Émile Zola’s climate history of the Second Empire

This article looks at Émile Zola’s novel cycle “Les Rougon-Macquart” and argues that it describes its subject, the Second Empire, as a warming climate tending toward climate catastrophe. Zola’s affinity to the notion of climate is shown to be linked to his poetic employment of the concept of ‘milieu,’ inspired by Hippolyte Taine. Close readings of selected passages from the “Rougon-Macquart” are used to work out the climatic difference between ‘the old’ and ‘the new Paris,’ and the process of warming that characterises the Second Empire. Octave Mouret’s department store holds a special place in the article, as it is analysed through what the article suggests calling a ‘meteorotopos’: a location of intensified climatic conditions that accounts for an increased interaction between human and non-human actors. The department store is also one of the many sites in the novel cycle that locally prefigure the ‘global’ climate catastrophe of Paris burning, in which the Second Empire perishes.El artículo hace una lectura del ciclo de novelas “Les Rougon-Macquart” de Émile Zola y sostiene la tesis de que el sujeto al que describe, el Segundo Imperio, puede leerse como un clima en proceso de calentamiento que se dirige hacia una catástrofe medioambiental. La afinidad de Zola con la noción de clima se ve expuesta al conectarla con su uso poetológico del concepto de ‘milieu’, inspirado en Hippolyte Taine. El artículo ofrece una lectura en profundidad de ciertos pasajes de las novelas “Rougon-Macquart” para mostrar las diferencias climáticas entre el “viejo” y el “nuevo París”, y el proceso de calentamiento que caracteriza el Segundo Imperio. El gran almacén de Octave Mouret es analizado, en particular, bajo el concepto de “meteorotopos” propuesto en el artículo: un lugar de condiciones climáticas intensificadas, un lugar de interacción aumentada entre actores humanos y no-humanos. El almacén es uno de varios espacios en el ciclo de novelas en los que ya se señala la catástrofe climática ‘global’ del París en llamas, momento en que se hunde el Segundo Imperio

Structural features and interactions of substrates complexed with molecular chaperones

Protein misfolding and aggregation perturbs cellular functions and is involved in aging and numerous medical disorders. In cells, the first line of defense is the association of deleterious aggregating proteins with small Heat shock proteins (sHsp). These oligomeric, ATP-independent chaperones sequester misfolded proteins into complexes and facilitate subsequent substrate solubilization and refolding by ATP-dependent chaperones. The cytosol of S. cerevisiae contains two sHsps: Hsp42 is constitutively active, while Hsp26 is activated at elevated temperatures. In my thesis, I wanted to elucidate how sHsps change the structure of aggregates, facilitating substrate reactivation. To this end, I studied the impact of Hsp26 and Hsp42 incorporation on the architecture of heat-induced aggregates by amide hydrogen exchange (HX). I established the experimental conditions for HX of heat-induced protein aggregates using thermolabile malate dehydrogenase (MDH) as model substrate. My data show that the formation of heat-induced Hsp26/MDH or Hsp42/MDH complexes has profound impact on the MDH structure. In the aggregated state formed in absence of sHsps, almost the entire MDH polypeptide becomes accessible to HX, reflecting global, large misfolding. In contrast, a more protected form of MDH is detected when complexed with Hsp26 or Hsp42. I observed that the mass spectra of many MDH peptides derived from sHsp/MDH complexes exist as a mixture of two populations after HX: a native-like and an aggregate-like population. Higher excess of sHsps promoted the native-like state. Single-molecule experiments confirmed the binding of sHsps to near native substrate folds. Furthermore, FRET experiments showed that sHsps increase the spacing between MDH molecules in sHsp/MDH complexes, preventing intermolecular contacts of misfolded MDH species. Finally, crosslinking approaches identified peripheral, surface-exposed MDH sites showing high HX as major sHsp binding sites. Summarized, these findings indicate that sHsps capture early unfolding intermediates of substrates and keep parts of the protein in a native-like state. This activity of sHsps might facilitate chaperone-dependent disaggregation. I then investigated how the two sHsps of yeast interact with their substrates. The N-terminal extensions (NTE) of both yeast sHsps were found to be the major substrate interaction sites. Compared to all known sHsps, the NTE of Hsp42 is unusually elongated and it was shown to be involved in the organized deposition of misfolded proteins at CytoQ (cytosolic quality control compartment). Hsp42 NTE harbors the two prototypes of intrinsically disordered domains (IDD): a prion-like and an unstructured subdomain. IDDs play important roles in the formation of membrane-free compartments due to their ability to self-associate and to coalesce into inclusions. In this study, the roles of both NTE subdomains in CytoQ formation and Hsp42 chaperone activity were investigated. We found that the prion-like domain of Hsp42 has a dual function: It binds misfolded substrate proteins and triggers CytoQ formation. The unstructured domain is dispensable for CytoQ formation, but it has a regulatory function, controlling Hsp42 localization and CytoQ numbers. Deletion of the unstructured domain increases Hsp42 substrate interaction and holdase activity, i.e. the prevention of tight contacts between misfolded species. Together, the presented data show that the prion-like domain of Hsp42 is essential for CytoQ formation, extending the role of prion-like domains in inclusion formation from RNA granules to protein aggregates and emphasizing their crucial contributions to protein phase transitions. In a second part of my thesis I studied how the Hsp70 chaperone system interacts with RepE, a dimeric replication initiation protein in E. coli. The disassembly of RepE seems mechanistically related to the disaggregation process. As a dimer RepE represses its own transcription, as a monomer it initiates the replication of the mini-F plasmid. Monomerization is mediated by the DnaK chaperone system. So far, it remained elusive, how components of the DnaK chaperone system interact with RepE and how they change its structure, leading to the disassembly of the RepE dimer. In this study the binding of DnaK and DnaJ to dimeric RepE wt and to RepE54, a constitutively monomeric variant, was studied by HX. HX analysis of RepE wt revealed a putative DnaK binding site and conformational changes induced by chaperones. Only dimeric RepE wt, but not monomeric RepE54, interacts with DnaJ. In contrast, both oligomeric states of RepE were able to bind DnaK – at least in absence of DNA. In presence of their respective DNA-binding elements, the binding of DnaK was prevented, most likely due to sterical hindrance as the DNA and the putative DnaK binding sites in RepE are in close proximity. The binding of DnaJ probably occurs in aa 96-116, and it destabilized parts of the DNA binding region in RepE, indicating conformational changes. Although interaction with DnaJ was shown to enhance the binding affinity of RepE to DNA, the DnaJ-induced conformational change might enable DnaK to access its binding site. Crosslinking experiments, however, showed that DnaJ binding is not sufficient to allow for interaction of DnaK with DNA-complexed RepE wt. Only concomitant presence of DnaJ and GrpE enabled DnaK to interact with DNA-bound RepE wt. HX revealed, that concerted binding of DnaJ and DnaK causes substantial conformational changes in RepE: Destabilization of the C-terminal region and stabilization in helix α4 near the dimer interface. The latter might be implicated in the monomerization of RepE wt. In summary, my results provide major contributions to elucidate the chaperone-mediated RepE monomerization process

RUNX3 transcript variants have distinct roles in ovarian carcinoma and differently influence platinum sensitivity and angiogenesis

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis

Mitochondria-Targeted Antioxidants SkQ1 and MitoTEMPO Failed to Exert a Long-Term Beneficial Effect in Murine Polymicrobial Sepsis

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n=90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments

Inactivation of the tyrosine phosphatase SHP-2 drives vascular dysfunction in sepsis

Background: Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence. Methods: Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay. Findings: We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1β as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1β-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88. Interpretation: Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. Fund: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research. Keywords: Endothelial cells, IL-1β, MyD88, ROS, SHP-2, Sepsi

A Tunable Narrowband Source in the Sub-THz and THz Range at DELTA

At DELTA, a 1.5-GeV electron storage ring operated as a synchrotron light source by the TU Dortmund University, an interaction of ultrashort laser pulses with electron bunches is used to generate broadband as well as tunable narrowband radiation in the frequency range between 75 GHz and 5.6 THz. The performance of the source was studied using two different Fourier-transform spectrometers. It was demonstrated that the source can be used for the characterization and comparison of Schottky-diode based detectors, e.g., an on-chip spectrometer enabling single-shot applications

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies