Design structure matrix ja sen hyödyntäminen ohjelmistotuotannossa

Työn tarkoituksena on selvittää, miten DSM ja siihen liittyvät analyysit toimivat. Työssä selvitetään myös, miten DSM:a voidaan hyödyntää tuotekehityksessä ja ohjelmistotuotannon kohtaamissa ongelmissa. DSM-neliömatriisiin sijoitetaan tarkasteltavan kohteen osat riveihin ja kolumneihin identtisessä järjestyksessä. Matriisin soluihin merkitään osien väliset riippuvuudet, joilla selvitetään kunkin osan lähettämä ja vastaanottama data suhteessa muihin osiin. DSM-analyysissa osien järjestystä muutetaan suoritus- tai toteutusjärjestyksen mukaisesti parhaaseen järjestykseen. Osat ryhmitellään moduuleihin, jolloin esimerkiksi tuotekehitys ohjautuu automaattisesti modulaarisuuteen. Tuotekehitysprojekteihin DSM on kompaktin muodon, yksinkertaisuuden ja automaattisen järjestelyn ansiosta erinomainen työkalu, jolla voidaan mallintaa käytännössä mitä tahansa, mikä voidaan jakaa toisistaan riippuviin osiin. DSM voi vähentää projektien suunnitteluun vaadittua työmäärää ja avustaa realististen budjettien ja aikataulujen luontia suurissa projekteissa.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 µg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

Effect of photodynamic therapy on the extracellular matrix and associated components

In many countries, photodynamic therapy (PDT) has been recognized as a standard treatment for malignant conditions (for example, esophageal and lung cancers) and non-malignant ones such as age-related macular degeneration and actinic keratoses. The administration of a non-toxic photosensitizer, its selective retention in highly proliferating cells and the later activation of this molecule by light to form reactive oxygen species that cause cell death is the principle of PDT. Three important mechanisms are responsible for the PDT effectiveness: a) direct tumor cell kill; b) damage of the tumor vasculature; c) post-treatment immunological response associated with the leukocyte stimulation and release of many inflammatory mediators like cytokines, growth factors, components of the complement system, acute phase proteins, and other immunoregulators. Due to the potential applications of this therapy, many studies have been reported regarding the effect of the treatment on cell survival/death, cell proliferation, matrix assembly, proteases and inhibitors, among others. Studies have demonstrated that PDT alters the extracellular matrix profoundly. For example, PDT induces collagen matrix changes, including cross-linking. The extracellular matrix is vital for tissue organization in multicellular organisms. In cooperation with growth factors and cytokines, it provides cells with key signals in a variety of physiological and pathological processes, for example, adhesion/migration and cell proliferation/differentiation/death. Thus, the focus of the present paper is related to the effects of PDT observed on the extracellular matrix and on the molecules associated with it, such as, adhesion molecules, matrix metalloproteinases, growth factors, and immunological mediators.

Prevalence of vascular-endothelial growth factor, matrix metalloproteinases and tissue inhibitors of metalloproteinases in primary breast cancer

Our objective was to determine the presence of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and MMP-9 and specific tissue inhibitors of matrix metalloproteinase (TIMP-1 and TIMP-2) in tumor samples obtained from patients with primary breast cancer. We attempted to correlate these findings with the status of the sentinel lymph node (SLN) and clinical-pathological characteristics such as age, tumor size, histological type, histological grade, and vascular invasion. Tumor samples from 88 patients with primary breast cancer were analyzed. The immunoreactivity of VEGF, MMP-2, MMP-9, TIMP-1, and TIMP-2 in tumors was correlated with clinical and pathological features, as well as SLN status. Nonparametric, Mann-Whittney, Kruskal-Wallis, and Spearmann tests were used. Categorical variables were analyzed by the Pearson test. No statistically significant correlation was found between the amount of VEGF, MMP-2, MMP-9, TIMP-1, and TIMP-2 and the presence of tumor cells in the SLN. However, larger tumor diameter (P < 0.01) and the presence of vascular invasion (P < 0.01) were correlated positively with a positive SLN. A significant correlation of higher VEGF levels (P = 0.04) and lower TIMP-1 levels (P = 0.04) with ductal histology was also observed. Furthermore, lower TIMP-2 levels showed a statistically significant correlation with younger age (<50 years) and larger tumor diameter (2.0-5.0 cm). A positive SLN correlated significantly with a larger tumor diameter and the presence of vascular invasion. Higher VEGF and lower TIMP-1 levels were observed in patients with ductal tumors, while higher TIMP-1 levels were observed in lobular tumors.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53%), TIMP-1 (-31.7%), TGF-β1 (-57.7%), and MMP-2 (-41.6%), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1%), ALT (-17.6%), and AST (-12.2%) serum levels; c) increased liver weight (11.3%), and reduced liver collagen (-37.1%), regenerative nodules size (-22.1%), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Role of the extracellular matrix in variations of invasive pathways in lung cancers

Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas (SqCC), is the massive degradation of the extracellular matrix. The remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan synthases (HAS), E-cadherin adhesion molecules, and the transforming growth factor β (TGF-β) may favor invasion, cellular motility, and proliferation. We examined HAase proteins (Hyal), HAS, E-cadherin, and TGF-β profiles in lung AD subtypes and SqCC obtained from smokers and non-smokers. Fifty-six patients, median age 64 years, who underwent lobectomy for AD (N = 31) and SqCC (N = 25) were included in the study. HAS-1, -2 and -3, and Hyal-1 and -3 were significantly more expressed by tumor cells than normal and stroma cells (P < 0.01). When stratified according to histologic types, HAS-3 and Hyal-1 immunoreactivity was significantly increased in tumor cells of AD (P = 0.01) and stroma of SqCC (P = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS-3 immunoreactivity in tumor cells (P < 0.01). Stroma cells of SqCC from non-smokers presented a significant association with HAS-3 (P < 0.01). Hyal, HAS, E-cadherin, and TGF-β modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. HAases in resected AD and SqCC were strongly related to the prognosis. Therefore, our findings suggest that strategies aimed at preventing high HAS-3 and Hyal-1 synthesis, or local responses to low TGF-β and E-cadherin, may have a greater impact in lung cancer prognosis.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Computational models for and from biology : simple gene assembly and reaction systems

The advancement of science and technology makes it clear that no single perspective is any longer sufficient to describe the true nature of any phenomenon. That is why the interdisciplinary research is gaining more attention overtime. An excellent example of this type of research is natural computing which stands on the borderline between biology and computer science. The contribution of research done in natural computing is twofold: on one hand, it sheds light into how nature works and how it processes information and, on the other hand, it provides some guidelines on how to design bio-inspired technologies. The first direction in this thesis focuses on a nature-inspired process called gene assembly in ciliates. The second one studies reaction systems, as a modeling framework with its rationale built upon the biochemical interactions happening within a cell. The process of gene assembly in ciliates has attracted a lot of attention as a research topic in the past 15 years. Two main modelling frameworks have been initially proposed in the end of 1990s to capture ciliates’ gene assembly process, namely the intermolecular model and the intramolecular model. They were followed by other model proposals such as templatebased assembly and DNA rearrangement pathways recombination models. In this thesis we are interested in a variation of the intramolecular model called simple gene assembly model, which focuses on the simplest possible folds in the assembly process. We propose a new framework called directed overlap-inclusion (DOI) graphs to overcome the limitations that previously introduced models faced in capturing all the combinatorial details of the simple gene assembly process. We investigate a number of combinatorial properties of these graphs, including a necessary property in terms of forbidden induced subgraphs. We also introduce DOI graph-based rewriting rules that capture all the operations of the simple gene assembly model and prove that they are equivalent to the string-based formalization of the model. Reaction systems (RS) is another nature-inspired modeling framework that is studied in this thesis. Reaction systems’ rationale is based upon two main regulation mechanisms, facilitation and inhibition, which control the interactions between biochemical reactions. Reaction systems is a complementary modeling framework to traditional quantitative frameworks, focusing on explicit cause-effect relationships between reactions. The explicit formulation of facilitation and inhibition mechanisms behind reactions, as well as the focus on interactions between reactions (rather than dynamics of concentrations) makes their applicability potentially wide and useful beyond biological case studies. In this thesis, we construct a reaction system model corresponding to the heat shock response mechanism based on a novel concept of dominance graph that captures the competition on resources in the ODE model. We also introduce for RS various concepts inspired by biology, e.g., mass conservation, steady state, periodicity, etc., to do model checking of the reaction systems based models. We prove that the complexity of the decision problems related to these properties varies from P to NP- and coNP-complete to PSPACE-complete. We further focus on the mass conservation relation in an RS and introduce the conservation dependency graph to capture the relation between the species and also propose an algorithm to list the conserved sets of a given reaction system.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.