No description available.

No description available.

Almost 2 billion people world-wide are infected with parasitic helminths. These complex multicellular eukaryotic organisms are capable of establishing long-term infections even in the face of an intact immune response. Typically, in these settings regulatory components of the immune response, such as Foxp3+ T regulatory cells (Tregs), become dominant, limiting protective effector responses towards the parasite. Helminths are thought to have evolved mechanisms, including release of immunomodulatory molecules termed excretory-secretory products (ES), to sway the balance between the regulatory and effector arms of the immune response to favour their persistence. In this thesis both the development of a protective immune response toward, and the potential manipulation of the immune response by, the rodent gastrointestinal nematode Heligmosomoides polygyrus have been studied. Firstly, the effects of H. polygyrus ES (HES) on bone-marrow derived dendritic cells (DCs) were analysed. Although HES did not alter the phenotype of the DC it was found to be able to suppress the ability of the DC to respond to inflammatory stimuli. This activity was lost when HES was heat-inactivated (hiHES). After adoptive transfer, HES-pulsed DCs were able to induce a HESspecific T helper (Th)2-type response even if co-treated with an inflammatory stimulus. Th2-type responses are protective against H. polygyrus infection. Surprisingly, the ability of HES to generate a Th2-response in a co-treatment situation was not related to its anti-inflammatory properties; DCs co-treated with hiHES and an inflammatory stimulus were able to drive an equivalent Th2-response to HES in this situation. Next, making use of mouse strains with different susceptibility phenotypes to primary H. polygyrus infection, potential mechanisms of resistance were characterised. Development of granulomas in the gut wall were found to be associated with reduced worm burdens. Furthermore, in highly susceptible C57BL/6 mice, production of IL-23 was shown to be counter-regulatory to this process, as mice on the same background but deficient in this cytokine have increased numbers of granulomas and dramatically enhanced resistance. Susceptibility to H. polygyrus was also considered at the level of epigenetic regulation. A protein that binds specifically to methylated DNA, methyl-CpG binding domain protein (MBD)2, was found to affect the proportion of Foxp3+ Tregs within the CD4+ T cell population in vivo. Additionally, in vitro induction of Foxp3 in response to TGF-β was enhanced in MBD2-/- CD4+ T cells. MBD2-/- mice had a trend towards increased worm burdens when infected with H. polygyrus, suggesting that the difference in proportion of Tregs may limit generation of an effector response. Finally, the ability of HES to directly affect the regulatory arm of the immune response was focussed upon. It was found that HES was able to induce Foxp3 expression in naïve peripheral T cells, and that this was mediated by stimulation of the TGF-β pathway. The TGF-β mimic was of parasite origin as a pan-vertebrate TGF-β antibody was unable to block its effects but sera from H. polygyrus infected animals was competent to do this. Activity of this type was not limited to HES as ES from the ovine helminth Haemonchus contortus was found to have the same property. These data imply that some helminth parasites have evolved mechanisms to support generation of Foxp3+ Tregs, thus favouring the regulatory arm of the immune response and hence their own persistence.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The activation of many pro-hormones occurs through cleavage at pairs of basic residues and is mediated by two serine proteases, PC2 and PC3. Like their substrates, they are also synthesised as inactive precurors (pro-PC2 and pro-PC3). Maturation is autocatalytic and requires removal of the N-terminal pro-peptide. Pro-PC3 matures within the endoplasmic reticulum (ER), whereas maturation of pro-PC2 proceeds within the later compartments of the Golgi network (TGN)/secretory vesicle (SV) and is thought to be regulated by 7B2. In this study the molecular basis for the differences in the maturation location and the interaction with 7B2 was examined by performing domain swap and site directed mutagenesis experiments. The mutant constructs were analysed within an <I>in vitro</I> cell free system. The results suggest that the oxyanion hole residue (Asp³¹⁰) of pro-PC2 restricts maturation to a late secretory compartment and is important for the interaction with 7B2. Mutation of this residue to resemble that of PC3 (Asp310Ans), altered pro-PC2 maturation from a TGN/SV like environment to an ER like environment. Maturation of pro-PC2, but not pro-PC3, was shown to be inhibited by 7B2. Residue Asp³¹⁰ of PC2 was necessary to mediate this interaction. When a similar mutant of PC3 was created to resemble PC2 (Asn309Asp), this residue was not sufficient to alter the maturation profile of pro-PC3 nor was it able to confer 7B2 sensitivity. The pro-region of PC2 was sufficient to alter maturation of PC3 from the ER-like compartment to a TGN/SV-like compartment, but was not able to confer 7B2 sensitivity onto PC3. This study also demonstrated that a basic cluster (HHKQQ⁸⁸) of pro-PC2 was important for delaying maturation to a late compartment and that the residue Phe¹⁰⁴, was important for efficient maturation. Mutational analysis of pro-7B2 revealed that a region within residues 1-151 was important for the interaction with pro-PC2.

Thesis--University of Helsinki, 1983. / Also published in: Proceedings of the Finnish Dental Society, v. 79, 1983, Supp. III. Includes bibliographical references (p. 47-55).

Thesis--University of Helsinki, 1983. / Also published in: Proceedings of the Finnish Dental Society, v. 79, 1983, Supp. III. Bibliography : p. 47-55.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The hamster model of experimental chronic bronchitis comprises a persistent increase in the proportion of bronchial granulated secretory cells after a single intratracheal instillation of elastase. This granulated secretory cell increase, which does not occur in the trachea, has been termed secretory cell metaplasia (SCM). Susceptibility of the bronchial epithelium may be due to a large population of elastase-responsive cells specific to this region. Three dimensional reconstruction of the major form of bronchial secretory cells revealed very little or no rough endoplasmic reticulum (RER), thus demonstrating significant regional heterogeneity since all epithelial secretory cells in the trachea have abundant RER. Animals with bronchial SCM were stimulated with pilocarpine to determine whether the cells subsequent to discharge would re-accumulate granules, thus indicating a permanent phenotypic change. However, bronchial secretory cells failed to discharge at doses equal to and greater than those claimed to be effective in rats. Elastase instilled intratracheally was immuno-localized in the hamster airways to assess the possibility of regional differences in cellular uptake of the enzyme. Elastase was not seen intracellularly in trachea or bronchus suggesting that initiation of bronchial SCM results from a cell surface effect, possibly because of elastase-specific sites on bronchial but not tracheal cells. Tracheal resistance was tested by challenging the epithelial cells in vivo and in vitro with very high doses of elastase. Light and electron microscopy revealed no evidence of a stimulation of the mucus synthetic apparatus, suggesting that tracheal epithelial cells are inherently resistant to proteolytic up-regulation. / 2031-01-01

The sheepshead has two readily isolatable humoral immunoglobulins, a 16S tetrameric form and a 6S monomeric form. The 16S tetrameric form is composed of two subpopulations, one being a disulfide linked form (~700,000 daltons) and the other a noncovalently linked population of predominantly disulfide linked dimers (~350,000 daltons). The 6s immunoglobulin (~140,000 daltons) is composed of two noncovalently linked units (~70,000 daltons) each having one heavy and one light chain. The 6S immunoglobulin is antigenically deficient to the 16S immunoglobulin, this deficiency may be due to the heavy chain of the 6s protein lacking a~25,000 dalton segment present in the heavy chain of the 16S molecule. Cutaneous mucus and bile also contain immunoglobulins. The mucus contains three proteins that can be considered immunoglobulins: a 6S form which is antigenically indistinguishable from the serum 6S immunoglobulin: a ~700,000 dalton form which does not have a "dimeric" subpopulation as observed with the serum 16S protein; and a dimeric form of ~350,000 daltons. The dimeric form may have a secretory piece since the reduced mucus dimeric protein shows an additional polypeptide chain at ~95,000 daltons. All of the cutaneous mucus high molecular weight immunoglobulins have heavy and light chains identical to the serum high molecular weight immunoglobulins (~70,000 and ~25,000 daltons). Bile immunoglobulin is dimeric and composed of two noncovalently linked monomers of ~l60,000 daltons. The bile heavy chains are ~55,000 daltons; the light chains are ~25,000 daltons. The bile immunoglobulin does not appear to be a different class of protein from that of the serum or mucus immunoglobulins. In vivo administration of radiolabeled 16S and 6S serum immunoglobulins indicates that the 6S protein is not a degradation product of the 16S form. The half lives of the 16S and 6s forms are both ~16 days. Furthermore, the secretory immunoglobulins of the bile and mucus are not due to simple transudation or active transport of the predominant serum immunoglobulins. This result suggests that the secretory immunoglobulins of the sheepshead may be locally synthesized.