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Coming theses from other universities

  • RsbX and stress response in Listeria monocytogenes Author: Ana Henriques de Oliveira Link: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-190851 Publication date: 2022-01-07 06:00

    Listeria monocytogenes is a ubiquitous foodborne Gram-positive bacterium. Despite being mainly a soil bacterium, it can reach the food processing environment and contaminate food destined for human consumption, causing outbreaks. Because of its pathogenicity, it poses a danger for certain high-risk groups, including children, elderly, and immune-compromised people, as well as pregnant women, being capable of causing a life-threatening systemic infection known as listeriosis.

    All bacteria require an efficient transcriptional response and its fine-tuned modulation in order to survive the different stresses it encounters. This is especially true for L. monocytogenes, which presents an impressive range of stress adaptions that allows it survival in certain extreme conditions such as low temperature, low pH and high osmolarity. The alternative Sigma factor B, SigB, is responsible for the expression of the general stress response of this bacterium and plays a key role in the survival and adaption to new environments. The activation of SigB requires an intricate system of partner switching mechanisms, involving anti-sigma and anti-anti-sigma factors, triggered by a number of phosphorylation and dephosphorylation events that culminates with SigB being available to interact with RNA polymerase and lead the transcription of the general stress response regulon. At the top of this signal transduction pathway lies a large multi-protein complex, known as the stressosome. It is formed by RsbR (and its paralogs), RsbS and RsbT and is believed to function as a sensory hub for environmental stimuli. After signal detection, the stressosome proteins are phosphorylated and the complex goes through conformational changes that will ultimately allow for SigB activation. The reset of the stressosome to its pre-stress conformation, is hypothesized to be exerted by a putative phosphatase, RsbX, which most likely dephosphorylates the stressosome proteins post-stress.

    The role of RsbX in modulating the activity and conformation of the stressosome as well as in subsequent regulation of SigB activity was investigated. RsbX was shown to be required for maintaining SigB levels and activity low in non-stressed conditions as well as for proper SigB mediated stress adaptation. A ΔrsbX mutant strain was shown to have a very slight growth defect, but it also exhibited impaired motility, reduced biofilm formation, as well as a more acid resistant phenotype. The absence of RsbX was shown to alter the composition of the stressosome without drastically affecting its phosphorylation pattern. In general, RsbX was shown to play a crucial role in modulating the signal transduction pathways by preventing SigB activation under non-stressed conditions.

    Strains that acquire sigB operon mutations have been shown to have a growth advantage under certain mild stress conditions recurrent in a laboratory set. These strains were shown to outcompete the wild-type strain when grown in these conditions, demonstrating how a deficient SigB activity poses an advantage to the cell. On the other hand, and the ΔrsbX mutant strain was shown to have a growth disadvantage, since it was outcompeted by the wild-type strain when co-cultured. The data highlights the significant cost stress protection presents to this pathogen, since deploying the general stress response is a burden on cellular resources, and in its absence the cell can redirect energy for growth. In contrast, in the presence of a lethal stress (low pH) the strains with impaired SigB activity showed a reduced survival and an overall increased sensitivity to the stress. Hence demonstrating that in a more stressful condition the high cost of the general stress response regulon is outweighed by the protection benefits it confers to the cell. The importance of RsbX, which prevents unnecessary SigB activation, is even more evident. RsbX is not only critical to shut down the general stress response post-stress and subsequent recovery of homeostasis, but it also keeps SigB activity to low levels in non-stressed conditions, avoiding unwarranted gene expression and contributing to important energy saving. 

    SigB also plays an important role in the transition of L. monocytogenes from a saprophytic to a pathogenic lifestyle. Even though most of the virulence factors are under the control of PrfA, the master regulator of virulence, SigB is fundamental in the survival of the bacteria inside the host’s gastro-intestinal tract (e.g., stomach high acidity and bile salt release in the duodenum), as well as in the early stages of infection, such as internalization into not phagocytic cells. Because of the importance of SigB for virulence, we speculated if RsbX, by controlling activity of SigB, would also impact the virulence of the bacteria. The data showed somewhat contradicting results, but in general it suggests that even though the expression of the virulence genes responsible for the uptake of the bacteria are increased in a strain lacking RsbX compared with the wild-type strain, the effect on the general infectivity of this strain was either minimal or not existent at all. A reason for this could be the suggested growth defect caused by the absence of RsbX, which could also jeopardize the bacteria’s ability to efficiently grow within infected cells or organisms.

    Overall, RsbX seems to play a crucial role for L. monocytogenes, since it is responsible to maintain a very important, but extremely costly, stress protection mechanism in an inactive mode in absence of stress. Its functions span from alteration of stressosome conformation and subsequent modulation of stress response, to homeostasis recovery, motility, biofilm formation, stress survival, and even to indirect impact in the bacteria’s infectivity. This shows the diversified, but impactful range of effects RsbX seems to have for the bacterial cell.

  • Studies of Regulatory T cells with Implications for Clinical Applications Author: Marcus Bergström Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-460270 Publication date: 2022-01-04 07:13

    Regulatory T cells (Tregs) are vital for regulating immune homeostasis and for preventing immunopathology. The immunosuppressive functions of Tregs have raised hope for their potential use in clinical applications. This thesis investigates features of Treg that may be relevant for their use in clinical applications and includes the first-in-man study of Treg infusion in clinical islet transplantation. In paper I we explored the immunological profile of haemodialysis patients and young healthy individuals; haemodialysis patients are a prospective target for adoptive Treg therapy following kidney transplantation. Flowcytometric gating strategies were analyzed to optimize the isolation of Tregs. We found that both groups presented a similar Treg profile, and sorting for CD25 in combination with CD127low was preferable in terms of Treg yield and purity. In paper II we compared the effects of mTOR inhibitors Azithromycin (AZM) and Rapamycin (RAP) on in vitro Tregs cultures, as compounds that improve the quality of Treg cultures are sought. While RAP can improve the purity of Treg expansions by suppressing the proliferation of non-Treg cells, the effects of AZM on Treg expansions had not been previously studied. We found that RAP induced a FoxP3+Helios + phenotype and increased suppressive function, but may also inhibit Treg expansion. In comparison, AZM promoted a FoxP3+ phenotype, but to a lesser extent than RAP and the AZM treated Tregs are possibly less suppressive. In Paper III we performed the first-in-man study of autologous Treg infusion in clinical allogenic pancreatic islet transplantation. Patients underwent leaukapheresis from which polyclonal Tregs were purified by magnetic-activated cell sorting (MACS) and cryopreserved until transplantation. The Tregs were thawed and co-infused with pancreatic islets in the portal vein. No negative effects were seen related to the Treg infusion, regardless of cell dose. This indicates the procedure is safe and feasible. Future efficacy studies can be performed based on these results, with aim of minimizing the need for chronic immunosuppressive medication in islet transplantation. In summary, the studies included in this thesis supports the development of clinical Treg applications.

  • Ulnar fractures and ligament injuries of the wrist Author: Maria Moloney Link: http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-181707 Publication date: 2021-12-23 07:44

    Falling over on an outstretched hand with the wrist extended is a common accident that can cause a multitude of injuries in the wrist. Research has mainly focused on distal radius fractures while injuries of the distal ulna and adjacent ligaments have taken a back seat. If not treated adequately, these injuries may result in ulnar-sided wrist pain, sometimes referred to as the “black box” of hand surgery. The distal radio-ulnar joint (DRUJ) enables  forearm rotation where the radio-carpal unit rotates around the fixed ulna, stabilised most importantly by the triangular fibrocartilage complex (TFCC). Painless rotation and stability of the forearm under load is important for upper limb function. If full function is to be restored after wrist injury, it is important to treat not only injury to the radius but also those to the ulna and TFCC.

    The aims of this thesis were to evaluate the long-term results of TFCC injury repair, and contribute to our knowledge on fractures of the distal ulna, their epidemiology, radiographic classification and results of treatment.

    The long-term outcome of 47 patients with a foveal TFCC tear was evaluated by patient-rated wrist evaluation (PRWE) and radiography 20 years after open repair. The median PRWE score was 22.6 (7-48.5), and 34 % had had further surgery after the initial repair, half of these due to recurrence of DRUJ instability. Signs of osteoarthritis were seen in the radiocarpal joint in 17.5 % and in the DRUJ in 34 %. Higher PRWE scores were seen in patients with osteoarthritis or those who had undergone further surgery. Radio-carpal osteoarthritis was less frequently seen in patients who had undergone initial arthroscopy. This study highlights the importance of arthroscopy in identifying associated injuries. Twenty years after foveal TFCC tear repair patient reported outcomes were similar to those of distal radius fractures.

    Apart from fractures of the styloid process, distal ulna fractures are rare. All distal ulna fractures in the county of Östergötland 2010-2012 were identified. A total of 766 fractures were found (incidence 74/100 000 person-years), the majority of which were fractures of the ulnar styloid process(79%) usually caused by a fall from standing height. Most patients were female (76%), mean age at the time of injury was 63 years and 92 % had a concomitant distal radius fracture. Second after styloid fractures were fractures of the ulnar neck. A retrospective study of 96 patients with 97 fractures of the distal ulna (excluding the styloid) showed that 40 % were treated by internal fixation. The median PRWE score was 15, with significantly worse scores in patients with an internally fixed distal ulna fracture compared to patients not operated. When classifying these fractures according to AO/OTA 2018, transverse extra-articular fractures (2U3A2.3) had a significantly better outcome when not treated surgically, and these probably do not require internal fixation if the distal radius provides stability and alignment. 

    All 97 fractures were classified by three independent observers according to three classification systems: Biyani, AO/OTA 2007, and AO/OTA 2018. Classification was repeated after a minimum of 3 weeks. Reliability and reproducibility were calculated. The reliability was judged as fair for AO/OTA 2007, and moderate for Biyani and AO/OTA 2018. The reproducibility was moderate for all three systems. These results together with the observers´ opinion that Biyani is an easier system to use, leads us to conclude that a slightly modified Biyani-system together with improved radiological techniques that provide more information about the fracture pattern, may well improve accuracy, reliability and reproducibility.

    A better classification of distal ulna fractures could help us in the evaluation of new and existing treatments as well as providing the information necessary for designing treatment algorithm. After careful consideration, internal fixation is probably the treatment of choice for certain types of fracture, but this needs further investigation.

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