A systematic development of 2D alloy catalyst with synergistic performance of high lithium polysulfide (LiPS) binding energy and efficient Li+ ion/electron conduction is presented. The first section of work found that Li+ ions can flow through the percolated ion transport pathway in polycrystalline MoS2, while Na+ and K+ ions can easily flow through the percolated 1D ion channel near the grain boundaries. An unusually high ionic conductivity of extrinsic Li+, Na+, and K+ ions in 2D MoS2 film exceeding 1 S/cm was measured that is more than two orders of magnitude higher than those of conventional solid ionic materials, including 2D ionic materials. The second section of this dissertation focus on catalyzing the transformation of LiPSs to prevent the shuttle effect during the battery cycling by synthesizing 2H (semiconducting) – 1T (metallic) mixed phase 2D Mo0.5W0.5S2 alloy on CNF paper, using two step sputtering and sulfurization method. The lithium sulfur (Li-S) battery cell assembled with the 2D Mo0.5W0.5S2/CNF/S cathode shows a high specific capacity of 1228 mAh g-1 at 0.1C and much higher cyclic stability over 4 times as compared to the pristine cathodes. The high LiPSs binding energy of catalyst efficiently prevents the shuttling effect and corrosion of Li anode after long term stability test for over 400 cycles. The defect engineered MoWS catalyst on CNF showed significantly enhanced polysulfide transformation resulting in specific capacity of 1586 mAh g-1 at 0.05C for the full cell Li-S battery and much higher cyclic stability over 1000 cycles. Stacked layers of D-MoWS-CNF-S cathodes can result in an increased sulfur loading up to 10 mg cm-2 with highest achievable areal capacity of 13.5 mAh/cm2. The efficient sulfur utilization and reduced negative-to-positive capacity (N/P) ratio by D-MoWS catalyst significantly increased the gravimetric energy density to the highest reported value of 1090 Wh kg-1 w.r.t the total weight of anode and cathode.

Zipper-Interacting Protein Kinase (ZIPK) is a known modulator of actin-myosin contractility in vertebrate species. Interestingly, rodent and mouse ZIPK has undergone a divergence in regulation in comparison to other vertebrate orthologs including human. Whereas the human ortholog of ZIPK requires phosphorylation of residues TT299/300 for nuclear exit, rodents and mouse require interaction with another protein termed PAR-4. In this project we completed several experiments to examine zebrafish ZIPK in development and its effect on acto-myosin contractility. It was found that zebrafish ZIPK was expressed ubiquitously in maternal stages. In zygotic stages, ZIPK expression dropped dramatically and localized to the anterior portions of the embryo. Zebrafish and human ZIPK, but not rodent ZIPK were able to increase stress fiber formation and myosin light chain-2 (MLC-2) phosphorylation in vitro. Human and zebrafish ZIPK underwent nucleocytoplasmic shuttling without PAR-4 interaction, unlike rodent ZIPK, which required PAR-4 for nuclear exit. Unlike human ZIPK, zebrafish ZIPK TT299/300AA mutants were able to undergo shuttling. Similar to human ZIPK, catalytic mutations to zebrafish ZIPK abolished or dramatically reduced activity. Through these experiments we were able to show human and zebrafish ZIPK homologs function and are regulated similarly, while the rodent ZIPK was much more unique. Although the exhibited phenotypes were similar between human and zebrafish ZIPK orthologs, the mechanism of regulation is not completely conserved.

Cell homeostasis is maintained via strictly regulated processes. One of the important regulation systems is ubiquitin-proteasome proteolytic pathway. Proteins to be degraded are posttranslationally modified with polyubiquitin chains and targeted to the proteasome for degradation. Ubiquitin-proteasome system consists of several processes: ubiquitination of target substrates via set of enzymes, substrate transfer and degradation in the 26S proteasome. There are two ways of ubiquitinated substrate recognition via proteasome. It is either directly by proteasomal receptors or by protein shuttles. Shuttling factors bind polyubiquitinated target substrate and transfer it to the entrance of proteasomal cavity thanks to their typical domain architecture. The N-terminal ubiquitin-like domain binds to regulatory particle of the proteasome and the C-terminal ubiquitin-associated domain binds polyubiqitinated chains on substrates. This thesis focuses on the human DNA damage-inducible protein homolog 2 (Ddi2), a potential member of protein shuttles of humans, and on the interaction of its ubiquitin-like domain with its putative interaction partner, a proteasomal subunit PSMD2. PSMD2 has been cloned, expressed and purified in sufficient yields for further experiments. "Cold" as well as isotopically labeled UBL domain of...

Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication. Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment. In addition, the features rendering the abundant plant DEAD-box helicases either antiviral or pro-viral functions in tombusvirus replication are intriguing. I found the reversion of the antiviral function of DDX17-like RH30 DEAD-box helicase and the coopted pro-viral DDX3-like RH20 helicase due to deletion of unique N-terminal domains. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts. Moreover, I discovered that Xpo1 possesses an anti-viral function and exports previously characterized cell-intrinsic restriction factors (CIRFs) from the nucleus to the replication compartment of tombusviruses. Altogether, in my PhD studies, I found plant RH30 DEAD-box helicase is a potent host restriction factor inhibiting multiple steps of the tombusvirus replication. In addition, I provided the evidence supporting that the Nterminal domain determines the functions of antiviral DDX17-like RH30 DEAD-box helicase and pro-viral DDX3-like RH20 DEAD-box helicase in tombusvirus replication. Moreover, I discovered the emerging significance of the Xpo1-dependent nuclear export pathway in tombusvirus replication.

<p>The mouse xenobiotic-inducible <i>Cyp2a5</i> gene is both transcriptionally and posttranscriptionally regulated. One of the most potent <i>Cyp2a5</i> inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.</p><p>The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the <i>Cyp2a5</i> expression and the significance of the 71 nt hairpin-loop region for the <i>Cyp2a5</i> expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in <i>Cyp2a5</i> expression.</p><p>The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for <i>Cyp2a5</i> expression.</p><p>The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the <i>Cyp2a5</i>. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.</p><p>Treating primary mouse hepatocytes with phenobarbital (PB), a <i>Cyp2a5</i> transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the <i>Cyp2a5</i> proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.</p><p>In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and <i>Cyp2a5</i> promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the <i>Cyp2a5</i> expression.</p><p>The presented studies are the first showing involvement of an hnRNP protein in the regulation of a <i>Cyp</i> gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the <i>Cyp</i> gene family.</p>

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA. The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression. The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for Cyp2a5 expression. The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the Cyp2a5. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA. Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment. In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression. The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

[ES] La ubiquitinación inducida por hormonas desempeña un papel crucial en la vida media de los reguladores negativos clave de la propia señalización hormonal. En la señalización por ABA, los reguladores negativos clave son las PP2Cs del clado A, como PP2CA o ABI1, y su degradación es un mecanismo complementario a la inhibición de su actividad mediada por PYR/PYL/RCAR. El ABA promueve la degradación de ABI1 a través de las E3 ligasas PUB12/13, y PP2CA a través de las E3 ligasas RGLG1/5. Sin embargo, se predice que otras E3 ligasas no identificadas también regularán la vida media de las PP2Cs del clado A. En pasos posteriores de la señalización por ABA, el ABA también induce la regulación positiva de los niveles de transcrito y proteína de las PP2Cs como un mecanismo de retroalimentación negativa. Por lo tanto, restablecer la señalización de ABA también requiere la degradación de las PP2Cs para evitar su acumulación excesiva inducida por el propio ABA. En este trabajo identificamos las proteínas BTB/POZ AND MATH DOMAIN (BPM), adaptadores del sustrato de las E3 ligasas multiméricas CULLIN3-RING E3 (CRL3), como proteínas que interactúan con las PP2Cs. BPM3 y BPM5 interactúan en el núcleo con PP2CA, así como con ABI1, ABI2 y HAB1. Además, BPM3 y BPM5 aceleran la degradación de las PP2Cs de una manera dependiente del ABA y su sobreexpresión conduce a una mayor sensibilidad al ABA. Además, las plantas mutantes bpm3 bpm5 mostraron una mayor acumulación de PP2CA, ABI1 y HAB1, lo que conduce a una sensibilidad global disminuida al ABA. Finalmente, utilizando ensayos bioquímicos y genéticos, demostramos que las BPM aumentaban la ubiquitinación de PP2CA. Dado que la formación de los complejos ternarios receptor-ABA-fosfatasa se ve notablemente afectada por la abundancia de sus componentes proteicos y la concentración de ABA, revelamos que las BPM y las E3 ligasas multiméricas CRL3 son moduladores importantes de los niveles del correceptor PP2C para regular la señalización temprana de ABA, así como durante los consiguientes pasos de restablecimiento. Al contrario que con PUB12/13, no se sabe cómo el ABA aumenta la degradación de PP2CA a través de RGLG1/5. En el caso de RGLG1, esta proteína se encuentra predominantemente en la membrana plasmática, mientras que PP2CA se encuentra predominantemente en el núcleo. Nosotros demostramos que el ABA modifica la localización subcelular de RGLG1, promoviendo la interacción nuclear con PP2CA. En primer lugar, encontramos que RGLG1 está miristoilado in vivo, lo que facilita su unión a la membrana plasmática, sin embargo, el ABA inhibe esta miristoilación. El ABA también regula negativamente a N-myristoyltransferase 1, la enzima activa y central en la miristoilación de proteínas, esto puede ayudar a promover la translocación de RGLG1 al núcleo. Allí, RGLG1 puede interactuar con ciertos receptores monoméricos del ABA, como PYL8. El reclutamiento nuclear de la E3 ligasa también fue promovido por el aumento de los niveles de proteína PP2CA y por la formación de complejos RGLG1-PYL8-PP2CA en presencia de ABA. Además, nosotros encontramos que RGLG1Gly2Ala, mutada en el sitio de miristoilación N-terminal, muestra localización nuclear constitutiva y provoca una respuesta más sensible al ABA y al estrés salino y osmótico. En resumen, proporcionamos evidencia de que una ligasa E3 puede reubicarse dinámicamente en respuesta al ABA, el estrés salino y osmótico, y el aumento de los niveles de su sustrato, lo que revela un mecanismo para explicar cómo el ABA mejora la interacción RGLG1-PP2CA y, por lo tanto, la degradación de PP2CA. / [CAT] L' ubiquitinació induïda per hormones té un paper crucial en la vida mitjana dels reguladors negatius clau de la pròpia senyalització hormonal. En la senyalització per ABA, els reguladors negatius clau són les PP2Cs del clado A, com PP2CA o ABI1, i la seva degradació és un mecanisme complementari a la inhibició de la seva activitat mediada per PYR/PYL/RCAR. El ABA promou la degradació de ABI1 a través de les E3 lligases PUB12/13, i PP2CA per mitja de les E3 lligases RGLG1/5. No obstant això, es prediu que altres E3 lligases no identificades també regularan la vida mitjana de les PP2Cs del clado A. En passos posteriors de la senyalització per ABA, l'ABA també indueix la regulació positiva de nivells de transcrit i proteïna de les PP2Cs com un mecanisme de retroalimentació negativa. Per tant, restablir la senyalització d'ABA també requereix la degradació de les PP2Cs per evitar la seva acumulació excessiva induïda pel propi ABA. En aquest treball identifiquem les proteïnes BTB/POZ AND MATH DOMAIN (BPM), adaptadors del substrat de les E3 lligases multimèriques CULLIN3-RING E3 (CRL3), com proteïnes que interactuen amb les PP2Cs. BPM3 i BPM5 interactuen en el nucli amb PP2CA, així com amb ABI1, ABI2 i HAB1. A més, BPM3 i BPM5 acceleren la degradació de les PP2Cs d'una manera dependent del ABA i la seva sobreexpressió porta a una major sensibilitat al ABA. A més, les plantes mutants bpm3 bpm5 van mostrar una major acumulació de PP2CA, ABI1 i HAB1, el que porta a una sensibilitat global disminuïda a ABA. Finalment, utilitzant assajos bioquímics i genètics, aconseguint que les BPM augmentaven l' ubiquitinació de PP2CA. Atès que la formació dels complexos ternaris receptor-ABA-fosfatasa es veu notablement afectada per l'abundància dels seus components proteics i la concentració d'ABA, revelem que les BPM i les E3 lligases multimèriques CRL3 són moduladors importants dels nivells del coreceptor PP2C per regular la senyalització primerenca de ABA, així com durant els consegüents passos de restabliment. Al contrari que amb PUB12/13, no se sap com el ABA augmenta la degradació de PP2CA a través d'RGLG1/5. En el cas de RGLG1, aquesta proteïna es troba predominantment en la membrana plasmàtica, mentre que PP2CA es troba predominantment en el nucli. Nosaltres vam demostrar que l'ABA modifica la localització subcelular de RGLG1, promovent la interacció nuclear amb PP2CA. En primer lloc, trobem que RGLG1 està miristoilado in vivo, el que facilita la seva unió a la membrana plasmàtica, però, el ABA inhibeix aquesta miristoilación. El ABA també regula negativament N-myristoyltransferase 1, l' enzim actiu i central en la miristoilación de proteïnes, això pot ajudar a promoure la translocació de RGLG1 al nucli. Allà, RGLG1 pot interactuar amb certs receptors monomèrics de l'ABA, com PYL8. El reclutament nuclear de l'E3 lligasa també va ser promogut per l'augment dels nivells de proteïna PP2CA i per la formació de complexos RGLG1-PYL8-PP2CA en presència d'ABA. A més, nosaltres trobem que RGLG1Gly2Ala, mutada en el lloc de miristoilació N-terminal, mostra localització nuclear constitutiva i provoca una resposta més sensible al ABA i l'estrès salí i osmòtic. En resum, proporcionem evidència que una ligasa E3 pot reubicar dinàmicament en resposta al ABA, l'estrès salí i osmòtic, i l'augment dels nivells de la seva substrat, el que revela un mecanisme per explicar com el ABA millora la interacció RGLG1-PP2CA i, per tant, la degradació de PP2CA. / [EN] Hormone-induced ubiquitination plays a crucial role to determine the half-life of key negative regulators of hormone signaling. In case of ABA signaling, the key negative regulators are the clade-A PP2Cs, such as PP2CA or ABI1, and their degradation is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of their activity. ABA promotes the degradation of ABI1 through the PUB12/13 E3 ligases, and PP2CA through the RGLG1/5 E3 ligases. However, other unidentified E3 ligases are predicted to regulate clade A PP2Cs half-life as well. At later steps of ABA signaling, ABA also induces upregulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. In this work we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric CULLIN3-RING E3 ligases (CRL3s), as PP2C-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2 and HAB1. Additionally, BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity. Moreover, bpm3 bpm5 mutant plants showed increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Finally, using biochemical and genetic assays we demonstrated that BPMs enhance the ubiquitination of PP2CA. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C co-receptor levels to regulate early ABA signaling as well as the subsequent resetting steps. In contrast to PUB12/13, it was not known how ABA enhances the degradation of PP2CA by RGLG1/5. RGLG1 is predominantly found in the plasma membrane whereas PP2CA is predominant in the nucleus. We demonstrate that ABA modifies the subcellular localization of RGLG1, promoting nuclear interaction with PP2CA. Firstly, we found that RGLG1 is myristoylated in vivo, which facilitates its attachment to the plasma membrane, nevertheless, ABA inhibits its myristoylation. ABA also downregulates N-myristoyltransferase 1, the central active enzyme of protein myristoylation, which may help to promote RGLG1 translocation to the nucleus. There, RGLG1 can interact with certain monomeric ABA receptors, as PYL8. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-PYL8-PP2CA complexes in the presence of ABA. Additionally, we found that RGLG1Gly2Ala protein, mutated at the N-terminal myristoylation site, shows constitutive nuclear localization and causes an enhanced response to ABA and salt and osmotic stresses. In summary, we provided evidence that an E3 ligase can dynamically relocalize in response to ABA, salt and osmotic stress, and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation. / Julián Valenzuela, J. (2020). Regulation of ABA signaling through degradation of clade A PP2Cs by the RGLG1 and CRL3 BPM E3 ligases [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137777 / TESIS

No description available.

Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...

<p>As the future of energy looks increasingly electrified, the development of safe and sustainable battery technologies has never been more relevant. This is particularly critical for applications in stationary energy storage and transportation, where batteries must be produced and stored at large scale. Sustainability is necessary to meet the volume of demand at reasonable cost without straining resources. Safety is also paramount since fires can easily spread from one cell to the next and result in catastrophe when batteries are stored in proximity for large power banks or EVs. The focus of this thesis is thus to design and engineer materials for rechargeable batteries, which improve safety and sustainability while still enhancing the electrochemical performance. Towards this end, polymers play a central role throughout this thesis work due to their tunable chemical and physical properties.</p>