Wound healing and carcinogenesis are defined as complex, adaptive processes which are controlled by intricate communications between the host and the tissue microenvironment. A number of phenotypic similarities are shared by wounds and cancers in cellular signaling and gene expression. Radiotherapy is the second most effective modality of cancer treatment after surgery and can be used, either alone or in combination with chemotherapy. Recent findings suggest that radiotherapy apart from tumor cell death also rapidly and persistently modifies the tissue microenvironment. These modifications affect cell phenotype, tissue metabolism, bidirectional exchanges and signaling events between cells. The complex interactions between stromal cells and cancer cells are of immense interest and in The First Part of My Thesis, I tried to explore the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts, irrespective of their RhoB status, do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms respectively, TGF-β1 and MMP-mediated. We also found that co-irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response. Secondly, our in vivo experiments, tends to confirm the in vitro data showing that irradiated tumor bed does not stimulate tumor growth and escape. Our results also challenges the view that irradiated stroma would promote migration of carcinoma cells as we show that independently from their genotype co-irradiation of fibroblasts and carcinoma cells repressed carcinoma cell migration and confirmations studies are currently performed in vivo. The Third Part of My Project, was dedicated to investigate the effect on CTC release after radiotherapy. Consistently with the results reported after surgery , the number of CTC increases in the blood stream after radiotherapy probably due to radiation-induced vascular injury induced or/and by EMT induction in tumor cells but these cells seemed to be entrapped into the cardiac cavity. The significance of these CTC to metastatic development is still under investigation but there is evidence for a metastasis-promoting effect of RT from animal studies.Thus the microenvironment can exert antagonist stimulatory or inhibitory effects on malignant cells.

Ο καρκίνος του μαστού αποτελεί τον πιο συχνό τύπο καρκίνου των γυναικών. Παρότι η σχετική έρευνα είναι αρκετά εκτεταμένη, οι υποκείμενοι μηχανισμοί δεν έχουν πλήρως αποσαφηνιστεί. Πρόσφατες μελέτες έχουν αναδείξει τη σημαντικότητα της διαντίδρασης των καρκινικών κυττάρων και του καρκινικού μικροπεριβάλλοντος ως μια κομβική συνιστώσα στη παθοφυσιολογίας της νόσου. Σκοπός της παρούσας μελέτης είναι η διερεύνηση της ανοσοϊστοχημικής έκφραση του υποδοχέα CXCR4, της χημοκίνης SDF-1, της μεταλλοπρωτεϊνάσης MMP-9 και του παράγοντα HIF-1α σε διηθητικά καρκινώματα του μαστού και στον παρακείμενο μη καρκινικό ιστό (τόσο στο επιθηλιακό όσο και στο στρωματικό στοιχείο), καθώς και οι συσχετίσεις των ποικίλων ανοσοεντοπίσεων με τις κλινικοπαθολογοανατομικές παραμέτρους και την επιβίωση. η επιλογή των μορίων έγινε βάσει της σημαντικότητάς τους σε ποικίλα στάδια της παθογένειας της νόσου (υποξία, νεοαγγείωση, ανάπτυξη κτλ). Η έκφραση όλων των υπό εξέταση μορίων ήταν στατιστικά σημαντικότερη στον καρκινικό ιστό σε σχέση με τον παρακείμενο μη νεοπλασματικό. Από τα αποτελέσματα προκύπτει επίσης συσχέτιση μεταξύ ανοσοϊστοχημικών εντοπίσεων της MMP-9 και των υπολοίπων υπό διερεύνηση μορίων. Προέκυψαν επίσης ποικίλες συσχετίσεις μεταξύ συγκεκριμένων προτύπων έκφρασης (pattern) και προγνωστικών παραγόντων. Η έκφραση της MMP-9 στο κυτταρόπλασμα των καρκινικών κυττάρων σχετίστηκε θετικά με τη λεμφαδενική προσβολή, αλλά αρνητικά με το μέγεθος του όγκου. Επίσης, η έκφραση του CXCR4 και της SDF-1 στα καρκινικά κύτταρα σχετίστηκε με την παρουσία οστικών μεταστάσεων και με τον ιστολογικό βαθμό κακοήθειας, αντίστοιχα. Επιπλέον, η ανοσοεντόπιση της SDF-1 στους ινοβλάστες του καρκινικού στρώματος συσχετίστηκε θετικά με την έκφραση του Ki67 και με το στάδιο κατά ΤΝΜ, ενώ η ανοσοεντόπιση της SDF-1 στα ενδοθηλιακά κύτταρα του καρκινικού στρώματος με την έκφραση του Her2. Η ανοσοϊστοχημική έκφραση του HIF-1α συσχετίστηκε αρνητικά με την έκφραση των στεροειδικών υποδοχέων ER και PR. Επιπλέον, η έκφραση της MMP-9 στους ινοβλάστες του καρκινικού στρώματος και η έκφραση της SDF-1 στα επιθηλιακά κύτταρα και στους ινοβλάστες του παρακείμενου μη καρκινικού ιστού συσχετίστηκαν με δυσμενέστερη επιβίωση. Το εύρημα αυτό τονίζει τη σημαντικότητα τόσο του στρώματος όσο και του ξενιστή στην παθογένεια του καρκίνου. Τα αποτελέσματα αυτά αναδεικνύουν τη σημαντικότητα των υπό μελέτη μορίων στην καρκινογένεση και στην εξέλιξη της νόσου. Για την επαλήθευση των αποτελεσμάτων αυτών απαιτείται η διενέργεια μελετών μεγαλύτερης κλίμακας ενώ προσεγγίσεις με λειτουργικές μεθοδολογίες θα μπορούσαν να αναδείξουν πιθανώς κοινά ή διαπλεκόμενα υποκείμενα μηνυματοδοτικά μονοπάτια στα οποία εμπλέκονται τα ως άνω μόρια. / Breast cancer is the most frequently diagnosed cancer in women. Despite the ongoing research in breast cancer tumorigenesis the underlying mechanisms are not yet well elucidated. In recent years, the interaction between tumour cells and tumour microenvironment has gained appreciation as an active participant in cancer pathophysiology. In the present study we attempt to investigate the immunohistochemical staining of CXCR4, SDF-1, MMP-9 and HIF-1a in invasive breast cancer and adjacent normal breast tissue (including epithelial and stromal components) and to determine the relationship between different expression patterns and various tumor clinicopathological parameters and survival. The understudy molecules where chosen due to their crucial role in different steps of breast cancer progression (tumor growth, hypoxia, neovascularisation, invasiveness etc). All molecules showed statistically significant higher expression in cancer tissue compared to expression in the adjacent noncancerous tissue. Our results reveal a correlation between expression patterns of MMP9 and the other understudy molecules (SDF1, CXCR4 and HIF-1a). Furthermore, MMP9 expression in fibroblasts of cancer stroma and SDF1 expression in normal epithelial cells and fibroblasts of adjacent normal stroma were associated with poorer survival, underscoring the importance of tumor microenvironment and host derived molecules in tumor progression. There were also various correlations between specific expression patterns and prognostic factors: MMP9 expression in cancer cells was positively correlated with lymph node involvement, but negatively with tumor size,¬ while CXCR4 and SDF-1 expression in cancer cells was positively correlated with bone metastases and tumor grade, respectively. Furthermore, SDF-1 immunoexpression of cancer stromal fibroblasts was positively correlated with Ki67 expression and TNM stage, whereas SDF1 immunoexpression in endothelial cells of cancer stroma was positively correlated with Her2 expression. HIF-1a expression in cancer cells was negatively correlated with expression of steroid receptors. The abovementioned results underline the importance of the understudy molecules in carcinogenesis and tumor progression. Larger scale studies are necessary to confirm our results, while functional approaches could possibly reveal common or interwoven molecular pathways for the understudy molecules.

Made available in DSpace on 2016-06-02T19:22:05Z (GMT). No. of bitstreams: 1 3128.pdf: 3115695 bytes, checksum: a3cd2a9e5289c0b15425f8203b8ca0c1 (MD5) Previous issue date: 2010-07-21 / INTRODUÇÃO: osteoporose é reconhecida mundialmente como sério problema de saúde pública, apresentando como característica principal a fragilidade óssea. A qualidade do osso é fator determinante nesse aspecto, e depende do colágeno. Um dos fatores intrínsecos que regula o colágeno é a atividade da metaloproteinase -2 (MMP-2). O treinamento de força é o melhor recomendado para preservar e/ou melhorar a qualidade da massa óssea. OBJETIVO: foi investigar a influência do treinamento de força na atividade da MMP-2 e nas propriedades mecânicas do osso de ratas ovariectomizadas (OVX) e intactas. MATERIAIS E MÉTODOS: 48 ratas maduras jovens foram distribuídas em 2 grupos distintos, ovariectomia (OVX) e Intactas (Int); a seguir, 3 subgrupos foram formados similarmente em cada grupo: sedentário (OVX-Sed e Int-Sed), exercício agudo (OVX-Ex-Ag e Int-Ex-Ag) e exercício crônico (OVX-Ex-Cr e Int-Ex-Cr) (n = 8 por grupo). Foi utilizado um treinamento de força de 12 semanas no qual os animais escalaram uma escada vertical de 1,1-m com pesos presos as suas caudas. As sessões foram realizadas com intervalo de três dias, 4-9 escaladas e 8-12 movimentos dinâmicos por escalada. Após o término do período experimental, foram realizadas análises da atividade da MMP-2 por zimografia e análises biomecânicas e biofísicas utilizando-se uma máquina de ensaio universal (Instron modelo 4444). RESULTADOS: a atividade da MMP-2 apresentou-se reduzida em 2 grupos OVX (OVXSed e OVX-Ex-Ag) comparada com todos os outros grupos (p ≤ 0,05). Em contrapartida, os grupos treinados cronicamente (OVX-Ex-Cr e Int-Ex-Cr) apresentaram aumento significativo da MMP-2. Esses resultados também foram observados nas análises biomecânicas e biofísicas, nas quais os grupos OVX-Sed e OVX-Ex-Ag apresentaram Densidade Mineral, Densidade Óssea, Carga Máxima e Carga de Fratura menores em relação à todos os outros grupos; na outra mão, os grupos treinados cronicamente, apresentaram índices maiores nas análises biomecânicas e biofísicas acima citadas. CONCLUSÕES: a ovariectomia reduziu a atividade da MMP-2, produzindo efeitos deletérios sobre a massa óssea. O treinamento de força proposto foi eficiente em combater esses efeitos, apresentando inclusive efeito modelador.

Abstract Cardiovascular diseases (CVD), and especially atherosclerotic vascular diseases (ASVD), are the largest cause of morbidity and premature death worldwide. Coronary heart disease (CHD) and cerebrovascular disease (stroke) are common and severe manifestations of ASVD. Atherosclerosis is a chronic inflammatory disease and lipoprotein metabolism disorder. If the regulation of inflammatory process is disturbed, the systemic release of pro-inflammatory mediators, including matrix metalloproteinases (MMPs), may lead to a low-grade systemic inflammation, which is a risk factor for CVDs. MMPs are enzymes that are responsible for the degradation of the extracellular matrix (ECM) during growth and tissue renewal but also in many pathological conditions. These ECM degrading proteases and their regulators play an important role in atherogenesis and subsequent plaque rupture, leading to acute cardiovascular manifestations. The pivotal role of MMPs in atherosclerosis has raised interest in the development of drug therapies targeting these proteases. Doxycycline has inhibitory effects on some MMPs in addition to its antimicrobial properties. The main objective of this thesis project was to investigate the potential of these inflammatory mediators as biomarkers, risk factors, and therapeutic targets in CVD. The special focus was on MMP-8 and its main regulator, tissue inhibitor of matrix metalloproteinase (TIMP)-1. The results of this study show that a high serum MMP-8 concentration indicates an acute cardiac condition and predicts a future CVD event. In addition to MMP-8, MMP-7 is a potential biomarker for incident CVD. The balance between these MMPs and their tissue inhibitor may indicate vulnerability to plaque rupture. Measurement of serum MMP-8 concentration is reliable, anti-invasive and inexpensive and can be done in hospital settings. We also show that regular-dose doxycycline decreases the systemic inflammatory burden in patients with earlier myocardial infarction and is a promising anti-inflammatory therapy in the prevention of CVDs with relatively minor side effects. In conclusion, MMP-8 and TIMP-1 can be considered inflammatory risk markers of CVD events and death, and they can be utilized both for diagnostic and screening purposes. The inhibition of MMP-8 by doxycycline may reduce the systemic inflammatory burden in patients with myocardial infarction. / Tiivistelmä Sydän- ja verisuonisairaudet, erityisesti ateroskleroottiset valtimosairaudet, ovat maailman yleisin sairastuvuuden ja ennenaikaisen kuoleman syy. Sepelvaltimotauti ja aivohaveri ovat ateroskleroottisen valtimosairauden yleisiä ja vakavia ilmenemismuotoja. Ateroskleroosi on krooninen tulehduksellinen sairaus ja lipoproteiiniaineenvaihdunnan häiriö. Jos tulehdustapahtuma häiriintyy, elimistöön vapautuvat tulehdusvälittäjäaineet, kuten matriksin metalloproteinaasit (MMP), voivat aiheuttaa elimistön matala-asteisen tulehduksen, joka on sydän- ja verisuonisairauksien riskitekijä. MMP:t ovat entsyymejä, jotka pilkkovat solunväliainetta kasvun ja kudosten uusiutumisen mutta myös monien tautitilojen yhteydessä. Nämä soluväliainetta hajottavat proteaasit ja niiden säätelijät ovat tärkeässä roolissa ateroskleroottisen plakin muodostumisessa ja repeämisessä, joka johtaa äkillisiin sydäntautitapahtumiin. Matriksin metalloproteinaasien keskeinen rooli ateroskleroosissa on herättänyt kiinnostusta niihin kohdistuvan lääkehoidon kehittämiseen. Doksisykliinillä on joidenkin MMP-entsyymien toimintaa estävä vaikutus antimikrobiaalisten ominaisuuksiensa lisäksi. Tämän väitöskirjatutkimuksen päätavoitteena oli tutkia näiden tulehdusvälittäjäaineiden mahdollisuuksia biomarkkereina, riskitekijöinä ja lääkehoidon kohteena sydän- ja verisuonisairauksissa. Erityinen kiinnostuksen kohde oli MMP-8 ja sen pääsäätelijä ja kudosestäjä, tissue inhibitor of matrix metalloproteinase (TIMP)-1. Tämän tutkimuksen tulokset osoittavat, että seerumin korkea MMP 8 pitoisuus viittaa akuuttiin sydäntautiin ja ennakoi tulevaa sydäntautitapahtumaa. MMP-8:n lisäksi MMP-7 on lupaava sydäntapahtuman biomarkkeri. Näiden matriksin metalloproteinaasien ja niiden kudossäätelijä TIMP-1:n välinen tasapaino voi liittyä ateroskleroottisen plakin haurauteen. Seerumin MMP-8:n mittaus on luotettavaa, kajoamatonta ja edullista, ja mahdollista toteuttaa myös sairaalaolosuhteissa. Näytämme myös, että doksisykliini vähentää elimistön tulehdustaakkaa sydäninfarktin sairastaneilla potilailla ja että se on sydäntautien ehkäisyssä lupaava anti-inflammatorinen lääke, jolla on suhteellisen vähän sivuvaikutuksia. Johtopäätöksenä on, että MMP-8:aa ja TIMP-1:tä voidaan pitää lupaavina sydän- ja verisuonitautien sekä kuoleman biomarkkereina sekä diagnostiikka- että seulontakäytössä. Lisäksi tutkimustulokset osoittavat, että MMP-8:n esto doksisykliinillä voi vähentää elimistön tulehdustaakkaa sydänkohtauksen sairastaneilla potilailla.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

No description available.

Collective cell migration is a defining feature of many morphogenetic processes. Diseases such as congenital heart diseases and cancer arise due to mis-regulation of collective migratory behaviour and animal models have played a pivotal role in dissecting the molecular mechanisms which underlie this process. During embryonic heart development, cardiac precursors undergo a stage of collective migration in both vertebrates and invertebrates. We developed a paradigm to quantitatively assess collective cell migration of cardiac precursors in live embryos of Drosophila, which is the simplest genetic model organism with a heart. Therefore, we studied processes which are commonly observed in most collective cell migration models such as guidance signalling and extracellular matrix remodelling. Our results demonstrate that leading edge of migrating cardioblasts is highly active and that this behaviour is regulated by guidance cues, Slit and Netrin and their respective receptors Robo/Robo2 and Frazzled/Uncoordinated5. These molecules cooperatively promote leading edge motility and epithelial characteristics of the cardioblasts. Next, we determined that matrix restructuring around the cardioblasts requires proteases Mmp1 and Mmp2, which are members of the highly conserved Matrix Metalloproteinase family. We demonstrate that Mmp1 and Mmp2 have distinct roles during lumen formation, however, both Mmp1 and Mmp2 are required for collective motility of the cardioblast leading edge. Hence, we propose that embryonic heart development in Drosophila is an effective and amenable model of collective cell migration which can be applied to discover unique mechanisms which coordinate cell movement in groups. / Thesis / Doctor of Philosophy (PhD)

No description available.

Syfte: Är att ta reda på om man med hjälp av immunhistokemi (IHC) med infärgning avantikroppar mot laminin-5 och C3d kan särskilja mellan slemhinnepemfigoid (MMP) ochorala lichenoida reaktioner (OLR) med epitelsläpp. Vidare undersöks graden inflammation för MMP och OLR för att fastställa om det går att se ett samband mellan grad av inflammation och antikroppsinfärgning.Material och metod: En pilotstudie utfördes på 10 prover med diagnosen MMP respektive 9 prover med OLR, som hämtades från Malmö universitets biobank. Proverna genomgickrutinfärgning respektive antikroppsinfärgning mot laminin-5 och C3d. Granskning av prover skedde i digitalmikroskop. Efteråt delades proverna in i grupper efter var infärgningen sågs. Sammanställning gjordes i Excel med stapeldiagram.Resultat: Ingen tendens till särskiljning ses mellan MMP och OLR avseende infärgning mot laminin-5 och C3d. Positiva utslag för infärgning mot Laminin-5 ses enhetligt på enbart en sida om epitelsläppet hos både snitten för MMP och OLR. Vidare kan det inte ses att snitt med diagnosen OLR har slumpartad infärgning mot laminin-5 på båda sidor om släppet. Det finns en tendens till att MMP-snitt får mer positiva utslag för infärgning mot C3d än för OLR snitt. Graden inflammation var högre i OLR snitt än för MMP snitt.Slutsats: Enligt studien går det inte att, med IHC, med infärgning av antikroppar mot laminin5 och C3d, kunna särskilja MMP och OLR med epitelsläpp. Större urval krävs för definitiva slutsatser. Vidare studier behövs för att utreda om det går att använda IHC för att särskilja MMP och OLR.Nyckelord: C3d, immunhistokemi (IHC), laminin-5, orala lichenoida reaktioner (OLR),slemhinnepemfigoid (MMP) / Aim: To investigate if it is possible to differentiate between the diagnoses mucous membrane pemphigoid (MMP) and oral lichenoid reactions (OLR) with epithelial detachment using immunohistochemistry (IHC) with antibodies against laminin-5 and C3d. Furthermore, the extent of inflammation was examined for MMP and OLR to determine if a correlation between the inflammation and immunostaining is evident.Material and method: A pilot study is conducted using 10 samples diagnosed with MMP and 9 samples diagnosed with OLR, collected from Malmö University’s biobank. H&E staining and immunostaining against laminin-5 and C3d is performed on the samples. Analysis is conducted using a microscope. Samples are then divided into groups depending on the staining. Excel is used to compile the results.Result: No differentiating tendencies are observed between MMP and OLR regardingimmunostaining against laminin-5 and C3d. Immunostaining against laminin-5 is positive for MMP and OLR and is seen continuously on only one side of the epithelial detachment.Furthermore, staining with laminin-5 is not seen as random staining on both sides of theepithelial detachment. Samples with MMP have a higher tendency to stain against C3dcompared to OLR samples. Inflammation is higher in OLR samples than those for MMP.Conclusion: According to this study it is not possible to differentiate between the diagnoses MMP and OLR with epithelial detachment using immunostaining against laminin-5 and C3d. A larger sample size is needed for a definitive conclusion. Additionally, further studies are required to conclude if IHC can be used to differentiate between MMP and OLR.Keywords: C3d, immunohistochemistry (IHC), laminin-5, mucous membrane pemphigoid(MMP), oral lichenoid reactions (OLR)

L'arthrose (OA) est la maladie musculosquelettique la plus courante. Elle se caractérise par la détérioration progressive du cartilage articulaire, entraînant des douleurs, des raideurs et une réduction de l'amplitude des mouvements. Les mécanismes sous-jacents de l'OA impliquent un déséquilibre physiologique entre la dégradation et la réparation du cartilage, sous l'influence de processus inflammatoires et d'altérations de la structure de la matrice extracellulaire. Le nicotinamide adénine dinucléotide (NAD+) est un cofacteur essentiel impliqué dans de nombreux processus physiologiques, et sa diminution dans l'organisme est associée au vieillissement et à certaines pathologies. Les précurseurs connus du NAD+, tels que la niacine (NA), le nicotinamide (NAM) et le nicotinamide mononucléotide (NMN), suscitent un intérêt croissant dans le domaine de l’OA. La supplémentation en NAD+ a donc le potentiel d'améliorer la santé des articulations, notamment en inhibant l'inflammation et la dégradation. Dans un premier temps, nous avons sélectionné trois précurseurs du NAD+ sur la base de la littérature antérieure et de leur pertinence clinique. Nous avons ensuite étudié l'effet de ces trois précurseurs du NAD+ sur la régulation des protéines dans les processus cataboliques et inflammatoires. Le traitement de chondrocytes primaires avec des concentrations élevées de NA ou de NAM a entraîné une diminution de l'expression des métalloprotéinases matricielles (MMP). En outre, nous avons observé des tendances à la hausse non significatives de l'oxyde nitrique synthase induit (iNOS), après traitement à la NA, et de la cyclooxygenase-2 (COX-2), après traitement au NAM. Le NMN, quant à lui, a produit des résultats contradictoires en termes d'expression protéique, avec une augmentation de l'expression de la MMP et de la COX-2 à une concentration élevée. Suite à ces résultats préliminaires, nous avons voulu connaître les effets du NMN en particulier sur d'autres aspects de la pathogenèse de l’OA. Nous avons donc testé l'effet antioxydant du NMM et de la NA sur la production d'oxyde nitrique (NO) dans des explants de cartilage humain. À notre surprise, nous avons constaté que la NA avait une tendance à la hausse non significative de la production de NO, suivant un schéma similaire à la régulation de la iNOS. En revanche, le NMN avait un effet inhibiteur substantiel sur la synthèse de NO. Nous avons également effectué des tests de prolifération, révélant que le NMN, à des doses élevées, était capable d'augmenter la prolifération des synoviocytes stimulée par l'IL-1β (Interleukine-1beta) in vitro. Nous avons ensuite démontré l'effet du NMN sur la migration cellulaire et la régénération des plaies. Nos résultats ont mis en évidence la capacité du NMN à promouvoir la migration cellulaire à la fois dans les synoviocytes et les chondrocytes, mais surtout dans les chondrocytes dans des conditions normales et inflammatoires. Parallèlement, nous avons montré par des études histologiques que le NMN peut protéger le cartilage articulaire humain de l'érosion induite par l'IL-1β. Nos données fournissent des informations précieuses sur la voie du NAD+ et soulignent sa nouveauté dans les maladies musculosquelettiques telles que l'OA. Bien que nos résultats soulèvent de nombreuses questions, ils laissent entrevoir un effet potentiellement bénéfique de la supplémentation du NAD+ en tant qu'approche thérapeutique ou préventive potentielle de l'OA. / Osteoarthritis (OA) is the most common musculoskeletal disease. It is characterized by the progressive deterioration of articular cartilage, leading to pain, stiffness, and a reduced range of motion. The underlying mechanisms of osteoarthritis involve a physiological imbalance between cartilage degradation and repair, influenced by inflammatory processes and alterations in the structure of the extracellular matrix. Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor involved in many physiological processes, and its depletion in the body is associated with aging and certain pathologies. Known NAD+ precursors, such as Niacin (NA), Nicotinamide (NAM), and Nicotinamide Mononucleotide (NMN), are attracting growing interest in the field of osteoarthritis. Therefore, NAD+ supplementation can potentially improve joint health, notably by inhibiting inflammation and degradation. First, we selected three NAD+ precursors based on previous literature and their clinical relevance. We then investigated the effect of these three NAD+ precursors on protein regulation in catabolic and inflammatory processes. Treatment of primary chondrocytes with high concentrations of NA or NAM resulted in a decrease in Matrix metalloproteinase (MMP) expression. In addition, we observed non-significant upward trends in induced nitric oxide synthase (iNOS), after NA treatment, and cyclooxygenase-2 (COX-2), after NAM treatment. NMN, on the other hand, produced contradictory results in terms of protein expression, with increased MMP and COX-2 expression at a high concentration. Following these preliminary results, we were interested to learn about the effects of NMN in particular on other aspects of osteoarthritis pathogenesis. Thus, we tested the antioxidant effect of NAM and NA on nitric oxide production in human cartilage explants. To our surprise, we found that NA had a non-significant upward trend in NO production, following a similar pattern to iNOS regulation. On the other hand, NMN had a substantial inhibitory effect on NO synthesis. We also performed proliferation assays, revealing that NMN, at high doses, could increase IL-1β (Interleukin-1beta)-stimulated synoviocyte proliferation in vitro. We then demonstrated the effect of NMN on cell migration and wound regeneration. Our results highlighted NMN's ability to promote cell migration in both synoviocytes and chondrocyte cell types, but especially in chondrocytes under normal and inflammatory conditions. In parallel, we have shown through histological studies that NMN can protect human articular cartilage from IL-1β-induced erosion. Our data provide valuable insights into the NAD+ pathway and highlight its novelty in musculoskeletal diseases such as osteoarthritis. Although our results raise many new questions, they point to a possible beneficial effect of NAD+ enhancement as a potential therapeutic or preventive approach to OA.