Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay

Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay

Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay

Influence of Leptin on the Secretion of Growth Hormone in Ewes under Different Photoperiodic Conditions

Leptin is an adipokine with a pleiotropic impact on many physiological processes, including hypothalamic-pituitary-somatotropic (HPS) axis activity, which plays a key role in regulating mammalian metabolism. Leptin insensitivity/resistance is a pathological condition in humans, but in seasonal animals, it is a physiological adaptation. Therefore, these animals represent a promising model for studying this phenomenon. This study aimed to determine the influence of leptin on the activity of the HPS axis. Two in vivo experiments performed during short- and long-day photoperiods were conducted on 12 ewes per experiment, and the ewes were divided randomly into 2 groups. The arcuate nucleus, paraventricular nucleus, anterior pituitary (AP) tissues, and blood were collected. The concentration of growth hormone (GH) was measured in the blood, and the relative expression of GHRH, SST, GHRHR, SSTR1, SSTR2, SSTR3, SSTR5, LEPR, and GH was measured in the collected brain structures. The study showed that the photoperiod, and therefore leptin sensitivity, plays an important role in regulating HPS axis activity in the seasonal ewe. However, leptin influences the release of GH in a season-dependent manner, and its effect seems to be targeted at the posttranscriptional stages of GH secretion

The Effect of Leptin on the Blood Hormonal Profile (Cortisol, Insulin, Thyroid Hormones) of the Ewe in Acute Inflammation in Two Different Photoperiodical Conditions

As a day animal with sensitivity to inflammation similar to that of humans, the sheep may highly outperform the rodent model in inflammation studies. Additionally, seasonality makes sheep an interesting model in endocrinology research. Although there are studies concerning inflammation’s influence on leptin secretion and vice versa, a ewe model, with its possible ‘long-day leptin resistance’, is still not examined enough. The present study aimed to examine whether leptin may modulate an acute inflammation influence on plasma hormones in two photoperiodical conditions. The experiment was conducted on 48 ewes divided into four groups (control, lipopolysaccharide (LPS), leptin, LPS + leptin) during short and long days. Blood sampling started 1 hour before and continued 3 h after LPS/saline administration for further hormonal analysis. The results showed that the photoperiod is one of the main factors influencing the basal concentrations of several hormones with higher values of leptin, insulin and thyroid hormones during long days. Additionally, the acute inflammation effect on cortisol, insulin and thyroid hormones was photoperiod-dependent. The endotoxemia may also exert an influence on leptin concentration regardless of season. The effects of leptin alone on hormone blood concentrations are rather limited; however, leptin can modulate the LPS influence on insulin or thyroxine in a photoperiod-dependent way

Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development

Development and Characterization of Novel Selective, Non-Basic Dopamine D-2 Receptor Antagonists for the Treatment of Schizophrenia

The dopamine D-2 receptor, which belongs to the family of G protein-coupled receptors (GPCR), is an important and well-validated drug target in the field of medicinal chemistry due to its wide distribution, particularly in the central nervous system, and involvement in the pathomechanism of many disorders thereof. Schizophrenia is one of the most frequent diseases associated with disorders in dopaminergic neurotransmission, and in which the D-2 receptor is the main target for the drugs used. In this work, we aimed at discovering new selective D-2 receptor antagonists with potential antipsychotic activity. Twenty-three compounds were synthesized, based on the scaffold represented by the D2AAK2 compound, which was discovered by our group. This compound is an interesting example of a D-2 receptor ligand because of its non-classical binding to this target. Radioligand binding assays and SAR analysis indicated structural modifications of D2AAK2 that are possible to maintain its activity. These findings were further rationalized using molecular modeling. Three active derivatives were identified as D-2 receptor antagonists in cAMP signaling assays, and the selected most active compound 17 was subjected to X-ray studies to investigate its stable conformation in the solid state. Finally, effects of 17 assessed in animal models confirmed its antipsychotic activity in vivo

Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation

Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own