Electron Spin Resonance detectors from 400 MHz to 360 GHz

Methods based on the electron spin resonance (ESR) phenomenon are non-invasive tools adopted to investigate paramagnetic systems at temperatures ranging from above 1000 K to below 1 K. Since 2008, the group of Dr. Boero has been working on a detection technique based on the integration of ESR sensors on single chips. The proposed methodology allowed to study samples in the nanoliter scale and reach a spin sensitivity at least two orders of magnitude better than the best commercially available spectrometers. The detection principle can be summarized as follows. An ESR sensitive sample is placed in close proximity to the planar inductor of an LC oscillator operating at microwave frequency. In presence of a suitable static magnetic field, the ESR phenomenon takes place. It causes a variation in the sample magnetization which translates to a variation of the inductance, leading to both a frequency shift of the oscillator (frequency detection) and a variation of the oscillation amplitude (amplitude detection). Consequently, the ESR phenomenon may be detected by tracking the operating point of the oscillator. In this thesis, I investigate the application of the aforementioned detection principle in the range from 400 MHz to 360 GHz. Firstly, a semi-integrated solution operating from 400 MHz to 610 MHz is developed for an industrial application (CTI project). In such context, the originality of the work stands in the implementation of a completely standalone portable scanner for contactless inspection which may also be used for ferromagnetic (FMR) applications and zero-field measurements. Secondly, a set of single-chip ESR detectors working from 10 GHz to 146 GHz and based on CMOS technologies are characterized from 300 K down to 10 K. Here, an ESR experiment at a frequency as high as 360 GHz can be performed thanks to the fourth harmonic signal generated by a 90 GHz detector. Conversely, the 10 GHz detector shows the best noise performance and allows to achieve the record distance resolution of 0.3 pm when used as a proximity sensor. After that, the possibility of using integrated technologies based on HEMTs is investigated so as to overcome the main limitations of the CMOS based detectors: (1) the high power consumption which denies their use below 10 K and (2) the saturation issue due to the magnitude of the intrinsic microwave magnetic field produced by the oscillators. In this context, two HEMT oscillators working at 11 GHz and 25 GHz are realized. In particular, the former achieves the record minimum power consumption (90 uW at 300 K and 4 uW below 30 K) currently reported in the literature for oscillators working in the same frequency range. Also, the proposed sensor achieves a minimum microwave magnetic field of less than 1 uT at 300 K and less than 0.1 uT below 30 K, i.e., orders of magnitude below the values achieved with previous CMOS detectors. Furthermore, an analytical model is carried out in order to estimate the minimum achievable power consumption for an LC single-ended Colpitts oscillator based on any single FET. Lastly, the DC characterization of a standing alone HEMT transistor is provided from 300 K down to 1.4 K, ranging from the standard Ids-Vs-Vds curves to the extraction of both the number of carriers and their effective mobility. The former comes from the analysis of the Shubnikov-de-Haas oscillations whereas the latter is calculated by means of Hall-effect based experiments

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role

Single chip dynamic nuclear polarization microsystem

The integration on a single chip of the sensitivity-relevant electronics of nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectrometers is a promising approach to improve the limit of detection, especially for samples in the nanoliter and subnanoliter range. Here we demonstrate the co-integration on a single silicon chip of the front-end electronics of an NMR and an ESR detector. The excitation/detection planar spiral microcoils of the NMR and ESR detectors are concentric and interrogate the same sample volume. This combination of sensors allows to perform dynamic nuclear polarization (DNP) experiments using a single-chip integrated microsystem having an area of about 2 mm$^2$. In particular, we report $^1$H DNP-enhanced NMR experiments on liquid samples having a volume of about 1 nL performed at 10.7 GHz(ESR)/16 MHz(NMR). NMR enhancements as large as 50 are achieved on TEMPOL/H$_{2}$O solutions at room temperature. The use of state-of-the-art submicrometer integrated circuit technologies should allow the future extension of the single-chip DNP microsystem approach proposed here up the THz(ESR)/GHz(NMR) region, corresponding the strongest static magnetic fields currently available. Particularly interesting is the possibility to create arrays of such sensors for parallel DNP-enhanced NMR spectroscopy of nanoliter and subnanoliter samples

PINK1 and Parkin:emerging themes in mitochondrial homeostasis

The Parkinson’s disease (PD)-associated protein kinase, PINK1, and ubiquitin E3 ligase, Parkin function in a common signalling pathway known to regulate mitochondrial network homeostasis and quality control including mitophagy. The multistep activation of this pathway, as well as an unexpected convergence between the post-translational modifications of ubiquitylation and phosphorylation, has added breadth to our understanding of cellular damage responses during human disease. In concert with these new insights in signal transduction, unique modalities and signatures of vertebrate mitophagy have been unravelled in vivo for the very first time. The cell biology of mammalian mitophagy, and the roles of PINK1-Parkin signalling in vivo have emerged to be more complex than previously thought

The making of a mammalian peroxisome, version 2.0: mitochondria get into the mix

This is the author accepted manuscript. The final version is available from Nature Publishing Group via the DOI in this record.A recent report from the laboratory of Heidi McBride (McGill University) presents a role for mitochondria in the de novo biogenesis of peroxisomes in mammalian cells (1). Peroxisomes are essential organelles responsible for a wide variety of biochemical functions, from the generation of bile, to plasmalogen synthesis, reduction of peroxides, and the oxidation of very long chain fatty acids (2). Like mitochondria, peroxisomes proliferate primarily through growth and division of pre-existing peroxisomes (3-6). However, unlike mitochondria, peroxisomes do not fuse (5,7); further, and perhaps most importantly, they can also be born de novo, a process thought to occur through the generation of pre-peroxisomal vesicles that originate from the endoplasmic reticulum (reviewed in (8,9). De novo peroxisome biogenesis has been extensively studies in yeast, with a major focus on the role of the ER in this process. Comprehensive studies in mammalian cells are, however, scarce (5,10-12). By exploiting patient cells lacking mature peroxisomes, Sugiura et al. (1) now assign a role to ER and mitochondria in de novo mammalian peroxisome biogenesis by showing that the formation of immature preperoxisomes occurs through the fusion of Pex3- / Pex14-containing mitochondriaderived vesicles with Pex16-containing ER-derived vesicles

Ubiquitin and Parkinson's disease through the looking glass of Genetics

Biochemical alterations found in the brains of Parkinson's disease (PD) patients indicate that cellular stress is a major driver of dopaminergic neuronal loss. Oxidative stress, mitochondrial dysfunction, and ER stress lead to impairment of the homeostatic regulation of protein quality control pathways with a consequent increase in protein misfolding and aggregation and failure of the protein degradation machinery. Ubiquitin signalling plays a central role in protein quality control; however, prior to genetic advances, the detailed mechanisms of how impairment in the ubiquitin system was linked to PD remained mysterious. The discovery of mutations in the α-synuclein gene, which encodes the main protein misfolded in PD aggregates, together with mutations in genes encoding ubiquitin regulatory molecules, including PTEN-induced kinase 1 (PINK1), Parkin, and FBX07, has provided an opportunity to dissect out the molecular basis of ubiquitin signalling disruption in PD, and this knowledge will be critical for developing novel therapeutic strategies in PD that target the ubiquitin system

Leishmania inhibits antigen crosspresentation by direct cleavage of the SNARE VAMPS

Phagosomes play a key role in immunity by killing microbes and processing their antigens for T cell activation. Sorne intracellular pathogens such as Leishmania inhibit these steps and prevent phagosome maturation through different mechanisms. ln the present study, we investigated the impact of infection on SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins involved in the trafficking ta and from the phagosome. We discovered that upon infection, VAMPS, VAMP3, SNAP23, and syntaxin-4 were cleaved by GPI-anchored zinc metalloprotease GP63. Using a L. major gp63-KO mutant, we showed that this parasite protease is responsible for the cleavage of VAMPS and other SNAREs in infected macrophages. We also found that Leishmania promastigotes inhibit antigen crosspresentation in a GP63-dependent manner. Using cells from VAMPS-deficient mice, we confirmed that this SNARE protein is required for antigen crosspresentation. Also, the phagosomal exclusion of Sec22b, one of the regulators in phagosome maturation and antigen cross presentation, was observed upon infection with L. major WT and gp63-KO add-back. Thus, we uncovered the existence of a novel mechanism used by Leishmania promastigotes to evade recognition by the immune system, whereby the parasites impair crosspresentation by degrading key regulators of vesicular trafficking.</p

Dendritic Cells Crosspresent Antigens from Live B16 Cells More Efficiently than from Apoptotic Cells and Protect from Melanoma in a Therapeutic Model

Dendritic cells (DC) are able to elicit anti-tumoral CD8+ T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8+ T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8+ T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy

Primary biliary cholangitis: pathogenic mechanisms

Purpose of review Primary biliary cholangitis (PBC) is characterized by autoimmune damage of intrahepatic bile ducts associated with a loss of tolerance to mitochondrial antigens. PBC etiopathogenesis is intriguing because of different perplexing features, namely: a) although mitochondria are present in all cell types and tissues, the damage is mainly restricted to biliary epithelial cells (BECs); b) despite being an autoimmune disorder, it does not respond to immunosuppressive drugs but rather to ursodeoxycholic acid, a bile salt that induces HCO3- rich choleresis; c) the overwhelming female preponderance of the disease remains unexplained. Here we present an etiopathogenic view of PBC which sheds light on these puzzling facts of the disease. Recent findings PBC develops in patients with genetic predisposition to autoimmunity in whom epigenetic mechanisms silence the Cl-/HCO3- exchanger AE2 in both cholangiocytes and lymphoid cells. Defective AE2 function can produce BECs damage as a result of decreased biliary HCO3- secretion with disruption of the protective alkaline umbrella that normally prevents the penetration of toxic apolar bile salts into cholangiocytes. AE2 dysfunction also causes increased intracellular pH (pHi) in cholangiocytes, leading to the activation of soluble adenylyl cyclase, which sensitizes BECs to bile salt-induced apoptosis. Recently, mitophagy was found to be inhibited by cytosolic alkalization and stimulated by acidification. Accordingly, we propose that AE2 deficiency may disturb mitophagy in BECs, thus, promoting the accumulation of defective mitochondria, oxidative stress and presentation of mitochondrial antigens to the immune cells. As women possess a more acidic endolysosomal milieu than men, mitophagy might be more affected in women in an AE2-defective background. Apart from affecting BECs function, AE2 downregulation in lymphocytes may also contribute to alter immunoregulation facilitating autoreactive T-cell responses. Summary PBC can be considered as a disorder of Cl-/HCO3- exchange in individuals with genetic predisposition to autoimmunity