Itch est une ligase de l’ubiquitine impliquée dans la reconnaissance et la dégradation des protéines par le protéasome. Itch contient trois sites phosphorylés par JNK et il a été démontré que la phosphorylation de ces résidus est nécessaire pour que Itch puisse reconnaître et ubiquityler les protéines c-Jun et JunB. Ces sites de phosphorylation se retrouvent dans le domaine PRD responsable des interactions de Itch avec les protéines à domaine SH3. Si la phosphorylation de Itch par JNK est importante pour réguler son activité avec c-Jun et JunB, on connaît peu de choses sur les interactions de Itch avec les protéines à domaine SH3 ainsi que l’implication de la phosphorylation dans leur régulation. Nous avons donc créé des mutants de Itch par mutagenèse dirigée où les sites de phosphorylation étaient remplacés par des alanines (mutant non phosphorylable) et où l’un des trois sites était remplacé par un acide aspartique (mutant constitutivement phosphorylé). Ces mutants sont utilisés dans des tests d’interaction et d’ubiquitylation, dans le but de déterminer l’impact de la phosphorylation de Itch dans la reconnaissance et l’ubiquitylation des protéines SH3. Nos résultats montrent que, contrairement au modèle proposé, la phosphorylation de Itch n’est pas essentielle à l’interaction de Itch avec l’endophiline, mais la phosphorylation de Itch module l’ubiquitylation ainsi que la dégradation de l’endophiline. La régulation de l’interaction de Itch avec ses substrats est donc différente selon le substrat. / Itch is an ubiquitin ligase involved in protein recognition and degradation by the proteasome. Itch has three phosphorylation sites targeted by JNK. These sites overlap a small proline rich domain responsible for Itch binding to SH3 domain proteins. Phosphorylation of Itch is important for Itch interaction with c-Jun and JunB. However, little is known about Itch interaction with SH3 proteins and the impact of phosphorylation on Itch ability to recognize and ubiquitinate SH3 proteins. We created a phosphomimic mutant of Itch and a mutant of Itch that cannot be phosphorylated by JNK. We tested these mutants in interaction and ubiquitination assays to determine their effect on Itch ability to bind and ubiquitinate Endophilin, an SH3 domain protein. Phosphorylation is not a prerequisite for Itch binding to Endophilin but phosphorylation of Itch modulates Itch ability to ubiquitinate Endophilin. Phosphorylation of Itch also modulates Endophilin fate, as phosphomimic Itch induces degradation of Endophilin compared to non-phosphorylated Itch. These results show that phosphorylation regulates Itch activity towards differents substrates in different ways.

Bakgrund: Klåda nämns vanligen i samma andetag som hudbesvär och har sedan länge fått stå i skuggan av begreppet smärta. Betydande forskning rörande klådans bakomliggande fysiologi har dock givit detta ämne ett uppsving och även distingerat det från smärta. Klåda är mer än ett trivialt symptom i samband med hudsjukdom och kan även vara ett tidigt tecken på många allvarliga sjukdomar, däribland HIV och cancer. Således kan klåda vara en källa till stor ohälsa i samhället, då den leder till lidande och sänkt livskvalitet – något som ofta underskattas av vårdpersonal. Sjuksköterskans attityd rörande detta är dock till synes outforskat, varför denna studie ämnade att undersöka detta. Syfte: Denna studies syfte var att undersöka attityd och kunskap rörande begreppet klåda hos sjuksköterskor verksamma vid vårdcentraler. Metod: Empirisk tvärsnittsstudie med enkät som instrument och deskriptiv statistik som dataanalys. Urvalet bestod av 31 sjuksköterskor från fem offentliga vårdcentraler i södra delen av Region Skåne. Resultat: Enkäten visade att patienter med klåda tas på allvar och att 14/31 informanter mer än tre gånger/månad träffar på patienter som primärt söker vård för detta besvär. Kunskapen om ämnet är i högre utsträckning ett resultat av praktisk erfarenhet, än lärdom från sjuksköterskeutbildningen. På vårdcentralen diskuterades dock inte området i någon större utsträckning, men majoriteten menade ändå att klåda som omvårdnadsområde förtjänar mer uppmärksamhet. Konklusion: Resultatet av denna enkätstudie gav en inblick i sjuksköterskors attityder gentemot klåda, vilket till synes är ett nytt perspektiv. Dock kvarstår fortfarande många frågetecken och forskningsområdet behöver utvidgas med både kvalitativa och kvantitativa studier för att fullt kunna förstå klådans komplexitet. / Background: Itch is often mentioned in relation to skin disorders and throughout the years it has received less attention in comparison to pain. However, new and important research regarding physiological mechanisms behind itch has given the subject a renewed importance and has even distinguished it from pain. Itch is more than a trivial symptom related to skin diseases and may even be an early warning sign of many serious conditions, including HIV and cancer. Accordingly itch is a major health problem in the society, causing suffering and a reduced quality of life –which is often underestimated by healthcare staff. Nurses’ attitudes regarding the subject remain unexplored and was thus the intentions of this thesis. Aim: The aim of this study was to investigate the attitudes towards itch among nurses at healthcare centers. Methods: Empirical cross-sectional study with a questionnaire as a tool and descriptive statistics as the data analysis. The sample consisted of 31 nurses from five public healthcare centers in the southern part of Skåne, Sweden. Results: The questionnaire indicated that patients with itch are taken seriously and that 14/31 respondents encounter patients who primarily seek treatment for the symptom more than three times per month. The knowledge obtained in regards to itch was largely due to practical experience and not to nursing education. Itch was not frequently discussed at the healthcare center, but the majority still agreed that itch, in relation to the science of nursing care, deserves more attention. Conclusion: The results of this questionnaire study gave insight into the attitudes among nurses in regards to itch, which was found to be a new perspective. Nevertheless, many questions remain unanswered and the research needs to be enlarged with both qualitative and quantitative studies in order to fully understand the complexity of itch.

Background: Measuring patient-reported outcomes is crucial to fully capture the burden of chronic hand eczema (CHE). - Objectives: To assess the effect of delgocitinib cream on itch, pain and nine additional key signs and symptoms reported by patients with CHE using the Hand Eczema Symptom Diary (HESD). - Methods: In a double-blind, phase IIb dose-ranging trial (NCT03683719), 258 adults with mild to severe CHE were randomized to delgocitinib cream 1, 3, 8 or 20 mg/g or cream vehicle twice daily for 16 weeks. Patients assessed 11 signs and symptoms of CHE daily through the HESD using an 11-point numeric rating scale; this was an exploratory endpoint. - Results: Delgocitinib cream 20 mg/g was associated with an early and sustained reduction in itch and pain, along with clinically relevant reductions of ≥4 points from baseline to Week 16 in 48.4% and 63.6% of patients, respectively (17.9% and 5.9% with cream vehicle). There were improvements versus cream vehicle in all assessed CHE signs and symptoms (20 mg/g, p < 0.05). - Conclusions: Delgocitinib cream reduced itch, pain and other signs and symptoms in patients with CHE. This data correlated with clinician-reported outcomes, indicating that the HESD may be a useful assessment tool for CHE management.

Itch est la seule ligase de l'ubiquitine de type C2-WW-HECT capable d'interagir avec les protéines à domaine SH3. Ce domaine est particulièrement représenté parmi les protéines régulatrices de l'endocytose. Les travaux présentés ici visaient à examiner la capacité d'Itch à interagir avec plusieurs protéines endocytiques. Nous avons utilisé la technique du BRET (Bioluminescence Resonance Energy Transfer) pour examiner quelques protéines candidates. Nous avons ensuite confirmé les résultats obtenus par BRET avec des tests d'interaction in vitro, puis déterminé la capacité d'Itch à ubiquityler les protéines liées via leurs domaines SH3. Nous avons ainsi découvert deux nouveaux partenaires d'interaction et substrats d'Itch parmi les protéines endocytiques, amphyphisine et pacsine. De plus, Itch interagit avec les domaines SH3 isolés d'intersectine, mais pas avec la protéine complète, suggérant que cette dernière n'est pas un substrat d'Itch. Itch est donc bien positionnée pour exercer un rôle régulateur de l'endocytose en ubiquitylant ses substrats. / Itch is the only C2-WW-HECT type ubiquitin ligase that can bind SH3 domain proteins. This domain is particularly frequent in accessory endocytic proteins. We have used Bioluminescent Resonance Energy Transfer to examine a few candidate endocytic proteins, in addition to the already known substrate of Itch, endophilin. We then used standard in vitro techniques to confirm these interactions, and tested Itch capacity to ubiquitylate these putative substrate proteins. We thus discovered two new substrates of Itch, amphiphysin and pacsin. We also determined that although Itch interacts with the isolated SH3 domains of intersectin, it does not recognize the full length protein, thus rulling out Intersectin as a substrate of Itch. Itch is thus a putatively important regulator of endocytosis, through its capacity to recognize and ubiquitylate several SH3-domain proteins.

Survival after severe burns has improved in recent decades, but there is limited information on the course of recovery after surviving a burn and on factors that can affect recovery. The aims of this thesis were to investigate the occurrence of physical and psychological problems after burns, and to examine the consequences of psychological problems for the clinical management of burn patients. Three groups of consecutive patients who were treated at the Burn Unit at Uppsala University Hospital between 1980 and 2005 were included in the studies. The Burn Specific Health Scale (BSHS) was used for self-report of burn-specific aspects of health. Personality traits and coping strategies as psychological factors during recovery were examined with the Swedish universities Scales of Personality (SSP) and the Coping with Burns Questionnaire (CBQ). Presence of symptoms of posttraumatic stress were assessed with the Impact of Event Scale-Revised (IES-R), and the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) was used to determine the concurrent validity of the IES-R as a measure of Posttraumatic Stress Disorder (PTSD). Furthermore, the effect of pre-injury psychiatric morbidity on perceived health one year after injury was assessed. Both pruritus and nightmares were common problems after burns; 59% of the individuals in the study reported pruritus and 43% reported nightmares. Neuroticism-related personality traits and avoidant coping strategies were associated with an increased risk of having pruritus or nightmares. The presence of nightmares could be used as a screening tool for high scores in the IES-R. The IES-R was in turn shown to be a good, although overly inclusive, test for the diagnosis of PTSD. Pre-injury psychiatric morbidity predicted perceived outcome in six out of nine burn-specific health domains. These studies show that psychological factors and psychiatric morbidity affect outcome after burns.

Vaginismus is a sexual pain disorder, where spasm of musculature of the outer third of the vagina interferes with intercourse. Vaginismus exists in two forms: total vaginismus, where intercourse is impossible, and the more seldom described partial vaginismus, in which intercourse is possible but painful. The aim of the thesis was to develop a useful definition of partial vaginismus for both clinical and scientific purposes; to describe the prevalence of partial vaginismus among women with superficial coital pain; to report on symptoms and clinical findings in women with partial vaginismus; and to present treatment results for women with vaginismus. In a clinical sample of 224 women with superficial coital pain, we found a great overlap of the clinical diagnoses of partial vaginismus (PaV) and vulvar vestibulitis (VVS) (nowadays called provoked vestibulodynia); 102 women had both PaV and VVS. All women with VVS had vaginismus. Partial vaginismus was more common in all our samples than total vaginismus. sEMG of pelvic floor muscles was found to be of no value in distinguishing women with partial vaginismus with or without vulvar vestibulitis (PaV+/-VVS) (n=47) from each other or from an asymptomatic group (n=27). Women with PaV+/-VVS (n=53) reported not only burning pain but also itch during a standardized penetration situation (sEMG of pelvic floor muscles), while asymptomatic women (n=27) did not. In most cases, the appearance of burning pain preceded the appearance of itch. In a retrospective interview study, 24 women with PaV+/-VVS reported pain after intercourse more often than pain during penetration at the onset of the problem. When the women ceased having intercourse, both symptoms were equally common. Intensity of pain during penetration increased dramatically from very low at onset of the problem to very high when the women ceased having intercourse, while intensity of pain after intercourse was already high at onset of the problem and increased to very high when the women ceased having intercourse. Pain after intercourse in women with PaV+/-VVS was described as burning and/or smarting and lasted in mean for two hours, while pain during penetration was described with words like sharp/incisive/bursting and lasted for one minute. At long-term follow-up (more than three years) of a group of women treated with cognitive behaviour therapy for vaginismus (n=59, response rate 44/59 on a questionnaire), a majority were able to have and enjoy intercourse. The proportion of women with positive treatment outcome was, however, associated to the definition of treatment outcome. An ability to have intercourse at end of therapy was maintained at follow-up. Every tenth women with vaginismus healed spontaneously after thorough assessment. Conclusion: Partial vaginismus was more common in our studies than total vaginismus, and all women with vulvar vestibulitis had partial vaginismus. Women with PaV+/-VVS reported not only burning pain during standardized penetration but also itch. When the problem started in women with PaV+/-VVS, pain after intercourse was more common than pain during penetration. Pain after intercourse was described as longlasting and burning and/or smarting, while pain during penetration was described as short and sharp/incisive/bursting. Long-term follow-up results of a series of women treated with CBT for vaginismus show good treatment outcome.

Itch est la seule ligase de l'ubiquitine de type C2-WW-HECT capable d'interagir avec les protéines à domaine SH3. Ce domaine est particulièrement représenté parmi les protéines régulatrices de l'endocytose. Les travaux présentés ici visaient à examiner la capacité d'Itch à interagir avec plusieurs protéines endocytiques. Nous avons utilisé la technique du BRET (Bioluminescence Resonance Energy Transfer) pour examiner quelques protéines candidates. Nous avons ensuite confirmé les résultats obtenus par BRET avec des tests d'interaction in vitro, puis déterminé la capacité d'Itch à ubiquityler les protéines liées via leurs domaines SH3. Nous avons ainsi découvert deux nouveaux partenaires d'interaction et substrats d'Itch parmi les protéines endocytiques, amphyphisine et pacsine. De plus, Itch interagit avec les domaines SH3 isolés d'intersectine, mais pas avec la protéine complète, suggérant que cette dernière n'est pas un substrat d'Itch. Itch est donc bien positionnée pour exercer un rôle régulateur de l'endocytose en ubiquitylant ses substrats. / Itch is the only C2-WW-HECT type ubiquitin ligase that can bind SH3 domain proteins. This domain is particularly frequent in accessory endocytic proteins. We have used Bioluminescent Resonance Energy Transfer to examine a few candidate endocytic proteins, in addition to the already known substrate of Itch, endophilin. We then used standard in vitro techniques to confirm these interactions, and tested Itch capacity to ubiquitylate these putative substrate proteins. We thus discovered two new substrates of Itch, amphiphysin and pacsin. We also determined that although Itch interacts with the isolated SH3 domains of intersectin, it does not recognize the full length protein, thus rulling out Intersectin as a substrate of Itch. Itch is thus a putatively important regulator of endocytosis, through its capacity to recognize and ubiquitylate several SH3-domain proteins.

ITCH est une ligase de l’ubiquitine impliquée dans différents processus cellulaires. Elle contient une région riche en prolines (PRR, proline rich region) qui lui permet de lier le domaine SH3 (Src homology 3) d’Endophiline et d’autres protéines à domaine SH3. Plusieurs de ces protéines sont impliquées dans l’endocytose clathrine-dépendante de récepteurs tel le récepteur du facteur de croissance épidermique (EGFR, epidermal growth factor receptor). Après activation, l’EGFR est internalisé dans des vésicules enrobées de Clathrine et un complexe protéique formé par CBL, CIN85 et Endophiline participe à cet évènement. ITCH lie l’ubiquitine à CBL et à Endophiline pour vraisemblablement modifier leurs fonctions, ce qui suggère un lien direct entre cette ligase et l’endocytose de l’EGFR. Afin de déterminer le rôle d’ITCH dans ce processus, plusieurs expériences furent réalisées. Premièrement, la modalité de liaison entre la ligase ITCH et les protéines à domaine SH3 a été étudiée en détails. Une série de mutations dans la région PRR d’ITCH nous a aidé à identifier trois résidus arginines (R252, R255, R258) nécessaires pour son interaction avec Endophiline et d’autres protéines à domaine SH3. Deuxièmement, des lignées cellulaires HeLa et Cos7 furent modifiées génétiquement par CRISPR pour empêcher l’expression d’ITCH. Ces lignées cellulaires knockout furent caractérisées et utilisées dans un essai d’endocytose de l’EGFR, puis examinées par spectrométrie de masse. L’internalisation d’EGFR fut suivie en microscopie confocale à l’aide d’un ligand EGF fluorescent -/- dans les deux types de cellules ITCH . En absence d’ITCH, nous observons une diminution significative du niveau d’EGF internalisé par rapport aux cellules parentales. La surexpression d’ITCH dans les cellules ITCH-/- rétablit l’internalisation normale de l’EGF, confirmant l’implication d’ITCH dans le processus, mais la surexpression des formes mutantes de ITCH incapable de lier Endophiline ou catalytiquement inactive ne rétablit pas l’internalisation de l’EGF. Ces résultats nous permettent de conclure que l’interaction ITCH-Endophiline et la fonction catalytique de ITCH sont nécessaires pour l’endocytose de l’EGFR. Ensemble, ces deux fonctions de ITCH régulent le trafic endocytique de l’EGFR. De plus, les cellules ITCH-/- montrent un délai de dégradation de l’EGFR phosphorylé ainsi qu’une prolongation du temps d’activation de la kinase MAPK (mitogen-activated protein kinase). Finalement, pour explorer l’influence de l’absence d’ITCH sur ses substrats et partenaires moléculaires nous avons effectué une comparaison protéomique des partenaires d’interaction et des protéines ubiquitylées à partir des lysats cellulaires ITCH-/- et WT. Les résultats ont montré que le manque d’expression de la ligase ITCH altère la présence peptidique des protéines liées à la signalisation de l’EGFR, à la voie protéolytique dépendante de l'ubiquitine et à l’adhésion cellulaire. Cette étude révèle pour une première fois que la protéine ITCH est requise pour l’endocytose dépendante de la Clathrine de l’EGFR. / ITCH is a ubiquitin ligase involved in various cellular processes including endocytosis. It contains a proline rich region (PRR) which allows it to bind the SH3 domain of endophilin and other SH3 domain-containing proteins involved in Clathrin-mediated endocytosis (CME). CME is an important regulatory mechanism for growth factor receptor activity. The epidermal growth factor receptor (EGFR) is actively internalized in Clathrin-coated vesicles after activation. This endocytosis is facilitated by a protein complex formed by CBL, CIN85 and Endophilin. ITCH is known to ubiquitinate both CBL and endophilin, providing a potential functional link between the ligase and receptor internalization. In order to determine the role of ITCH in this process, several experiments were performed. First, the mapping of molecular binding sites between the ligase ITCH and SH3 domain proteins has been studied in detail. A series of mutations in the PRR region of ITCH helped us identify three arginine residues (R252, R255, R258) as necessary for its interaction with endophilin and all the tested SH3-domain containing proteins. Secondly, HeLa and Cos7 cell lines were genetically modified by CRISPR to prevent ITCH expression. These knockout cell lines were characterized for use in an EGFR endocytosis assay and for mass spectrometry analysis. EGFR internalization was monitored by confocal microscopy using fluorescent EGF ligand in both ITCH-/- cell types. In the absence of ITCH, a significant decrease in the level of internalized EGF compared to parental cells is visible. Overexpression of WT ITCH in the knockout cells restores normal internalization of EGF, confirming the involvement of ITCH in the process. Overexpression of Endophilin-binding defective or catalytically inactive ITCH does not restore the internalization of EGF in ITCH-/- cells. These results show that the ITCH- Endophilin interaction as well as the catalytic function of ITCH are necessary for the endocytosis of EGFR. In addition, ITCH-/- cells show a delay in the degradation of phosphorylated EGFR accompanied with an extended period of mitogen-activated protein kinase (MAPK) activation. In a last set of experiments, we explored the influence of the absence of ITCH on its substrates and molecular partners. We performed a proteomic comparison of ITCH-binding partners and potential substrates using the ITCH-/- and WT cell lysates. The results showed that the lack of ITCH expression alters the peptide count of proteins mainly related to EGFR signaling, theubiquitin-dependent proteolytic pathway and cell adhesion. This study shows for the first time that the protein ITCH is required for the clathrin-dependent endocytosis of EGFR.

MPH / Department of Public Health

No description available.