SHIP2 is a phosphatase that belongs to the family of the phosphoinositide 5-phosphatases. It is known to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2 imparting a tight control of the PI 3-kinase pathway. Over the last decade, SHIP2 has been described as a tumor promotor or tumor suppressor in several cancer types such as glioblastoma, colorectal cancer or breast cancer cells. Several studies have proposed a role of SHIP2 in breast cancer cells, but its tumor promoting function was unclear at the beginning of this thesis especially in terms of its mode of regulation. In 2013, the INPPL1 gene that encodes SHIP2 has been found to be mutated in opsismodysplasia (OPS), a rare autosomal recessive disease characterized by delayed bone maturation but no molecular mechanism was provided to explain the mechanism. In this thesis, we first contributed to establish a negative regulation of SHIP2 on cell migration in 1321 N1 glioblastoma (GBM) cells. Our studies revealed a dephosphorylation activity of SHIP2 on PI(4,5)P2 at the plasma membrane to control cell migration. This study was done in collaboration with Dr. Elong Edimo in the lab. We have also shown that the regulation of cell motility cannot be generalized to all the GBM cells. In LN229 and U-251 GBM cells we observed a positive regulation of cell migration by SHIP2. We next took advantage of a unique model comparing fibroblasts derived from non-affected and OPS patients (in collaboration with Dr. Valérie Cormier-Daire). We have shown that the fibroblasts from the OPS patients are SHIP2 deficient and migrate slower as compared to fibroblasts from non-affected individuals. Finally, the major part of the thesis was the study of breast cancer cells: in the model MDA-MB-231 cells, we established a positive regulation of SHIP2 on cell migration. We extended this regulation on cell migration to different breast cancer cell models using a SHIP2 inhibitor AS1949490. We confirmed that this inhibitor blocks the phosphatase activity of SHIP2 and showed its selectivity towards SHIP2 in cell migration assay. In MDA-MB-231 cells we deciphered a second messenger role of PI(3,4)P2 to control cell migration. Our data in this model rely on the use of SHIP2 depleted cells obtained by lentiviral infection and shRNA. We confirmed the positive role of SHIP2 on cell migration in the model of rat chondrosarcoma SHIP2CRISPR cells (in collaboration with Dr. Pavel Krejci).A major goal of this thesis was achieved thanks to in-vivo studies: using MDA-MB-231 cells injected in SCID mice, we found a tumor promoting role of SHIP2 by determining the tumor weight. We also observed less lung metastasis of SHIP2 depleted injected cells as compared to control cells suggesting SHIP2 to be important for invasiveness of triple negative breast cancers. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Abstract Type 1 neurofibromatosis (NF1) syndrome is caused by a mutation of the NF1 gene. NF1 protein (neurofibromin) contains a domain which is related to the GTPase activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The clinical symptoms of NF1 patients include e.g. the formation of benign neurofibroma tumors and hyperpigmented lesions of the skin. The NF1 protein has been referred to as a tumor suppressor since cells of malignant schwannomas of NF1 patients may display loss of heterozygosity of the NF1 gene. In the present study, the expression of the NF1 gene was investigated during tissue repair in human skin. Elevated NF1 protein levels were seen in a fibroblastic cell population of healing wounds. In vitro studies were designed to investigate NF1 expression in dermal fibroblasts under the influence of growth factors that are operative during wound healing. Platelet-derived growth factor (PDGF) isoforms AB and BB and transforming growth factor β1 (TGFβ1) elevated NF1 mRNA levels in cultured dermal fibroblasts. In further studies, histological examination on apparently healthy skin of NF1 patients revealed frequently small masses of neurofibromatous tissue at the vicinity of hair follicles. Thus, action of the NF1 gene appears to be an integral part of normal tissue repair. Enhanced NF1 tumor suppressor expression may serve to limit excessive fibrosis in wound healing. As Ras proteins play a role in the regulation of cell differentiation and formation of cell junctions, the functional expression of NF1 protein was elucidated using differentiating keratinocytes as an in vitro model system. The results demonstrate that an intense NF1 tumor suppressor signal on intermediate filaments was temporally limited to the period in which the formation of desmosomes takes place. In analogy to NF1 protein, a rapid elevation of NF1 mRNA level was detected following initiation of differentiation. Interestingly, NF1 mRNA hybridization signal polarized towards cell-cell contact zones. This finding recognizes a potential way for post-transcriptional modification of NF1 expression and targeting of translation to subplasmalemmal location. The results demonstrate that the function of NF1 protein is associated with the formation of cell junctions, and thus to cellular communication.

Problems with musculoskeletal system, such as of developmental disorders, fractures or damage of the bone by age, inflammatory or tumor diseases, are still increasing in orthopaedics. Sometimes the bone tissue is not capable to completely regenerate to exert its physiological function in the organism. For this reason, using the bone replacements is necessary and common nowadays. Despite of an intensive research and testing of a wide range of the potential biomaterials and their combinations, the usage of metal materials for construction of the bone implants, still remains to be the gold standard. Ti-6Al-4V alloy is one of the commercialy used metal materials, which is known for the high mechanical and chemical resistance and a good biocompatibility. For a good biological response of the patient's organism for the bone implant, is an ability of osteointegration into the surrounding bone tissue, the key. This ability can be influenced in the case of the metals, by their surface structure. As it is known from earlier studies, the surface topography of the material is very important for the adhesion and proliferation of the bone cells, which are able to discriminate, very sensitively, between various stages of the material surface roughness. For this reason we have focused on studying of an influence...

Doctor of Philosophy / Department of Entomology / Michael R. Kanost / James R. Nechols / Cellular encapsulation is the immune response in which insects protect themselves from multicellular parasites such as nematodes or parasitoids. During an encapsulation episode, certain insect hemocytes become attracted to a foreign invader and aggregate on its surface. In short order, the invading entity will become entrapped within a capsule comprised of thousands of hemocytes, thus rendering the parasite harmless to the insect host. Although the process of cellular encapsulation has been known for a great many years, very little knowledge yet exists regarding the biochemistry underlying capsule formation. It would seem likely that cell surface adhesion proteins mediate this immune response. In a series of in vivo encapsulation assays in the tobacco hornworm, Manduca sexta, a collection of anti-hemocyte monoclonal antibodies (mAbs) was screened for their ability to inhibit cellular encapsulation. Two of the mAbs that inhibited this immune response and incidentally specifically bind plasmatocytes, MS13 and MS34, were used to isolate a ≈ 90 kDa protein. Several short peptide sequences contained within this protein were acquired via Edman degradation. Degenerate primers based on two of these peptide sequences and total RNA from M. sexta hemocytes were used to perform RT-PCR and 5´ and 3´ RACE. This resulted in a full-length cDNA sequence of 2426 bp. A 2301 bp open reading frame within this cDNA sequence codes for a protein of 767 residues. This protein, denominated [Beta]Ms1, exhibits significant sequence homology to the [Beta]-subunits of integrins, which are a family of transmembrane, heterodimeric glycoproteins that possess adhesive properties. Analysis of recombinant segments of [Beta]Ms1 showed that the protein produced from the PCR product is the antigen to MS13 and MS34 and that these mAbs bind to the region of the integrin that contains the extracellular binding site. Northern blot analysis of various M. sexta tissues together with immunofluorescence labeling with MS13 and MS34 shows that [Beta]Ms1 is solely expressed in plasmatocytes. The totality of these experiments demonstrates that integrins are essential for the cellular immune response of encapsulation.

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Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

INTRODUÇÃO: A interação entre leucócitos e plaquetas é fundamental para o início e a progressão da reestenose e da aterosclerose. Recentemente foi evidenciado em estudos in vitro que a integrina Mac-1 dos leucócitos se liga à glicoproteína Ibalfa (GP Ibalfa) das plaquetas e que esta interação possui uma função central na firme adesão e transmigração de leucócitos em locais de deposição de plaquetas. Entretanto, não há estudos in vivo que avaliam a importância da interação entre a integrina Mac-1 dos leucócitos e a GP Ibalfa das plaquetas (alfaMbeta2-GP Ibalfa) em modelo experimental de lesão vascular. MÉTODO: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação da integrina Mac-1 dos leucócitos com a GP Ibalfa das plaquetas, visando, deste modo, inibir a adesão de leucócitos na superfície do vaso coberta por plaquetas, a proliferação celular e a hiperplasia neointimal. Este peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos à lesão vascular da artéria femoral com corda-guia. Um dia (controle: n= 6; anti-M2: n= 6), 5 dias (controle: n= 9; anti-M2: n= 9) ou 28 dias (controle: n= 9; anti-M2: n= 9) após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imunohistoquímica. RESULTADOS: O bloqueio da interação alfaMbeta2-GP Ibalfa promoveu redução estatisticamente significativa de 75% do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9 ± 5,0% do total de células na camada média; versus anti-M2: 2,0 ± 1,6%; p=0,021), bem como determinou diminuição estatisticamente significativa de 42% em 5 dias (controle: 42,3 ± 12,9% do total de células na neoíntima; versus anti-M2: 24,6 ± 10,8%; p=0,047) e de 58% em 28 dias do acúmulo de leucócitos na neoíntima em desenvolvimento (controle: 7,9 ± 3,0% versus anti-M2: 3,3 ± 1,3%; p=0,012). A proliferação celular na camada média do vaso em 5 dias pós-lesão vascular apresentou redução estatisticamente significativa de 64% com o bloqueio da interação alfaMbeta2-GP Ibalfa (controle: 5,0 ± 2,9% do total de células na camada média; versus anti-M2: 1,8 ± 0,5%; p=0,043), assim como houve diminuição significativa de 47% da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8 ± 1,7% do total de células na camada íntima; versus anti-M2: 2,0 ± 1,2%; p=0,047). O bloqueio da interação alfaMbeta2-GP Ibalfa também determinou redução estatisticamente significativa de 56% do espessamento intimal em 28 dias (controle: 10.395 ± 3.549um2; versus anti-M2: 4.561 ± 4.915um2; p=0,012). CONCLUSÕES: O recrutamento de leucócitos após a lesão vascular é dependente da interação alfaMbeta2-GP Ibalfa e a neutralização desta interação inibe a proliferação celular e a formação neointimal. / INTRODUCTION: The interaction between leukocytes and platelets is fundamental for the beginning and the progression of restenosis and atherosclerosis. Recent in vitro studies have shown that the leukocyte integrin Mac-1 binds to the platelet glycoprotein (GP) Ibalfa, and this interaction plays a central role in the leukocyte firm adhesion and transmigration at sites of platelet deposition. However, there is no in vivo study evaluating the importance of the integrin Mac-1 and GP Ibalfa (alfaMbeta2-GP Ibalfa) interaction in experimental models of vascular injury. METHODS: A peptide termed M2 or anti-M2 antibody was developed to block the leukocyte Mac-1 and platelet GP Ibalfa interaction, aiming to inhibit the adhesion of leukocytes to the platelet-coated surface of vessels as well as the cellular proliferation and the neointimal hyperplasia. The peptide was injected and compared with a control-antibody in C57B1/6J mice subjected to wire-induced femoral artery injury. One day (control: n= 6; anti-M2: n= 6), 5 days (control: n= 9; anti-M2: n= 9) or 28 days (control: n= 9; anti-M2: n= 9) after vascular injury, the femoral arteries were harvested for morphometry and immunohistochemistry. RESULTS: The alfaMbeta2-GP Ibalfa interaction blockade promoted a statistically significant 75% reduction in leukocytes in the medial layer on the first day after vascular injury (control: 7.9 ± 5.0% out of the total cells in the medial layer versus anti-M2: 2.0 ± 1.6%; p=0.021), as well as determined a statistically significant 42% decrease 5 days later (control: 42.3 ± 12.9% out of the total cells in the neointima versus anti-M2: 24.6 ± 10.8%; p=0.047), and a 58% decrease in leukocyte accumulation in the developing neointima 28 days later (control: 7.9 ± 3.0% versus anti-M2: 3.3 ± 1.3%; p=0.012). The cellular proliferation in the vessel medial layer 5 days after vascular injury presented a statistically significant 64% reduction by the alfaMbeta2-GP Ibalfa interaction blockade (control: 5.0 ± 2.9% out of the total cells in the medial layer versus anti-M2: 1.8 ± 0.5%; p=0.043), and there was also a significant 47% decrease in the vessel intimal layer cellular proliferation 28 days later (control: 3.8 ± 1.7% out of the total cells in the intimal layer versus anti-M2: 2.0 ± 1.2%; p=0.047). Furthermore, the alfaMbeta2-GP Ibalfa interaction blockade determined a statistically significant 56% reduction in the intimal thickening 28 days after vascular injury (control: 10,395 ± 3,549um2 versus anti-M2: 4,561 ± 4,915um2; p=0.012). CONCLUSIONS: The leukocyte recruitment after vascular injury depends on the alfaMbeta2-GP Ibalfa interaction, and its neutralization inhibits cellular proliferation and neointimal formation.

A relativa raridade de casos graves de malária no Brasil sugere que os isolados locais de Plasmodium falciparum tenham menor virulência que os parasitas africanos e asiáticos. Este trabalho investigou os padrões de aderência de sete isolados de P. falciparum, provenientes de uma área da Amazônia brasileira em que a malária grave é rara, a dois receptores do endotélio vascular, CD36 e ICAM-1. Também analisamos a resposta de anticorpos de indivíduos locais contra oitos antígenos de superfície, incluindo isolados de campo e a cepa 3D7. Mostramos que: (a) de modo geral, os isolados locais de P. falciparum expressam VSAs capazes de aderir tanto a ICAM-1 quanto a CD36; (b) detectamos anticorpos contra antígenos apresentados por isolados de campo e pela cepa 3D7 entre moradores de uma área endêmica próxima à origem dos isolados; (c) vimos que alguns dos soros testados foram capazes de bloquear a adesão de hemácias parasitadas a ICAM-1 e CD36, in vitro; (d) detectamos uma baixa prevalência do alelo S (hemoglobina S) na população de estudo. / The relative rarity of severe malaria in Brazil suggests that the local Plasmodium falciparum isolates are less virulent than African and Asian parasites. This work investigated adherence patterns to CD36 and ICAM-1, two receptors of the vascular endothelium, in P. falciparum isolates from an area of the Brazilian Amazonia, where severe malaria is rare. We also analyzed, in the same area, the antibody responses of people against eight VSAs. We found that: (a) local P. falciparum isolates express VSAs capable to adhere to both receptors, CD36 and ICAM-1; (b) we detected antibodies against VSAs in a human population exposed to malaria, expressed from local parasites and the 3D7 control; (c) we found in vitro that some sera contained naturally acquired antibodies which blocked the adherence of the parasitized RBCs to ICAM-1 and CD36; (d) we detected a low frequency of the S allele (hemoglobin S) in the study population.

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