Traditionally, language was proposed to be mediated by various left hemisphere perisylvian structures and the associated role of the motor cortices was limited to tasks such as articulation. Recent theoretical models have proposed that effector specific words with motor related meaning are represented somatotopically on the primary motor (Brodmann’s Area 4) and premotor (Brodmann’s Area 6) cortices. For example, it has been reported that when verbs associated with the hand (e.g., pick) are processed, the primary and premotor areas involved with moving the hand are engaged. However, fundamental methodological problems exist within the reported research. This thesis aimed to address and correct the inconsistencies and methodological limitations within the existing literature to provide more conclusive evidence regarding the involvement of the primary and premotor cortices in processing verbs with motor related meaning. This thesis also aimed to investigate whether the names of effectors (nouns) also involve processing by the motor cortices, either generally or somatotopically. Three behavioural dual task experiments and one fMRI experiment were conducted. Results indicated no evidence of somatotopically organised overlapping activation in the primary or premotor cortex between the various semantic categories of words and related effector movements. However, in the fMRI experiment, motor related verbs in general yielded significant overlapping activity between reading all effector related verbs and moving all effectors in the pre-supplementary motor area of the premotor cortex. These findings indicate that an embodied language involving somatotopic representations of effector specific verbs on the primary or premotor cortex is unlikely to be the case. Rather there appears to be a more general representation of effector related verbs in a more cognitive than motor area of the premotor cortex. The findings of this thesis are consistent a wealth of evidence supporting the motor cortices being generally associated with motor related language and with the idea that semantic representations are distributed throughout the brain according to the embodied cognitive framework, rather than being localised to amodal regions that process all words.

Traditionally, language was proposed to be mediated by various left hemisphere perisylvian structures and the associated role of the motor cortices was limited to tasks such as articulation. Recent theoretical models have proposed that effector specific words with motor related meaning are represented somatotopically on the primary motor (Brodmann’s Area 4) and premotor (Brodmann’s Area 6) cortices. For example, it has been reported that when verbs associated with the hand (e.g., pick) are processed, the primary and premotor areas involved with moving the hand are engaged. However, fundamental methodological problems exist within the reported research. This thesis aimed to address and correct the inconsistencies and methodological limitations within the existing literature to provide more conclusive evidence regarding the involvement of the primary and premotor cortices in processing verbs with motor related meaning. This thesis also aimed to investigate whether the names of effectors (nouns) also involve processing by the motor cortices, either generally or somatotopically. Three behavioural dual task experiments and one fMRI experiment were conducted. Results indicated no evidence of somatotopically organised overlapping activation in the primary or premotor cortex between the various semantic categories of words and related effector movements. However, in the fMRI experiment, motor related verbs in general yielded significant overlapping activity between reading all effector related verbs and moving all effectors in the pre-supplementary motor area of the premotor cortex. These findings indicate that an embodied language involving somatotopic representations of effector specific verbs on the primary or premotor cortex is unlikely to be the case. Rather there appears to be a more general representation of effector related verbs in a more cognitive than motor area of the premotor cortex. The findings of this thesis are consistent a wealth of evidence supporting the motor cortices being generally associated with motor related language and with the idea that semantic representations are distributed throughout the brain according to the embodied cognitive framework, rather than being localised to amodal regions that process all words.

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Nous avons adapté avec succès un protocole de transformation médiée par PEG pour D. coniospora. En collaboration avec le ccdB et la méthode de construction de plasmide à base de Gibson, nous avons établi un système pour manipuler génétiquement ce champignon qui sert comme un outil important pour l'hôte-pathogène étude d'interaction. Nous avons identifié le mode de vie pathogène spécifique de D. coniospora sur la base de sa séquence génomique. analyse génomique comparative a révélé une liste des effecteurs fongiques potentiels qui se livrent à l'immunité de l'hôte, par exemple SAPA (G3895). Nous avons également construit une souche rapporteuse pour SAPA et identifié sa cible hôte SPP-5, un peptide antimicrobien. Notre étude se concentrant particulièrement sur l'agent pathogène fournit un aperçu de l'interaction hôte-pathogène entre C. elegans et D. coniospora.En dépit de la génération réussie de 5 D. coniospora souches transgéniques. Néanmoins, les problèmes qui subsistent, tels que le transfert multiple lors de protoplastes préparation et la croissance lente de D. coniospora après transformation doivent encore être résolus. L'une des solutions est de remplacer le moyen général avec un milieu qui ressemble à l'environnement hôte. / We have successfully adapted a PEG-mediated transformation protocol for D. coniospora. Together with the ccdB and Gibson based plasmid construction method, we established a system to genetically manipulate this fungus which serves as an important tool for host-pathogen interaction study. We identified the specific pathogenic lifestyle of D. coniospora based on its genomic sequence. Comparative genomic analysis revealed a list of potential fungal effectors which engage with the host immunity, for instance SapA (G3895). We further constructed a reporter strain for SapA and identified its host target SPP-5, an antimicrobial peptide. Our study focusing particularly on the pathogen provides an insight for the host-pathogen interaction between C. elegans and D. coniospora. Despite the successful generation of 5 D. coniospora transgenic strains. Nevertheless, the remaining problems such as multiple transferring during protoplasts preparation and slow growth of D. coniospora after transformation still need to be resolved. One of the solutions is to substitute the general medium with a medium resembling the host environment.We show that D. coniospora SapA protein interacts with worm immune effector, SPP-5 in vitro indicating its potential role to suppress the host immunity. Due to the fact that SapA is also highly expressed at the late stage of infection, we cannot rule out the other possible functions of this protein. We could employ Mass spectrometry technique to identify other host proteins which interact with SapA in vivo.

Historiquement, les LT CD8 cytotoxiques ont été considérés comme la seule composante cellulaire du système immunitaire nécessaire et suffisante pour l’élimination de cellules infectées par des virus ou transformées, les LT CD4 ne jouant qu’un rôle auxiliaire, dans le développement et le maintien de la réponse immune effectrice, ou modulateur par la fonction suppressive des T-Reg. Aux côtés, de ces fonctions auxiliaires ou suppressive, nombre de données indiquaient que les LT CD4 pouvaient également exercer une activité cytotoxique directe. Nos travaux ont permis par l’analyse chez l’Homme, de l’expression des récepteurs α à l’IL-2 (CD25) et à l’IL-7 (CD127) à la surface des LT CD4 du sang périphérique d’identifier une population singulière de LT CD4 caractérisée par l’absence de ces deux molécules. Ces LT CD4, CD25-CD127-, faiblement représentées chez les sujets sains, entre 0,2-2% des LT CD4 totaux du sang périphérique, étaient fortement augmentées, entre 2-20% dans les infections chroniques VIH et Tuberculose, et notamment dans les cancers incluant les mélanomes uvéaux métastatiques (Mum) et les cancers du sein. Puisque prédominant dans des situations de stimulation chronique, ces LT CD4 ont été définis comme des LT CD4 chroniquement stimulés : chCD4. Dans le sang périphérique de patients atteints de cancer comme chez les sujets sains, la majorité de ces chCD4 arboraient un phénotype mémoire/effecteur (CD45RO+). Cependant, dans les Mum et les cancers du sein la proportion de chCD4 effecteurs (CD45RO+CD27-) était fortement augmentée. Par ailleurs, si la plus part de ces cellules effectrices apparaissaient à un stade de différenciation terminale (CD57+), elles présentaient toutes les mêmes caractéristiques phénotypiques distinctes, définies par l’absence d’expression de la molécule de co-stimulation CD28 coordonnée à l’expression à leur surface de l’intégrine CD11b et du récepteur NK, 2B4. Dans les chCD4 effecteurs, nous avons également mis en évidence la présence spécifique de granules cytoplasmiques concentrant granzyme B et perforine, molécules impliquées dans la cytotoxicité directe de cellules cibles. Cette propriété fonctionnelle a été démontrée par des tests de cytotoxicité redirigée et était restreinte aux chCD4 effecteurs en comparaison aux autres sous-populations effectrices de LT CD4 conventionnels et T-Reg. L’analyse du profile de sécrétion de cytokines, révèle l’absence total de production d’IL-17 et un profile orienté Th1, soulevant la question du lignage de cette population particulière. L’absence d’expression de Ki67, marqueur des cellules en cycle, au sein de cette population de LT CD4 cytotoxiques, parallèlement à leur accumulation, suggérait qu’elles seraient capables de persister à l’état quiescent chez les patients. Par ailleurs, dans les Mum, nous avons mis en évidence que l’augmentation importante du nombre de chCD4 chez les patients, concordait avec la présence d’expansions oligoclonales au sien de cette population, et démontré une corrélation positive entre le pourcentage des cellules effectrices, chCD4 et LT CD8, LT CD8 parmi lesquels nous avons déterminé une fréquence élevée de cellules répondeuses spécifiques d’antigènes tumoraux associés à la tumeur. Nos travaux ont également permis d’évaluer l’impact de la chimiothérapie sur les populations lymphocytaires dans le sang périphérique. Chez les patientes atteintes de cancer du sein, traitées par chimiothérapie néo-adjuvante, c’est-à-dire préopératoire, nous avons constaté une augmentation du nombre de chCD4 chez 17/22 patientes. Nous avons mis en évidence que cette augmentation sous traitement était fortement corrélée au pourcentage de régression tumorale. L’ensemble de ces résultats apporte une nouvelle vision des LT CD4 dans l’immunité tumorale. (...) / Historically, CD8 positive Cytotoxic T Lymphocytes (CTL) have been associated with an effector immune response, while T cells with a CD4 phenotype where considered helper T cells. More recent data suggest that CD4 positive T cells are also capable of a direct cytotoxic activity. Through a systematic analysis of the IL-2 (CD25) as well as IL-7 (CD127) receptors α on the surface of CD4+ CTL in peripheral blood of patients before during and after treatment we were able to identify a specific CD4+ T cell population devoid of expression for these 2 molecules. These CD4+, CD25-CD127-, T cells only represent 0,2-2% of the total CD4+ pool in peripheral blood of healthy donors, while in the presence of a chronic infectious disease such as HIV or tuberculosis they were increased, representing up to 2-20% of all CD4+ T cells. Similarly, high numbers of CD4+, CD25-CD127-, T cells could be identified in the circulation in patients with metastatic uveal melanoma (muM) or with breast cancer. These chronically stimulated T cells (chCD4) demonstrate a memory effector phenotype (CD45RO+); while the majority shows a terminally differentiated phenotype (CD57+), these T cells all arbore distinct phenotypic characteristics as defined by the absence of expression of CD28 together with the presence of a surface expression of integrin CD11b and of the NK receptor, 2B4. The presence of cytoplasmic granules concentrating granzyme B and perforin, known to be responsible for T cell cytotoxicity, were identified in effector chCD4 while they were absent in conventional CD4+ T cells as well as in Tregs. This cytotoxic potential was demonstrated through redirected cytotoxicity assays that functionally confirm this feature of these chronically activated CD4+ T cells. The secretory cytokine profile showed absent IL-17 levels and a Th1 orientation, asking questions as regards to the lineage of this particular T cell population. Ki67 expression, a marker of cell proliferation, was absent, suggestive of their ability to persist quiescently in patients. However in muM patients we were able to demonstrate a vast oligoclonal increase in chCD4+ T cells, which correlated positively with CD8+ T cells. We were able to detect a high frequency of T cells responding against a specific tumor antigen among these CD8+ T cells. We furthermore studied the effects of chemotherapy on peripheral lymphocyte populations. In breast cancer patients who had been treated with preoperative (neoadjuvant) chemotherapy we detected high levels of effector chCD4 in 17/22 patients. Of particular interest was the fact that this increase through a course of chemotherapy treatment was strongly correlated to the percentage of regression of the original tumor. Together, these results cast new light on the role and function of CD4+ T cells in tumor immunity. Our observations show that CD4+ cytotoxic T lymphocytes do exist and suggest for the first time in human that they may have an important role in response to treatment and in particular in the establishment of a durable protective immune response.

The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

The definitive endoderm gives rise to several specialized organs, including the thymus. Improper development of the definite endoderm or its derivatives can lead to human disease; in the case of the thymus, immunodeficiency or autoimmune disorders. Human pluripotent stem cells (hPSCs) have emerged as a system to model human development, as study of their differentiation allows for elucidation of the molecular basis of cell fate decisions, under both healthy and impaired conditions. Here, we first developed a CRISPR-effector system to control endogenous gene expression in hPSCs, a novel approach to manipulating hPSC state. Next, the human-specific, loss-of-function phenotypes of candidate transcription factors driving hPSC-to-definitive endoderm differentiation were analyzed through combined CRISPR-perturbation and single-cell RNA-sequencing. This analysis revealed the importance of TGFβ mediators in human definitive endoderm differentiation as well as identified an unappreciated role for FOXA2 in human foregut development. Finally, as the differentiation of definitive endoderm to thymic epithelial progenitors (TEPs) is of particular interest, a single-cell transcriptomic atlas of murine thymus development was generated in anticipation of identifying factors driving later stages of TEP differentiation. Taken together, this dissertation establishes a CRISPR-effector system to interrogate gene and regulatory element function in hPSC differentiation strategies, details the role of specific transcription factors in human endoderm differentiation, and sets the groundwork for future investigations to characterize hPSC-derived TEPs and the factors driving their differentiation.

This diploma thesis deals with the design of a virtual model of a robotic workplace. Robot and robotic workplaces are researched. Further, the design and safety phases of these workplaces are discussed. A conceptual model of the robotic workplace with robot IRB 4400/60 is designed, which is placed in the machine laboratory C1 of the Institute of Production Machines, Systems and Robotics at the Faculty of Mechanical Engineering of the Brno University of Technology. The virtual model is created in Process Simulate 13.0. It is designed to manipulate the dice, weld and operate the vertical machine tool.