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Defining the molecular basis of dynamic localization of type VI secretion system assembly in "Pseudomonas aeruginosa"
No full textInterbacterial competition is a highly resource-intensive and physiologically complex undertaking. Consequently, gene regulation and the functions of large macromolecular complexes are often driven by environmental signals. One particularly crucial complex for inter-species competition in Gramnegative bacteria is the type VI secretion system (T6SS), commonly used to deliver toxins across bacterial membranes into other cells. The expression of type VI secretion system (T6SS) assemblies varies across species and is commonly regulated on a transcriptional and post-translational level. Studies in different model organisms have found a variety of phenotypes associated with T6SS assembly.
Pseudomonas aeruginosa, encodes three separate T6SS on distinct loci. The main anti-bacterial T6SS, H1-T6SS, is tightly regulated on a post-translational level and is assembled in response to outer membrane damage, producing a characteristic “tit-for-tat” phenotype wherein adjacent cells appear to be dueling with their T6SS. This response was previously shown to depend on a threonine phosphorylation pathway (TPP) consisting of a Thr kinase/phosphatase pair (PpkA/PppA) as well as the proteins TagQ/TagR/TagS and TagT, which act upstream of kinase activation. TagQ is an outer membrane (OM) lipoprotein that sequesters TagR to the OM, while TagR has been shown to promote PpkA activity. TagST forms an ATP-binding cassette (ABC)-transporter like complex that has some ATPase activity in vitro but does not seem to affect the localization of either TagQ or TagR. In particular, it remains unknown if any of these proteins interact directly or form complexes either before or during signal transduction. Moreover, the mechanism by which a signal is passed on to PpkA has remained elusive.
In this thesis, I used a combination of biochemical, structural and microscopy approaches to describe a mechanism for activation of H1-T6SS assembly by membrane damage. I set out first to characterize the function of TagQ, which I found to form a 1:2 complex with TagR in vivo using coimmunoprecipitation (Co-IP) approaches valdiated by mass spectrometry (MS) and mass photometry. Analysis of TagR by mass photometry revealed that it can form a dimer in the absence of TagQ but only at supraphysiological concentrations. Subsequently, I found that TagQR complex formation is induced upon membrane damage using several different methods of inducing damage, validated by live cell microscopy. Following up on previously reported roles of TagR in stimulating PpkA activity, I wondered if TagQ could potentially be involved as well through its binding to TagR. Strikingly, I was able to detect a supercomplex formed by TagQR binding directly to PpkA using Co-IP and in vitro pulldowns. Using in silico modeling approaches supported by live cell imaging of mutants, I was able to show a potential mechanism for PpkA dimerization by the TagQR complex. Similarly, I was able to shed light on the role of TagS/T by studying its own interactions by in vitro and in vivo approaches. Finally, I was curious to see whether PpkA would have any phosphorylation targets that could produce the gene regulation effects seen in other envelope stress responses. Using MS and Co-IP approaches I found that the TagQRST-PpkA pathway is exclusive to H1-T6SS activation, making it a truly unique membrane damage response in bacteria
Gremlin1 controls digit numbers and identities by spatial regulation of two early limb bud mesenchymal progenitor populations
Full text linkThe developing vertebrate limb bud is an excellent model to study the mechanisms that provide the vertebrate embryo with robustness during pattern formation and organogenesis. We have previously shown that the core of this signaling system consists of SHH/GREM1/AER-FGF feedback signaling. The BMP antagonist GREM1 is a key node for the control of patterning, proliferation, and survival of limb mesenchymal progenitors (LMPs). Inactivation of Grem1 in mice limb bud results in skeletal defects, including a severe reduction in digit numbers. Whole limb RNA-sequencing (RNA-seq) for several distinct mutations in Grem1-enhancers that reduce Grem1 expression revealed that genotypes with a visible digit phenotype, have many differentially expressed genes in key developmental pathways while mutants with no apparent phenotype are able to compensate the Grem1 deficiency. Amongst the mutants with a phenotype, specific deletion of multiple enhancers has shown to alter the spatial expression of Grem1 in such a way that it causes the loss of one digit, resulting in stable tetradactyly. With the use of specific recombinant Cre alleles and lineage tracing, we determined that the missing digit is d2.
In order to understand the digit loss on a molecular and cellular level, single cell RNA-seq analysis of wildtype and two distinct tetradactyl mutants (E1C5Δ/Δ, E1C8Δ/Δ) was performed at E10.75 (37 – 39 somites), which revealed molecular signatures of two LMP populations. In the mutants, both populations exhibit altered cell numbers and levels of expression, compared to the wildtype. RNA-Fluorescence in situ hybridization (RNA-FISH) analysis of key signature markers for these progenitor populations confirmed a reduction of the most distal population (dLMP) at the expense of an expansion of the peripheral one (pLMP). This result points to a problem in specification of the cells that will give rise to the handplate, including the digits. The reduction in dLMPs detected at E10.75 persists during progression of limb bud outgrowth and onset of handplate development (E10.75-E11.25, 37-43 somites) showing that mutants fail to catch up to wildtypes and eventually end up with one digit less. To gain insight into how these populations behave in the context of digit loss/gain, mutants that are models for human malformations were analyzed, namely Tbx3Δ/Δ, ShhΔ/Δ and Gli3Δ/Δ. Genetic inactivation of Shh disrupts Grem1 expression and results in digit agenesis such that only a rudimentary condensation forms. In contrast to wildtype forelimb buds, the pLMP signature genes are expressed uniformly in the peripheral mesenchyme of ShhΔ/Δ forelimb buds at E10.75, whereas expression of dLMP marker genes is not detected. By E10.75, Grem1 expression is expanded anteriorly in Gli3Δ/Δ forelimb buds. Concurrent with this anterior expansion, the anterior bias in pLMPs is lost, while markers of dLMPs are also anteriorly expanded. This analysis of Gli3Δ/Δ limb buds shows that this early peripheral distal-anterior expansion of dLMP signature foreshadows anterior digit duplications, while loss of the anterior bias in pLMP signatures correlates with the loss of anterior digit identities and asymmetry. Furthermore, single cell analysis and RNA-FISH were done on mutant forelimb buds without Grem1 expression (Grem NULL) in comparison to wildtype. dLMPs were severely reduced with some of their makers depleted while pLMPs were increased and had a symmetric expression, corroborating the conclusions of previous analysis.
Tetradactyl mice exhibit a paraxonic skeletal phenotype, resembling that of pigs at corresponding developmental stages. We further investigated gene expression patterns by analyzing mRNA levels of key progenitor population markers. This analysis revealed similarities in the expression of pLMPs but also identified an expanded dLMP domain. Taken together, our findings provide new insights into the molecular mechanisms of digit specification and show how the balance of two distinct progenitor populations can shift the limb axis asymmetry and influence digit numbers and identities
Early nuclear power plant retirement and policy choices in the New York electricity market
Full text linkThe U.S. nuclear industry has overcome a challenging period during which low wholesale market prices threatened the survival of nuclear power plants (NPPs). From 2017 to 2019, several U.S. states initiated out-of-market support schemes to bolster the financial conditions of NPPs. This paper provides a comparative cost assessment between the preservation of three upstate New York NPPs under the zero-emission credit (ZEC) support scheme or an early retirement. In addition, the paper explores future market development scenarios with a carbon price mechanism. A bespoke cost-minimization dispatch model is developed for the New York electricity market along with four neighboring electricity markets. The comparative cost assessment of a nuclear phaseout and ZEC expenditures is not definitive. Results indicate that phasing out upstate NPPs in 2018 and 2021 incurred a slightly higher cost burden for New York consumers compared to the total ZEC expenditures. In contrast, phasing out upstate NPPs in 2030 incurs a lower cost burden compared to the total ZEC expenditure, mainly due to a high credit price. Furthermore, results show that a low carbon price of USD 51/ton would raise average NYISO prices by USD 24.1/MWh, thereby improving the long-term income conditions of NPPs, and ensuring sufficient accumulation of nuclear decommissioning funds. The study provides policymakers with a sequence of optimal policy options taking into account the pace of renewable development
Substrate adaptors are flexible tethering modules that enhance substrate methylation by the arginine methyltransferase PRMT5
Full text linkProtein arginine methyltransferase (PRMT) 5 is an essential arginine methyltransferase responsible for the majority of cellular symmetric dimethyl-arginine (SDMA) marks. PRMT5 uses substrate adaptors such as pICln, RIOK1, and COPR5, to recruit and methylate a wide range of substrates. Although the substrate adaptors play important roles in substrate recognition, how they direct PRMT5 activity towards specific substrates remains incompletely understood. Using biochemistry and cryogenic electron microscopy (cryo-EM), we show that these adaptors compete for the same binding site on PRMT5. We find that substrate adaptor and substrate complexes are bound to PRMT5 through two peptide motifs, enabling these adaptors to act as flexible tethering modules to enhance substrate methylation. Taken together, our results shed structural and mechanistic light on the PRMT5 substrate adaptor function and the biochemical nature of PRMT5 interactors
Modulation of human adipose derived stromal cell chondrogenesis for controlled endochondral ossification and efficient bone formation
Full text linkEndochondral ossification (ECO), is the primary ossification process during embryogenesis and fracture repair. It involves the formation of a cartilaginous tissue (CT) intermediate which progressively remodels into a mature bone organ. Adipose-derived stromal cells (ASC), non-skeletal multipotent progenitors derived from the stromal vascular fraction (SVF) of human adipose tissue, hold promise for bone regeneration via ECO. Few studies have shown the potential of ASC to recapitulate ECO and form a bone organ in vivo when pre-differentiated into cartilage tissue in vitro. However, the reproducibility of this process remains a challenge and the major triggers are not fully understood. Furthermore, ex vivo processing and expansion of ASC negatively impact their functionality. Therefore, a deeper understanding of ASC response to chondrogenic cues in vitro and how they modulate their microenvironment in vivo is crucial. Thus, this thesis aims to get better insights into the different stages of ASC chondrogenesis in vitro to ultimately control the associated endochondral ossification process and therefore, enhance their bone-forming capacity. We found that both SVF-cells and expanded-ASC can reproducibly generate cartilage tissue (CT) with varying degrees of maturation in a time-dependent manner. However, SVF cells exhibited accelerated maturation compared to expanded-ASC. Proteomic analysis revealed a quiescent state of SVF-ASC characterized by enrichment in lipid metabolism pathways, which transitions to a proliferative state upon monolayer expansion. We also showed that the metabolic signature can be restored by inducing quiescence at the end of the expansion phase. Furthermore, we could preserve and enhance the chondrogenic potential during monolayer expansion of ASC by modulating their metabolic signature (by quiescence induction) or by modulating their TGFb3 receptors expression profile (by TGFβ3 supplementation). Our findings indicate that the host response, specifically osteoclast recruitment, is dependent on the maturation level of the implanted CT, leading to various remodelling outcomes. Consequently, we identified a specific in vitro maturation window that leads to endochondral ossification and ultimately the formation of a mature bone organ in vivo. Moreover, we demonstrated that adipose-derived hypertrophic cartilage grafts (Adiscaf) outperformed clinical standard biomaterials in maxillofacial surgery, exhibiting enhanced bone formation and osteointegration. In summary, this work provides a detailed characterization of the stages of ASC endochondral ossification, highlighting the critical roles of monolayer expansion and chondrogenic maturation in modulating ECO. These findings offer a simple, yet reproducible and effective strategy for enhanced bone formation that warrants further investigation in clinical settings
Engineering an antibody-discernible and functional CD45 variant on hematopoietic stem and progenitor cells via base editing
Full text linkHematopoietic stem cell transplantation (HCT) stands as the unique curative recourse for various hematological malignancies. However, the current standard-of-care relies on untargeted toxic conditioning regimens to deplete malignant cells and remove cells from host’s bone niches. These untargeted regimens limit HCT’s widespread use due to severe off-target toxicity effects. To counteract this roadblock, antigen-specific cell-depleting agents have emerged as promising targeted alternatives. Pairing antigen-specific conditioning regimens with transplantation of depleter-shielded HSPCs has shown promise in the treatment of malignancies characterized by the overexpression of specific cell-surface markers (e.g. CD123 or CD33). Indeed, by rendering transplanted HSPCs resistant to antigen-specific depleters, we can replenish the depleted cell subsets (e.g CD123+ or CD33+ cells) without risking to eradicate them if the need for retreatment arises due to minimal residual disease (MRD) leading to relapse. Nevertheless, focusing solely on CD123 or CD33 limits this strategy to specific cell subtypes. It would therefore be desirable to develop a similar system for a cell surface marker present on all hematopoietic cells, such as CD45. Engineering a CD45 variant shielded from antigen-specific depleters would enable protection of the entire blood system post-HCT while still permitting eradication of all CD45+ cells (e.g. host’s blood cells and hematological cancer cells). In this study, we screened CD45’s extra-cellular domain regions with base editors and generated multiple CD45 protein variants altering the binding of antibodies. We selected the CD45-K352E/G variant profile and improved its base editing rate as it showcased loss of binding of a unique anti-CD45 antibody while still maintaining the surface marker’s expression, stability, and function in human hematopoietic stem and progenitor cells (HSPCs). The resulting loss of antibody binding prompted the modification and humanization of the said anti-CD45 antibody, culminating in the development of an anti-CD45 antibody- drug conjugate (CD45-ADC; CIM053-SG3376). Transplantation of the base edited HSPCs into immunodeficient mice showcased their long-term engraftment and multi-lineage reconstitution ability in sequential host mice. Importantly, in AML mouse models xenografted with HSPCs, administration of the CD45-ADC selectively depleted human leukemia and HSPCs-wt cells while preserving the healthy hematopoietic system derived from the transplanted base edited HSPCs. This approach of generating de novo antigens through gene editing to evade targeted-killing represents a robust strategy for creating cell-specific antigens, address the current limitations of HCT but also suggests broader implications beyond hematological malignancies, offering a promising avenue for future therapeutic development
Understanding configurational entropy and polymorphism: a computational study of lithium alanate and molecular crystals
Full text linkThis thesis investigates complex energy landscapes and phase transitions of two different classes of materials; alanates and molecular crystals. With the use of the Minima Hopping structure search method, the effects of a structurally tolerant phase are studied in the case of lithium alanate and the molecular crystal consisting of N-(4'Methylbenzylidene)-4-methylalanine. A structurally tolerant phase allows for many variations of its basic structural motif that change the energy only marginally.
Firstly, a new implementation of Minima Hopping method is discussed. The Minima Hopping method combines short molecular dynamics trajectories with local geometry optimizations to efficiently explore the potential energy surface and locate the global minimum. With the new implementation in the Python language, different existing and new features have been brought together into one software package. Furthermore, the code is interfaced to the atomic simulation environment which offers a broad variety of energy and force evaluation routines. The method's effectiveness is demonstrated through its application to broad range of materials, highlighting its capability to overcome the challenges posed by the vast and high-dimensional search spaces typical of potential energy surfaces.
Combined with a machine learned potential, the Minima Hopping method is applied to lithium alanate, a promising candidate for hydrogen storage applications, to explore the configurational density of states. Lithium alanate exhibits an ionic phase which, unlike its polymeric counterpart, demonstrates high structural tolerance. The configurational density of states derived from Minima Hopping runs reveals that the ionic phase is stabilized through configurational entropy. A detailed analysis shows that despite the polymeric form being lower in energy, the ionic form remains prevalent due to its higher configurational entropy, explaining the absence of the polymeric phase in experiments.
Finally, the polymorphism in molecular crystals is studied, with a particular focus on N-(4'-Methylbenzylidene)-4-methylalanine. The configurational density of states, derived from Minima Hopping runs, indicates that Form II is the most structurally tolerant despite being higher in energy compared to Form III, the form lowest in energy. The phenomenon of disappearing polymorphism, where a stable polymorph transforms into another, rendering the original form challenging to reproduce, is explored for the case of Form I of N-(4'-Methylbenzylidene)-4-methylalanine. Such transformations had significant implications in pharmaceutical applications, where the stability of the polymorphic form directly impacts drug efficacy. The Boltzmann probabilities of different polymorphic forms are calculated to understand their thermodynamic stability, revealing that structural tolerance plays a critical role in the persistence of certain phases
Bayesian spatial-temporal modelling to assess the impact of climate variability and control interventions on the burden of malaria in Kenya
Full text linkMalaria, one of the oldest and most persistent infectious diseases, continues to pose a significant public health challenge, particularly in sub-Saharan Africa (SSA), where it disproportionately affects children under 5 years of age. The risk of malaria in Kenya is heterogeneous; with western Kenya experiencing a high burden. Contributing factors include a favourable climate for mosquitoes, weak health systems, and socioeconomic challenges.
This research aimed to evaluate the influence of climatic, environmental, and non-climatic factors, alongside control interventions, on malaria incidence and mortality in Kenya. Using data from Kisumu's Health and Demographic Surveillance System (HDSS) (2008–2022) and Kenya Malaria Indicator Surveys (2015 and 2020), the study employed advanced statistical and geostatistical models. It analysed trends in malaria incidence, parasitemia prevalence, and mortality, considering factors such as temperature, rainfall, bed net use, socioeconomic status, and proximity to health facilities. Additionally, the study applied empirical dynamic modelling to establish causal links and forecast malaria transmission.
The findings enhance our understanding of malaria epidemiology and highlight the significant and varied effects of climatic factors on malaria transmission. Results underscore the protective role of bed nets, the influence of socioeconomic disparities, and the spatial and temporal heterogeneity of malaria risk. The work provides critical tools for mapping and targeting malaria control efforts, with implications for the National Malaria Control Program (NMCP). Additionally, the forecasting model offers actionable insights for localized, short- and long-term malaria prediction, supporting more effective resource allocation and intervention strategies
Influence of additional weight carrying on load-induced changes in glenohumeral translation in patients with rotator cuff tear - an experimental glenohumeral simulator study
Full text linkThe rotator cuff muscles are critical in providing the necessary compressive forces in the joint to maintain stability during glenohumeral motion. In shoulders with rotator cuff tears, this mechanism may be compromised, and unfortunately, this is a common shoulder pathology in the general population. Untreated rotator cuff tears can lead to secondary pathologies such as osteoarthritis and fatty infiltration of the torn muscle. To study glenohumeral biomechanics, in vivo studies can provide physiological data, but often require invasive or ionizing radiation measurements, while ex vivo studies allow an in-depth study of biomechanics while facing the challenge of reproducing physiological behavior.
The purpose of this thesis was twofold: first, to develop a control strategy for a glenohumeral simulator to simulate the abduction motion of shoulders with rotator cuff tears, and second, to study the glenohumeral biomechanics of weight-bearing shoulders with rotator cuff tears during 30° glenohumeral abduction.
We thoroughly reviewed the state-of-the-art of glenohumeral simulators and their limitations. We then developed a musculoskeletal model-based simulator to simulate abduction motion in weight-bearing shoulders with rotator cuff tears. Compared to in vivo measurements, we showed that our simulators replicated similar muscle activation and joint reaction forces during 30° abduction. Furthermore, our experiments showed that glenohumeral translations were affected by weight-bearing and critical shoulder angle, but not by rotator cuff tears. In addition, we found that the center of force in the glenohumeral joint was located in areas where glenoid erosion has been reported in patients with osteoarthritis following massive rotator cuff tears.
The remaining intact tendons of the rotator cuff muscles were able to compensate for torn rotator cuff tendons and maintain glenohumeral translation comparable to intact shoulders. Our findings suggest that patients with multiple rotator cuff tears are at risk for developing secondary osteoarthritis and that strengthening the remaining rotator cuff muscles with intact tendons may be beneficial for these patients
Design and evaluation of bioinspired lipid nanoparticles for optimized gene therapy
No full textGene therapy offers the possibility to prevent and treat numerous diseases, thereby improving the well-being of millions of individuals. The success of such treatments requires efficient delivery of nucleic acids to cells. For this, various gene delivery strategies, including modified oligonucleotides, viruses, and lipid nanoparticles (LNPs), have been widely explored and used in clinical practice. LNPs have demonstrated their effectiveness with the success of the messenger ribonucleic acid vaccines from Pfizer-BioNTech and Moderna against the coronavirus disease 2019. However, despite this monumental achievement, the LNP technology still faces challenges, including limited efficacy and potency, safety concerns, and regulatory considerations.
In the first part of this thesis, the aim was to optimize the performance of LNPs in terms of efficacy and potency. To achieve this, bioinspired and ionizable cationic lipid modifications were explored. Two bioinspired formulations were subsequently developed: phosphatidylserine-LNPs and hybrid LNPs. The first formulation incorporates phosphatidylserine, a lipid typically found in viruses, while the second results from the fusion of LNPs with extracellular vesicles, which are a naturally occurring gene delivery system. Impressively, these formulations surpassed the performance of standard LNPs, exhibiting up to 14-fold higher reporter gene expression in vitro and in vivo in zebrafish larvae and mice. Further investigations highlighted the potential of ionizable cationic lipid modifications to boost the performance of LNPs. The improvements of these formulations were primarily linked to enhancements in cellular uptake and intracellular processing, especially endosomal escape.
In the second part, aiming to bridge the gap between in vitro and in vivo, an endosomal escape reporter cell line, zebrafish larvae, and intravital imaging were employed to assess the behavior and performance of various nanomedicines. The insights gained from these experiments were invaluable, helping to determine certain in vitro to in vivo prediction aspects. Notably, the in vitro endosomal escape capability of LNPs and their biodistribution and circulation behavior in zebrafish larvae have emerged as important indicators of their in vivo behavior and performance in rodents.
In conclusion, modifying the lipid composition of LNPs significantly improves their performance with respect to efficacy and potency. However, persisting challenges related to cell specificity and cellular uptake emphasize the potential of active targeting strategies, as indicated by preliminary studies. Ultimately, the combination of lipid composition modifications, active targeting strategies, and both in vitro and in vivo models, will serve as a firm foundation for the design of efficacious and potent LNP-based gene therapies