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.

Apicomplexan parasites are responsible for some of the most devastating human and veterinarian diseases and are parasites of great economic importance. Apicomplexa include Plasmodium, Toxoplasma and Babesia species. The pathogenic mechanisms developed by Apicomplexa parasites, in particular those that reside in a parasitophorous vacuole, involve considerable changes to the host cell, including the expression of variable surface proteins required for immune evasion. In Plasmodium falciparum infections, host cell remodeling is responsible for disease symptomology and severity in the human host. This work represents a multi-faceted study of antigenic secretion and the role of secreted antigens in pathogenesis. We study in detail the mechanisms of antigen secretion in Apicomplexa parasites. By use of comparative genomics, we find Plasmodium Export Element (PEXEL)-like motifs in a subset of Cryptosporidium and Babesia secreted proteins. However, in Babesia the motif functions as a spherical body targeting sequence, suggesting that secretory mechanisms in Apicomplexa are adapted to the parasite's intracellular lifestyle. To elucidate the relationship and function of exported antigens, we first focused on P. falciparum to determine gene co-expression modules. We found that in vivo, export modules are composed of constitutively or variably expressed genes, the latter group associated with patient clinical phenotypes. We then focused on a novel gene family called "phist" and show, using transcriptional expression profiling, its role in P. falciparum cytoadherence. In total, we demonstrate that antigen secretion is an evolutionary mechanism in Apicomplexa parasites and that variant expression of the genes encoding these antigens may allow parasites to adapt to environmental stresses.

published_or_final_version / Physiology / Master / Master of Philosophy

Childhood maltreatment is extremely common in patients diagnosed with psychotic disorders. Moreover, it has been linked with impaired functioning of the Hypothalamic-Pituitary-Adrenal axis. Furthermore, abnormality of the HPA has been found in psychotic patients. Presence of childhood maltreatment could then explain why the HPA axis is dysfunctional in these subjects. Our objective was to clarify the role of childhood trauma in the cortisol profiles of psychotic patients. Thirty-one patients underwent assessments of childhood maltreatment. Diurnal cortisol and cortisol after a controlled psychosocial stress were also collected. Our results show that childhood trauma is associated with lower cortisol levels during the morning and during 24 hours. In men diagnosed with psychosis, childhood trauma is also associated with a higher cortisol response during psychosocial stress. This suggests an alteration of the HPA axis in psychotic patients, resulting from early trauma. Moreover, our results suggest that looking at specific types of childhood abuse may also be important.

In 1968, a study demonstrated that consolidated memories can be affected again if they are reactivated. Given the importance of the stress hormones glucocorticoids (GCs) on memory consolidation, the goal of the current study was to assess whether GCs had the capacity to affect a reactivated long-term memory and whether neutral and emotional memories were affected differently. At the first session, participants encoded a movie containing neutral and emotional scenes. Two days later, they recalled the story. Half of them were then exposed to a psychosocial stressor. Memory performance was assessed again right after the stressor and five days later. The stressed group recalled less neutral material five days after the stressor compared to controls. Immediately after the stressor, the stressed group recalled more emotional material than controls. Moreover, this enhanced memory trace was maintained across time. This highlights the importance of minimizing exposure to stressful contexts when reactivating emotional memories.

This Master's dissertation explored whether a rapid orienting of attention toward or away from social stress information during a restful state, relates to the magnitude of glucocorticoids (GC) released in response to a stressor, the Trier Social Stress Test (TSST). It also assessed whether childhood rearing in a low socioeconomic status (SES) context mediates this relationship. Subjects rested for 45 minutes during which time they completed a modified version of Posner's attention paradigm, comprising social stress words. Immediately following, participants were exposed to the stressor. Results indicated that a rapid attentional engagement toward social stress words associated with pronounced GC responses to the TSST. Fast engagers displayed lower self-esteem and did not differ in terms of their past SES. These findings demonstrate that attentional biases for social stress information at rest combine with diminished self-esteem to predict the magnitude of GC released during psychological stress irrespective of early SES conditions.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

Enteropathogenic Escherichia coli (EPEC) is an enteric pathogen that causes potentially fatal infantile diarrhea. A type III secretion system is employed by EPEC to inject bacterial effector proteins directly into host intestinal epithelial cells. The multivalent chaperone, CesT, interacts with nine effectors and is a significant contributor to EPEC pathogenesis. A putative transcriptional promoter region was identified directly upstream of cesT. In silico analyses identified conserved elements that suggest the cesT promoter is recognized by ?70. Using transcriptional fusions to lux reporter genes I showed that the cesT promoter region is active under conditions known to induce virulence-gene expression. I conclude that the cesT promoter is active early during an in vitro assay, and regulated by different mechanisms than those affecting the Ptir operon promoter.

Shigellae are pathogenic bacteria that cause the disease shigellosis. Two methods for studying secreted effectors encoded by this pathogen’s virulence plasmid are described. First, protein microarrays were used to identify substrates of an E3 ubiquitin ligase called IpaH7.8. Second, a deletion collection containing mutants for every gene on the virulence plasmid was used in two screens: one to identify mutants that elicit atypical levels of Interleukin-8 (IL-8) from U937 cells, and one to identify mutants that bind the dye Congo red abnormally. Although protein microarrays were an ineffective tool, the deletion collection proved valuable. Most mutants were less effective at sequestering Congo red than wild-type S. flexneri, although this ability was enhanced in several mutants. Four mutants, ?ospB, ?orf186, ?mxiH and ?mxiK, elicited higher levels of IL-8 from U937 cells than wild type S. flexneri. These results validate the use of the deletion collection as a tool for studying bacterial pathogenesis.