Kidney Induction: control by Notch, Wnt and GDNF/Ret signalling

The permanent mammalian kidney (metanephros) develops as a result of complex reciprocal tissue interactions between a ureteric epithelium and the renal mesenchyme. The overall goal of the research in this thesis was to gain data that will eventually help in elucidating the formation of congenital renal malformations. The experiments in my thesis aimed to reveal the mechanisms by which Notch, Wnt and GDNF/Ret signalling pathways regulate the development of functional kidney. The function of Notch pathway was studied by a transgenic mouse model, where it was shown that overactivation of Notch signalling disturbs kidney development and alters the expression of Gdnf and Ret/GFRa1. This indicates that Notch signalling interplays with GDNF/Ret in the regulation of the primary ureteric budding and its subsequent branching. The data also suggested that strict spatio-temporal regulation of these two pathways is required for determination of ureteric tip-identity, which appeared to be crucial for the branch formation. The function of Wnt signalling in the ureteric morphogenesis was studied by in vivo and in vitro methods to show that a canonical pathway is required for ureteric branching. Stabilisation and deletion of the canonical pathway mediator, b-catenin specifically in the ureteric epithelium result in renal aplasia/hypodysplasia. These defects originate from severe blockage of ureteric branching due to the disrupted Ret signalling. Consequently, ureteric tip specific markers are lost and ureteric stalk identity is expanded throughout the whole epithelium. Thus, the data demonstrates that the Wnt/b-catenin pathway plays an essential role in the patterning and branching of the ureteric epithelium. A novel in vitro method was generated and utilised in nephron induction studies to reveal the mechanisms through which nephrogenesis is induced. Transient GSK3 inhibition results in stabilisation of b-catenin in the isolated renal mesenchyme, which efficiently triggers nephron formation. Also genetic stabilisation of b-catenin specifically in the mesenchyme results in spontaneous nephrogenesis. The results show that activation of the canonical Wnt pathway is sufficient to initiate nephrogenesis, and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes. Taken together, this thesis demonstrates Notch and Wnt signalling pathways as novel regulators of ureteric branching morphogenesis, and that activation of the canonical Wnt pathway is sufficient for nephron induction. The studies also indicate that the Notch and Wnt pathways cross-talk with GDNF/Ret signalling in the patterning of ureteric epithelium.

CADASIL: molecular studies on the most common hereditary vascular dementing disorder

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia. CADASIL is a systemic disease of small and medium-sized arteries although the symptoms are almost exclusively neurological, including migraineous headache, recurrent ischemic episodes, cognitive impairment and, finally, subcortical dementia. CADASIL is caused by over 170 different mutations in the NOTCH3 gene, which encodes a receptor expressed in adults predominantly in the vascular smooth muscle cells. The function of NOTCH3 is not crucial for embryonic development but is needed after birth. NOTCH3 directs postnatal arterial maturation and helps to maintain arterial integrity. It is involved in regulation of vascular tone and in the wound healing of a vascular injury. In addition, NOTCH3 promotes cell survival by inducing expression of anti-apoptotic proteins. NOTCH3 is a membrane-spanning protein with a large extracellular domain (N3ECD) containing 34 epidermal growth factor-like (EGF) repeats and a smaller intracellular domain with six ankyrin repeats. All CADASIL mutations are located in the EGF repeats and the majority of the mutations cause gain or loss of one cysteine residue in one of these repeats leading to an odd number of cysteine residues, which in turn leads to misfolding of N3ECD. This misfolding most likely alters the maturation, targetting, degradation and/or function of the NOTCH3 receptor. CADASIL mutations do not seem to affect the canonical NOTCH3 signalling pathway. The main pathological findings are the accumulation of the NOTCH3 extracellular domain on degenerating vascular smooth muscle cells (VSMCs), accumulation of granular osmiophilic material (GOM) in the close vicinity of VSMCs as well as fibrosis and thickening of arterial walls. Narrowing of the arterial lumen and local thrombosis cause insufficient blood flow, mainly in small arteries of the cerebral white matter, resulting in tissue damage and lacunar infarcts. CADASIL is suspected in patients with a suggestive family history and clinical picture as well as characteristic white matter alterations in magnetic resonance imaging. A definitive verification of the diagnosis can be achieved by identifying a pathogenic mutation in the NOTCH3 gene or through the detection of GOM by electron microscopy. To understand the pathology underlying CADASIL, we have generated a unique set of cultured vascular smooth muscle cell (VSMC) lines from umbilical cord, placental, systemic and cerebral arteries of CADASIL patients and controls. Analyses of these VSMCs suggest that mutated NOTCH3 is misfolded, thus causing endoplasmic reticulum stress, activation of the unfolded protein response and increased production of reactive oxygen species. In addition, mutation in NOTCH3 causes alterations in actin cytoskeletal structures and protein expression, increased branching and abnormal node formation. These changes correlate with NOTCH3 expression levels within different VSMCs lines, suggesting that the phenotypic differences of SMCs may affect the vulnerability of the VSMCs and, therefore, the pathogenic impact of mutated NOTCH3 appears to vary in the arteries of different locations. Furthermore, we identified PDGFR- as an immediate downstream target gene of NOTCH3 signalling. Activation of NOTCH induces up-regulation of the PDGFR- expression in control VSMCs, whereas this up-regulation is impaired in CADASIL VSMCs and might thus serve as an alternative molecular mechanism that contributes to CADASIL pathology. In addition, we have established the congruence between NOTCH3 mutations and electron microscopic detection of GOM with a view to constructing a strategy for CADASIL diagnostics. In cases where the genetic analysis is not available or the mutation is difficult to identify, a skin biopsy is an easy-to-perform and highly reliable diagnostic method. Importantly, it is invaluable in setting guidelines concerning how far one should proceed with the genetic analyses.

Studies on the assessment of the adequacy of anesthesia

Several hypnosis monitoring systems based on the processed electroencephalogram (EEG) have been developed for use during general anesthesia. The assessment of the analgesic component (antinociception) of general anesthesia is an emerging field of research. This study investigated the interaction of hypnosis and antinociception, the association of several physiological variables with the degree of intraoperative nociception, and aspects of EEG Bispectral Index Scale (BIS) monitoring during general anesthesia. In addition, EEG features and heart rate (HR) responses during desflurane and sevoflurane anesthesia were compared. A propofol bolus of 0.7 mg/kg was more effective than an alfentanil bolus of 0.5 mg in preventing the recurrence of movement responses during uterine dilatation and curettage (D C) after a propofol-alfentanil induction, combined with nitrous oxide (N2O). HR and several HR variability-, frontal electromyography (fEMG)-, pulse plethysmography (PPG)-, and EEG-derived variables were associated with surgery-induced movement responses. Movers were discriminated from non-movers mostly by the post-stimulus values per se or normalized with respect to the pre-stimulus values. In logistic regression analysis, the best classification performance was achieved with the combination of normalized fEMG power and HR during D C (overall accuracy 81%, sensitivity 53%, specificity 95%), and with the combination of normalized fEMG-related response entropy, electrocardiography (ECG) R-to-R interval (RRI), and PPG dicrotic notch amplitude during sevoflurane anesthesia (overall accuracy 96%, sensitivity 90%, specificity 100%). ECG electrode impedances after alcohol swab skin pretreatment alone were higher than impedances of designated EEG electrodes. The BIS values registered with ECG electrodes were higher than those registered simultaneously with EEG electrodes. No significant difference in the time to home-readiness after isoflurane-N2O or sevoflurane-N2O anesthesia was found, when the administration of the volatile agent was guided by BIS monitoring. All other early and intermediate recovery parameters were also similar. Transient epileptiform EEG activity was detected in eight of 15 sevoflurane patients during a rapid increase in the inspired volatile concentration, and in none of the 16 desflurane patients. The observed transient EEG changes did not adversely affect the recovery of the patients. Following the rapid increase in the inhaled desflurane concentration, HR increased transiently, reaching its maximum in two minutes. In the sevoflurane group, the increase was slower and more subtle. In conclusion, desflurane may be a safer volatile agent than sevoflurane in patients with a lowered seizure threshold. The tachycardia induced by a rapid increase in the inspired desflurane concentration may present a risk for patients with heart disease. Designated EEG electrodes may be superior to ECG electrodes in EEG BIS monitoring. When the administration of isoflurane or sevoflurane is adjusted to maintain BIS values at 50-60 in healthy ambulatory surgery patients, the speed and quality of recovery are similar after both isoflurane-N2O and sevoflurane-N2O anesthesia. When anesthesia is maintained by the inhalation of N2O and bolus doses of propofol and alfentanil in healthy unparalyzed patients, movement responses may be best avoided by ensuring a relatively deep hypnotic level with propofol. HR/RRI, fEMG, and PPG dicrotic notch amplitude are potential indicators of nociception during anesthesia, but their performance needs to be validated in future studies. Combining information from different sources may improve the discrimination of the level of nociception.

Les fleuves dans l'œuvre romanesque de Jean-Marie Gustave Le Clézio

Relying on Merleau-Ponty's phenomenology of perception and on Mircea Eliade's works on the Sacred and the Profane, this study explores the river as a perceptual space and as the sacred Center in a cosmic vision of the world in twelve of Jean-Marie Gustave Le Clézio's fictional works, from The Interrogation (1963) to Revolutions (2003). In the first chapter, after introducing the field of study, I discuss the relation between the radical subjectivity and the evasiveness of perceiving subjects in Le Clézio's fiction. Next are some thoughts on the relation between Merleau-Ponty's and Le Clézio's ideas. The second chapter studies the river as an experience in the text, first as a topographical space, then as a sound world. The investigations move on to its water as a visual and a tactile phenomenon. Then follows the human use of the river, the (absence of) baths, and the river as a traveling space. The chapter closes with the study of the metaphorical use of the word, occurring mainly in urban space and for phenomena in the sky. The third chapter is organized around the river as the Center of the world in a religious cosmogony, where the river represents the origin of the world and of the human race. The core analysis shows how the middle of the river is a symbolic space of a new beginning. As a sacred space, the river abolishes time as the object of contemplation and as relative immobility from the point of view of a person drifting downstream. The functions of a new beginning and of abolition of time are combined in the symbolic immersions in the water. Finally, the dissertation explores other symbolical spaces, such as the unknown destination of the drift, and the river as the Center of a utopia. The chapter closes with the existential agony as a result of the elimination of the Center in the urban environment. In the final chapter, the river is compared to other watercourses : the creek, the brook and the rapids. The river is more of a spatial entity, whereas the actual water is more important in the smaller watercourses. The river is more common than the other watercourses as a topographical element in the landscape, whereas the minor watercourses invite the characters to a closer contact with their element, in immersions and in drinking their water. Finally, the work situates the rivers in a broader context of different fictional spaces in Le Clézio's text.