A practical, unitary simulator for non-Markovian complex processes

Stochastic processes are as ubiquitous throughout the quantitative sciences as they are notorious for being difficult to simulate and predict. In this letter we propose a unitary quantum simulator for discrete-time stochastic processes which requires less internal memory than any classical analogue throughout the simulation. The simulator's internal memory requirements equal those of the best previous quantum models. However, in contrast to previous models it only requires a (small) finite-dimensional Hilbert space. Moreover, since the simulator operates unitarily throughout, it avoids any unnecessary information loss. We provide a stepwise construction for simulators for a large class of stochastic processes hence directly opening the possibility for experimental implementations with current platforms for quantum computation. The results are illustrated for an example process.Comment: 12 pages, 5 figure

An engineered mammalian band-pass network

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of ‘low-threshold' morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a ‘high-threshold' concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing application

pQuattro vectors allow one-step multigene metabolic engineering and auto-selection of quattrocistronic artificial mammalian operons

Based on internal ribosomal entry sites (IRES) of picornaviral origin we constructed a novel family of mammalian expression vectors. pQuattro vectors contain quattrocistronic artificial eukaryotic operons which link, in a single transcript, the simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest to a terminal neomycin (neo) resistance marker. Due to the strict genetic linkage of the transgenes and the terminal selection marker, this genetic configuration enables, by the selection on neomycin, multigene metabolic engineering of mammalian cells in a single step (one-step metabolic engineering). Furthermore, selection on the terminal cistron of multicistronic expression units enforces cocistronic expression of all upstream encoded genes and maximises genetic integrity of the eukaryotic operon in stable mammalian cell lines, since clones harbouring damaged multicistronic expression units become neomycin-sensitive and are automatically counterselected (auto-selection). The modular set-up and the abundance of restriction sites in pQuattro vectors facilitate the movement of individual genes between multicistronic expression vectors and guarantees high compatibility with genetic elements of a wide variety of existing mammalian expression vector

An engineered mammalian band-pass network

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of ‘low-threshold’ morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a ‘high-threshold’ concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing applications

Dual‐regulated expression of C/EBP‐α and BMP‐2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts

CCAAT/enhancer‐binding proteins (C/EBPs) as well as bone morphogenic proteins (BMPs) play essential roles in mammalian cell differentiation in shaping adipogenic and osteoblastic lineages in particular. Recent evidence suggested that adipocytes and osteoblasts share a common mesenchymal precursor cell phenotype. Yet, the molecular details underlying the decision of adipocyte versus osteoblast differentiation as well as the involvement of C/EBPs and BMPs remains elusive. We have engineered C2C12 cells for dual‐regulated expression of human C/EBP‐α and BMP‐2 to enable independent transcription control of both differentiation factors using clinically licensed antibiotics of the streptogramin (pristinamycin) and tetracycline (tetracycline) classes. Differential as well as coordinated expression of C/EBP‐α and BMP‐2 revealed that (i) C/EBP‐α may differentiate C2C12 myoblasts into adipocytes as well as osteoblasts, (ii) BMP‐2 prevents myotube differentiation, (iii) is incompetent in differentiating C2C12 into osteoblasts and (iv) even decreases C/EBP‐α's osteoblast‐specific differentiation potential but (v) cooperates with C/EBP‐α on adipocyte differentiation, (vi) osteoblast formation occurs at low C/EBP‐α levels while adipocyte‐specific differentiation requires maximum C/EBP‐α expression and that (vii) BMP‐2 may bias the C/EBP‐α‐mediated adipocyte versus osteoblast differentiation switch towards fat cell formation. Dual‐regulated expression technology enabled precise insight into combinatorial effects of two key differentiation factors involved in adipocyte/osteoblast lineage control which could be implemented in rational reprogramming of multipotent cells into desired cell phenotypes tailored for gene therapy and tissue engineerin

Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression

Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmySΔS, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-α) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (PPIR8) placed 5′ of desired transgenes; (iii) within modified enhancer-free 3′-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct PPIR-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell line

Synthetic mammalian trigger-controlled bipartite transcription factors

Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), γ-butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and γ-butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapie

Identification of a novel proliferation‐inducing determinant using lentiviral expression cloning

One of the major challenges in the post‐genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation‐modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation‐inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP‐expressing oncoretroviruses to target proliferation‐competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd‐uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today's untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation‐modulating genes is essential for developing new strategies for gene therapy and tissue engineerin

Sprachidentität - Identität durch Sprache

a) Zusammenfassung der Diplomarbeit Die Sprache ist für den Menschen Teil seiner Identität, durch diese definiert er sich, drückt sich aus, stellt er sich der Öffentlichkeit dar. Zudem ist die Sprache ein wichtiger Teil der Identität von Gruppierungen. Viele Völker oder ethnische Gruppen, insbesondere Minderheiten definieren sich unter anderem durch die Sprache, so auch die drei Sprachgruppen im dreisprachigen Südtirol. Sprache dient dort nicht nur als Kommunikationsmittel, sondern auch als Zeichen, um die Eigenständigkeit der Minderheit, die Verbundenheit des Einzelnen mit der Volksgruppe, auch mit deren Geschichte und damit die eigene Identität zu präsentieren. Vorliegende Diplomarbeit versucht zu zeigen, wie wichtig die Sprache für die deutschsprachige Minderheit in Südtirol im 20. Jahrhundert war, wie verschiedene Regierungen des Mehrheitsvolks der Italiener versuchten, durch das Verbot der deutschen Sprache und danach durch die Ansiedelung italienischsprachiger Bürger und schließlich durch die Aussiedelungen der Südtiroler mithilfe Nazideutschlands ein Minderheitengebiet ethnisch umzuwandeln und wie sich die Geschichte Südtirols noch anfangs des 21. Jahrhunderts auf die Sprache und die Identität der Bewohner der Provinz Bozen auswirkte. Des Weiteren werden folgende Fragen behandelt: Trägt die aktuelle Sprachpolitik zu einer gemeinsamen Identität aller Südtiroler bei oder verhindert gerade die aufgezeigte Sprachpolitik eine „Gesamt-Südtiroler-Identität“? Herrscht in Südtirol ein sprachliches Mit- oder Nebeneinander? Welche Folgen hat Südtirols Geschichte des 20. Jahrhunderts auf das Verhältnis der Bevölkerung zu ihrer Sprache und für die Entwicklung einer mehrsprachigen Gesellschaft? Kann Südtirol als Modellregion/Vorbild für andere mehrsprachige Regionen Europas wie Kärnten, das Baskenland oder den Kosovo dienen? Welche großen Chancen bietet die sprachliche Vielfalt für Südtirol in einem vereinten Europa und wie sieht die sprachliche Zukunft dieses Landes an der Schnittstelle dreier Kulturen und Sprachen aus

Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene network