Proper functioning of endocrine cells is crucial for organismal homeostasis. The underlying mechanisms that fine-tune the amount, and the timing of hormone secretion are not clear. JIP1 / MAPK8IP1 (JNK interacting protein 1) is a scaffold protein that mediates cellular stress response, and is highly expressed in endocrine cells, including insulin secreting b-cells in pancreas islets. However, the role of JIP1 in b-cells is unclear. This study demonstrates that b-cell specific Jip1 ablation results in decreased glucose-induced insulin secretion, without a change in Insulin1 and Insulin2 gene expression. Inhibition of both JIP1-kinesin interaction, and JIP1-JNK interaction by genetic mutations also resulted in decreased insulin secretion, suggesting that JIP1 may mediate insulin vesicle trafficking through interacting with kinesin and JNK. Autophagy is a cellular recycling mechanism and implicated in the b-cell function. Both JIP1 and JNK are proposed to regulate autophagy pathway. However, it is unclear whether JNK plays a role in the promotion or suppression of autophagy. The findings of this study show that JNK is not essential for autophagy induction, but can regulate autophagy in a cell and context specific manner. The results in this thesis implies a mechanism that link cellular trafficking and stress signaling pathways in the regulated hormone secretion. In addition to the known role of JIP1 in metabolism and insulin resistance, this finding may also be relevant to endocrine pathologies.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative anaerobe that induces severe inflammation resulting in gastroenteritis. In the case of S. Typhimurium infection, induction of an inflammatory response has been linked to its primary virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host’s cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. One such effector, SifA, is a bi-functional T3SS effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. In the present study, we demonstrate that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for T3SS mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

Indiana University-Purdue University Indianapolis (IUPUI) / BACKGROUND: Dental erosion is a multifactorial condition that causes irreversible loss of dental hard tissues. Its development is highly influenced by saliva, with higher prevalence in hyposalivatory patients. There is no scientific consensus regarding the restorative treatment of choice for eroded teeth in such highly erosive conditions and to date, this has not been studied under in-vitro conditions. OBJECTIVES: To investigate the effect of erosion on direct tooth-colored restorations and adjacent enamel/dentin under different simulated salivary flow rates. METHODS: Bovine enamel and dentin specimens were prepared (n =16) and restored with the testing restorative materials, resin-composite (Filtek Z250), resin-modified glass ionomer (Fuji II LC), high viscosity glass ionomer cement (Fuji IX), and low viscosity glass ionomer cement (Fuji II).Then, submitted to an in-vitro erosion cycling model simulating different salivary flow rates (normal 0.5 ml/min and low 0.05 ml/min) and dental erosion protocols for 5 days. Surface loss of the restorative material and surrounding enamel/dentin surfaces were analyzed. A mixed-model ANOVAs and Sidak adjustment were used for statistical comparisons (p < 0.05). RESULTS: The surface loss was lower at 0.5 than at 0.05 ml/min, for all tested restorative materials except resin composite. Surface loss was higher in enamel and dentin adjacent to Filtek Z250 compared to Fuji II LC and Fuji IX, with no significant difference in enamel and dentin surface loss adjacent to Filtek Z250 and Fuji II. The restorations surface degradation was significantly lower for Filtek Z250 than for Fuji II, Fuji II LC, and Fuji IX, at both 0.5 and 0.05 ml/min; moreover, the surface loss was significantly lower for Fuji II LC than for Fuji II and Fuji IX, which did not differ from each other. CONCLUSION: Within the limitations of this study, it can be concluded that low salivary flow promoted higher erosive conditions. The use of Fuji II LC and Fuji IX may reduce erosive effects on enamel and dentin adjacent to restoration. Of the materials evaluated, resin-modified glass ionomer restoration may be the most suitable for restoration for patients at higher risk of erosion with low exposure to fluoride.

Indiana University-Purdue University Indianapolis (IUPUI) / Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.

The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most successful and commonly used secretion signal leader in Pichia pastoris has been the MAT α prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently even with the MAT α prepro secretion signal. Some strategies to enhance secretion efficiency involved modifying the secretion signal leader. Based on the knob-socket model and Jpred3 ( a secondary structure predictor), eleven deletions of MAT α prepro secretion signal and one MAT α pre double pro-peptide mutant was engineered and assayed with either horseradish peroxidase (HRP), or Candida antarctica lipase B reporter protein to evaluate the correlation between secondary structure and secretion level. In addition, structural analysis through circular dichroism was completed for the wild type pro-peptide and a mutant pro-peptide to evaluate differences in secondary structure. Results suggest pro-peptide amino acids 75-78 play an important role in determining secretion level and a higher secretion level tends to associate with secondary structures that are less defined. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

In effort to understand and isolate genes that are associated with protein secretion, the Lin-Cereghino laboratory at University of the Pacific created mutant strains of Pichia pastoris using the restriction enzyme mediated integration method. The mutants exhibited an unusual ability to supersecrete beta-galactosidase, due to the effects of a randomly disrupted gene by pREMI-Z. To learn more about the novel effects of the gene disruption, nine beta-galactosidase supersecreters ( bgs ) have been characterized for their phenotypes such as growth rate, cell wall integrity, and ability to produce and secrete various types of recombinant proteins. The mutants showed various population doubling times, which ranged from 1.7 to 2.4 hours. Generally, the mutants with severely diminished growth rates had much lower secretion of the reporter proteins. The mutants also showed different levels of cell wall (osmotic) defect, indicated by moderate to severe leakage of alkaline phosphatase from the vacuole. It was revealed that the cell wall defect was not necessarily associated with increased protein secretion, which suggests that the cell wall may not be a limiting barrier for the secretion of most reporter proteins. The result of the reporter study suggests that the secretion phenotypes of bgs mutants were protein specific and likely to be dependent upon the structure of the secreted protein rather than the size.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20616号 / 医博第4265号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 渡邊 直樹, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, microbial aspects related to Salmonella as a food pathogen are investigated. A bioluminescent reporting system was developed for Salmonella to monitor the attachment and growth of the pathogen on food products. Twelve and eleven Salmonella strains from the broiler production continuum were tagged with bioluminescence by plasmid and integration of the lux operon into the chromosome, respectively. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, an attachment model using chicken skin was developed. Variables including washing and temperature were tested in the attachment model to determine the effects on attachment of Salmonella strains to chicken skin, a characteristic that enhances persistence during processing. Additionally, the invasion process for two serovars of Salmonella with differing host tropism was examined with emphasis on the initial establishment of the bacterium in the host. The major facilitator for invasion, type III secretion system, was inactivated through deletion mutation to evaluate invasion of human epithelial cell line by additional means. The difference in host tropism between the two subspecies of Salmonella was also taken into account when evaluating invasion. Results showed that invasion of human epithelial cells can be initiated despite inactivation of the type III secretion system. A serovar of Salmonella that is not typically associated with human illness was also shown to initiate invasion of human epithelial cells, a result that carries public health implication as this serovar has recently been shown to be multi-drug resistant.

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