Recruitment of circulating cells towards target sites is primarily dependent on E-selectin receptor/ligand adhesive interactions. Glycosyltransferase (GTs) are involved in the creation of E-selectin ligands. A sialofucosylated terminal tetrasaccharide like glycan structure known as sialyl Lewis x (sLex), is the most recognized ligand by selectins. This structure is found on the surface of cancer cells and leukocytes but is often absent on the surface of many adult stem cell populations. In order to synthesize sLex, GTs must be endogenously expressed and remain active within the cells. Generally, these stem cells express terminal sialylated lactosamine structures on their glycoproteins which require the addition of alpha-(1,3)-fucose to be converted into an E-selectin ligand. There are a number of fucosyltransferases (FUTs) that are able to modify terminal lactosamine structures to create sLex such as FUT6. In this work we focused on expressing and purifying active recombinant FUTs as a tool to help create sLex structures on the surface of adult stem cells in order to enhance their migration.

The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2a) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2a with an ablated C1P interaction site (KI) to examine the cPLA2a/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was dramatically enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (e.g., PGE2) and increased levels of specific HETE species (e.g., 5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2a was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key in vivo biological mechanisms by a defined protein:lipid interaction and provide new insights into cPLA2a function.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Orientadores: Ronei Luciano Mamoni, Maria Heloisa de Souza Lima Blotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T12:51:02Z (GMT). No. of bitstreams: 1 Paiao_MunirRegini_M.pdf: 7191608 bytes, checksum: f8a5e81d9fd9e880ec841934fe773e2d (MD5) Previous issue date: 2012 / Resumo: A participação de células produtoras de IL-17 (Th17) na resposta imunológica contra infecções causadas por fungos tem sido recentemente objeto de grande interesse. A principal função da IL-17 é a ativação e atração de neutrófilos para o local de infecção. Lesões de pacientes com paracoccidioidomicose (PCM), a micose sistêmica mais importante no Brasil, são caracterizadas por um infiltrado inflamatório rico em neutrófilos. Entretanto, neutrófilos de pacientes com PCM apresentam uma resposta fungicida diminuída, quando comparada à observada em indivíduos saudáveis. O objetivo desse estudo foi avaliar o efeito do tratamento com IL-17 e IL-23 sobre a atividade fungicida, bem como sobre a capacidade migratória e inflamatória de neutrófilos. Neutrófilos de indivíduos saudáveis e de pacientes com a forma adulta multifocal da doença foram isolados e estimulados com citocinas recombinantes (rh-IL17 e/ou rh-IL23). Após o estímulo as células foram avaliadas quanto a expressão de receptores de quimiocinas (CXCR1 e CXCR2) e moléculas de adesão (CD54 e CD62L). Os neutrófilos também foram avaliados quanto a sua capacidade de adesão em Células Endoteliais Pulmonares Humanas (HLEC), sua resposta migratória a diferentes concentrações de CXCL8 e sua capacidade fungicida, e sobre a produção de H2O2, matriz metaloproteinase 9 (MMP-9) e citocinas inflamatórias (IL-6, TNF-? e IL-1?). Os resultados demonstraram que células estimuladas com IL-17 e/ou IL-23 apresentaram uma expressão aumentada de receptores de quimiocina e moléculas de adesão, associadas com uma capacidade aumentada de adesão e migração. Além disso, observamos que neutrófilos estimulados com ambas as citocinas exibiram uma antifúngica diminuída relacionada com produção diminuída de H2O2, ao lado de uma produção aumentada de MMP-9, IL-6 e IL-1?. Os dados obtidos nos permitem concluir que citocinas relacionadas à resposta Th17 (IL-17 e IL-23) podem desempenhar um papel importante na contenção da infecção causada pelo P. brasiliensis, uma vez que predominam em sua forma mais branda e localizada, a forma adulta. Essas citocinas poderiam atuar de forma semelhante àquela observada em outras infecções fúngicas, induzindo uma resposta inflamatória local e ativando células do sistema imunológico inato. Apesar de sua importância, a produção crônica dessas citocinas, e sua atuação sobre os neutrófilos, podem induzir uma resposta inflamatória exacerbada que em última instância seria responsável pela destruição tecidual observada principalmente nas formas crônicas da doença / Abstract: The participation of IL-17 producing cells in the immune response to fungi has been recently described. The main function of IL-17 is the activation and attraction of neutrophils to infection sites. Lesions of patients with paracoccidioidomycosis (PCM), the most important systemic mycosis in Brazil, are characterized by an inflammatory infiltrate rich in neutrophils, that shows an impaired fungicidal activity when compared to cells from healthy individuals. The aim of this study was to evaluate the effect of IL-17 and IL-23 on the fungicidal activity, as well as on the migratory and inflammatory ability of neutrophils. Neutrophils from healthy controls and patients with the multifocal adult form of PCM were isolated and stimulated with rhIL-17 and/or rhIL-23. Stimulated cells were evaluated for their expression of chemokine receptors (CXCR1 and CXCR2), and adhesion molecules (CD54 and CD62L), as well as, for their capacity of adhesion on Human Lung Endothelial Cells (HLEC) and for their migratory ability in response to CXCL8. We also evaluated the effect of these cytokines treatment on fungicidal activity and on the production of H2O2, matrix metalloproteinase-9 (MMP-9) and inflammatory cytokines (IL-6, TNF-alpha and IL-1-beta). Neutrophils exposed to IL-17 and/or IL-23 increased their expression of CXCR1 and CXCR2 and adhesion molecules, which were associated with an increased adhesive and migratory capacity. Furthermore, the treatment of neutrophils with IL-17 and IL-23 had a suppressive effect on their fungicidal activity, characterized by a diminished production of H2O2, but increased their production of MMP-9 and inflammatory cytokines. These data indicate that Th17 related cytokines (IL-17 and IL-23) can participate in the initial resistance to the infection caused by P. brasiliensis, once they are predominant in the milder and localized form of the disease, the adult form. As in other fungal infections, IL-17 (and IL-23) can induce a local inflammatory response and the activation of innate immune cells. However, when chronically produced these cytokines can induce an exacerbated inflammatory response, mainly acting on neutrophils, which can be responsible for the tissue damage observed in the chronic form of the disease / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Cell-substrate interactions are important to study for development of accurate in vitro research platforms.  Recently it has been demonstrated that physical microenvironment of cells directly affects cellular motility and cytoskeletal arrangement.  Specifically, previous studies have explored the role of material stiffness (Young's modulus: N/m2) on cell behavior including attachment, spreading, migration, cytoskeleton arrangement (stress fiber and focal adhesion distribution) and differentiation. In this study using our recently described non-electrospinning fiber manufacturing platform, customized scaffolds of suspended nanofibers are developed to study single cell behavior in a tunable structural stiffness (N/m) environment.  Suspended fibers of three different diameters (400, 700 and 1200 nm) are deposited in aligned configurations in two lengths of 1 and 2 mm using the previously described STEP (Spinneret based Tunable Engineered Parameters) platform.  These fibers present a gradient of structural stiffness to the cells at constant material stiffness.   Single cells attached to fibers are constrained to move along the fiber axis and with increase in structural stiffness are observed to spread to longer lengths, put out longer focal adhesions, have elongated nucleus with decreased migration rates. Furthermore, more than 60% of cell population is observed to migrate from areas of low to high structural stiffness. Additionally dividing cells are observed to round up and daughter cells are observed to migrate away from each other after division. Interestingly, dividing rounded cells are found to be anchored to the fibers through thin protrusions emanating from the focal adhesion sites. These results indicate a substrate stiffness sensing mechanism that goes beyond the traditionally accepted modulus sensing that cells have been shown to respond to previously.  From this work, the importance of structural stiffness in cellular mechanosensing at the single cell-nanofiber scaled warrants consideration of the above factors in accurate design of scaffolds in future. / Master of Science

Le récepteur ERRα (Estrogen Receptor-Related Receptor alpha) appartient à la superfamille des récepteurs nucléaires. Une forte expression de ERRα est corrélée à un mauvais pronostic, suggérant l’implication de ce récepteur dans le processus métastatique. Mon projet est d'analyser le rôle de ERRα dans les mouvements cellulaires. J’ai montré qu’inhiber ERRα perturbe la migration cellulaire. L’étude des mouvements montre que l’absence de ERRα induit une perturbation de l’orientation cellulaire, du nombre des fibres de stress et des protrusions membranaires. Les cellules migrent de façon désorientée. J’ai démontré l’existence d’une cascade de régulation où ERRα stimule transcriptionnellement l'expression de la protéine BACURD2/TNFAIP1, elle-même régulant la stabilité de RhoA. Inhiber ERRα induit également une surexpression de RhoA, sa suractivation et une perturbation de la migration orientée. Cette cascade a été confirmée par des expériences de complémentation. J’ai vérifié ces résultats par des expériences in vivo et ex vivo, chez les souris KO pour ERRα. L’absence de ce récepteur induit une diminution de l’expression de TNFAIP1 inhibant la dégradation de RhoA et entrainant finalement une perturbation des mouvements cellulaires. Ainsi ERRα régule positivement la migration cellulaire conduisant à une forte potentialité métastatique dans les tumeurs surexprimant ce récepteur.L’ensemble de mes résultats pourrait faire de ERRα une nouvelle cible en vue de nouvelles thérapies anticancéreuses et nous pourrions proposer BACURD2/ TNFAIP1 comme un nouveau marqueur de pronostic dans les cancers. / High expression of the orphan nuclear receptor ERRα is strongly correlated with poor prognosis in various types of tumors, including those of the breast. The fact that high ERRα expression in tumors is also correlated with elevated invasiveness suggests that this nuclear receptor positively regulates cell migration and invasiveness.This possibility was investigated using MDA-MB231 breast cancer cell line as a model. Inactivating ERRα impairs cell migration. Using time-lapse-based cell tracking analysis and Golgi positioning, we show that this impairment is not due to reduced migration speed but rather to cell disorientation. The enhanced number of cell protrusions present in migrating cells and disorganized actin fibers confirm this. In summary cells do migrate but do not sustain persistent linear movement. We observed that upon ERRα inactivation, RhoA, which is instrumental in oriented movement, is overexpressed at the protein level. Further analysis showed that the stability and proteasome-dependent degradation of the protein is affected. To analyze the relationship between ERRα (as a transcription factor) and RhoA protein stability we performed a transcriptomic analysis comparing (by RNA-Seq) wt cells to ERRα-depleted ones. We identified genes regulated by ERRα that are involved in both cell migration (as a biological process) and in protein stability and degradation, more specifically that of RhoA protein (as a molecular process). TNFAIP1/Bacurd2 is stimulated by ERRα and fits these criteria: this protein mediates the Culin3-based, proteasome-dependent of RhoA and its inactivation leads to defects in cell migration.TNFAIP1/RhoA cascade is a major downstream effector of ERRα in cell migration

Hokkaido University (北海道大学) / 博士 / 医学

From the developing embryo to the evacuation of football stadiums, the migration and movement of populations of individuals is a vital part of human life. Such movement often occurs in crowded conditions, where the space occupied by each individual impacts on the freedom of others. This thesis aims to analyse and understand the effects of occupied volume (volume exclusion) on the movement of the individual and the population. We consider, as a motivating system, the rearrangement of individuals required to turn a clump of cells into a functioning embryo. Specifically, we consider the migration of cranial neural crest cells in the developing chick embryo. Working closely with experimental collaborators we construct a hybrid model of the system, consisting of a continuum chemoattractant and individual-based cell description and find that multiple cell phenotypes are required for successful migration. In the crowded environment of the migratory system, volume exclusion is highly important and significantly enhances the speed of cell migration in our model, whilst reducing the numbers of individuals that can enter the domain. The developed model is used to make experimental predictions, that are tested in vivo, using cycles of modelling and experimental work to give greater insight into the biological system. Our formulated model is computational, and is thus difficult to analyse whilst considering different parameter regimes. The second part of the thesis is driven by the wish to systematically analyse our model. As such, it concentrates on developing new techniques to derive continuum equations from diffusive and chemotactic individual-based and hybrid models in one and two spatial dimensions with the incorporation of volume exclusion. We demonstrate the accuracy of our techniques under different parameter regimes and using different mechanisms of movement. In particular, we show that our derived continuum equations almost always compare better to data averaged over multiple simulations than the equivalent equations without volume exclusion. Thus we establish that volume exclusion has a substantial effect on the evolution of a migrating population.

Disulfide bridges play a significant role in protein-folding as well as en- zyme activity and thus regulate many intra- and extracellular processes. Sulfhydryl oxidase QSOX1 forms S-S bridges de novo, modulating the activity of its substrates and thus directly or indirectly influences vital cel- lular processes. The first part of this thesis focuses on characterization of the role of QSOX1 in cancerogenesis, using breast cancer cell lines (MCF7, MDA-MB-231) and pancreatic cancer cell line (Panc-1), while the second part emphasizes the regulation of QSOX1 expression by different oxygen concentrations. To study the effect of QSOX1 on proliferation of triple-negative cancer cells MDA-MB-231, two genetically modified cell lines - QSOX1-overexpressing and QSOX1 knockout cell lines - were constructed. While increased QSOX1 protein levels do not have a significant effect, the absence of QSOX1 leads to a decreased cellular growth. Lack of QSOX1 also results in visible change in cellular morphology. QSOX1 knockout cells can be mostly characterized as more round-shaped with less noticeable or completely missing lamellipo- dia. This finding is with agreement with to-date literature suggesting that QSOX1 is important not only for cellular proliferation but also for migration and invasiveness. While authenticating the theory of...