Biological Activity of Pyrrole-Imidazole Polyamides in vivo

This thesis focuses on biological activity of pyrrole-imidazole polyamides in vivo. The work presented includes experiments underlining sequence selectivity of these compounds in living cells and potential methods to improve it. A large fraction of this thesis is devoted to activity of Py-Im in murine models of cancer. We investigated the pharmacokinetics and biodistribution of two compounds – targeted to 5'-WGGWCW-3' and 5'-WTWCGW-3' sequences – and characterized their activity by measuring their effects on tumor growth, gene expression in vivo and in tissue culture, and their effects on physiology of tumors. The initial theoretical studies suggested that a large fraction of genomic sites are bound by Py-Im polyamides non-specifically and experimental data shows that the programmed binding sequence is not a sole determinant of the patterns of gene regulation. Despite the likely presence of non-specific effects of Py-Im polyamides in living cells, in vivo administration of Py-Im polyamides resulted in tolerable host toxicity and anti-tumor activity. Py-Im polyamide targeted to Estrogen Receptor Response Element showed downregulation of ER-driven gene expression in tumor cells, while the compound targeted to hypoxia response element reduced vascularization of tumors and their growth rate, induced apoptosis of cells in hypoxic areas and reduced expression of proangiogenic and prometastatic factors. Further studies, showed that polyamides distributed to many of the tested tissues and their FITC-conjugates showed nuclear uptake. The gene expression effects were also present in murine tissues, such as liver and kidneys, indicating a potential for use for Py-Im polyamides in non-cancerous diseases

Density operators that extremize Tsallis entropy and thermal stability effects

Quite general, analytical (both exact and approximate) forms for discrete probability distributions (PD's) that maximize Tsallis entropy for a fixed variance are here investigated. They apply, for instance, in a wide variety of scenarios in which the system is characterized by a series of discrete eigenstates of the Hamiltonian. Using these discrete PD's as "weights" leads to density operators of a rather general character. The present study allows one to vividly exhibit the effects of non-extensivity. Varying Tsallis' non-extensivity index $q$ one is seen to pass from unstable to stable systems and even to unphysical situations of infinite energy.Comment: 22 page

An HRE-binding Py-Im polyamide impairs hypoxic signaling in tumors

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1 has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1 in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1 distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1 show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response

Att predicera arbetsprestation hos telekommunikatörer: personlighet och specifika kognitiva förmågor

Callcenterbranschen växer kraftigt och flera forskare har uppmärksammat behovet av att identifiera vad som kännetecknar lämpliga kandidater för arbete på callcenters. Syftet med denna studie var att studera huruvida verbal förmåga, numerisk förmåga och personlighet, utifrån femfaktormodellen, predicerar typisk arbetsprestation hos anställda (N=100) på ett inkommande callcenter. Arbetsprestation operationaliserades genom måtten försäljning, effektivitet och first time resolution. Verbal förmåga, numerisk förmåga och personlighet operationaliserades genom de tre arbetspsykologiska testen scales verbal instruct, scales eql och shapes basic. Studien baserades på en reell rekryteringsprocess på ett callcenter. Bivariata regressionsanalyser genomfördes med testresultaten och arbetsprestationsmåtten. Gällande verbal och numerisk förmåga hittades endast ett signifikant reslutat, där numerisk förmåga predicerade arbetsprestation, vilket avvek från tidigare forskning som har identifierat dessa förmågor som starka prediktorer till arbetsprestation. Resultaten gällande personlighet visade att prediktiv validitet delvis förelåg och att personlighetsfaktorn emotionell stabilitet var den starkaste prediktorn i förhållande till arbetsprestation. Studien har ett praktisk värde för testleverantörer och testanvändare. Detta i form av kunskap kring egenskaper som utmärker framgångsrika telekommunikatörer.Call centres is a rapidly growing industry and several researchers have recognized the need to identify the characteristics of suitable candidates for work in call centres. The purpose of this study was to study whether verbal ability, numerical ability and personality based on the five factor model, predicts typical job performance of employees (N=100) in an inbound call centre. Job performance was operationalized by the measures sales, efficiency and first time resolution. Verbal ability, numerical ability and personality were operationalized by three psychometric tests; scales verbal instruct, scales eql and shapes basic. The study was based on a real recruitment process to a call centre. Bivariate regression analyzes were conducted with the test results and the job performance measures. Concerning verbal and numerical ability, only one significant relation was identified, between numerical ability and job performance. This differs from earlier research which has identified these abilities as strong predictors of job performance. Results concerning personality showed that predictive validity partially exists and that the personality factor emotional stability was the strongest predictor of job performance. The study has a practical value for test providers and test users consisting of knowledge about what characterizes successful call centre operators

Tumor Xenograft Uptake of a Pyrrole−Imidazole (Py-Im) Polyamide Varies as a Function of Cell Line Grafted

Subcutaneous xenografts represent a popular approach to evaluate efficacy of prospective molecular therapeutics in vivo. In the present study, the C-14 labeled radioactive pyrrole–imidazole (Py-Im) polyamide 1, targeted to the 5′-WGWWCW-3′ DNA sequence, was evaluated with regard to its uptake properties in subcutaneous xenografts, derived from the human tumor cell lines LNCaP (prostate), A549 (lung), and U251 (brain), respectively. Significant variation in compound tumor concentrations was seen in xenografts derived from these three cell lines. Influence of cell line grafted on systemic polyamide elimination was established. With A549, a marked variation in localization of 1 was determined between Matrigel-negative and -positive xenografts. An extensive tissue distribution analysis of 1 in wild-type animals was conducted, enabling the comparison between the xenografts and the corresponding host organs of origin

A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma has Potent Anti-tumor Activity

Multiple myeloma is incurable and invariably becomes resistant to chemotherapy. Although the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in contributing to multiple myeloma progression, angiogenesis, and resistance to chemotherapy. These effects occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIF), and targeting HIFs can have anticancer effects in both solid and hematologic malignancies. Here, it was found that in most myeloma cell lines tested, HIF1α, but not HIF2α expression was oxygen dependent, and this could be explained by the differential expression of the regulatory prolyl hydroxylase isoforms. The anti–multiple myeloma effects of a sequence-specific DNA-binding pyrrole-imidazole (Py-Im) polyamide (HIF-PA), which disrupts the HIF heterodimer from binding to its cognate DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and binds specific regions of DNA with an affinity comparable with that of HIFs. Most of the multiple myeloma cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could overcome this resistance in vitro. Using xenograft models, it was determined that HIF-PA significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a rationale for targeting the adaptive cellular hypoxic response of the O_2-dependent activation of HIFα using polyamides

Acoustically Targeted Chemogenetics for Noninvasive Control of Neural Circuits

Neurological and psychiatric disorders are often characterized by dysfunctional neural circuits in specific regions of the brain. Existing treatment strategies, including the use of drugs and implantable brain stimulators, aim to modulate the activity of these circuits. However, they are not cell-type-specific, lack spatial targeting or require invasive procedures. Here, we report a cell-type-specific and non-invasive approach based on acoustically targeted chemogenetics that enables the modulation of neural circuits with spatiotemporal specificity. The approach uses ultrasound waves to transiently open the blood–brain barrier and transduce neurons at specific locations in the brain with virally encoded engineered G-protein-coupled receptors. The engineered neurons subsequently respond to systemically administered designer compounds to activate or inhibit their activity. In a mouse model of memory formation, the approach can modify and subsequently activate or inhibit excitatory neurons within the hippocampus, with selective control over individual brain regions. This technology overcomes some of the key limitations associated with conventional brain therapies

Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function, based on fluorescent or light-controlled proteins, work best in cultured cells, transparent model species, or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for actuation. These capabilities are widely used in clinical techniques such as diagnostic ultrasound, magnetic resonance imaging, focused ultrasound ablation, and magnetic particle hyperthermia. Each of these modalities offers spatial and temporal precision that could be used to study a multitude of cellular processes in vivo. However, connecting these techniques to cellular functions such as gene expression, proliferation, migration, and signaling requires the development of new biochemical tools that can interact with sound waves and magnetic fields as optogenetic tools interact with photons. Here, we discuss the exciting challenges this poses for biomolecular engineering and provide examples of recent advances pointing the way to greater depth in in vivo cell biology