Screening compounds with image-based analysis is an important part in the processof drug discovery. It is an efficient way to screen compounds as it gives moreinformation than for example HTS. High-content screening as it is also called, hasreally progressed in recent years, as the field of data science evolves, and with it sodoes the efficiency of how images can be processed into information. Anotherimportant part of the drug discovery field is the family of receptors GPCRs, a largefamily of over 800 different receptors in humans. The reason GPCRs are importantin drug discovery is because of the large number of drugs targeting them. In thisexperiment we wanted to use image-based analysis to challenge drugs orcompounds that were said to be specific and see if they actually are that specific, orif we can see indications of the drug also working somewhere else. While the drugswe tested did not appear to cause any morphological perturbations large enough todistinguish them from the control, some drugs appear to cluster differently. Thismight suggest that they affect multiple targets, but it needs to be followed up upon inorder to draw any substantial conclusions.

Giardia intestinalis is a protozoan parasite causing the diarrheal disease called giardiasis that infects millions of people worldwide each year. The life cycle of Giardia intestinalis is characterized by two stages; the sturdy, infectious cyst and the vegetative, motile trophozoite. This project sought to investigate the regulation of the encystation process where trophozoites transform into cysts. Twelve genes with interesting transcriptomic profiles were chosen to study as putative transcription factors and regulators of encystation. These genes were cloned onto a plasmid with a Strep-Tag and transfected into Giardia intestinalis. The protein expression and localization was studied using immunofluorescence microscopy with antibodies against the Strep-Tag at different time points after inducing encystation. Although the project did not fully characterize these genes, protein expression was seen in all cases except two. Some proteins were seen localized to the nuclei and others had a localization pattern similar to the localization of cyst wall protein. In addition, a dramatic phenotype resembling cells going through programmed cell death was observed in one of the transfectants early in encystation and would be interesting to study further. The transfectant strains generated from this project remain interesting candidates to investigate as putative transcription factors.

The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell. By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell. Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner. To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome. / <p>QC 20170512</p>

Primär myelofibros (PMF) är en typ av myeloproliferativ neoplasm (MPN) som leder till en progressiv och irreversibel benmärgsfibros. En somatisk mutation, Jak2V617F, har hittats hos 50 % av patienterna med MPN i hematopoetiska stamceller. Nyligen har man upptäckt grupp 2 av medfödda lymfoida celler (ILC2) som tillhör det medfödda systemet. De är T-cellernas motsvarighet men saknar TCR-receptorn. ILC2 reagerar på IL-33 och producerar Il-13. Under de senaste åren har man upptäckt att dessa två cytokiner är inblandade i PMF. För att undersöka ILC2:s roll i utvecklingen av benmärgsfibros in vivo producerade vi retrovirus som uttrycker Jak2 vildtyp (JAK2_WT) eller Jak2V617F (JAK2_V617F) och transducerade benmärg vildtyp (BM_WT) eller benmärg ILC2KO (BM_ILC2KO). Benmärgen transplanterades till subletalt bestrålade immunbristande möss (NOG). Klinikopatologiska drag som är karakteristiska för sjukdomens första stadier, som förhöjda hemoglobinnivåer, megakaryocythyperplasi och betydande trombocytos, uppstod inte under studieperioden. Ökade vita blodkroppar uppstod dock på grund av avsaknaden av ILC2 i JAK2_V617F-expressiva möss. Flödescytometeranalys visade ursprunget till den markerade leukocytosen som ett resultat av expansionen från lymfocytlinjen, mer specifikt B-celler, men resultaten är inte entydiga eftersom de förhöjda nivåerna av B-celler kan vara en följd av ILC2 knock-out fenotypen som förvärras av närvaron av mutationen. Granulocytnivåerna från de inympade cellerna hölls låga till följd av att stamcellerna i värdens benmärg var inblandade på grund av subletal bestrålning. Vi drar slutsatsen att frånvaron av ILC2 i JAK2_V617F-uttryckta benmärgsprogenitorer har en tendens att förvärra den myeloproliferativa fenotypen i sjukdomens tidiga skeden, vilket tyder på en möjlig skyddande roll för ILC2 vid utvecklingen av MPN. / Primary myelofibrosis (PMF) is one type of myeloproliferative neoplasm (MPN) that leads to a progressive and irreversible bone marrow fibrosis. A somatic mutation, Jak2V617F has been found in 50% of patients with MPN in hematopoietic stem cells. Group 2 innate lymphoid cells (ILC2) belonging to the innate system has been recently discovered. They are the counter part of T cells but lacking the TCR receptor. ILC2 response to IL-33 producing Il-13. In recent years, the involvement of these two cytokines in the PMF has been uncovered. To investigate the role of ILC2 in the progression of bone marrow fibrosis in vivo we produced retrovirus expressing Jak2 wild-type (JAK2_WT) or Jak2V617F (JAK2_V617F) and transduced bone marrow wild type (BM_WT) or bone marrow ILC2KO (BM_ILC2KO). The bone marrow was transplanted into sub-lethally irradiated immunodeficient mice (NOG). Clinicopathologic features characteristic from the first stages of the disease, as elevated hemoglobin levels, megakaryocyte hyperplasia and significant thrombocytosis did not emerge during the study period. However, increased in white blood cells arise from the absence of ILC2 in JAK2_V617F expressing mice. Flow cytometer analysis revealed the origin of the marked leukocytosis as a result of the expansion from the lymphocyte lineage, more specifically B cells, but the results are inconclusive as the elevated levels of B-cells could be a consequence of the ILC2 knock-out phenotype aggravated by the presence of the mutation. Granulocyte levels from engrafted cells were kept low because of the involvement of host bone marrow stem cells due to sublethal irradiation. We conclude that the absence of ILC2 in JAK2_V617F-express bone marrow progenitors has a tendency to aggravate the myeloproliferative phenotype in the early stages of the disease, indicating a possible protective role of ILC2 in the development of MPNs.

No description available.

<p>Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺]_i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺]_i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺]_i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺]_i oscillations. The [Ca²⁺]_i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺]_i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺]_i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺]_i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺]_i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺]_i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺]_i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺]_i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.</p>

<p>Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca²⁺ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. </p><p>A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.</p>

<p>During development of type 1 and type 2 diabetes plasma insulin patterns are altered. Since the islet insulin release pattern has been implicated in this development, insulin secretion from single islets was studied and linked to the islet protein levels of uncoupling protein-2 (UCP2). Islets were isolated from NOD- and KKA^y- mice, GK- and GK-derived congenic rats, which are animal models of diabetes, and three human subjects with type 2 diabetes. At basal glucose (3 mM), insulin release from such islets was pulsatile and the amount released was comparable to that of control islets. When the glucose concentration was raised to 11 mM insulin release was essentially unchanged in islets isolated from older NOD- and KKA^y- mice, GK- and Niddm1i congenic rats, and NIDDM persons. In islets from Niddm1f congenic rats, younger NOD- and KKA^y-mice, control animals and normal human donors the secretion rate increased 2-9 fold when the glucose concentration was raised. This rise in secretion was manifested as increase of the amplitude of the insulin oscillations without affecting their frequency. Impaired glucose-induced insulin release was associated with reduction in glucose oxidation measured in NOD-islets, unaffected respiration measured in GK-islets and higher protein level of UCP2 measured in KKA^y-islets. When the UCP2 amounts in KKA^y-islets were reduced by culture to those of control islets, glucose-induced insulin secretion was essentially normalized. Our studies suggest that the deranged plasma insulin patterns observed in diabetes are related to decrease in the amplitude of insulin oscillations from the islets rather than loss of the oscillatory activity. This reduction of pulse amplitude may be related to impaired glucose metabolism and/or increased mitochondrial uncoupling. </p>

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca2+ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.