Rozprawy doktorskie na temat „S100 proteins”

The S100 family of calcium binding proteins exhibits a unique pattern of cell type specific expression. These proteins are found in the cytoplasm and/or nucleus of a variety of cells, and involved in the control of a wide range of cellular processes such as cell cycle progression and differentiation. S100A4 and S100A6 are members of the S100 protein family that interact with several molecular targets including the heavy chain of non-muscle myosin IIA (NM IIA) and annexin II, respectively. NM IIA is a major actin-associated motor protein, which is involved in cell motility and cytokinesis. Assembly/disassembly of myosin filaments is primarily controlled by myosin light chain phosphorylation. However, small calcium-binding proteins of the S100 family also play an active role in the dynamics of actin-myosin filaments, leading to an increase in the dissemination of tumour cells. Accordingly, the main aim of this work was to study the molecular mechanism underlying S100A4/A6 function in epithelial mesenchymal transition (EMT) and provide in vivo data highlighting their role in the regulation of myosin dynamics. Intriguingly, we employed a novel transition electron microscopy approach to study the function of non-muscle IIA isoforms and their interactions with S100A4/A6 in A431/ZEB2 cells undergoing an EMT. Our data confirmed that both 6S and 10S myosin isoforms do exist in cells and directly interact with S100A4/A6 in vivo. Depletion of S100A4 resulted in the disappearance of the peaks corresponding to monomeric myosin indicating that S100A4 is required for balancing monomer-polymer equilibrium in cells. In blot overlay, both S100A4 and S100A6 showed similar binding site on myosin fragment 4 (C-terminus). However, a new S100A6 binding site was mapped on myosin heavy chain represented in M53 fragment which is a part of rod domain. In addition to the solubility of myosin in high ionic buffer, S100A4 and S100A6 are able to solubilise the myosin which was measured by the turbidity assay. Moreover, a decrease in ATPase activity of actomyosin complex in cells undergoing EMT was observed in the presence of S100A4/A6. In conclusion: This study shows that S100A4/A6 protein interacts with NM IIA. There is no redundancy and both proteins promote myosin dynamics, cell migration and invasion. S100A4 and S100A6 are up-regulated by ZEB2 and is implicated in the dynamic regulation of myosin filaments by switching the balance towards monomeric myosin.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death worldwide with the survival rate less than 5% because of late diagnosis. Development of PC is complex, it is promoted by the tumour microenvironment and often accompanied by inflammation. Epithelial mesenchymal transition (EMT) is an embryonic genetic program reactivated in cancer. EMT is implicated in the escape from senescence, tumour cell invasiveness, cancer metastasis, and drug resistance. EMT encompasses global reorganisation of the gene expression profiles, loss of epithelial markers and activation of mesenchymal genes. Among the genes affected during EMT are those coding for the members of the S100 protein family. Regulation and function of these genes in PC are, however, insufficiently studied. To link S100 proteins with EMT programs in PC, expression of eleven S100 proteins, a number of epithelial and mesenchymal markers, and several EMT-inducing transcription factors was analysed in a panel of the PC cell lines and clinical samples. I found that two S100 family members, namely S100A4 and S100A6 are induced during EMT in PC. In contrast, S100A14 expression was repressed by EMT-inducing transcription factors. Consistent with this observation, S100A4&A6 and S100A14 respectively activated and repressed invasion of PC cells in zebrafish xenografts. Exosomes isolated from the actively migrated MIA PaCa-2 cells contained high levels of S100A4&A6 proteins, stimulated invasion of the slowly migrating BxPC-3 cells. In a cohort of PC samples, it was observed a trend towards enhanced expression of S100A4 protein in most aggressive tumours. The mechanism of S100A4 activation was investigated in PC. Our data show that in the course of an EMT” mesenchymal” genes, S100A4 and S100A6 are induced via IL-6 and IL-11/STAT3 pathway. Treatment of cells with the STAT3 inhibitor, Stattic, inhibited expression of these genes, and blocked cell invasion in zebrafish xenografts. It has been proposed that uncoupling inflammatory IL/STAT3 signalling from the activation of S100A4 and S100A6 genes may decrease the mortality rate of PC patients.

Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

The multigene family of Ca2+-binding S100 proteins comprises 22 members that have various important intra- and extracellular roles. The three inflammation-associated members of this family???S100A8, S100A9 and S100A12 (collectively termed "calgranulins")???are constitutive neutrophil and monocyte proteins also expressed by macrophages within acute and chronic inflammatory lesions, but not in tissue macrophages. They are expressed in human/murine wounds and by appropriately activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The " calgranulins" are implicated in leukocyte activation/deactivation, fatty acid transport, leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound healing, protection against oxidants and antibacterial defence. Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with important regulatory immune-inflammatory functions in wound healing, tissue remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their synthetic repertoire are modulated by various factors including extracellular matrix components, growth factors, prostaglandins, reactive oxygen species and cytokines. Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-? (TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ? (TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2 (25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36 hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts. Dexamethasone (10???6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9 mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression depended on de-novo protein synthesis and was partially mediated by the mitogenactivated protein kinase pathway of activation. Preliminary promoter deletion analyses indicated that FGF-2-responsive elements in the gene promoter were distinct from those responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8- positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also regulate intracellular redox equilibrium and antioxidant defence. Atherosclerosis is a progressive chronic disease with complex aetiology and pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in plaques of ApoE???/??? mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis presents novel evidence that, in contrast to the murine ApoE???/??? model, the three human " calgranulins" were expressed in human atherosclerotic plaques, but not in normal arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9 staining was also evident on the extracellular matrix. Patterns of expression of S100A8, S100A9 and S100A12 were overlapping in serial sections, except that only smooth muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather than passive protein uptake. Western blotting of plaque extracts revealed monomeric S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction, suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an in-vitro system, molar ratios of HOCl of >1 generated stable complexes of S100A8 and S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in plaque, levels of oxidised proteins in plaque are likely to be significantly greater than described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers. However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and extracellular matrix components may contribute to plaque pathogenesis. These studies support key roles for the " calgranulins" in chronic inflammation, wound healing and atherogenesis possibly by regulating cellular differentiation, activation and modulation of redox-dependent mechanisms.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2019
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
The human host is continually exposed to potentially harmful organisms and the innate immune response is the first line of defense against microbial invasion. One strategy employed by the human innate immune system includes the release of antimicrobial host-defense proteins (HDPs). The goal of this thesis is to understand the antimicrobial functions of four host-defense proteins of the S100 family of proteins: calprotectin (CP), S100A12, S100A7, and S100A15. In the first half of this thesis, we elucidate the Zn(lI)-binding and antimicrobial properties of S100A12 and S100A7 through the use of solution and microbiology studies. We evaluate the affinity of S100A12 for Zn(ll), the scope of its antimicrobial activity, and put forward a model whereby S100A12 uses Ca(ll) ions to tune its Zn(Il)-chelating properties and antimicrobial activity. Our work with S1 00A7 demonstrates that the protein may exist in more than one redox state under physiological conditions, and that unlike CP and S100A12, the antimicrobial properties of S100A7 are not directly modulated by Ca(ll) ions. We report a model whereby the local redox environment of S100A7 tunes its Zn(ll)-sequestration capacity through intramolecular disulfide-bond redox chemistry, and that Ca(II) ions exert an indirect modulatory effect on the Zn(Il)-binding properties of this protein. In the second half of this thesis, we examine the bactericidal properties of the four S100 proteins. Our results agree with prior work on the bactericidal properties of S100A7. Furthermore, we show that CP and S100A15, but not S100A12, possess bactericidal activity at pH 5, and that CP is a broad-spectrum Gram-negative bactericidal factor that functions through a mechanism of membrane permeabilization. Taken together, our studies provide new insights into the multifunctionality of the antimicrobial S100 HDPs.
by Lisa Stephanie Cunden.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemistry

Kinesiology
Ph.D.
In this prospective longitudinal investigation of Division-I collegiate football players, the acute and longer-term effects of repetitive subconcussive impacts on plasma S100β and PINCH levels and concussion-related symptom score were examined. The first aim was to investigate the acute repetitive subconcussive impact effect by comparing the biomarker levels at pre and post full-gear practice, followed by examining the relationship of head impact magnitude and frequency of on acute increases in S100β and PINCH levels and symptom score. Hypotheses for the first aim were that there would be acute increases in plasma S100β and PINCH levels, but no change would be observed in symptom score. A significant relationship between subconcussive impact kinematics and acute changes in outcome measurements would be observed only in S100β. The second aim was to examine the longer-term effect of subconcussive effects on plasma S100β and PINCH levels as well as symptom score compared to the pre-season baseline. It was hypothesized that the players who sustained high frequency and magnitude of subconcussive impact would induce chronically high levels of plasma PINCH compared to the baseline. However, chronic effect would not be found in plasma S100β and symptom score. Independent variables were time (pre vs. post-practice), days (baseline, 1st Pads-OFF, 1st Pads-ON, 2nd Pads-ON, 3rd Pads-ON, 4th Pads-ON, and post-season), and group (higher vs. lower impact group). Dependent variables were the plasma expression of S100β and PINCH and symptom scores at each time point, pre-post differences in the plasma expression of S100β and PINCH and symptom scores, and head impact kinematics (frequency, sum of peak linear and rotational acceleration). This prospective observational study of 22 Division-I collegiate football players included pre-season baseline, pre-season practices [1 helmet-only and 4 full-gear], and post-season follow-up. Acute subconcussive effects were examined using the data from the first full-gear practice. Cumulative subconcussive effects were examined across the study duration (total 12 time points per player). Blood samples and self-reported symptom scores were obtained and blood biomarkers were assessed for pre-post practices and pre-post season. Plasma S100β expression level was assessed using a sandwich-based enzyme-linked immunosorbent assay. Plasma PINCH expression level was assessed using western blot analysis. An accelerometer-embedded mouth guard was employed to measure impact kinematics including number of impacts (hits), peak linear acceleration (PLA), and peak rotational acceleration (PRA). For examining cumulative effects, based on the previously established cut-off value of 173.5 g, players who were exposed average impact magnitudes below 173.5 g per practice were categorized into lower (n = 8) or greater than 173.5 g were categorized into higher (n = 14) impact groups. Data analysis consisted of descriptive and inferential statistics. Student’s t-tests were used to assess group differences in demographic and head impact kinematic data, acute effects using pre-post practice change in concussion-related symptom scores and biomarker levels, and longer-term effects using pre-post season change in concussion-related symptom scores and biomarker levels. Pearson r correlations were used to examine potential relationship between acute increase in outcome measures and head impact kinematics data. Two-way repeated measures ANOVAs were used to identify cumulative subconcussive effects over time in concussion-related symptoms scores and biomarker levels. If necessary, one-way ANOVA as a function of group was used to identify where cumulative effect began compared to the baseline, using Dunnett’s host-hoc correction. The alpha level was set at p < 0.05. A total of 721 head impacts were recorded from the 22 players during the 5 training camp practices. There were significant differences in head impact kinematics per practice between lower and higher impact groups [number of impacts per practice, 1.3 vs. 10.0 (p < .001); linear acceleration, 36.4 vs. 285.6 g (p < .001); rotational acceleration, 2,048.4 vs. 16,497.31 rad/s2 (p < .001), respectively]. There were no changes in self-reported concussion symptoms across the study duration. While there was no change in longer-term effect between pre-season baseline and post-season follow-up in plasma S100β level, robust and acute increase was observed in post-full gear practice (0.111 + 0.01 ng/ml) compared to pre-practice S100β level, (0.048 + 0.01 ng/ml; p < .0001). The acute increase in plasma S100β was significantly and positively correlated to the number of hits (r = 0.636, p = 0.001), sum of peak linear acceleration (r = 0.570, p = .006), and sum of peak rotational acceleration (r = 0.655, p = 0.001) sustained. For plasma PINCH level, there was a 4-fold increase at post-practice compared to that of pre-practice (p = .037), indicating the acute effect of subconcussive impacts. However, the acute increase in plasma PINCH level was independent from frequency and magnitude of impacts sustained, demonstrated by no statistically significant correlations with the number of hits (r = 0.222, p = .333), sum of peak linear acceleration (r = 0.289, p = .204), and sum of peak rotational acceleration (r = 0.297, p = .191). When players were categorized into the lower and higher impact groups and assessed across the 5 training-camp practices, consistently higher levels of plasma S100β and PINCH were found only in the higher impact group at post-practice compared to the baseline. However, plasma level of S100β and PINCH at pre-practice remained stable from the baseline, suggesting the absence of chronic effect from repetitive head impacts. When season-long effects on plasma S100β and PINCH levels were examined, 10 out of 16 players showed increase in plasma PINCH level at post-season compared to the baseline (p = .039) while no significant difference in plasma S100β level. Results from the current study suggest that subconcussive head impacts do not exert self-claimed concussion-related symptoms; however, blood biomarkers detected noticeable acute changes following repetitive subconcussive impacts. Plasma level of S100β protein can be a potential diagnostic measurement to track acute brain burden, and plasma level of PINCH protein may be reflective of the longer-term cumulative brain damage from repetitive head impacts.
Temple University--Theses

As ações da atividade física na neuroquímica dos neurônios, com enfoque às proteínas ligantes de cálcio (Ca2+), e o estado de ativação de células gliais da medula espinal do rato foram investigadas em preparados imuno-histoquímicos através da análise morfométrica e microdensitométrica com auxílio do computador. Ratos machos adultos foram divididos em dois grupos: treinado, cujos animais foram expostos à roda de corrida onde realizava atividade física espontânea, por um período de 4 e 14 noites; e sedentário, onde os animais foram mantidos em caixas individualizadas, sem a roda de corrida. Após os períodos determinados, os animais sofreram eutanásia e suas medulas espinais foram processadas para imunohistoquímica. Os ligantes de Ca2+ neuronal e glial foram avaliados pela imunorreatividade das proteínas calbindina e parvalbumina e, ainda, pela imunorreatividade da proteína S100 astrocitária. A atividade física voluntária promoveu uma diminuição na imunorreatividade da proteína calbindina em nível torácico no corno posterior (lâminas I e II de Rexed), assim como no núcleo espinal lateral após 14 dias. No nível lombar, também se observou uma diminuição da imunorreatividade no corno posterior (lâminas I e II de Rexed). Contudo os animais submetidos à atividade física voluntária por 4 dias apresentaram um aumento na área imunorreativa da proteína parvalbumina em relação ao seu controle. Efeito semelhante ocorreu no núcleo dorsal nos grupos que treinaram por 4 e 14 dias. Entretanto, no fascículo cuneiforme ocorreu uma diminuição da imunorreatividade à parvalbumina. Já em relação à proteína S100, os animais treinados apresentaram um aumento na imunorreatividade (spMGV) no corno anterior. Assim, conclui-se que a atividade física voluntária modificou a imunorreatividade das proteínas ligantes de Ca2+ na medula espinal, o que pode estar associado aos mecanismos de ativação intracelular realizados pelo cálcio, bem como a liberação de neurotransmissores na fenda sináptica.
Actions of the physical activity in the neurochemistry focuzing calcium-bindin proteins and the activation of the glial cells in the spinal cord of the rat were investigated with imunohistochemistry over. Male wistar adult rats were divided in two groups: trained, which animals exercised in the wheel running for 4 and 14 nigths; and sedentary, which animals were maintained in private box without wheel running. After that period rats were sacrificed and their spinal cords were processed to imunohistochemistry. Calcium-bindin proteins neuronal (parvalbumin and calbindin) and glial (S100) were evaluted. The activity promoted a decrese in the imunoreativite of the calbindin protein in the torácic level of the posterior horn (lamina I and II of Rexed), and lateral spine nucle after 14 days. In the lombar level, decrese in the posterior horn was also found. Animals submited to physic activity for 4 days showed an increased in the imunoreatived area of parvalbumin. Similar effect was observed all of groups that were treineds for 4 e 14 days. However, in the cuneiforne fascicule, parvalbumin decreased. The S100 protein showed decresed in the anterior horn. In conclusion volunteer phisical activity changed the pattern of the calcium-bindin protein immunoreactivity in the spinal cord, effect than can be associated to neuroplasticity.

Die Interaktion von S100-Proteinen mit dem Rezeptor für advanced glycation endproducts (RAGE) wird als hoch relevant bei der Entstehung, Manifestation und Progression verschiedener entzündlicher Erkrankungen sowie bei der Tumorigenese gewertet. Das tiefergehende Verständnis der Interaktion von S100-Proteinen mit RAGE in vivo stellt eine wissenschaftliche Herausforderung dar und ist ein Ansatz für therapeutische Interventionen. Darüber hinaus stellen Untersuchungen zum Metabolismus von extrazellulär zirkulierenden S100-Proteinen in vivo einen vielversprechenden Forschungsansatz zur Analyse von S100-Protein-assoziierten Erkrankungen dar. Die einzigartigen Eigenschaften der Positronen-Emissions-Tomographie (PET) als nicht-invasives bildgebendes Verfahren erlauben die Darstellung und quantitative Erfassung biochemischer Prozesse mit der Möglichkeit zelluläre und molekulare Reaktionswege aufzuzeigen sowie in vivo-Mechanismen von Krankheiten im Kontext eines physiologischen Umfeldes darzulegen. Ziel der vorliegenden Arbeit war es, Fluor-18-markierte S100-Proteine (18F-S100) herzustellen, diese biochemisch, radiochemisch und radiopharmakologisch zu charakterisieren und deren Metabolismus und Interaktion mit RAGE in vivo mittels Kleintier-PET am Tiermodell zu untersuchen. Es wurden die mit RAGE interagierenden S100-Proteine S100A1, S100A12 und S100B in biologisch funktioneller Form hergestellt. Dazu wurden die entsprechenden S100-Gene in den prokaryotischen Expressionsvektor pGEX-6P-1 kloniert. Mit diesen Konstrukten wurden E. coli-Zellen transformiert, aus denen nachfolgend die S100-Proteine isoliert und gereinigt werden konnten. Es konnte eine Reinigung unter nativen, milden Bedingungen etabliert werden, die es ermöglichte, S100A1, S100A12 und S100B in biologisch aktiver Form und in hohen Reinheitsgraden (&gt; 95%) für die nachfolgenden Experimente bereitzustellen. Diese S100-Proteine wurden über den 18F-tragenden Aktivester N-Succinimidyl-4-[18F]fluorbenzoesäure ([18F]SFB) radioaktiv markiert und charakterisiert. Dabei konnte sichergestellt werden, dass die 18F-S100-Proteine in vitro und in vivo stabil sind. Weiterhin konnte nachgewiesen werden, dass die radioaktive Markierung keine Beeinträchtigung auf die biologische Funktionalität der S100-Proteine hat. Dies wurde anhand von sRAGE-Bindungsuntersuchungen sowie Zell-Interaktionsuntersuchungen an konfluenten Endothelzellen (HAEC) und an zu Makrophagen differenzierten THP-1-Zellen (THP-1-Makrophagen) verifiziert. Für die Untersuchung der RAGE-Bindung war die Produktion des löslichen sRAGE bzw. die Generation von flRAGE-berexprimierenden Zellen erforderlich. Beide Konstrukte wurden in geeigneten Zellsystemen exprimiert und das sRAGE-Protein wurde in biologisch aktiver Form synthetisiert und gereinigt (Reinheitsgrad &gt; 97%). Die 18F-S100-Bindung an THP-1-Makrophagen und HAEC wurde in Gegenwart von glykierten LDL (glykLDL) sowie sRAGE signifikant inhibiert, was auf eine RAGE-Interaktion hinweist. Weiterhin konnten durch den Einsatz von Scavenger-Rezeptor-Liganden, wie z. B. Maleinanhydrid-modifiziertes BSA (malBSA) bzw. von Lektinen inhibierende Effekte erzielt werden. Dies ist ein Indiz für die 18F-S100-Interaktion mit Scavenger-Rezeptoren und Glykokonjugaten an der Zelloberfläche. Durch die Untersuchungen mittels konfokaler Laserscanning-Mikroskopie an THP-1-Makrophagen wurde eine Zellaufnahme des Fluoreszein-markierten S100A12 festgestellt. Weiterhin konnten Kolokalisationen mit Lektinen detektiert werden. Das metabolische Schicksal extrazellulär zirkulierender 18F-S100-Proteine in vivo wurde mit Hilfe dynamischer PET-Untersuchungen bzw. anhand von Bioverteilungs-Untersuchungen in männlichen Wistar-Ratten analysiert. Die Hauptakkumulation der Radioaktivität wurde in der Leber und in den Nieren detektiert. In diesen Organen findet der Metabolismus bzw. die glomeruläre Filtration der 18F-S100-Proteine statt. In den Untersuchungen zur Genexpression mittels Echtzeit-PCR sowie im immunchemischen Proteinnachweis am Western Blot wurde eine hohe Expression und Proteinbiosynthese des RAGE in der Lunge ermittelt. Die Lunge eignet sich daher als „Referenz“-Organ für eine funktionelle in vivo-Charakterisierung von RAGE mit 18FS100-Proteinen. Bei den durchgeführten PET-Untersuchungen konnte eine temporäre 18F-S100-Interaktion mit dem Lungengewebe festgestellt werden. Die Retention des 18FS100A12 in der Lunge wurde in Gegenwart von sRAGE inhibiert. Dies ist ein Hinweis dafür, dass 18F-S100-Proteine auch in vivo an RAGE binden können. Die Radioaktivitäts-Akkumulation in den Organen Leber und Milz, die eine Vielzahl von sessilen Makrophagen aufweisen, wurde durch die Applikation von malBSA inhibiert. Dies ist ein Indiz dafür, dass 18F-S100-Proteine in vivo mit Scavenger-Rezeptoren interagieren können. Die vorliegende Arbeit liefert deutliche Hinweise darauf, dass RAGE nicht der alleinige Rezeptor für 18F-S100-Proteine ist. Der Einsatz von 18F-S100-Proteinen als experimentelles Werkzeug in dynamischen PET-Untersuchungen birgt das Potential einer Charakterisierung von S100-Protein-assoziierten, pathophysiologischen Prozessen
Members of the S100 family of EF-hand calcium binding proteins play important regulatory roles not only within cells but also exert effects in a cytokine-like manner on definite target cells once released into extracellular space or circulating blood. Accordingly, increased levels of S100 proteins in the circulating blood have been associated with a number of disease states, e.g., diabetes, cancer, and various inflammatory disorders. As the best known target protein of extracellular S100 proteins, the receptor for advanced glycation endproducts (RAGE) is of significant importance. However, the role of extracellular S100 proteins during etiology, progression, and manifestation of inflammatory disorders still is poorly understood. One reason for this is the shortage of sensitive methods for direct assessment of the metabolic fate of circulating S100 proteins and, on the other hand, measurement of functional expression of extracellular targets of S100 proteins, e.g., RAGE in vivo. In this line, small animal PET provides a valuable tool for noninvasive imaging of physiological processes and interactions like plasma or vascular retention, tissue-specific receptor binding, accumulation or elimination in vivo. To address this question, human S100 proteins were cloned in the bacterial expression vector pGEX-6P-1, expressed in E. coli BL21, and purified by affinity chromatography and anion exchange chromatography. Purified S100A1, S100B and S100A12 proteins were then radiolabeled with the positron emitter fluorine-18 (18F) by N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabeling of S100 proteins resulted in radiochemical yields of 3-10% (corrected for decay) and effective specific radioactivities of 1 GBq/µmol, respectively. For investigations about RAGE binding soluble RAGE (sRAGE) was expressed and purified using pSecTag2B. A radioligand binding assay confirmed specific binding of 18F-S100A12, 18F-S100A1, and 18F-S100B to immobilized sRAGE, also showing an order of affinity with S100A12 &gt; S100A1 &gt; S100B. These results indicate that radioactive labelling of S100 proteins did not affect their overall affinity to RAGE. Cellular association studies in human THP-1 macrophages and human aortic endothelial cells (HAEC) showed specific binding of all 18F-S100 proteins to the non-internalizing RAGE as confirmed by inhibitory effects exerted either by other RAGE ligands, e.g., glycated LDL, or by soluble RAGE. Of interest, 18F-S100 proteins were also shown to interact with other putative binding sites, e.g. scavenger receptors as well as proteoglycans. In this line, uptake of 18F-S100 proteins in THP-1 and HAEC could be inhibited by various scavenger receptor ligands, in particular by maleylated BSA as well as by lectines (e.g. ConA and SBA). Confocal laser scanning microscopy analysis showed a major part of the fluoresceinated S100A12 bound to the surface of THP-1 macrophages. Beyond this, uptake of S100A12 could be determined indicating an interaction of S100A12 with both non-internalizing, e.g., RAGE, and internalizing receptors, e.g. scavenger receptors. By evaluation of the relative contribution of 18F-S100A12 association to RAGE-overexpressed CHO cells (using pIres2-AcGFP1), 18F-S100A12 showed a significantly higher association to CHO-RAGE cells compared with CHO-mock cells. Based on these findings and due to their crucial role in inflammatory disorders the metabolic fate of S100 proteins was further investigated in dynamic small animal Positron emission tomography (PET) studies as well as in biodistribution studies in Wistar rats in vivo. For interpretation of in vivo investigations in rats, expression of RAGE was analyzed by quantitative real time RT-PCR as well as western blotting in various organs. Lung tissue expressed the highest level of RAGE protein compared to the other tissues. PET studies in rats revealed a comparatively long mean residence time of circulating 18F-S100 proteins. A major contributor to this phenomenon seems to be a sustained temporary interaction with tissues overexpressing RAGE, e.g., the lung. On the other hand, renal clearance of 18F-S100 via glomerular filtration is a major elimination pathway. However, scavenger receptor-mediated pathways in the liver, the spleen and, to a minor extent, in the kidneys, also seem to contribute to the overall clearance. The presence of sRAGE revealed a decreased retention of 18F-S100A12 in the lung, indicating in vivo binding to RAGE. In vivo blocking studies using maleylated BSA demonstrated a strong inhibition of putative binding sites in rat tissues enriched in cells expressing scavenger receptors like liver and spleen. In conclusion, 18F-labeling of S100 proteins and the use of small animal PET provide a valuable tool to discriminate the kinetics and the metabolic fate of S100 proteins in vivo. Furthermore, the results strongly suggest an involvement of other putative receptors beside RAGE in distribution, tissue association and elimination of circulating proinflammatory S100 proteins. Moreover, the approach provides novel probes for imaging of functional expression of RAGE and scavenger receptors in peripheral inflammatory compartments

Diabetes melito ou intolerância à glicose estão presentes em até 80% dos pacientes com adenocarcinoma de pâncreas. Portadores desta neoplasia têm resistência à insulina e alteração na secreção de insulina em resposta à glicose, o que pode levar ao aparecimento ou piora de diabetes. Para identificar genes diferencialmente expressos, que podem representar fatores diabetogênicos produzidos pelo adenocarcinoma de pâncreas, utilizou-se a comparação de microarranjos de oligonucleotídeos hibridizados com RNA complementar (cRNA) de tumores pancreáticos de pacientes com e sem diabetes melito no pré-operatório. Uma lâmina foi hibridizada com cRNA de dois pacientes portadores de diabetes melito, e outra com cRNA de dois pacientes com tolerância normal à glicose pelo teste oral. Considerando a expressão ajustada para os controles internos dos microarranjos, 293 genes estavam duas ou mais vezes mais expressos na lâmina dos portadores de diabetes melito; destes, 25 genes estavam pelo menos cinco vezes mais expressos. Duzentos e noventa e sete genes estavam pelo menos duas vezes mais expressos na lâmina dos pacientes com tolerância normal à glicose, dos quais 54 genes estavam cinco ou mais vezes mais expressos nestes indivíduos. Dos genes mais expressos nos tumores dos indivíduos portadores de diabetes melito, três deles, FAM3D, do inglês Family with Sequence Similarity number 3 member D, neuropeptídeo Y (NPY), e proteína de ligação do cálcio S100A8, foram estudados por reação em cadeia da polimerase em tempo real. A expressão do FAM3D foi 4070 (1000-37588) nas amostras de tumores de pacientes com diabetes melito, contra 109 (10-1112) nas de pacientes não-diabéticos (com intolerância à glicose ou com tolerância normal à glicose) (p<0,05). A expressão do NPY foi 0,46 (0,19-0,91) nos tumores dos portadores de diabetes, contra 0,32 (0,21- 0,58) nos tumores dos não-diabéticos (p = NS). Quanto à expressão de S100A8, foi 0,52 (0,27-0,60) nos tumores dos diabéticos, e 0,34 (0,16-1,44) nos não-diabéticos. Estudo imunohistoquímico mostrou que o FAM3D está expresso no núcleo e no citoplasma de células de tumores pancreáticos, tanto de indivíduos com diabetes melito quanto de não-diabéticos, assim como no citoplasma de células de ilhotas pancreáticas e de células ductais normais do pâncreas. Concluímos que o FAM3D é uma proteína expressa em tecido pancreático normal e tumoral, e que existe maior conteúdo do mRNA do FAM3D nos adenocarcinomas de pâncreas de portadores de diabetes melito do que nos de não-diabéticos.
Pancreatic ductal adenocarcinoma is closely related to diabetes mellitus; up to 80% of pancreas adenocarcinoma patients have diabetes or impaired glucose tolerance. Pancreas adenocarcinoma patients have both insulin resistance and altered insulin secretion in response to glucose, and impaired glucose metabolism has been reported in muscle of tumor patients, involving glycogen metabolism and post-receptor insulin signaling. But despite progress in research about this issue, precise mechanisms responsible for the interaction of pancreatic adenocarcinoma and diabetes mellitus remain unknown. The aim of this study was to identify differentially expressed genes between pancreas adenocarcinoma of patients who had and who did not have diabetes mellitus before surgery. Clinical and laboratorial data of 33 patients with pancreatic adenocarcinoma were evaluated, and tumor gene expression was analyzed by microarray method between two patients who had diabetes mellitus and two who did not have glycemic homeostasis impairment, and later used quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in twelve tumor fragments mRNA to confirm obtained data. Pancreatic adenocarcinoma patients who had diabetes mellitus had higher HOMA-IR (p < 0.05) and a trend to lower HOMA-beta indexes than non-diabetic patients. icroarray revealed 293 genes twice more expressed in the pool of diabetic patients as compared to the pool of normal glucose tolerance patients. Of these, 25 were five times more expressed in diabetic patients? pancreatic adenocarcinomas. Three genes were chosen for RT-qPCR: Family with Sequence Similarity number 3 member D (FAM3D), neuropeptide Y (NPY), and calcium-binding protein S100A8. FAM3D expression was 4070 (1000-37588) in diabetic patients tumors versus 109 (10-1112) in non-diabetic (impaired glucose and normal glucose tolerance) patients? tumors (p<0.05). NPY expression was 0.46 (0.19- 0.91) in diabetic patients and 0.32 (0.21-0.58) in non-diabetic patients? tumors (p=NS). Calcium-binding protein S100A8 expression was 0.52 (0.27-0.60) in diabetic and 0.34 (0.16-1.44) in non-diabetic patients (p=NS). Immunohistochemistry revealed that FAM3D protein was expressed in pancreatic adenocarcinoma cells in a diffuse nuclear and cytoplasmic pattern. It was also expressed in the cytoplasm of islets of Langerhans and normal pancreatic ducts cells. The present study indicates that cytokine-like FAM3D protein is expressed in normal and tumoral pancreatic tissue, and that FAM3D mRNA content is higher in pancreatic adenocarcinoma in diabetic than in non-diabetic patients.

S100B-proteiini on gliasoluspesifinen proteiini, jota vapautuu verenkiertoon aivovaurioiden yhteydessä. Kohonneet pitoisuudet korreloivat aivovaurion vaikeusasteen ja lopputuleman kanssa. S100B-proteiini toimii myös merkkiaineena veri—aivoesteen toimintahäiriöille. Lapsilla kohonneet proteiinipitoisuudet on liitetty astrosyyttivaurion markkereiksi jopa lievän aivovamman jälkeen. On mahdollista, että kuumekouristusten yhteydessä seerumin ja likvorin S100B-proteiinipitoisuudet nousevat ollen merkkinä aivoissa käynnissä olevasta patologisesta prosessista ja ennustavat kohtausten uusiutumistodennäköisyyttä. Tässä tutkimuksessa mittasimme seerumin ja likvorin S100B-proteiinipitoisuudet 103 lapselta, jotka olivat sairastaneet ensimmäisen kuumekouristuksensa. 33 lapsipotilasta, joilla oli akuutti kuumeinen infektio ilman kouristuksia, toimivat kontrolleina seerumipitoisuuksien suhteen. Kuumekouristajapotilailla S100B-proteiinipitoisuuksien keskiarvo likvorissa oli 0,21 μg/l ja seerumissa 0,12 μg/l. Kontrollipotilailla vastaava seerumipitoisuuksien keskiarvo oli 0,11 μg/l (ero 0,01 μg/l, 95 %:n luottamusväli -0,02—0.04 μg/l, P = 0,46). Seerumin S100B-proteiinipitoisuus oli riippuvainen potilaiden iästä (r = -0,28, P = 0,008) alle neljävuotiailla lapsilla. Pitoisuus ei kuitenkaan ennustanut kuumekouristuksen vaikeusastetta eikä kohtauksen uusiutumista. Aikaviiveellä kuumekouristuskohtauksen alkamisesta sairaalaan saapumiseen ei ollut korrelaatiota S100B-pitoisuuksiin seerumi- (r = -0,130, P = 0,28) eikä likvorinäytteissä (r = -0,091, P = 0,52). Tutkimustuloksemme tukevat aiempaa käsitystä siitä, että kuumekouristukset ovat vaarattomia kehittyville aivoille.

Les annexines (ax) sont des proteines liant les phospholipides charges negativement en presence de ca2+ et impliquees dans des processus de transport membranaire comme les phenomenes d'agregation precedant la fusion des membranes. Plusieurs annexines peuvent s'associer avec des membres de la famille des proteines s100, petites proteines de 10 kd de poids moleculaire formees d'une paire de mains ef, un motif de liaison au ca2+ en helice-boucle-helice. Ainsi, l'annexine i se lie a la s100c en presence de ca2+ et l'annexine ii a la p11. Cette derniere est insensible au ca2+. Les proteines s100 forment generalement des homodimeres tres stables. Les etudes biochimiques de la liaison de la p11 et de la s100c avec des peptides derives respectivement de l'axii et de l'axi, ont montre que la region des annexines necessaire a la liaison aux proteines s100 se limite a la partie n-terminale, predite en helice alpha amphiphile, et que la n-acetylation du premier residu est determinante. Les structures des complexes entre la p11 et le peptide correspondant a la region n-terminale de l'axii d'une part et de la s100c avec la region n-terminale de l'axi d'autre part, ont ete determinee par cristallographie des rayons x. Les structures des deux complexes sont tres similaires, ce qui montre que la p11 mime une structure de proteine s100 chargee en ca2+. Les peptides se logent dans un sillon hydrophobe a la surface des proteines s100, forme par la region reliant les deux mains ef et l'helice c-terminale d'une molecule du dimere et l'helice n-terminale de l'autre molecule. L'etat dimerique des proteines s100 est donc necessaire a la fixation du ligand. Le groupement acetyle des peptides vient renforcer la structure helicale. Des contacts specifiques s'etablissent entre deux residus glu conserves dans la p11 et la s100c et les nh peptidiques en position 2 et 3 du peptide ainsi que la chaine laterale en position 4 qui est une his dans la p11 et une ser dans la s100c. Les types d'interaction dans le site de fixation sont donc tres conserves. Ces structures constituent la base d'un travail de modelisation moleculaire de l'heterotetramere (axii)2-(p11)2 les modeles obtenus sont compatibles avec les donnees de la microscopie electronique et d'experiences de diffusion des rayons x aux petits angles et pourraient constituer des modeles de jonction membranaire.

S100B is a member of the S100 calcium binding protein family and is highly expressed within astrocytes in the brain. Elevated levels of S100B are associated with brain and central nervous system disorders, due to the breakdown of the blood brain barrier. Therefore S100B is routinely used as a marker of disease. Traditionally S100B was thought only as a cell breakdown product but increasing evidence suggests that it may play a role in exacerbating inflammation, however this role is not clear. S100B is known to be present within the eye but its role in retinal inflammation has not been investigated. The aim of this project was therefore to examine the role of S100B using the animal model experimental autoimmune uveoretinitis (EAU). This is a well-established model for the sight-threatening human condition posterior endogenous uveoretinitis. In this disease model an autoimmune response is induced leading to retinal inflammation. Using S100B knockout mice, I have shown a significantly reduced level of disease, as determined by clinical and histological grading. Real-time PCR array analysis of diseased matched retinas indicated down regulation of cytokines and chemokines in S100B knockout mice. In vitro experiments on a macrophage cell line confirmed S100B to have a pro-inflammatory effect on macrophages, the main effector cell in EAU, with up-regulation of cytokine and chemokine expression. In particular IL-1β, CCR1 and CCL22 showed a marked increase in gene expression in response to S100B which was confirmed by real-time PCR. Increased protein production of IL-1β (pro-form), CCR1 and CCL22 was also confirmed. S100B inhibited activation of T cells separated from spleens, as shown by reduced CD25+ expression and IL-2 production. IFN-γ and IL-17 production however was not affected. CCL2 and IL-6 are main inflammatory mediators produced by retinal pigment epithelial cells which are known to be elevated during retinal inflammation. S100B promoted CCL2 and IL-6 production in retinal pigment epithelial cells at different concentrations. The work carried out in this thesis provides additional understanding of the actions of extracellular S100B on immune system cells and its potential role in posterior uveitis.

Master of Science
Department of Biochemistry
Jianhan Chen
Paul E. Smith
Intrinsically disordered proteins have been shown to be important in many physiological processes, including cell signaling, translation, and transcription. They are also associated with cancer, and neurodegenerative diseases. The tumor suppressor p53 contains several disordered regions, including the C-terminal negative regulatory domain (NRD). In cancer the function of p53 has been shown to be repressed by S100B(ββ) binding to p53-NRD. Binding of S100B(ββ) blocks acetylation and phosphorylation sites in the p53-NRD, which leads to tetramer dissociation and prevents p53 activation. NMR studies have shown that p53-NRD binds S100B(ββ) in a stable α-helix conformation. Interestingly, despite the well-converged and apparent rigid nature of the NMR structure ensemble, a majority of intermolecular NOEs used to calculate the NMR ensemble are very weak (≥6 Å). The final NMR structures also contains unsatisfied buried charged residues at the binding interface. It’s plausible that the p53-S100B(ββ) complex is more dynamic than previously believed. The goal of the study is to determine the potential conformational heterogeneity in p53-S100B(ββ) complex using molecular modeling. For this, five diverse structures were selected from the 40-member NMR ensemble. For each initial conformation, we performed 100 ns molecular dynamic simulations in explicit solvent to explore the structure and dynamics of the p53-NRD in complex with S100B(ββ). Several analytical tools were used to characterize the p53-NRD conformation, including root-mean squared deviation (RMSD), root-mean squared fluctuation (RMSF), and residue helicity. The accuracy of the simulations was mainly assessed by comparing with experimental NOEs. The results show that, even though the ensemble is heterogeneous it satisfies 82% of the experimental NOEs. Clustering analysis further suggests that many conformational sub-states coexist for this complex, and individual clusters appear to satisfy only subsets of NOE distances. Importantly, the buried surface analysis demonstrates that the heterogeneous ensemble generated from MD provides similar shielding of key residues, which include post-translational modification residues needed for p53 activation. This study also demonstrates that atomistic simulations can provide important insights into structure and dynamics of IDPs for understanding their biological function.

Traumatic brain injuries are common (hospitalization incidence: 250-300 per 100.000 inhabitants/year) and a great majority of these injuries (80-85%) are classified as mild traumatic brain injury (MTBI/concussion). Many patients with MTBI (20-80%) suffer from subsequent persistent and often disabling symptoms. In previous studies serum levels of biochemical markers of brain tissue damage (S-100B and neuron-specific enolase, NSE) have been propounded to serve as predictors of persisting symptoms.In the present studies serum concentrations of S-100B, NSE and cortisol in acute phase and post-concussion symptoms, post-traumatic stress-related symptoms, disabilities and life satisfaction one year after the trauma, were investigated in 88 patients (53 men and 35 women) with MTBI. Serum concentrations of S-100B and NSE were also assessed in elite players (n=54) of typical contact sports (ice-hockey and soccer), which are known to be high risk activities with respect to head injury. Basketball players (n=18) were used as a control group. A majority of patients with MTBI showed higher serum concentrations of S-100B, NSE and cortisol on admission compared with a second blood sample obtained about 7 hours later (p<0.001 for all analyses). Sequelae were common one year after the injury. Postconcussion symptoms were encountered in 45 % of the patients, stress-related symptoms in 17 % and disabilities in 48 %, but only 3 patients (4 %) were on sick-leave on follow-up due to the head trauma. There was a statistically significant negative correlation between the total score of life satisfaction and the total score of disability (r= -0.514, p<0.001). Symptoms on admission (dizziness, nausea) and S-100B were statistically significantly associated with disabilities (p<0.024, multiple logistic regression analysis). Nausea on admission was also statistically significantly associated with life satisfaction (p=0.004). A statistically significant association was found only for S-100B with early (0-1 week postinjury, p=0.008) and only for cortisol with late (more than 52 weeks post-injury, p=0.022) post-traumatic stress-related symptoms. Concentrations of S-100B after game were statistically significantly increased in comparison with the levels before game (soccer, p<0.001; ice-hockey, p<0.001; basketball (p<0.001). Concentrations of NSE were only raised after soccer play (p<0.001). Increases in S-100-B (post-game minus pre-game values) were correlated to the number of jumps in basketball play (r=0.706, p=0.002). For soccer, increases in S-100B were correlated to the number of headers (r=0.428, p=0.02) and to the number of acceleration/deceleration events other than heading (r=0.453, p=0.02). The findings provide support for the idea that injury of brain tissue is involved in the genesis of persisting disabilities and long-term changes of life satisfaction in MTBI. Since S-100B increases in serum were correlated to the number of headers and since soccer play also increased serum levels of NSE (in contrast to ice hockey and basketball), it seems that heading may have an impact on brain tissue. The studies have also shown that ordinary playing of the team sports in question (i.e. soccer, ice hockey and basketball) increases S-100B serum concentrations, which has to be taken into consideration when S-100B is used for the detection of injury of brain tissue in sportsmen with acute/overt head trauma during sport practice. An analysis of the biochemical markers of brain damage (in particular S-100B) may be an additional source of valuable information in the management of patients and sportsmen with MTBI. S-100B also seems to be promising for the prediction of impairments and disabilities after MTBI.

S100A4 is a member of the S100 family of proteins and increases the motility of many cell types. This is also thought to explain its association with the epithelial-mesenchymal transition (EMT), a developmental program re-activated during tumourigenesis. Mechanistically, S100A4 interacts with a number of targets including Smad3 and liprin-β1; however, the best characterised is non-muscle myosin IIA (NMIIA) which regulates many aspects of the cytoskeleton. There is a large body of in vitro data indicating that S100A4 promotes the monomeric state of NMIIA; however, in vivo evidence for the interaction in cells is lacking. Accordingly, the first aim of this study was to determine if S100A4 interacts with, and promotes the monomeric state of NMIIA in A431 cells undergoing SIP1-induced EMT. Intriguingly, co-localisation analysis of S100A4 and NMIIA in A431-SIP1 cells using immunoelectron microscopy indicated that NMIIA is present in a folded, 10S state, and unfolded 6S state, and S100A4 interacts with both. This represents the first evidence of 10S and 6S states of NMIIA in non-muscle cells. In addition, FRAP analysis demonstrated that cells with attenuated expression of S100A4 turned over NMIIA with a slower rate, consistent with S100A4 promoting the monomeric state. The second part of the study explored the mechanism of the S100A4-NMMIA interaction. In vitro analysis of phosphomimetic S1916D and S1943D NMIIA showed no differences in binding affinity with S100A4 compared to WT NMIIA, contrary to the published literature. Based on the NMR structure of S100A4 and NMIIA, V77 and C81 were identified as key S100A4 residues that mediated the interaction with NMIIA. Mutation of these sites abolished the interaction with NMIIA, an effect reflected in null-phenotypes for both proteins when over-expressed in A431 cells compared to WT S100A4. In conclusion, this study suggests S100A4 is an important regulator of NMIIA dynamics in cells.

A S100B é uma proteína ligante de cálcio produzida e secretada por astrócitos e tem sido relacionada à interação neurônio-glia. Nosso primeiro objetivo foi investigar um possível efeito autócrino da S100B na captação de glutamato. A utilização do anticorpo anti-S100B reduziu a captação de glutamato após 30 minutos de incubação, sem afetar a integridade e viabilidade celular. Além disso, baixas concentrações de S100B (menos de 0,1 ng/mL) estimularam a captação de glutamato avaliada imediatamente após a troca de meio. Este dado reforça a importância dos astrócitos na transmissão glutamatérgica, particularmente, a função da S100B na neuroproteção contra dano excitotóxico. De fato, a S100B extracelular protege os neurônios quanto ao dano excitotóxico, enquanto concentrações tóxicas de glutamato têm demonstrado reduzir a secreção de S100B em astrócitos e fatias cerebrais, por mecanismos desconhecidos. Nosso segundo objetivo foi investigar os mecanismos possivelmente envolvidos neste efeito em cultura primária de astrócitos hipocampais utilizando agonistas glutamatérgicos e inibidores da captação de glutamato. Nossos resultados sugerem que a secreção de S100B está inversamente ligada à captação de glutamato. Além disso, a secreção da S100B parece ser estimulada por serotonina e estudos clínicos têm sugerido que a elevação da S100B está positivamente relacionada à resposta terapêutica dos antidepressivos, particularmente, os inibidores da recaptação da serotonina. Desta forma, nosso terceiro objetivo foi medir a secreção da S100B em culturas de astrócitos expostas à fluoxetina. Nós observamos um significativo aumento da secreção da S100B pela fluoxetina, efeito aparentemente dependente de PKA. Este dado reforça a importância da fluoxetina, independente da serotonina e receptores serotoninérgicos, para a atividade antidepressiva, bem como a função putativa da S100B nas doenças depressivas. Em paralelo, a padronização de uma técnica de ELISA para GFAP (a principal proteína marcadora de astrócitos), nós observamos que a fosforilação in vitro da GFAP purificada ou de amostras biológicas com PKA, indicam que a fosforilação aumenta o reconhecimento pelo anticorpo policlonal anti GFAP da DAKO. Estes resultados favorecem o entendimento de rápidas alterações na imunoreatividade da GFAP.
S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Our first aim was to investigate a possible autocrine role of S100B in glutamate uptake activity. Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affect cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement. This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage. In fact, extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by unknown mechanisms. Our second aim was to investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. Our findings suggest that S100B secretion is inversely coupled to glutamate uptake. Moreover, S100B secretion appears to be stimulated by serotonin and clinical studies have suggested that serum elevation of S100B is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors. Our third aim was to measure S100B secretion is astrocyte cultures exposed to fluoxetine. We observed a significant increment of S100B release by fluoxetine, apparently dependent on PKA. These data reinforce the importance of fluoxetine, independent of serotonin and serotonin receptors, for antidepressant activity, as well as the putative role of S100B in depressive disorders. In parallel, standardizing an ELISA for GFAP (the main protein marker for mature astrocytes) we found that in vitro phosphorylation of purified GFAP or biological samples with PKA indicate that GFAP phosphorylation improves the recognition by the polyclonal antibody anti-GFAP from DAKO. These results provide support to the understanding of fast changes in the GFAP-immunoreactivity.

A estimulação neonatal ocasiona alterações no desenvolvimento neuroendócrino, morfológico e comportamental de ratos. O procedimento de manipulação neonatal promove uma ruptura na relação mãe-filhote que pode perturbar o desenvolvimento do SNC. Uma das alterações morfológicas apresentadas em ratos machos e fêmeas manipulados no período neonatal é a diminuição do número de neurônios no locus coeruleus. A S100B é uma proteína ligante de cálcio secretada por astrócitos que pode apresentar ações extracelulares neurotróficos e neurotóxicas. Diversos estudos relacionam alterações dos níveis de S100B a eventos estressores. O estudo objetivou avaliar o efeito da estimulação neonatal, através da manipulação repetida, em ratos machos e fêmeas de 10 dias de idade sobre a concentração das proteínas S100B e GFAP no locus coeruleus, córtex frontal e bulbo olfatório, além da secreção de S100B no líquor. Os resultados revelaram um aumento da concentração de S100B no líquor apenas nas ratas fêmeas manipuladas, e uma diminuição da concentração de S100B no locus coeruleus em ratos machos e fêmeas manipulados. Ocorreu também, uma diminuição da concentração de S100B no córtex frontal em ratos machos manipulados. Em relação à proteína GFAP foi verificado um aumento no seu conteúdo no locus coeruleus em ratos machos e fêmeas manipulados. Foram encontrados diferenças entre os sexos, os machos não-manipulados têm um maior nível de S100B comparados com fêmeas nãomanipuladas no locus coeruleus. Os resultados sugerem que a manipulação neonatal pode interferir sobre o funcionamento das células gliais e através delas alterar o desenvolvimento das células nervosas. Além disso, os resultados mostram um efeito do sexo dos animais sobre as concentrações de S100B já no período neonatal.

Pancreatic cancer is relatively uncommon. Despite its relative scarcity, it is the fourth-ranked cancer killer in the Western world with less than a 5% 5-year survival rate. The high mortality rate is due to the asymptomatic nature of the disease and the advanced stage at which it is usually diagnosed. S100P is a calcium-binding protein that has been shown to be highly expressed in the early stages of pancreatic cancer and has been proposed as a potential therapeutic target via the blocking of its interaction with its receptor RAGE, the receptor for advanced glycation end-products. In this thesis, computational techniques were employed on the NMR ensemble of S100P (PDB Accession code 1OZO) to identify potential inhibitors of the S100P-RAGE interaction in the hope of identifying a series of novel leads that could be developed into clinical candidates for the treatment of pancreatic cancer. In silico studies identified putative binding sites at the S100P dimeric interface capable of accommodating cromolyn, an anti-allergy drug shown to bind to the protein both in vitro and in vivo. Virtual screening of >1 million lead-like compounds using 3D pharmacophore models derived from the predicted binding interactions between S100P and cromolyn, identified 9,408 'hits'. These were hierarchically clustered according to similarities between chemical structures into 299 clusters and 77 singletons. Biological screening of 17 of the 'hits' identified from virtual screening stuidies, 4 of which were synthesised in-house, against pancreatic cancer cell lines identified five compounds that demonstrated an equal or greater capacity to reduce BxPC-3 S100P-expressing pancreatic cells' metastatic potential in vitro relative to cromolyn. Compound 24 in particular, showed significant (p<0.05) inhibition of invasion of these cells at a concentration of 100 μM that was comparable to cromolyn at the same concentration. This compound, structurally distinct from cromolyn, was successfully synthesised, purified and characterised in-house alongside 39 of its analogues. Biological screening of compound 24 and four of its analogues for anti-proliferative activity against BxPC-3 and Panc-1 pancreatic cancer cell lines showed all five compounds significantly (p < 0.0001) inhibiting proliferation in both cell lines at a concentration of 1 μM relative to the non-treated control. Hence, structurally distinct compounds that show promising inhibitory activity on the metastasis and proliferation of pancreatic cancer cells have been identified using a structure-based drug design methodology. These compounds, with further optimisation, could provide good starting points as therapeutic lead candidates for the treatment of pancreatic cancer.

Fragestellung: Gegenstand dieser Untersuchung ist der diagnostische Nutzen der Neuro-Biomarker S100B-Protein und Neuronenspezifische Enolase (NSE) bei leichten Schädel-Hirn-Verletzungen im Kindesalter. Es wird untersucht, ob anhand der posttraumatischen S100B- und NSE-Serum-Konzentrationen Kinder mit einer Schädelprellung und einem leichten Schädel-Hirn-Trauma (SHT) differenziert werden können. Material und Methode: In einer prospektiven, klinischen Studie werden die posttraumatischen S100B- und NSE-Serum-Konzentrationen von Kindern im Alter zwischen 6 Monaten und 15 Jahren mit einer Schädelprellung oder einem leichten SHT untersucht. Kinder mit extrakraniellen Begleitverletzungen und Begleiterkrankungen sind ausgeschlossen. Die Blutentnahme erfolgt innerhalb von 6 Stunden nach dem Trauma. Es werden 2 diagnostische Gruppen definiert: Gruppe 1: asymptomatische Schädelprellungen (Glasgow-Coma-Scale [GCS] 15 Punkte), Gruppe 2: leichte SHT (GCS 13-15 Punkte) mit klinischen Zeichen einer Gehirnerschütterung (kurze Bewusstlosigkeit, Amnesie, Übelkeit, Erbrechen, Somnolenz, Kopfschmerzen, Schwindel, Sehstörungen, Kreislaufreaktion). Die S100B- und NSE- Konzentrationen werden zwischen beiden Diagnosegruppen verglichen. Die Korrelation zwischen S100B und NSE sowie zwischen den Markern und dem Alter der Kinder, dem Zeitraum zwischen Trauma und Blutentnahme, dem GCS-Wert und dem Vorhandensein von Kopfplatzwunden wird analysiert. Ergebnisse: 148 Kinder sind in die Studie eingeschlossen (53 Kinder mit einer Schädelprellung und 95 mit einem leichten SHT). Nach Adjustierung der gemessenen Marker-Konzentrationen auf Unterschiede im Alter und Zeitraum zwischen Trauma und Blutentnahme unterscheiden sich die S100B- und NSE-Konzentrationen nicht signifikant zwischen Kindern mit einer Schädelprellung und einem leichten SHT. Zwischen den S100B- und NSE-Konzentrationen besteht eine signifikant positive Korrelation. Beide Marker korrelieren signifikant negativ mit dem Alter und dem Entnahmezeitraum. Der GCS-Wert und das Vorhandensein von Kopfplatzwunden zeigen keinen Effekt auf die Marker-Konzentrationen. Schlussfolgerung: Die posttraumatischen S100B- und NSE-Serum-Konzentrationen zeigen keinen diagnostischen Nutzen bei der Differenzierung zwischen Kindern mit einer Schädelprellung und Kindern mit einem leichten SHT. S100B und NSE sind altersabhängige Marker.

A proteína S100b no cérebro é produzida e secretada pela célula da glia astrócito, e exerce de acordo com sua quantidade extracelular, ação trófica ou tóxica sobre os neurônios. Investigamos a distribuição da proteína S100b, no animal em condição basal, realizando o mapeamento em diferentes áreas do encéfalo, com a técnica imuno-histoquímica, explorando a hipótese do aparecimento de S100b em áreas encefálicas preferenciais. A distribuição da proteína foi analisada pela técnica do imuno-histoquímica, com utilização de anticorpo anti-S100 (b subunidade). O mapeamento da proteína S100b ao longo do eixo AP, permitiu observar marcação de elementos gliais distribuídos pelo telencéfalo, diencéfalo, e tronco encefálico, onde a proteína apresentou-se preferencialmente distribuída, na comparação dos animais. Nossos resultados sugerem que a proteína pode estar relacionada ao fato dessa distribuição ser conservada como padrão dentro da espécie.
S100b protein is expressed primarily by astroglia in the brain, and practice functional implication of S100b secretion by astrocytes into the extracellular space is scant but there is substantial evidence that secreted glial S100b exerts trophic or toxic effects depending on its concentration. We provide here a detailed description of the distribution of the calcium-binding protein S100b in and glial elements in the encefalo of rats. The distribution of S100-like immunoreactivity was analyzed by antisera: monoclonal, the b subunit (S100b) of this protein. All sera showed glial positive elements, which were more abundant in the brainstem than in the prosencephalon. S100-immunoreactive was detected in glial elements, in different regions of the telencephalon, diencephalon and mesencephalon. This distribution appears very similar to that, as well as to sparse observations on different vertebrates. Therefore, our results suggest that the distribution pattern of this protein in glial elements is highly conserved between the species.