Cell motility is a fundamental process, enabling cells to migrate, for instance during embryogenesis, tissue repair and defense. Force is generated by two protein systems, which also participate in cell proliferation, control macromolecular and organelle distribution and determine the fine structure of the cell interior. The major components of these are actin and tubulin, respectively, and they are referred to as the microfilament and the microtubule systems. This thesis focuses on tropomyosin, one of many microfilament associated proteins coupled to actin dynamics and organization and expressed in several isoform variants. Altered distribution and isoform expression of tropomyosin are signatures of malignant cells and are dealt with in the current thesis. The presence of tropomyosin isoforms in protruding lamellipodia of migrating cells is demonstrated, and a method to fractionate tropomyosin depending on its organization in an easily extractable, and a more tightly bound cytoplasmic form is presented. Analysis of the loosely associated tropomyosin fraction by gel filtration chromatography revealed that most of the tropomyosins in this fraction exist in a multimeric form. It was also observed that the distribution of tropomyosin varied between non-transformed and transformed cells with most of the isoforms enriched in the loosely bound fraction in the latter category of cells. Possibly this reflects the extensive reorganization of the microfilament system observed in cancer cells and which, depending on the context, can be normalized by introduction of certain tropomyosin isoforms. Many anti-cancer drugs target the microtubule system, inhibit cell division and promote apoptosis. Here it is shown that picropodophyllin, which has promising anticancer properties has a destabilizing effect on microtubules and via the microfilament system causes cells to detach from their substratum. Furthermore, picropodophyllin interferes with stimulation of the insulin-like growth factor receptor, which is involved in growth stimulation, differentiation and survival and whose expression is up-regulated in cancer cells.

Inom området för naturvetenskap, och inte minst inom biologi, används visualiseringar som ett hjälpmedel för att förklara olika fenomen. Syftet med den här litteraturstudien har därför varit att undersöka hur vi som blivande biologilärare kan använda oss av visuella representationer i undervisning om cellen. För att ta reda på detta har vi genom en litteraturstudie undersökt elevers preferenser av cellbiologiska bilder, hur olika typer av visuella representationer av cellen påverkar elevers lärande, hur animationer påverkar elevers lärande om cellen och hur visuella hjälpmedel bör användas i biologiundervisning. Urvalet av studierna gjordes på ett systematiskt sätt, men med något hårdare urvalskriterier än vad som brukar användas vid en systematisk litteraturstudie. Resultatet av vår litteraturstudie indikerar att visualiseringar är kraftfulla verktyg, som i fel sammanhang kan skapa problem för elevers lärande. Elevers visuella förmåga bör inte tas förgivet då det direkt påverkar det visuella lärandet. Det finns därför anledning att träna elever i att tolka bilder. Biologilärare bör därför ha en bred repertoar av visualiseringar som representerar cellen för att kunna bemöta olika elevers visuella förmågor och förkunskaper.

Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

Establishing a definitive cell cycle progression has been one of the fundamental aims of cellular biology. Its importance lies in gaining insight into the basic processes of life as well as the functions of mutant cell cycle pathways in promoting cancer by replication deficiencies and loss of checkpoint control. Currently used methods to control cell cycle and synchronize cells, function by halting cell cycle progression. Such harsh methods are detrimental to the cell and insufficient to provide an accurate reflection of the cell cycle. This study focused on replicating and confirming the efficiency of a technique developed by Helmstetter, called the “Baby Machine,” that can produce new born cells with little to no perturbations. Using this in conjunction with a short pulse RNA labelling technique, called Bru-seq, allowed the capture and RNA sequencing of synchronized cells and its nascent RNA. Here we show the first glimpse into the transcriptional profile of newly divided cells as well as novel rapid exon splicing and transcription read-through processes.

Inflammasomes are multiprotein complexes that play a critical role in the regulation of inflammation and the inflammatory responses against pathogens. NLRP3 (NOD-, LRR-and pyrin domain-containing protein 3) is among the molecules involved in the homonymous and well-studied NLRP3 inflammasome that accounts for the release of the pro-inflammatory cytokines interleukin (IL) 1-β and 18. Two signals (priming and activation) that include molecular and cellular events lead to the activation of the complex; the main events involved during the activation phase are ionic fluxes, mitochondrial dysfunction, and lysosomal damage. Calcium mobilisation belongs to the signalling events of ionic fluxes associated with the complex assembly initiation. Although no consensus has been established regarding the ionic Ca2+ fluxes and the exact mechanisms contributing to NLRP3 activation, several sources agree that Ca2+ mobilisation homeostasis is essential for the canonical function of the NLRP3 inflammasome, and other cellular processes associated with it. This systematic review aimed to determine the factors and mechanisms related to Ca2+ mobilisation contributing to inflammasome activation, examine NLRP3-associated pathologies, and propose potential therapeutic targets. The literature sources found were evaluated using the CASP tool. The obtained information revealed an intertwined relation of Ca2+ flux with the calcium-sensing receptor, reactive oxygen species (ROS) generation, lysosome rupture, Ca2+-permeable channels and K+ efflux contributing to NLRP3 inflammasome activation. The summarised knowledge in this review has led to the proposal of future studies through references to different NLRP3-related diseases such as Alzheimer’s and diabetes type II, while potential therapeutic targets were also discussed.

Inflammation is a defensive process that allows immune cells to be mobilized to help with infection removal and tissue regeneration. Inflammasomes are multiprotein oligomers in the cytoplasm and components of the innate immune system that have a role in inflammation. Glycogen synthase kinase 3 (GSK3) is a critical molecule involved in a wide range of inflammatory reactions. It has been reported to suppress the production of pro-inflammatory mediators in response to LPS when it is inhibited. The aim of this project was to study the effect of GSK3 inhibition in a concentration-dependent manner on the production of IL-6 as well as ASC-speck formation in THP1 ASC GFP cells stimulated with LPS and activated using nigericin. Using the cell culture supernatant ELISA was performed to quantify the IL-6 protein secreted by THP-1 macrophages. Using reverse-transcribed cDNA, qPCR was performed to measure the IL-6 gene expression. Finally, live-cell imaging was done to visualize the ASC-speck formation. It was found that upon stimulation of THP-1 cells a remarkable increase in the production of IL-6 was observed, however, the inhibitor did not suppress the production of IL-6 as hypothesized. This could be primarily due to the presence of another NF-κB pathway which is not mediated by GSK3 and therefore could not be inhibited using the GKS3 inhibitor. Future studies could decrease the LPS concentration to see if the uninhibited pathway can be observed at lower stimulation. Another probable solution could be lowering the FBS percentage to avoid potential inhibition.

No description available.

<p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity <i>in vitro</i>. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of <i>Yersinia pseudotuberculosis</i>. We found that Ras was ADP-ribosylated <i>in vivo</i> and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily <i>in vivo</i>, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by <i>P. aeruginosa</i>. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 <i>in vivo</i>, and that ExoT shows GAP activity towards RhoA <i>in vitro</i>. </p><p>The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids ⁴²⁴DALDL⁴²⁸ on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS <i>in vivo</i>.</p>

<p>The kidney is the major organ responsible for the regulation of the composition and volume of the body fluids, which is essential for homeostasis. The glycosaminoglycan hyaluronan (HA), with extreme water-binding capacity, is present in the interstitium of the kidney with a heterogenous distribution. The importance of HA in renal water-handling is unknown and was the focus of the present investigation.</p><p>Acute water-loading in rats caused the amount of papillary HA to increase and during water deprivation, the amount was reduced. Gerbils, with extreme urine concentrating capacity, have less HA in the renal papilla in normal conditions and responded diametrically different to water-loading (reduction in HA). Renomedullary interstitial cells (RMICs), which are probably the main producers of HA in the renal medulla, were cultured at different media osmolalities to mimic the milieu of the medulla during variations in the water balance. The amount of HA found in the media was decreased at high osmolalities and increased at low osmolalities, thereby strengthening the <i>in vivo</i> results. CD44, an HA-receptor involved in the uptake and degradation of HA, was expressed on RMICs in an osmolality dependent manner. During high media osmolality, the CD44 expression increased and at lower osmolalities, the opposite occurred, probably due to the need for uptake and degradation of HA.</p><p>Renal ischemia-reperfusion injury causes a cortical accumulation of HA, up-regulation of CD44, and a depression of functional parameters. The time periods of ischemia correlated with the accumulation of HA which, in turn, was inversely correlated to GFR. Hyaluronidase injections in this setting failed to reduce HA levels and significantly improve renal function.</p><p>In conclusion, the results from the present study suggest an important role for HA and RMICs in renal water-handling and that the intrarenal distribution of HA is altered after ischemia-reperfusion injury, which correlates with renal dysfunction.</p>

<p>The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important.</p><p>In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect.</p><p>The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP >> 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. </p><p>In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells. </p>