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Spreadsheet applications are widely adopted by millions of end users from several application domains and provide strategic support to many business, scientific, industrial, and organizational processes. These applications are usually developed by rapid application development processes, exploiting host scripting languages allowing the basic spreadsheets to provide complex functionality, business rules, and user interfaces. Several factors complicate the comprehension of these applications because they are usually developed and maintained by end users without specific software engineering skills, grow over time, are not adequately documented, and do not present explicit separation between data, business logic, and user interface layers. This paper presents a reverse engineering tool intended to support the comprehension of Excel spreadsheet applications developed using the Visual Basic for Application programming language. The tool has been implemented as an add-in that extends the Excel working environment by providing analysis and visualization features. It is able to extract information about the elements composing the analyzed Excel spreadsheet application, the functionality it exposes through its user interface, and the dependencies among its cells. This information is provided by means of interactive views. The validity of the tool has been assessed by a qualitative case study performed with professional end users from an automotive industrial domain.

Familial combined hyperlipidemia (FCHL) is the most frequent dislipidemic syndrome in our population, and it is strictly associated to the insurgence of coronary artery diseases in affected patients. In a previous project we have carried out a “genome wide” gene expression profiles analysis in FCHL patients as compared to normal control individuals, showing that the expression levels of approx. 300 genes are significantly altered (increased or reduced) in association to the disease. Statins are the elective drugs for treatment of this syndrome. The gene expression profiles of FCHL patients before and after statin treatment reveal that administration the drug for a minimum of 4 weeks "restores” the expression levels of most of the altered genes to "normal" values, in some cases causing hypo-expressed genes to became hyper-expressed. These results reveal the existence of regulatory interconnections among subsets of genes affected by FCHL syndrome. In order to identify putative common cis-acting sequence elements involved in the transcriptional regulation by statins, we searched the 5' upstream region of these genes for enriched short sequence motifs. We have analysed the -400/-200, -200/+1 and +1/+200 (relative to the transcriptional start site) human genomic sequences corresponding to both up- and down-regulated genes, obtaining six dataset. Each dataset has been searched with the MEME algorithm, setting the program to display up to 10 motifs, and using three different width windows (5-10, 6-15 or 10-20 nucleotides). We have identified some specifically enriched consensus sequences. One of these motifs is present in either the -400/-200 or the -200/+1 region of the majority (70%) of the up-regulated genes. We have synthesised double strand DNA fragments containing this consensus sequence in order to carry out EMSA (electrophoretic mobility shift assays) experiments with nuclear extracts from statin-treated cell cultures. These experiments are in presently in progress.

We have constructed SV40 derived episomic vectors for gene therapy able to undergo limited replication in human cells. In our vectors, DNA replication relies on a mutated version of the LT viral protein in which the ability to interact with p53 and pRB (and consequently its transforming activity) has been selectively eliminated. Even with a non-transforming LT, eccessive replication of SV40-derived vectors in human cells may result in target cells toxicity. In order to overcome this problem we have constructed: • “Low replication rate” vectors carrying deletions of the SV40 ORI/ses regions. • “Low copy number” vectors carrying anti-LT shRNA expression cassettes. We have achieved the tissue-specificity of our vectors in two ways, by constructing: • “Wide-range replication” vectors in which the selective expression of the transgene is driven by tissue-specific promoters. • “Restricted-range replication” vectors in which the viral LT gene is under the control of tissue-specific synthetic promoters, while the transgene expression is driven by the SV40 early promoter. In both cases, the tissue-specificity of our vectors depends on short synthetic modular promoters, obtained by polymerization of naturally occourring cis-acting elements. In the most successful constructs we have combined the binding domains for tissue-specific and cell-cycle-specific transcription factors, thus obtaining promoters that are selectively active in replicating cells (regenerating tissues, stem cells and tumor cells).

Manlio Cipriani, Vincenzo De Simone, Luciana D'Angelo, Enrico Perna, Marzia Lilliu, Virginia Bovolo, Fabrizio Oliva, Maria Frigerio Cardiovascular and Thoracic Department, A De Gasperis Niguarda Ca' Granda Hospital, Milan, Italy Abstract: Heart transplantation represents the “gold standard” for the treatment of patients with end-stage heart failure, but remains challenged by inadequate donor supply, finite graft survival, and long-term complications arising from immunosuppressive therapy. In addition, a lot of patients waiting for a heart transplant experience clinical deterioration, and other patients become ineligible to undergo this treatment due to their age or relevant comorbidities. Left ventricular assist devices have emerged as a valid therapeutic option for advanced heart failure. In recent years, we have seen significant advances not only in the technologies available, but also in patient selection, indications for use, and management after implantation. Consequently, there has been an increase in the number of implants and an improvement in the survival rate and quality of life for these patients. At the same time, there are new challenges on the horizon. Patient selection is a difficult process, based on clinical and imaging parameters and risk scores, and more data are needed to refine patient selection criteria and the timing of the implant. Left ventricular assist device-related complications are still a serious problem, causing adverse events and hospital readmissions. Continuous progress in the development of these implantable devices, such as a further reduction in size and hopefully the abolition of the external driveline, will probably make ventricular assist devices an option also for less advanced stages of heart failure. Here, we discuss the current indications for left ventricular assist device implantation, patient selection criteria, and the most frequent complications associated with these devices. Keywords: mechanical circulatory support, heart failure, destination therapy, bridge to transplantation, heart transplant

In previous expression profile studies by DNA microarrays we have identified several potato genes whose mRNAs are hyper- or hypo-expressed following of potato protoplast growth in culture conditions that simulate in vivo hydric deprivation. In order to understand whether groups of these genes are transcriptionally co-regulated via common cis-acting sequence elements, we undertook the comparison of their transcriptional regulatory regions by searching the 5’ upstream region for enriched short sequence motifs. Unfortunately, the potato genomic sequence of several of the selected genes is not yet available. Therefore, we decided to perform this analysis by using the genomic sequences of A. thaliana homologous gene counterparts, relying on the conservation across these organisms of transcriptional regulatory networks. By using the MEME/MAST motif discovery tools, we have analysed separately 3 consecutive 200 bp sequence blocks from the 5’ region of each gene. The MEME analysis reveals several short sequence motifs that are significantly enriched in the first two sequence blocks, corresponding to: a) upstream transcription regulatory elements; b) Pol II positioning promoter elements / 5’-UTRs.

We have identified and characterized two transcriptional modules derived from the promoters of liver genes whose expression is specific for proliferating hepatocytes: A) An enhancer module from the HNF1β promoter. HNF1α and β are two trans-acting factors that play a central role in the expression of liver-specific genes. We reported that, during mouse and rat development, HNF1β expression precedes that of HNF1α and is inducible by retinoic acid treatment. Moreover HNF1β (but not HNF1α) is expressed in undifferentiated hepatocytes. A combined strategy of deletions and site-specific mutagenesis allowed us to identify the cis-acting elements of the HNF1β promoter. In our transient expression assay, the -435/+33 region of the promoter is sufficient to reproduce the expression pattern of the endogenous gene. This region can be further subdivided into a tissue-specific enhancer and a proximal promoter element. The transcriptional activity of the enhancer element, as measured by co-transfection assays and band-shift experiments, depends on the binding of two transcriptional activators: the liver-specific HNF-4 factor (-255/-235), and the RARα/RXRα heterodimeric protein, that recognize a RARE element partially overlapping with the HNF-4 binding site. B) The proximal element of the alpha-foetoprotein promoter. The alpha-fetoprotein (AFP) gene is expressed in the prenatal life in the yolk sac and in the foetal liver. At birth, the AFP gene is "shut down" at the transcriptional level, and it can be re-expressed in the adult liver only in pathologic conditions, like hepatic parenchyma regeneration or liver tumours. Therefore, the AFP gene must contain cis acting regulatory elements able to activate transcription only in proliferating hepatocytes. We have isolated and individually analysed the key elements of the AFP transcriptional regulation located in the proximal promoter region, where two partially overlapping AP1 and GRE elements dictate the activation or repression of the gene, respectively. Our results suggest that, while two adjacent HNF1α/β binding sites restrict the AFP promoter activity to liver cell, changes in the GR/AP1 ratio can account for the AFP reactivation in the regenerating hepatocyte. We have constructed a series of artificial promoters by assembling different combinations of these transcriptional modules. The most efficient combinations have been used to construct SV40-derived episomal vectors, that we plan to use for gene therapy of proliferating hepatocytes. These experiments are currently in progress.

Alzheimer’s disease (AD), like other multifactorial diseases, is the result of a systemic breakdown of different physiological networks. As result, several lines of evidence suggest that it could be more efficiently tackled by molecules directed toward different dysregulated biochemical targets or pathways. In this context, the selection of targets to which the new molecules will be directed is crucial. For years, the design of such multitarget-directed ligands (MTDLs) has been based on the selection of main targets involved in the “cholinergic” and the “β-amyloid” hypothesis. Recently, there have been some reports on MTDLs targeting the glycogen synthase kinase 3β (GSK-3β) enzyme, due to its appealing properties. Indeed, this enzyme is involved in tau hyperphosphorylation, controls a multitude of CNS-specific signaling pathways, and establishes strict connections with several factors implicated in AD pathogenesis. In the present Miniperspective, we will discuss the reasons behind the development of GSK-3β-directed MTDLs and highlight some of the recent efforts to obtain these new classes of MTDLs as potential disease-modifying agents.

The Internet of Things (IoT) is rapidly increasing its diffusion, posing great challenges to the research community. IoT systems are composed by smart objects (Things) that are interconnected in order to provide new products and services. The interaction of heterogeneous and distributed smart things guided by software with the physical world brings new sources of safety issues. To this reason, providing valuable and effective solutions to support the verification and validation of such systems is needed. In this paper we introduce a model-driven Thing-In-the-Loop verification and validation approach that transfers the best practices adopted in different embedded system domains towards the IoT world. Starting from models and scenarios representing the structure and behaviors of the IoT system as well as models of its context our approach generates appropriate test cases that are executed in accordance with Model-in-the-Loop, Software-in-the-Loop and Hardware-in-the-Loop techniques. We preliminarily evaluated the feasibility of our approach by applying it in the context of a Smart Mobility case study.

Software has gained a critical role in the automotive domain that is becoming more and more complex. The ever-growing complexity in automotive software development is due to its high variability. In this scenario, automotive companies need to adopt cost-effective development processes in order to manage the variability of the produced software. A well-known solution for dealing with this problem is the adoption of Software Product Lines (SPL). In this paper we report an experience we performed in collaboration with the Fiat Chrysler Automobiles (FCA) company for the application of the SPL in one of its Model-Based Design (MBD) processes. SPL were supported by AutoMative, a software infrastructure we implemented for the semi-automatic generation of Product Architectures from specification documents.

Modern spreadsheet systems can be used to implement complex spreadsheet applications including data sheets, customized user forms and executable procedures written in a scripting language. These applications are often developed by practitioners that do not follow any software engineering practice and do not produce any design documentation. Thus, spreadsheet applications may be very difficult to be maintained or restructured. In this position paper we present in a nutshell two reverse engineering techniques and a tool that we are currently realizing for the abstraction of conceptual data models and business logic models., In Proceedings of the 2nd Workshop on Software Engineering Methods in Spreadsheets (http://spreadsheetlab.org/sems15/)

Nowadays Spreadsheet-based Information Systems are widely used in industries to support different phases of their production processes. The intensive employment of Spreadsheets in industry is mainly due to their ease of use that allows the development of Information Systems even by not experienced programmers. The development of such systems is further aided by integrated scripting languages (e.g. Visual Basic for Applications, Libre Office Basic, JavaScript, etc.) that offer features for the implementation of Rapid Application Development processes. Although Spreadsheet-based Information Systems can be developed with a very short time to market, they are usually poorly documented or in some case not documented at all. As a consequence, they are very difficult to be comprehended, maintained or migrated towards other architectures, such as Database Oriented Information Systems or Web Applications. The abstraction of a data model from the source spreadsheet files represents a fundamental activity of the migration process towards different architectures. In our work we present an heuristic- based reverse engineering process for inferring a data model from an Excel based information system. The process is fully automatic and it is based on seven sequential steps. Both the applicability and the effectiveness of the proposed process have been assessed by an experiment we conducted in the automotive industrial context. The process was successfully used to obtain the UML class diagrams representing the conceptual data models of three different Spreadsheet-based Information Systems. The paper presents the results of the experiment and the lessons we learned from it.

Potato cDNA microarray slides (TIGR 1 K) were used to compare changes in gene expression of two potato cell populations subjected to shock or gradual exposure to PEG-mediated water stress. A total of 180 transcripts were found to be statistically up- or down-regulated in PEG-shocked or gradually stressed cells, compared to untreated control cells. Up-regulated genes in shocked cells were prevalently involved in carbohydrate metabolism, cellular communication, and signal transduction. In cells stressed gradually, the most represented induced genes were those involved in signal transduction, in response to environmental signals and in the regulation of transcription and translation machineries, including the heat shock protein 90-2, ribosomal and RNA-binding proteins. The expression of selected up-regulated genes was also tested in potato plants subjected to slow soil-drying conditions. Interestingly, the rgga gene, encoding an RNA-binding protein, was strongly induced in both leaves and roots of water-stressed potato plants, supporting its putative function in adaptive mechanisms to water stress. Pointing to differences in gene expression between shock-like and adaptive response, our findings might contribute to the controversial debate on the biological function of stress-responsive genes in cell damage repair or in restoring homeostasis and elaborating longer term responses for adaptation.

Background A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed. Results By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes. Conclusions Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype. See: http://www.biomedcental.com/1741-7007/8/125

Hereditary haemochromatosis is an autosomal recessive disorder of iron regulation that results in abnormal intestinal iron absorption with progressive iron overloading of parenchymal cells. Two specific, single point mutations of the HFE gene (C282Y and H63D) have been described in haemochromatosis patients. Epidemiological studies have revealed a strict association between hereditary haemochromatosis and C282Y homozygosis or C282Y/H63D compound heterozygosis, suggesting that these mutations may provide a useful tool for diagnosis. However, recent investigations from southern Europe have reported lower allelic frequencies of the C282Y mutation among haemochromatosis patients, apparently depending on the geographical area of the population analysed. To assess the predictive value of the detection of the C282Y and H63D HFE mutations in our geographical area, we have evaluated their occurrence in 46 haemochromatosis patients from southern Italy. We found that only 19.6% of our patients were homozygous for the C282Y mutation and 21.7% were compound C282Y/H63D heterozygotes. Among the remaining 59%, approximately 40% did not display any of the known HFE mutations. We conclude that, in southern Italy, another genetic determinant/s must be responsible for many haemochromatosis cases and that a genetic screening for the C282Y and H63D HFE mutations is not sufficient for hereditary hae- mochromatosis diagnosis.

A human clone corresponding to the gene for the DNA-binding factor LFB3, a protein highly homologous to the liver-specific transcription factor LFB1, has been isolated and partially sequenced. This gene is designated TCF2. Oligonucleotide primers have been designed for LFB3 and used to amplify specifically the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this means, the human LFB3 gene has been mapped to the long arm of chromosome 17, between the centromere and the APL breakpoint.

The transcription start site and promoter of the rat gene coding for the transcription factor NF-1 have been identified. The NF-1 promoter was fused to the chloramphenicol acetyltransferase-coding sequence, and the resulting plasmid was transcriptionally active in the HepG2 cell line. Footprinting and gel retardation analysis indicated that the transcription factor Sp1 binds to the NF-1 promoter. Mutants in the Sp1-binding site displayed a strong reduction in transcriptional activity.