Strontium (Sr2+) är det aktiva ämnet i läkemedel som används för att reducera frakturrisken hos patienter som lider av osteoporos. På senare tid har Sr2+ kombinerats med olika biomaterial i syfte att gynna benbildning, emellertid är ämnet vagt studerat avseende effekten på parodontala vävnader. Trots omfattande användning, är verkningsmekanismer av Sr2+ inte helt klarlagda. Denna studie syftar därmed till att utvärdera effekten av Sr2+ på humana parodontalligament-celler (PDL-celler) och osteoblaster avseende proliferation och osteogen aktivitet. Odlade humana PDL celler och osteoblastcellinjerna MG63 och hFOB 1.19 behandlades med SrCl2 (0,1-10 mM) eller vehikel under 72 h. Manuell cellräkning utfördes med en Bürker kammare. Det totala proteininnehållet fastställdes med en kolorimetrisk mätning genom användning av Bio-Rad proteinanalys. Aktiviteten av alkaliskt fosfatas bestämdes enzymatiskt och normaliserades till totalt proteininnehåll. SrCl2 hade ingen signifikant effekt på PDL celler (p> 0.05), däremot observerades en tendens till inducerade osteoblastiska egenskaper. I motsats till det, ökade 5 mM SrCl2 den totala proteinhalten i MG63 celler med 37% (p <0,01) jämfört med vehikel, medan en lägre koncentration (0,1 mM) inte hade någon påverkan. 5 mM SrCl2 ökade MG63 cellantalet med 38% (p <0,001), medan en högre koncentration (10 mM) inte uppvisade en signifikant ökad effekt jämfört med 5 mM (+54%, jämfört med vehikel, p<0.05). Resultaten visar att 72 h administrering av ≥ 5 mM SrCl2 ger en pro-proliferativ effekt på humana osteoblastliknande MG63 celler och uppvisar en tendens till att stimulera osteogena egenskaper hos primära humana PDL-celler. / Strontium (Sr2+) is the active substance of pharmaceuticals used for reducing fracture risk in osteoporotic patients. Lately, Sr2+ is combined with biomaterials to enhance osteogenesis, which has been vaguely studied considering periodontal tissue regeneration. Despite extensive use, the mechanisms of action of Sr2+ are not fully understood. The present study assesses the impact of Sr2+ on primary human periodontal ligament cells (PDL cells) and human osteoblasts in regard to proliferation and pro-osteogenic activity. Cultured human PDL cells and osteoblast cell lines MG63 and hFOB 1.19 were treated with SrCl2 (0.1-10 mM) or vehicle for 72 h. Cells were counted manually using a Bürker chamber. Total protein content was determined by colorimetric analysis using Bio-Rad protein assay. Alkaline phosphatase activity was determined enzymatically and normalized to total protein content. SrCl2 had no significant effect on PDL cells (p>0.05), but a tendency towards induced osteogenic characteristics was observed. In contrast, 5 mM SrCl2 enhanced total MG63 cell protein content by 37% (p<0.01), compared to vehicle, whereas a lower concentration (0.1 mM) did not. 5 mM SrCl2 increased MG63 cell number by 38% (p<0.001), while a higher concentration (10 mM) did not have a significant additional effect over the 5 mM (+54%, compared to vehicle, p<0.05). The results demonstrate that 72 h administration of ≥ 5 mM SrCl2 exerts a pro-proliferative effect on human osteoblast-like MG63 cells and display a tendency to induce osteogenic characteristics in primary human PDL cells.

Mechanical interactions of cells with their immediately surrounding extracellular matrix (ECM) is now known to be critical in pathophysiology. For example, during cancer progression, while uncontrollable cell division leads to tumor formation, the subsequent metastatic migration of cells from the primary tumor site to distant parts of the body causes most cancer-related deaths. The metastatic journey requires cells to be able to adopt different shapes and move persistently through the highly fibrous native ECM, thereby requiring significant spatiotemporal reorganization of the cell cytoskeleton. While numerous studies performed on flat 2-dimensional culture platforms and physiological 3D gels have elucidated cytoskeletal reorganization, our understanding on how cells adapt to natural fibrous microenvironments and regulate their behavior in response to specific ECM biophysical cues including fiber size, spacing, alignment and stiffness remains in infancy. Here, we utilize the non -electrospinning Spinneret tunable engineered parameters (STEP) technique to manufacture ECM mimicking suspended fibrous matrices with precisely controlled fiber diameters, network architecture, inter-fiber spacing and structural stiffness to advance our fundamental understanding of how external cues affect cytoskeleton-based cellular forces in 3-distinct morphological processes of the cell cycle starting from division to spreading and migration. Mechanobiological insights from these studies are implemented to deliver intracellular cargo inside cells using electrical fields. Holistically, we conclude that fibrous environments elicit multiple new cell behaviors never before reported. Specifically, our new findings include (i) design of fiber networks regulates actin networks and cell forces to sculpt nuclei in varying shapes: compressed ovals, tear drop, and invaginations, and drive the nuclear translocation of transcription factors like YAP/TAZ. In all these shapes, nuclei remain rupture-free, thus demonstrating the unique adaptability of cells to fibers, (ii) dense crosshatch networks are fertile environments for persistent 1D migration in 3D shapes of rounded nuclei and low density of actin networks, while sparse fiber networks induce 2D random migration in flattened shapes and well-defined actin stress fibers, (iii) actin retraction fiber-based stability regulates mitotic errors. Cells undergoing mitosis on single fibers exhibit significant 3D movement, and those attached to two fibers can have rotated mitotic machinery, both conditions contributing to erroneous division, and (iv) a bi-phasic force response to electroporation that coincides with actin cytoskeleton remodeling. Cells on suspended fibers can withstand higher electric field abuse, which opens opportunities to deliver cargo of varying sizes inside the cell. Taken altogether, our findings provide new mechanobiological understanding of cell-fiber interactions at high spatiotemporal resolution impacting cell migration, division and nuclear mechanics-key behaviors in the study of pathophysiology. / Doctor of Philosophy / Cancer, one of the major pathophysiological conditions, progresses within the living body through spreading of malignant cells from the primary tumor to distant secondary sites, ultimately leading to life-ending outcomes. Such spreading of cancer also known as cancer metastasis requires mechanical interactions of cells with their immediately surrounding microenvironment or the extracellular matrix (ECM). Cells utilize their cytoskeleton, a dynamic internal network of filamentous proteins, to adopt various morphologies, exert mechanical forces and physically remodel their local environment as they navigate through the highly fibrous native ECM. While previous research has elucidated how biochemical factors and bulk matrix properties regulate such cytoskeletal organization and single cell behavior, our understanding of how cells adapt to fibrous environments and respond to local biophysical cues like fiber diameter, spacing, alignment and stiffness remains in infancy. Here we use the non -electrospinning Spinneret tunable engineered parameters (STEP) to generate suspended nanofiber networks of tunable geometric and mechanical properties to mimic the native cellular environment. We discover that cells elongated within these ECM-mimicking environments utilize a unique cytoskeletal caging structure to regulate the shape and response of their nuclei in a fiber -diameter and organization-dependent manner. Additionally, we demonstrate that these elongated cell morphologies often observed during metastatic cancer cell movements, is achievable not only in aligned fibers but can also be induced by dense networks of fibers in a crossing organization. Specifically, such dense crosshatch networks allow cells to migrate persistently at high speeds while cells on sparsely spaced networks demonstrate slower and random movements. As cells elongated during interphase rounded up to undergo division, we find that the underlying fiber-geometry modulates mitotic dynamics through differential levels of actin retraction fiber-mediated stability, leading to significant alterations in orientation of mitotic machinery and mitotic spindle defects. Finally, we utilize these mechanobiological insights on cytoskeletal organization and cell shape control to optimize intracellular delivery of cargo using high-voltage electric fields. We demonstrate suspended cells are capable of withstanding higher electric fields and identify multistage cell contractility recovery dynamics, which correlate with cytoskeletal disruption and reassembly. Taken altogether, our findings provide a comprehensive understanding of the fibrous ECM-mediated regulation of the cytoskeletal organization and its impact in cell migration, division and nuclear mechanics. Knowledge obtained from this study will improve our understanding of cancer metastasis and provide predictive data for in vivo cellular response, essential for cytoskeleton-targeting cancer therapies.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

G proteins coupled receptors (GPCRs) play essential developmental roles with functions in all of the immune, olfactory sensory systems amongst other systems as well as exhibiting essential roles in the central and peripheral nervous system. GPCRs are also major targets of pharmaceutical drugs currently used to treat a vast number of conditions. Despite their clear importance, the function of many GPCRs is still obscure. Identifying the physiological role of more GPCRs provides a niche for more drugs to be developed and thus more conditions to be treated. The C.elegans lat-1 gene encodes the latrophilin vertebrate homolog; it is a member of the adhesion GPCR family and is structurally related to the flamingo/CELSR, an essential component of planar cell polarity pathway. This study aims to phenotypically characterise lat-1 mutants in C.elegans to provide insights into the physiological role of this important member of adhesion GPCRs. lat-1 mutants exhibit several morphological defects throughout development and during vulva development. Analysing the embryonic development of such mutants also identified an anterior-posterior polarity defect. The results implicate a second evolutionary conserved subfamily of adhesion GPCRs in the control of tissue polarity and morphogenesis.

La division cellulaire asymétrique est un des processus clefs pour générer la diversité cellulaire au cours du développement. La régulation de l’orientation du fuseau mitotique est essentielle pour la production de divisions asymétriques. Elle détermine l’héritage asymétrique des déterminants cellulaires entre les cellules filles. Les mécanismes qui contrôlent l’orientation du fuseau mitotique sont bien caractérisés chez le nématode C. elegans et la mouche Drosophile. Ces modèles ont permis d'identifier les protéines clefs impliquées dans ce processus et conservées chez les mammifères, comme celles du complexe d’orientation du fuseau mitotique : Gαi-LGN-NuMA, et celles du complexe de polarité apicale : PAR3-PAR6-aPKC. Chez les vertébrés, la localisation cortico-latérale de LGN-NuMA dans les progéniteurs neuraux en division est déterminante pour l'orientation planaire du fuseau mitotique, mais son mécanisme de régulation reste inconnu. Nous utilisons la rétine de souris en développement comme modèle expérimental pour sa simplicité et son accessibilité. Des résultats antérieurs de notre laboratoire ont démontré que l'asymétrie de la division dépend de l’orientation du fuseau mitotique, et que cette orientation change au cours au développement. Les rares divisions verticales apparaissent surtout aux stades tardifs de la rétinogenèse. Le but de cette thèse est d'élucider les mécanismes régulateurs du changement d'orientation au cours de la rétinogenèse, et de déterminer le rôle des divisions asymétriques dans les phases prolifératives et neurogéniques du développement de la rétine. Avec nos collaborateurs, le laboratoire du Dr Stéphane Angers, nous avons identifié SAPCD2 (suppressor APC domain containing 2), comme un nouvel interacteur des protéines Gαi, LGN et PAR3. Le rôle du gène Sapcd2 est peu décrit jusqu'à ce jour. Son expression protéique varie au cours du cycle cellulaire, avec un pic d'expression en mitose, et sa surexpression est observée dans plusieurs cas de cancers. Dans un premier temps, nous avons analysé l'expression de Sapcd2 et Lgn dans la rétine de souris en développement. Leur expression varie selon les phases de la mitose et du stade développemental. À P0, soit le premier jour postnatal, SAPCD2 et LGN ont une localisation cellulaire complémentaire dans les progéniteurs en division, avec un enrichissement à la membrane apicale et au cortex cellulaire latéral respectivement, dans des proportions corrélées au nombre de divisions planaires. Tandis qu'au jour embryonnaire 14.5 (E14.5), SAPCD2 et LGN sont toutes deux localisées aux pôles du fuseau mitotique, suggérant des rôles dynamiques au cours du développement rétinien. Nous avons ensuite analysé l’orientation du fuseau mitotique à E14.5 et P0, dans des souris en absence de Sapcd2 et/ou Lgn. En parallèle de l'étude de la souris mutante Sapcd2, j'ai participé à l'étude de la souris mutante Lgn à P0, menée par Dre Marine Lacomme, post-doctorante au laboratoire. En absence de Lgn à P0, les divisions horizontales augmentent, à l'inverse de l'absence de Sapcd2, où les divisions verticales augmentent. Pour savoir si ces changements d’orientation affectent le destin cellulaire, nous avons réalisé une analyse clonale des divisions terminales dans des explants rétiniens. Cette approche permet de suivre l'effet d'une délétion clonale d'un gène dans le lignage d’un seul progéniteur. Comme attendu, l'ablation clonale de Sapcd2 augmente les divisions asymétriques terminales, produisant deux cellules postmitotiques différentes, et inversement sans Lgn. En termes de mécanisme, SAPCD2 compétitionne avec NuMA pour se lier à LGN, dont elle régule négativement la localisation au cortex de la cellule. Le phénotype rétinien des souris doubles mutantes Lgn;Sapcd2 est sévère, avec une quasi-exclusivité de divisions verticales, une augmentation de la prolifération globale, du nombre de cellules mitotiques non apicales, et une drastique expansion de la population neuronale avec une couche cellulaire supplémentaire, composée de presque tous les types cellulaires rétiniens. Cette hyperprolifération pourrait être due à l'augmentation des divisions verticales, engendrant une asymétrie de l'héritage du déterminant cellulaire NUMB, antagoniste de Notch. Nous faisons l'hypothèse que le progéniteur basal qui n'hérite pas de NUMB, a une capacité proliférative supérieure aux progéniteurs apicaux. Pour la première fois, nous suggérons que les progéniteurs rétiniens ne sont pas équipotents. Ces travaux ont permis d'identifier un nouveau régulateur de l’orientation du fuseau mitotique, et d'élucider la régulation de la localisation cortico-latérale de LGN dans les progéniteurs rétiniens des vertébrés. Ils suggèrent que SAPCD2 et LGN interagissent différemment et changent de rôle au cours de la rétinogenèse. Ces découvertes contribuent à mieux comprendre les mécanismes moléculaires et cellulaires qui contrôlent la taille du lignage neuronal et régulent la formation de la diversité cellulaire au cours du développement du système nerveux central des vertébrés. / The control of cell division orientation is an integral processing during asymmetric cell division, a critical process ensuring cell diversity by asymmetrically distributing cell fate determinants between daughter cells. Cell fate determinants in invertebrate model organisms such as the C. elegans nematode and Drosophila fruit fly have been well characterized, and genetic analyses in these organisms has identified the evolutionarily conserved ternary protein complex Gai-LGN-NuMA as essential molecules involved in mitotic spindle orientation, as well as the polarity protein complex PAR3-PAR6-aPKC. Precisely how the Gai-LGN-NuMA complex achieves proper sub-cellular localization in vertebrate neural progenitors to induce planar cell division remains unclear. We used the developing vertebrate retina as a model system to study the role of cell division orientation in cell fate decisions. We have previously demonstrated a link between cell division orientation and daughter cell outcome in neural retina. Specifically, vertical cell divisions have a tendency to give rise to asymmetric pairs of daughter cells, and appear in later stages of retinogenesis. We wished to elucidate the mechanism underlying the switch from planar to vertical cell divisions over time during the neurogenic vs. proliferative developmental phases of retinogenesis. With our collaborators from the University of Toronto in the lab of Dr Stéphane Angers, we identified a novel Gai, LGN and PAR3 interacting protein, named SAPCD2 (suppressor APC domain containing 2). SAPCD2 is a poorly characterized protein, but known to be expressed in a cell cycle-dependent manner with higher expression during mitosis and elevated expression in many human cancers. We first analyzed Sapcd2 and Lgn expression in the developing retina, and found strong expression during proliferative phases, with its subcellular localization dependent on mitotic phase and developmental stage. During mitoses at P0 (birth), SAPCD2 and LGN display complementary localization with an apical or cortico-lateral enrichment, respectively, suggestive of a role in planar cell division induction. However, at E14.5, SAPCD2 and LGN have a highly similar localization, independent of spindle orientation, suggesting different roles during retinal development. We then analyzed mitotic spindle orientation in Sapcd2 and Sapcd2/Lgn DKO mice at E14.5 and P0, and Lgn mutant mice studied in parallel by Dre Marine Lacomme, post-doc in the Cayouette lab. In the absence of Sapcd2, vertical divisions drastically increased, whereas in the absence of Lgn, horizontal cell divisions increased. To test if this reorientation affects cell fate outcome, we analyzed the lineage of individual progenitor cells. As expected, in absence of Sapcd2, we observed a drastic increase in terminal asymmetric cell divisions, leading to two different neurons; whereas in the absence of Lgn, we observed an increase in terminal symmetric cell divisions, leading to two photoreceptors. Mechanistically, we showed that SAPCD2 negatively regulates LGN cortical localization, by competing with NuMA for its binding. In Lgn;Sapcd2 DKO mice, the mitotic spindle reorientation phenotype is even more drastic, containing almost exclusively vertical cell divisions, combined with an increase of proliferation and non-apical mitoses. This leads to a drastic expansion of the neuronal population, which forms an extra-layer containing many different retinal cell types. This over-proliferation could be due to the increase of vertical cell divisions, leading to an asymmetrical distribution of cell fate determinant, NUMB, an antagonist of Notch, between daughter cells. We hypothesize that the retinal basal progenitor, without NUMB, has a higher proliferative potential than the apical progenitor. Contrary to previous studies, this suggests that retinal progenitors are not equipotent. This work identifies a new regulator of mitotic spindle orientation and clarifies the sub-cellular localization of the LGN-NuMA complex. Our results also suggest that SAPCD2 and LGN change their role and the way they interact throughout the course of retinogenesis. This research contributes to an understanding of both how neural number is regulated, and how cell diversity is generated during vertebrate central nervous system development.

The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW₃ proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.

Les tissus épithéliaux présentent deux caractéristiques majeures, ils sont robustes (rôle de barrière) mais également plastiques lors de la morphogénèse. L'homéostasie des tissus épithéliaux repose sur la régulation de la balance prolifération/mort cellulaire. Dans ma thèse, je décris tout d'abord, les mécanismes moléculaires permettant à la cellule épithéliale de se diviser tout en maintenant l'intégrité du tissu. J'ai ensuite altéré cette intégrité, en utilisant le système de génération de clônes mosaïques, afin de comprendre comment la cohésion du tissu est maintenue. Ce travail m'a alors permis de comprendre comment l'adhérence est modulée, puis restaurée, au cours de la division cellulaire. Ainsi, j'ai montré que l'intégrité des tissus est assurée par l'action concomitante des forces d'adhésion et des forces de tension. Enfin, mon travail apporte également des éléments clés pour l'étude de la perte d'adhérence des cellules tumorales responsable en partie, de la progression des tumeurs solides en métastases. / Tissue homeostasis relies on the tight regulation of cell proliferation and cell death. Epithelial tissues are robust tissues that support the structure of developing embryos and adult organs and are effective barriers that physically protect the organism against pathogens. In my thesis, I have first described the molecular mechanisms responsible for maintaining tissue integrity during epithelial cell division. I have then abrogated this integrity by inducing mosaic clones within tissues to understand how tissue cohesion is maintained. This work shows how the continuity of adhesive properties is ensured during cell division. It also reveals new key elements that result in loss of adhesion in tissues and thus may be responsible for the progession from solid cancer to metastasis.

Dissialogangliosídios, como GD3 e derivados são marcadores da progressão de melanomas. Para avaliar as possíveis funções desta molécula, transfectamos células de melanócitos com o gene da enzima ST8Sia I, que converte GM3 em GD3. Mostramos que GD3 não interfere na capacidade proliferativa dessas células, porém a expressão de GD3 mostrou-se associada à sobrevivência celular. Melanomas adquirem autonomia quanto às vias dependentes do fator de crescimento de fibroblastos (FGF-1 e -2). A expressão de GD3 não interfere na resposta proliferativa a estes fatores, porém GD3 e outros glicoesfingolipídios de membrana modulam a resposta migratória induzida por FGF-2. A expressão de GD3 sensibiliza as células à morte celular induzida por diferentes quimioterápicos, como cisplatina e vimblastina; porém, torna as células resistentes ao tratamento com temozolamida. A sensibilização ao tratamento com vimblastina, mas não às outras drogas, depende da presença de GD3, como observado por ensaios de depleção metabólica / Disialoganglioside GD3 and its derivatives are melanoma progression markers. To evaluate the possible roles of these molecules along melanoma progression, we have transfected the GD3 synthase gene (ST8Sia I) in a melanocyte cell line. Accumulation of GD3 did not confer any proliferative advantage to melanocytes. However, GD3 expression was associated with cell survival. The autonomic growth of melanomas is in part related to a constitutive activation of fibroblast growth factor dependent pathways. GD3 expression did not alter the proliferative response to either FGF-1 or FGF-2. However, GD3 and other membrane glycospingolipids modulate the motogenic activity of FGF-2. GD3 expression sensitizes melanocytes to chemotherapeutic agent-induced cell death, as cisplatin and vimblastin. On the other hand, GD3 turned melanocytes more resistant to temozolomide. Chemosensitization to vimblastin, but not to the other drugs, was dependent on the presence of GD3 within the cells, as shown by metabolic depletion of glycosphingolipids

Em procariotos, a principal forma de reprodução é a divisão binária, que permite à célula-mãe dar origem a duas outras células-filhas, com conteúdo genético idêntico ao da progenitora. Em Bacillus subtilis este processo acontece graças ao divisomo, um complexo formado por aproximadamente dezesseis proteínas, que leva à constrição da membrana e da parede, formando o septo de divisão. A montagem do divisomo é coordenada por FtsZ, um homólogo de tubulina, que polimeriza na região central da bactéria e serve de arcabouço para a montagem do divisomo. Partindo de um levantamento detalhado da distribuição dos genes envolvidos em divisão em genomas completos de procariotos detectamos que divIVA, um gene de divisão já bem caracterizado, apresentava um gene parálogo em B. subtilis, conhecido como ypsB. Para determinarmos se YpsB seria um novo componente do divisomo foi realizada uma caracterização citológica e funcional desta proteína. Utilizamos microscopia de fluorescência e fusões de YpsB a GFP para determinar a localização subcelular de YpsB. Estes experimentos revelaram que YpsB está presente no divisomo, apresentando um padrão de localização semelhante mas não idêntico ao de DivIVA. Medindo-se a taxa de co-localização entre o anel Z e YpsB ficou demonstrado que estas proteínas co-localizam em aproximadamente 50%, sugerindo que YpsB é recrutada depois que o anel Z é montado. Para determinar quando YpsB chega ao divisomo, usamos mutantes termo-sensíveis das proteínas de divisão que revelaram a dependência de YpsB pelo sub complexo DivIB-DivIC-FtsL-FtsW-PBP2B. Já na ausência de DivIVA, YpsB continua associado ao divisomo, indicando que não depende do seu parálogo para localizar. Além disso, análises de deleções de YpsB mostraram que a porção N-terminal da proteína é a mais importante para o seu recrutamento ao divisomo. Para determinarmos o papel de YpsB durante a divisão foi construído um mutante com deleção completa do gene. DivIVA é uma proteína responsável por localizar o sistema Min nos pólos da bactéria e assim contribui para a precisão espacial da divisão. Apesar de serem parálogos, a função de YpsB, no entanto, parece ser diferente da de DivIVA. Análise do mutante ypsB- mostrou que na sua ausência, o divisomo é montado e o seu posicionamento tanto em fase vegetativa como em esporulação não são afetados. Como a ausência de YpsB não afeta perceptivelmente a divisão, combinamos a mutação em ypsB com mutações em outros genes envolvidos em divisão. A análise destes duplos mutantes revelou que a ausência simultânea de YpsB e FtsA produz exacerbada lise celular e letalidade. Com base neste fenótipo e em evidências evolutivas, sugerimos que YpsB esteja envolvida na regulação da síntese de peptideoglicano do septo. Mais especificamente, YpsB seria responsável por modular a atividade de PBP1, uma enzima necessária para a síntese de peptideoglicano septal. / In prokaryotes, the main form of reproduction is binary fission, which allows the mother-cell to give origin the two daughter-cells, with identical genetic material. In Bacillus subtilis, this process is performed by the divisome, a complex formed for approximately sixteen proteins that leads to the constriction of the membrane and the wall, creating the division septum. The assembly of the divisome is coordinated by FtsZ, a homolog of tubulin, that polymerizes in the central region of the bacteria and serves as the base for the assembly of the divisome. From a detailed survey of the distribution of the genes involved in division in complete genomes of prokaryotes, we detected that divIVA, a well characterized division gene, showed a paralog in B. subtilis, known as YpsB. To determine if YpsB would be a new component of the divisome, a cytological and functional characterization of this protein was carried out. We used fluorescence microscopy and fusion of YpsB to GFP to determine the subcellular localization of YpsB. These experiments displayed that YpsB is present in the divisome, with similar but not identical localization as DivIVA. Measuring co-localization between the Z ring and YpsB demonstrated that this happened in approximately 50% of the cells, suggesting that YpsB go to the divisome after the Z ring is formed. To determine when YpsB goes to the divisome, we used temperature-sensitive mutants of the division proteins. This showed that YpsB depends on the DivIB-DivIC-FtsL-FtsW-PBP2B sub-complex to associate with the divisome. In the absence of DivIVA, YpsB is still present in the divisome, indicating that it does not depend on its paralog to localize. Moreover, deletion analyses of YpsB showed that the N-terminal portion of the protein is the most important for its recruitment to the divisome. To determine the role of YpsB during division, we constructed a ypsB- mutant. DivIVA is the protein responsible for localization of the Min system in polar regions of B. subtilis and, thus, contributes for the spatial precision of division. Our results showed that the function of YpsB must be different from that of DivIVA, since analysis of the ypsB- mutant showed that in the absence this protein the divisome is assembled and septum position in vegetatively growing or sporulating cells is not affected. Since the absence of YpsB does not affect division, we combined the ypsB- mutant with mutants involved in division. Analysis of these double mutants showed that the simultaneous absence of YpsB and FtsA caused cellular lysis and lethality. Based on this phenotype and evolutionary evidences, we suggest that YpsB is involved in the regulation of peptidoglycan synthesis in the septum. More specifically, YpsB would be responsible for modulating the activity of PBP1, a necessary enzyme for septum peptidoglycan synthesis.