesicles into the medium was also found to be minimal. We have identified a hydrophobic α–helical domain within enolase that contributes to the secretion specificity. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid-encoded mutant enolases is totally blocked, while that of the wild-type chromosomal enolase is not affected in the same cultures. However, mutations on the conserved basic residues flanking the hydrophobic core region show no effect. GFP fusion experiments demonstrate that minimal length of N-terminus 140 amino acids and its tertiary structure are required to serve as a functional signal for the export of enolase. Transcriptome analysis revealed several interesting patterns in gene expression when the cell growth switches from exponential phase into stationary phase. As expected, once cell growth enters the stationary phase, expressions of most SigA-dependent house-keeping genes (for syntheses of ATP, amino acids, nucleotides, ribosomes), and surprisingly secY and yidC homolog in the Sec-dependent general protein secretion system were significantly decreased; however, secA and sipT were found progressively induced in the stationary phase. The sigB gene and the SigB regulon exhibited a distinct pattern of transient induction with a peak in transition phase. A total of 62 genes were induced by three fold after cessation of SigB-dependent surge, which includes sigW and many of SigW-depedent genes specifically for antitoxin resistant genes, and some unknown function genes. In addition, oxidative stress response and damage repair genes also dominantly induced in stationary phase implied a high level of oxidant or thio-depleting agents in stationary phase. Besides, induction of fruRAB at T40 and gap operon at T100 suggested a sequential switch of carbon utilization from glucose to fructose. These results indicate a complex adaptation physiology as Bacillus cells change from the fast growing exponential phase toward the stationary phase.

The exeC gene, found in the gram-negative bacteria Aeromonas hydrophila codes for a 31 kDa, three domain, bitopic inner membrane protein. The components of the ExeC protein include an amino-terminal cytoplasmic domain, a trans-membrane helix and two periplasmic domains. The two periplasmic domains are involved in recognition and selection of protein substrates which are subsequently transported across the outer membrane and free of the cell. This study focuses exclusively on the two periplasmic domains referred to hereafter as the HR and the PDZ domains. Three constructs were used throughout the course of this study. Two of them were designed, cloned and expressed for this study. The third is a result of previous work. Two constructs contained both the HR and PDZ domains while the other consists of the amino-terminal periplasmic HR domain. Only one construct was used to grow single crystals for analysis by X-ray crystallography. Crystals comprised of the PDZ domain from a degraded construct grew in a hexagonal space group with a hexagonal bi-pyramidal morphology. Crystals diffracted anisotropically to a maximum resolutions of 2 Å along the c axis and 3 Å in the a/b plane. Anisotropy in combination with twinning drastically complicated structure solution. Efforts toward elucidating the crystal structure will be discussed.

Genetically engineered cells have the potential to solve the cell availability problem in developing a pancreatic tissue substitute for the treatment of insulin-dependent diabetes (IDD). These cells can be beta-cells genetically engineered so that they can be grown in culture, such as the betaTC3 and betaTC tet mouse insulinomas developed by Efrat et al; or, they can be non-beta cells genetically engineered to secrete insulin constitutively or under transcriptional regulation. The aim of this work was to thoroughly characterize and improve the secretion dynamics of pancreatic substitutes based on genetically engineered cells. One issue involved with the continuous beta-cell lines is the remodeling of the cells inside an encapsulated cell system, which may affect the insulin secretion dynamics exhibited by the construct. To evaluate the effect of remodeling on the secretion properties of the construct, we used a single-pass perfusion system to characterize the insulin secretion dynamics of different alginate beads in response to step-ups and downs in glucose concentration. Results indicated that the secretion dynamics of beads indeed changed after long-term culture. On the other hand, data with a growth-regulated cell line, betaTC tet cells, showed that the secretion profile of beads can be retained if the cell growth is suppressed. A major concern associated with genetically engineered cells of non-beta origin is that they generally exhibit sub-optimal insulin secretion characteristics relative to normal pancreatic islets. Instead of relying on molecular tools such as manipulating gene elements, our approach was to introduce a glucose-responsive material acting as a control barrier for insulin release from a device containing constitutively secreting cells. Proof-of-concept experiments were performed with a disk-shaped prototype based on recombinant HepG2 hepatomas or C2C12 myoblasts, which constitutively secreted insulin, and concanavalin A (con A)-based glucose-responsive material as the control barrier. Results demonstrated that the a hybrid pancreatic substitute consisting of constitutively secreting cells and glucose-responsive material has the potential to provide a more physiologic regulation of insulin release than the cells by themselves or in an inert material.

Pseudomonas syringae is a pathogen of numerous plant species, including several economically important crops. P. syringae pv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This dissertation demonstrates that a 29.9-kb cluster of genes in the B728a genome encodes a novel secretion pathway, the type VI secretion system (T6SS), that functions to deliver at least one protein outside of the bacterial cell. Western blot analyses show that this secretion is dependent on clpV, a gene that likely encodes an AAA+ ATPase, and is repressed by retS, which apparently encodes a hybrid sensor kinase. RetS and a similar protein called LadS are shown to collectively modulate several virulence-related activities in addition to the T6SS. Plate assays demonstrate that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation in retS affects B728a population levels on the surface of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed, and possible roles for the B728a T6SS are addressed.

Tuberculosis is an enormous global health problem. Despite decades of research, the mechanism(s) by which Mycobacterium tuberculosis (Mtb) mediates virulence remains incompletely understood. The ESX-1 secretion system is critical for Mtb to survive and cause disease in vivo, but its primary function and mechanism of action are unclear. The many inherent challenges of working with this slow-growing pathogen often limit the experimental approaches that can be used to address these questions. Thus, we have developed a model system in the nonpathogenic bacterium Bacillus subtilis to study ESX-type secretion systems. Here, we demonstrate that the B. subtilis yuk operon encodes an ESX-type secretion system responsible for the secretion of YukE. Additionally, we demonstrate that the yuk system is active in B. subtilis during conditions of nutrient deprivation and is required for normal biofilm formation. Interestingly, this is similar to our findings that the Mtb ESX-1 system plays dual roles in protein secretion and modulating cell wall integrity. One defining feature of all ESX loci is the presence of an FtsK/SpoIIIE family ATPase. Interestingly, these ATPases have a domain structure unique to ESX-associated ATPases, where each protein contains multiple (2-3) enzymatic domains. We used our B. subtilis system to dissect the mechanism of action of this unique class of motor proteins. We find that the yuk-encoded ATPase YukBA dimerizes to form a hexamer of enzymatic subunits that are differentially required for secretion. Strikingly, we find a unique requirement for rotational symmetry in the nucleotide binding activity of the subunits. Finally, we compared the energy requirements of the Mtb ESX-1 system and the B. subtilis yuk system. We find that these systems have some overlapping ATPase requirements for protein secretion and cell wall integrity/biofilm formation, suggesting that there is a conservation of function among ESX-type systems. We also find that some ATPase domains are differentially required for function between these two systems, which we postulate is due to the split protein architecture of the ESX-1-encoded ATPases. Together, these findings highlight the power of using a B. subtilis model system to understand the function and mechanism of action of ESX-type secretion systems.

During the synthesis of secretory and membrane proteins, polypeptides move through a universally conserved protein-conducting channel, formed by the Sec61/SecY complex that is located in the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. The channel operates in two different modes depending on its binding partners. In co-translational translocation, a pathway found in all organisms, the channel associates with a translating ribosome. In post-translational translocation, the channel cooperates with either the Sec62–Sec63 complex in eukaryotes or the SecA ATPase in bacteria. Despite tremendous progress in our understanding of protein translocation over the past decades, many questions about its mechanism remain to be answered. These include (1) how the channel maintains the membrane barrier for small molecules while transporting large proteins, (2) what is the functional implication of channel oligomerization, and (3) how the channel interacts with binding partners and polypeptide substrates during translocation. To address these questions, we developed a novel in vivo method to generate both co- and post-translation translocation intermediates in intact Escherichia coli cells, such that polypeptide chains are only partially translocated through the channel. Using this method, we first demonstrated that a translocating polypeptide itself blocks small molecules from passing through an open SecY channel. A hydrophobic pore ring surrounding the polypeptide chain is vital for maintaining the membrane barrier during translocation. Next, we examined the importance of SecY oligomerization in protein translocation. Crosslinking experiments showed that SecY molecules interact with each other in native membranes, but that this self-association is greatly decreased upon insertion of polypeptide substrates. We also showed that SecY mutants that cannot form oligomers are still functional in vivo. Collectively, our data indicate that a single copy of SecY is sufficient for protein translocation. Finally, we isolated an intact co-translational translocation intermediate from E. coli cells and analyzed its structure by cryo-electron microscopy. An initial map shows a translating ribosome containing all three tRNAs is bound to one copy of the SecY channel. Analysis of a large dataset is ongoing in order to understand the structural basis of how the channel interacts with the ribosome and translocating nascent chain.